INDIAN PHARMACOPOEIA 2007

MONOGRAPHS

VACCINES AND IMMUNOSERA FOR HUMAN USE

General Requirements ..................................................................................................... Monographs ..................................................................................................................... Adsorbed Diphtheria, Tetanus and Hepatitis B (rDNA) Vaccine ........................................ Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component) and Haemophilus Type b Conjugate Vaccine ........................................................................ Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component) and Hepatitis B (rDNA) Vaccine ........................................................................................... Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component), Inactivated Poliomyelitis Vaccine and Haemophilus Type b Conjugate Vaccine ................. Adsorbed Diphtheria, Tetanus, Pertussis (Acellular Component) and Inactivated Poliomyelitis Vaccine ................................................................................... Adsorbed Diphtheria, Tetanus, Pertussis and Poliomyelitis (Inactivated) Vaccine .................... Adsorbed Diphtheria, Tetanus, Pertussis, Poliomyelitis (Inactivated) and Haemophilus Type b Conjugate Vaccine ........................................................................ Adsorbed Pertussis Vaccine (Acellular Component) Adsorbed Pertussis Vaccine (Acellular, Co-purified) ......................................................... .........................................................

Bacillus Calmette-Guerin Vaccine (Freeze-Dried) ............................................................... Diphtheria and Tetanus Vaccine (Adsorbed) ........................................................................ Diphtheria and Tetanus Vaccine (Adsorbed) for Adults and Adolescents .............................. Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed) ..................................................... Diphtheria, Tetanus, Pertussis (Whole Cell), Hepatitis B (rDNA) and Haemophilus Type b Conjugate Vaccine (Adsorbed) .................................................... Diphtheria, Tetanus, Pertussis (Whole Cell) Hepatitis B (rDNA) Vaccine (Adsorbed) ................................................................................................... Diphtheria, Tetanus, Pertussis (Whole Cell) and Haemophilus Type b Conjugate Vaccine (Adsorbed) ...................................................................................... Diphtheria Vaccine (Adsorbed) ........................................................................................... Haemophilus Type b Conjugate Vaccine ............................................................................. Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine (Adsorbed) ................................. Hepatitis B Vaccine (rDNA) ............................................................................................. Inactivated Hepatitis A Vaccine (Adsorbed) ....................................................................... Inactivated Hepatitis B Vaccine ...........................................................................................
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INDIAN PHARMACOPOEIA 2007

Inactivated Influenza Vaccine (Split Virion) ........................................................................... Inactivated Influenza Vaccine (Surface Antigen) .................................................................... Inactivated Influenza Vaccine (Whole Virion) .................................................................... Japanese Encephalitis Vaccine (Human) .............................................................................. Measles and Rubella Vaccine (Live) ..................................................................................... Measles Vaccine (Live) ...................................................................................................... Measles, Mumps and Rubella Vaccine (Live) ....................................................................... Meningococcal Polysaccharide Vaccine .............................................................................. Mumps Vaccine (Live) ...................................................................................................... Pertussis Vaccine ................................................................................................................ Pneumococcal Polysaccharide Vaccine ................................................................................ Poliomyelitis Vaccine (Inactivated) ...................................................................................... Poliomyelitis Vaccine, Live (Oral) ....................................................................................... Rabies Vaccine, Human ...................................................................................................... Rubella Vaccine (Live) ....................................................................................................... Tetanus Vaccine (Adsorbed) ............................................................................................... Tick-borne Encephalitis Vaccine (Inactivated) ..................................................................... Tuberculin Purified Protein Derivative ................................................................................... Typhoid (Strain Ty 21a) Vaccine, Live (Oral) .................................................................... Typhoid Polysaccharide Vaccine ......................................................................................... Typhoid Vaccine ................................................................................................................ Typhoid Vaccine (Freeze Dried) ......................................................................................... Varicella Vaccine (Live) ....................................................................................................... Viper Venom ........................................................................................................................ Yellow Fever Vaccine (Live) ...............................................................................................

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VACCINES : GENERAL REQUIREMENTS

Vaccines : General Requirements

Vaccines are preparations of antigenic substances that are administered for the purpose of inducing in the recipient a specific and active immunity against the infective agent or toxin produced by it. Vaccines may contain living micro-organisms suitably treated to attenuate their virulence but retain their antigenic potency or they may consist of pathogenic organisms which have been killed or inactivated. Some vaccines consist of antigenic fractions or substances produced by the same pathogenic organisms but rendered harmless whilst retaining their antigenic efficiency. Vaccines may be prepared from one species only or from a mixture of two or more species. Vaccines may be prepared by the method described in the individual monographs or by the general methods given below or in any other manner provided the identity of the antigens is maintained and the vaccines are free from microbial contamination and extraneous agents. Suitable adjuvants may be added during the preparation but streptomycin, penicillin or other -lactam antibiotics may not be added at any stage of manufacture or in the final vaccine. A suitable bactericide may be added to sterile and inactivated vaccines. The final products are distributed aseptically into sterile containers which are then sealed to exclude extraneous micro-organisms. Unless otherwise indicated in the monograph, the final vaccine may be filled into single dose or multiple dose containers but vaccines in multiple dose containers must invariably contain a bactericide. Bacterial Vaccines. Bacterial vaccines are either sterile suspensions of live or killed bacteria or sterile extracts of derivatives of bacteria. They may be simple vaccines prepared from one species or may be mixed vaccines prepared by blending two or more simple vaccines from different species or strains. Bacterial vaccines may be prepared from cultures grown on suitable solid or liquid media. The whole culture or parts of it may be used in preparing the vaccine. The identity, antigenic potency and purity of each bacterial culture must be carefully controlled. Vaccines containing killed organisms may be prepared by killing the organisms by chemical or physical means provided the antigenic potency of the vaccine is preserved. Vaccines containing living bacteria may be prepared from strains which are avirulent for humans but which stimulate the production of antibodies active against pathogenic strains of the same species. The final vaccines must be free from any substance known to cause toxic, allergic or other undesirable immunological reactions in humans. Bacterial vaccines are suspensions of varying degrees of opacity in colourless or slightly coloured liquids or they may 1

be freeze-dried so that the water content is not more than 3.0 per cent w/w unless otherwise stated in the individual monograph. They may be standardized in terms of interopacity units or, where appropriate, by numbers of living or killed bacteria determined by direct cell count or by viable count. Bacterial toxoids. Bacterial toxoids are toxins or material derived therefrom, the toxicity of which has been reduced to a very low level or completely eliminated by chemical or physical means without destroying their immunizing potency. The toxins are obtained from selected strains of specific micro-organisms, grown in media free from ingredients known to cause toxic, allergic or other undesirable immunological reactions in humans. Toxoids produced by the action of formaldehyde are known as formol toxoids. Bacterial toxoids may be liquid or may be prepared by adsorbing on mineral carriers such as aluminium phosphate, aluminium hydroxide or any other suitable adsorbent; the adsorbed product may be separated, washed and suspended in a saline or other appropriate solution isotonic with blood. Bacterial toxoids are clear or slightly opalescent liquids, colourless or slightly yellow. Adsorbed toxoids may be white or greyish white suspensions or pale-yellow liquids with a sediment at the bottom of the container. Freeze-dried preparations are greyish white or yellowish white powders or pellets. Viral and rickettsial vaccines. Viral and rickettsial vaccines are suspensions of viruses or rickettsiae and are prepared from infected tissues or blood obtained from artificially infected animals, from cultures in fertile eggs, or from cell or tissue culture. Viral vaccines may be live or killed and they may be freeze-dried. Live vaccines are usually prepared using attenuated strains of the specific organisms. Killed vaccines may be inactivated by suitable chemical or physical means. Mixed Vaccines. Mixed vaccines are mixtures of two or more vaccines. A suitable antibacterial substance may be added to inactivated or live viral and rickettsial vaccines provided that it has no action against the specific organisms.

Production
General provisions. Requirements for production including in-process testing are included in individual monographs. Where justified and authorized, certain tests may be omitted where it can be demonstrated, for example by validation studies, that the production process consistently ensures compliance with the test. Unless otherwise justified and authorized, vaccines are produced using a seed-lot system. The methods of preparation are designed to maintain adequate immunogenic properties, to render the preparation harmless and to prevent contamination with extraneous agents.

VACCINES : GENERAL REQUIREMENTS

IP 2007

Unless otherwise justified and authorized, in the production of a final lot of vaccine, the number of passages of a virus, or the number of subcultures of a bacterium, from the master seed lot shall not exceed that used for production of the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. Vaccines are as far as possible free from ingredients known to cause toxic, allergic or other undesirable reactions in man. Suitable additives, including stabilizers and adjuvants may be incorporated. Penicillin and streptomycin are neither used at any stage of production nor added to the final product; however, master seed lots prepared with media containing penicillin or streptomycin may, where justified and authorized, be used for production. Substrates for propagation. Substrates for propagation comply with the relevant requirements of the Pharmacopoeia or in the absence of such requirements with those of the competent authority. Processing of cell banks and subsequent cell cultures is done under aseptic conditions in an area where no other cells are being handled. Serum and trypsin used in the preparation of cell suspensions shall be shown to be free from extraneous agents. Seed lot. The strain of bacterium or virus used in a master seed lot is identified by historical records that include information on the origin of the strain and its subsequent manipulation. No micro-organism other than the seed strain shall be present in a seed lot. Culture media. Culture media are as far as possible free from ingredients known to cause toxic, allergic or other undesirable reactions in man; if inclusion of such ingredients is necessary, it shall be demonstrated that the amount present in the final lot is reduced to such a level as to render the product safe. Approved animal (but not human) serum may be used in the growth medium for cell cultures but the medium used for maintaining cell growth during virus multiplication shall not contain serum, unless otherwise stated. Cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration although it is preferable to have a medium free from antibiotics during production. Propagation and harvest. The seed cultures are propagated and harvested under defined conditions. The purity of the harvest is verified by suitable tests as defined in the monograph. Control cells. For vaccines produced in cell cultures, control cells are maintained and tested as prescribed. In order to provide a valid control, these cells must be maintained in conditions that are rigorously identical with those used for the production cell cultures, including use of the same batches of media and media changes. 2

Control eggs. For live vaccines produced in eggs, control eggs are incubated and tested as prescribed in the monograph. Purification. Where applicable, validated purification procedures may be applied. Inactivation. Inactivated vaccines are produced using a validated inactivation process whose effectiveness and consistency have been demonstrated. Where there are recognised potential contaminants of a harvest, for example in vaccines produced in eggs from healthy, non-SPF flocks, the inactivation process is also validated with respect to the potential contaminants. A test for inactivation is carried out as soon as possible after the inactivation process, unless otherwise justified and authorised. Intermediates. Where applicable, the stability of intermediates in given storage conditions shall be evaluated and a period of validity established. Final bulk. The final bulk is prepared by aseptically blending the ingredients of the vaccine. Adsorbents. Vaccines may be adsorbed on aluminium hydroxide, aluminium phosphate, calcium phosphate or other suitable adsorbent; the adsorbents are prepared in special conditions which confer the appropriate physical form and adsorptive properties. Antimicrobial preservative. A suitable antimicrobial preservative may be included in sterile and inactivated vaccines and is invariably added if these preparations are issued in multidose containers, unless otherwise stated. If an antimicrobial preservative is used, it shall be shown that it does not impair the safety or efficacy of the vaccine and its effectiveness throughout the period of validity shall be demonstrated. Final lot. For vaccines for parenteral administration, the final lot is prepared by aseptically distributing the final bulk into sterile tamper-proof containers which, after freeze-drying where applicable, are closed so as to exclude contamination. For vaccines for administration by a non-parenteral route, the final lot is prepared by distributing the final bulk under suitable conditions into sterile, tamper-proof containers. Stability. Maintenance of potency of the final lot throughout the period of validity shall be demonstrated by validation studies; the loss of potency in the recommended storage conditions is assessed and excessive loss even within the limits of acceptable potency may indicate that the vaccine is unacceptable. Degree of adsorption. During development of an adsorbed vaccine, the degree of adsorption is evaluated as part of the consistency testing. A release specification for the degree of adsorption is established in the light of results found for batches used in clinical testing. From the stability data

IP 2007

ADSORBED DIPHTHERIA, TETANUS AND HEPATITIS B (rDNA) VACCINE

generated for the vaccine it must be shown that at the end of the period of validity the degree of adsorption will not be less than for batches used in clinical testing.

Adsorbed Diphtheria, Tetanus and Hepatitis B (rDNA) Vaccine

Diphtheria, Tetanus and Hepatitis B (rDNA) Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid; tetanus formol toxoid; hepatitis B surface antigen (HBsAg); a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively. HBsAg is a component protein of hepatitis B virus; the antigen is obtained by recombinant DNA technology.

Tests
Vaccines, reconstituted where necessary, comply with the following requirements unless otherwise stated in the individual monograph. Phenol (If present) (2.3.36). Not more than 0.25 per cent w/v. Thiomersal (If present) (2.3.48). Between 0.005 per cent w/v and 0.02 per cent w/v. Free formaldehyde (If present) (2.3.20). Maximum 0.02 g/l. Aluminium (If present) (2.3.9). Not more than 1.25 mg per dose. Sterility (2.2.11). Unless otherwise stated all vaccines comply with tests for sterility, except that for living bacterial vaccines, growth of the organism from which the vaccine was prepared is permitted (sterility means abesence of becterial and fungal contaminants except where specified in the individual monograph). Abnormal toxicity (2.2.1). Unless otherwise stated, all vaccines comply with the test for abnormal toxicity, Method B. In vaccines containing phenol as preservative, the test in mice may be inappropriate. NOTE The statements given in this general chapter is intended to be read in conjunction with the monographs on the individual vaccine in this Pharmacopoeia which refer to preparations for human use; they do not necessarily apply to vaccines for use in veterinary practice. Storage. Liquid vaccines must be stored at a temperature between 2o and 8o and should not be allowed to freeze unless otherwise specified in the individual monograph. Freeze-dried preparations must be stored at temperatures below 20o or as specified in the individual monograph. At higher temperatures vaccines deteriorate rapidly. Labelling. The label states (1) for liquid vaccines, the total number of ml in the container and, for dried vaccines, the number of doses in the container; (2) unless otherwise indicated the minimum number of Units per dose or per ml or, for viral vaccines, the minimum viral titre; (3) the dose and route of administration; (4) the name and proportion of any antibacterial preservative or other auxiliary substances added to the vaccine; (5) the date after which the vaccine is not intended to be used; (6) the conditions under which it should be stored; (7) for dried vaccines, the liquid to be used for reconstitution and its volume; (8) that the vaccine should be used immediately after reconstitution; (9) unless otherwise directed, that the vaccine should be shaken well before use; (10) any contraindication to the use of the vaccine. 3

Production
General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for antisera and vaccines, and with the following test for specific toxicity of the diphtheria and tetanus components: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal dies in the second test, the vaccine does not comply with the test. The content of bacterial endotoxins in the bulk purified diphtheria toxoid and tetanus toxoid is determined to monitor the purification procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxin is less than the limit approved for the particular vaccine and in any case the contents are such that the final vaccine contains less than 100 IU per single human dose. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of

ADSORBED DIPHTHERIA, TETANUS AND HEPATITIS B (rDNA) VACCINE

IP 2007

a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed) and Hepatitis B Vaccine (rDNA). FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, tetanus toxoid and HBsAg onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the test for antimicrobial preservative and the assays for the diphtheria and tetanus components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. If an in vivo assay is used for the hepatitis B component, provided it has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine is within the limits approved for the particular preparation.

vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant is obtained. The clear supernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant liquid obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The assay or, where applicable, the electrophoretic profile, serves also to identify the hepatitis B component of the vaccine.

Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject the equivalent of 1 human dose into each rabbit. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Hepatitis B component It complies with the assay of Hepatitis B Vaccine. Labelling. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose; (2) the amount of HBsAg per single human dose; (3) the type of cells used for production of the HBsAg component; (4) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily 4

Identification
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain

IP 2007

A. D. T. P. (ACELLULAR COMPONENT) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE

suitable for reinforcing doses or for administration to adults; (5) the name and the amount of the adsorbent; (6) that the vaccine must be shaken before use; (7) that the vaccine is not to be frozen.

consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. If the vaccine is presented with the haemophilus component in a separate vial, as part of consistency studies the assays of the diphtheria, tetanus and pertussis components are carried out on a suitable number of batches of vaccine reconstituted for use. For subsequent routine control, the assays of these components may be carried out without mixing with the haemophilus component. The content of bacterial endotoxins in bulk purified diphtheria toxoid, tetanus toxoid, pertussis components and PRP conjugate is determined to monitor the purification procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxins is less than the limit approved for the particular vaccine; if the vaccine is presented with the haemophilus component in a separate container, the contents of the diphtheria, tetanus and pertussis antigens are in any case such that the final vial for these components contains less than 100 IU per single human dose. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for antisera and vaccines. During development studies and wherever revalidation is necessary, it shall be demonstrated by tests in animals that the vaccine induces a T-cell dependent B-cell immune response to PRP. Reference vaccine Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of the components The production of the components complies with the tests of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Acellular Component, Adsorbed) and Haemophilus Type b Conjugate Vaccine. FINAL BULK VACCINE Different methods of preparation may be used: a final bulk vaccine may be prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, tetanus 5

Adsorbed Diphtheria, Tetanus, Pertussis (Acellular Component) and Haemophilus Type B Conjugate Vaccine
Diphtheria, Tetanus, Pertussis (Acellular Component) and Haemophilus Type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid; tetanus formol toxoid; individually purified antigenic components of Bordetella pertussis; polyribosylribitol phosphate (PRP) covalently bound to a carrier protein; a mineral absorbent such as aluminium hydroxide or hydrated aluminium phosphate. The product may be presented with the haemophilus type b component in a separate container, the contents of which are mixed with the other components immediately before use. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties produced by expression of a genetically modified form of the corresponding gene. Pertussis toxoid is prepared from pertussis toxin by a method that renders the toxin harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. The acellular pertussis component may also contain filamentous haemagglutinin, pertactin (a 69 kDa outermembrane protein) and other defined components of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. The latter two antigens may be copurified. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended. PRP is a linear copolymer composed of repeated units of 3--D-ribofuranosyl-(11)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. The carrier protein, when conjugated to PRP, is capable of inducing a T-cell-dependent B-cell immune response to the polysaccharide.

Production
General provisions The production method shall have been shown to yield

A. D. T. P. (ACELLULAR COMPONENT) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE

IP 2007

toxoid, acellular pertussis components and PRP conjugate onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate; or 2 final bulks may be prepared and filled separately, one containing the diphtheria, tetanus and pertussis components, the other the haemophilus component, which may be freeze-dried. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality shown below and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for absence of residual pertussis toxin, irreversibility of pertussis toxoid and antimicrobial preservative and the assays have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the free formaldehyde content has been determined on the bulk purified antigens or the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine, reconstituted where applicable, is within the limits approved for the particular preparation. pH (2.4.24). The pH of the vaccine, reconstituted if necessary, is within the range approved for the particular product. Free PRP. Unbound PRP is determined after removal of the conjugate, for example by anion-exchange, size-exclusion or hydrophobic chromatography (2.4.16), ultrafiltration or other validated methods. The amount of free PRP is not greater than that approved for the particular product.

w/v solution. Maintain at 37 for about 16 h and centrifuge until a clear supernatant is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear s u p e r n a t a n t obtained as described in Identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis components are identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained as described in Identification test A reacts with a specific antisera to the pertussis components of the vaccine. D. The haemophilus component is identified by a suitable immunochemical method (2.2.14) for PRP.

Tests
If the product is presented with the haemophilus component in a separate container: the tests for absence of residual pertussis toxin, irreversibility of pertussis toxoid, aluminium, free formaldehyde, antimicrobial preservative and sterility are carried out on the container with the diphtheria, tetanus and pertussis components; the tests for PRP content, water (where applicable), sterility and pyrogens are carried out on the container with the haemophilus component. If the haemophilus component is freeze-dried, some tests may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not less than 5 histaminesensitive mice. Inject intraperitoneally into each mouse of the first group twice the single human dose of the vaccine stored at 2 to 8. Inject intraperitoneally into each mouse of the second group twice the single human dose of the vaccine incubated at 37 for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 h. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5.0 per cent of the total number of mice die following histamine challenge. 6

Identification
If the vaccine is presented with the haemophilus component in a separate vial: identification tests A, B and C are carried out using the vial containing the diphtheria, tetanus and pertussis components; identification test D is carried out on the vial containing the haemophilus components. A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent

IP 2007

A. D. T. AND P. (ACELLULAR COMPONENT) AND HEPATITIS B (rDNA) VACCINE

The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as above; the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine shows symptoms of sensitisation. PRP. Minimum 80.0 per cent of the amount of PRP stated on the label. PRP is determined either by assay of ribose (2.7.1) or phosphorus (2.7.1), by an immunochemical method (2.2.14) or by anion-exchange liquid chromatography with pulsedamperometric detection. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20 ). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Water (2.3.43). Maximum 3.0 per cent for the freeze-dried haemophilus component. Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject per kg of the rabbits mass a quantity of the vaccine equivalent to: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier; 0.1 mg of PRP for a vaccine with tetanus toxoid as carrier; 0.025 mg of PRP for a vaccine with OMP as carrier. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than the minimum potency stated on the label. Unless otherwise justified and authorised, the minimum potency stated on the label is 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis component The vaccine complies with the assay as the stated Adsorbed Pertussis Vaccine (Acellular Component). 7

Labelling. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose; (2) the names and amounts of the pertussis components per single human dose; (3) the number of micrograms of PRP per single human dose; (4) the type and nominal amount of carrier protein per single human dose; (5) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults; (6) the name and the amount of the adsorbent; (7) that the vaccine must be shaken before use; (8) that the vaccine is not to be frozen (9) where applicable, that the vaccine contains a pertussis toxinlike protein produced by genetic modification.

Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component) and Hepatitis B (rDNA) Vaccine
Diphtheria, Tetanus, Pertussis (Acellular Component) and Hepatitis B (rDNA) Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid; tetanus formol toxoid; individually purified antigenic components of Bordetella pertussis; hepatitis B surface antigen; a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively. The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties, produced by expression of a genetically modified form of the corresponding gene. Pertussis toxoid is prepared from pertussis toxin by a method that renders the latter harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. The vaccine may also contain filamentous haemagglutinin, pertactin (a 69 kDa outer-membrane protein) and other defined components of B. pertussis such as fimbrial2 and fimbrial-3 antigens. The latter 2 antigens may be copurified. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended. Hepatitis B surface antigen is a component protein of hepatitis B virus; the antigen is obtained by recombinant DNA technology.

Production
General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven

A. D. T. AND P. (ACELLULAR COMPONENT) AND HEPATITIS B (rDNA) VACCINE

IP 2007

clinical efficacy and safety in man. The content of bacterial endotoxins in the bulk purified diphtheria toxoid, tetanus toxoid and pertussis components is determined to monitor the purification procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxins is less than the limit approved for the particular vaccine. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Acellular Component, Adsorbed) and Hepatitis B Vaccine (rDNA). FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, tetanus toxoid, acellular pertussis components and hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. 8

Provided the tests for absence of residual pertussis toxin, irreversibility of pertussis toxoid and antimicrobial preservative and the assays for the diphtheria, tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. If an in vivo assay is used for the hepatitis B component, provided it has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.2.23). The osmolality of the vaccine is within the limits approved for the particular preparation.

Identification
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 h and centrifuge until a clear supernatant is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained as described in identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis components are identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained as described in identification test A reacts with a specific antisera to the pertussis components of the vaccine. D. The assay or, where applicable, the electrophoretic profile, serves also to identify the hepatitis B component of the vaccine.

Tests
Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not less than 5 histaminesensitive mice. Inject intraperitoneally into each mouse of the first group twice the single human dose of the vaccine stored at 2 to 8. Inject intraperitoneally into each mouse of the second group twice the single human dose of the vaccine incubated at 37 for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine

IP 2007

A. D. T. P. (ACELL. COMPONENT), INACTIVATED POLIOMYLITIS VACCINE AND HAEMOPHILUS TYPE B CONJUGATE

base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 h. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5 per cent of the total number of mice die following histamine challenge. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as above; the strain is suitable if more than 50.0 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine shows symptoms of sensitisation. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility ( 2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject the equivalent of 1 human dose into each rabbit. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than the minimum potency stated on the label. Unless otherwise justified and authorised, the minimum potency stated on the label is 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis component 9

The vaccine complies with the assay as stated under Adsorbed Pertussis Vaccine (Acellular Component). Hepatitis B component The vaccine complies with the assay as stated under Hepatitis B Vaccine (rDNA). Labelling. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose; (2) the names and amounts of the pertussis components per single human dose; (3) the amount of HBsAg per single human dose; (4) the type of cells used for production of the hepatitis B component; (5) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults; (6) the name and the amount of the adsorbent; (7) that the vaccine must be shaken before use; (8) that the vaccine is not to be frozen (9) where applicable, that the vaccine contains a pertussis toxin-like protein produced by genetic modification.

Adsorbed Diphtheria, Tetanus, Pertussis (Acellular Component), Inactivated Poliomyelitis Vaccine and Haemophilus Type b Conjugate Vaccine
Diphtheria, Tetanus, Pertussis (Acellular Component) and Haemophilus Type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid; tetanus formol toxoid; individually purified antigenic components of Bordetella pertussis; polyribosylribitol phosphate (PRP) covalently bound to a carrier protein; a mineral absorbent such as aluminium hydroxide or hydrated aluminium phosphate. The product may be presented with the haemophilus type b component in a separate container, the contents of which are mixed with the other components immediately before use. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties produced by expression of a genetically modified form of the corresponding gene. Pertussis toxoid is prepared from pertussis toxin by a method that renders the toxin harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. The acellular pertussis component may also contain filamentous haemagglutinin, pertactin (a 69 kDa outer-

A. D. T. P. (ACELL. COMPONENT), INACTIVATED POLIOMYLITIS VACCINE AND HAEMOPHILUS TYPE B CONJUGATE IP 2007

membrane protein) and other defined components of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. The latter 2 antigens may be co-purified. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended. PRP is a linear copolymer composed of repeated units of 3--D-ribofuranosyl-(11)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. The carrier protein, when conjugated to PRP, is capable of inducing a Tcell-dependent B-cell immune response to the polysaccharide.

Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of the components

Production
General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The content of bacterial endotoxins in bulk purified diphtheria toxoid, tetanus toxoid, pertussis components, purified, inactivated monovalent poliovirus harvests and bulk PRP conjugate is determined to monitor the purification procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxins is less than the limit approved for the particular vaccine and, in any case, the contents are such that the final vaccine contains less than 100 IU per single human dose. The production method is validated to demonstrate that the product, if tested, would comply with the following test. Inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria, toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal dies in the second test, the vaccine does not comply with the test. During development studies and wherever revalidation is necessary, it shall be demonstrated by tests in animals that the vaccine induces a T-cell dependent B-cell immune response to PRP. As part of consistency studies the assays of the diphtheria, tetanus, pertussis and poliomyelitis components are carried out on a suitable number of batches of vaccine reconstituted for use. For subsequent routine control, the assays of these components may be carried out without mixing with the haemophilus component. 10 The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Acellular Component, Adsorbed), Poliomyelitis Vaccine (Inactivated) and Haemophilus Type b Conjugate Vaccine. FINAL BULK VACCINE The final bulk of the diphtheria, tetanus, pertussis and poliomyelitis components is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, bulk purified tetanus toxoid and bulk purified acellular pertussis components onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate and admixture of suitable quantities of purified, monovalent harvests of human polioviruses 1, 2 and 3 or a suitable quantity of a trivalent pool of such monovalent harvests. Suitable antimicrobial preservatives may be added. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent. A stabiliser may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Bovine serum albumin. Determine on the poliomyelitis components by a suitable immunochemical method (2.2.14) during preparation of the final bulk vaccine, before addition of the adsorbent, the amount of bovine serum albumin is such that the content in the final vaccine will not be more than 50 ng per single human dose. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium.

IP 2007

A. D. T. P. (ACELL. COMPONENT), INACTIVATED POLIOMYLITIS VACCINE AND HAEMOPHILUS TYPE B CONJUGATE

FINAL LOT The final bulk of the haemophilus component is freeze-dried. Only a final lot that is satisfactory with respect to the test for osmolality shown below and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the test for absence of residual pertussis toxin and irreversibility of pertussis toxoid, the test for antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided that the free formaldehyde content has been determined on the bulk purified antigens and the purified monovalent harvests or the trivalent pool of polioviruses or the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Provided that the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine, reconstituted where applicable, is within the limits approved for the particular preparation. Free PRP Unbound PRP is determined on the haemophilus component after removal of the conjugate, for example by anion-exchange, size-exclusion or hydrophobic chromatography (2.4.16), ultrafiltration or other validated methods. The amount of free PRP is not greater than that approved for the particular product.

immunochemical methods (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained during identification test A reacts with specific antisera to the pertussis components of the vaccine. D. The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14), such as determination of D-antigen by enzyme-linked mmunosorbent assay (ELISA). E. The haemophilus component is identified by a suitable immunochemical method (2.2.14) for PRP.

Tests
The tests for absence of residual pertussis toxin, irreversibility of pertussis toxoid, aluminium, free formaldehyde, antimicrobial preservative and sterility are carried out on the container with the diphtheria, tetanus, pertussis and poliomyelitis components; the tests for PRP content, water, sterility and pyrogens are carried out on the container with the haemophilus component. Some tests for the haemophilus component may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not fewer than 5 histaminesensitive mice. Inject intraperitoneally into each mouse of the first group twice the single human dose of the vaccine stored at 2 to 8. Inject intraperitoneally into each mouse of the second group twice the single human dose of the vaccine incubated at 37 for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 hours. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5 per cent of the total number of mice die following histamine challenge. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as above; the 11

Identification
Identification tests A, B, C and D are carried out using the vial containing the diphtheria, tetanus, pertussis and poliomyelitis components; identification test E is carried out on the vial containing the haemophilus component. A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis components are identified by suitable

A. D. T. P. (ACELLULAR. COMPONENT) AND INACTIVATED POLIOMYLITIS VACCINE

IP 2007

strain is suitable if more than 50.0 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. PRP. Minimum 80.0 per cent of the amount of PRP stated on the label. PRP is determined either by assay of ribose ( 2.7.1) or phosphorus ( 2.7.1), by an immunochemical method ( 2.2.14) or by anion-exchange liquid chromatography with pulsedamperometric detection. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Water (2.3.43). Maximum 3.0 per cent for the haemophilus component. Sterility (2.2.11 ). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject per kg of the rabbits mass a quantity of the vaccine equivalent to: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier; 0.1 mg of PRP for a vaccine with tetanus toxoid as carrier; 0.025 mg of PRP for a vaccine with OMP as a carrier. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). Unless otherwise justified and authorised, the lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis component It complies with the assay as stated under Adsorbed Pertussis Vaccine (Acellular Component). Poliomyelitis component D-antigen content As a measure of consistency of production, determine the Dantigen content for human polioviruses 1, 2 and 3 by a suitable 12

immunochemical method (2.2.14) using a reference preparation calibrated in units of D-antigen. For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits approved for the particular product. Poliomyelitis vaccine (inactivated) reference preparation is calibrated in Units and intended for use in the assay of D-antigen. The Unit and the International Unit are equivalent. In vivo test The vaccine complies with the in vivo assay as stated under Inactivated Poliomyelitis Vaccine. Labelling. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose (2) the names and amounts of the pertussis components per single human dose; (3) the nominal amount of poliovirus of each type (1, 2 and 3), expressed in units of D-antigen per single human dose; (4) the type of cells used for production of the poliomyelitis component; (5) the number of micrograms of PRP per single human dose; (6) the type and nominal amount of carrier protein per single human dose; (7) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults; (8) the name and the amount of the adsorbent; (9) that the vaccine must be shaken before use; (10) that the vaccine is not to be frozen; (11) where applicable, that the vaccine contains a pertussis toxin-like protein produced by genetic modification.

Adsorbed Diphtheria, Tetanus, Pertussis (Acellular Component) and Inactivated Poliomyelitis Vaccine
Diphtheria, Tetanus, Pertussis (Acellular Component) and Poliomyelitis (Inactivated) Vaccine is a combined vaccine containing: diphtheria formol toxoid; tetanus formol toxoid; individually purified antigenic components of Bordetella pertussis; suitable strains of human polioviruses 1, 2 and 3 grown in suitable cell cultures and inactivated by a validated method; a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties produced by expression of a genetically modified form of the corresponding gene. Pertussis toxoid is prepared from pertussis toxin by a method that renders the toxin harmless while maintaining

IP 2007

A. D. T. P. (ACELLULAR. COMPONENT) AND INACTIVATED POLIOMYLITIS VACCINE

adequate immunogenic properties and avoiding reversion to toxin. The vaccine may also contain filamentous haemagglutinin, pertactin (a 69 kDa outer-membrane protein) and other defined components of B. pertussis such as fimbrial2 and fimbrial-3 antigens. The latter 2 antigens may be copurified. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended.

bulk purified diphtheria toxoid, tetanus toxoid, acellular pertussis components and admixture of suitable quantities of purified monovalent harvests of human polioviruses 1, 2 and 3 or a suitable quantity of a trivalent pool of such purified monovalent harvests. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Bovine serum albumin. Determine on the poliomyelitis components by a suitable immunochemical method (2.2.14) after virus harvest and before addition of the adsorbent in the preparation of the final bulk vaccine, the amount of bovine serum albumin is such that the content in the final vaccine will be not more than 50 ng per single human dose. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for absence of residual pertussis toxin, irreversibility of pertussis toxoid and antimicrobial preservative and the assays for the diphtheria, tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the free formaldehyde content has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Provided that the determination of D-antigen content has been carried out with satisfactory results during preparation of the final bulk before addition of the adsorbent, it may be omitted on the final lot. Provided that the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine is within the limits approved for the particular preparation.

Production
General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for antisera and vaccines. The content of bacterial endotoxins in bulk purified diphtheria toxoid, tetanus toxoid, pertussis components and purified, inactivated monovalent poliovirus harvests is determined to monitor the purification procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxins is less than the limit approved for the particular vaccine and, in any case, the contents are such that the final vaccine contains less than 100 IU per single human dose. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Acellular Component, Adsorbed) and Poliomyelitis Vaccine (Inactivated). FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate, separately or together, of suitable quantities of 13

Identification
A. Diphtheria toxoid is identified by a suitable immunochemical

A. D. T. P. (ACELLULAR. COMPONENT) AND INACTIVATED POLIOMYLITIS VACCINE

IP 2007

method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 h and centrifuge until a clear supernatant is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained as described in Identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis components are identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained as described in Identification test A reacts with a specific antisera to the pertussis components of the vaccine. D. The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14) such as the determination of D-antigen by enzyme-linked immunosorbent assay (ELISA).

sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine shows symptoms of sensitisation. Aluminium (2.3.9). Maximum 1.25 mg per single human dose if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than the minimum potency stated on the label. Unless otherwise justified and authorised, the minimum potency stated on the label is 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis component The vaccine complies with the assay as stated under Adsorbed Pertussis Vaccine (Acellular Component). Poliomyelitis component D-antigen content As a measure of consistency of production, determine the Dantigen content for human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14) following desorption using a reference preparation calibrated in units of D-antigen. For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits approved for the particular product. Poliomyelitis vaccine (inactivated) reference preparation is calibrated in Units and intended for use in the assay of D-antigen. The Unit and the International Unit are equivalent. In vivo test The vaccine complies with the in vivo assay as stated under Inactivated Poliomyelitis Vaccine. 14

Tests
Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not less than 5 histaminesensitive mice. Inject intraperitoneally into each mouse of the first group twice the single human dose of the vaccine stored at 2 to 8. Inject intraperitoneally into each mouse of the second group twice the single human dose of the vaccine incubated at 37 for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 hours. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5.0 per cent of the total number of mice die following histamine challenge. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as above; the strain is suitable if more than 50.0 per cent of the animals are

IP 2007

ADSORBED DIPHTHERIA TETANUS PERTUSSIS AND POLIOMYLITIS (INACTIVATED) VACCINE

Labelling. The label complies with the requirements stated under Vaccine and also states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose (2) the names and amounts of the pertussis components per single human dose; (3) the nominal amount of poliovirus of each type (1, 2 and 3), expressed in units of Dantigen per single human dose; (4) the type of cells used for production of the poliomyelitis component; (5) the number of micrograms of PRP per single human dose; (6) the type and nominal amount of carrier protein per single human dose; (7) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults; (8) the name and the amount of the adsorbent; (9) that the vaccine must be shaken before use; (10) that the vaccine is not to be frozen; (11) where applicable, that the vaccine contains a pertussis toxin-like protein produced by genetic modification.

specific causes, repeat the test once; if more than 1 animal dies in the second test, the vaccine does not comply with the test. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of the components

Adsorbed Diphtheria, Tetanus, Pertussis and Poliomyelitis (Inactivated) Vaccine

Diphtheria, Tetanus, Pertussis and Poliomyelitis (Inactivated) Vaccine (Adsorbed) is a combined vaccine containing: diphtheria formol toxoid; tetanus formol toxoid; an inactivated suspension of Bordetella pertussis; suitable strains of human polioviruses 1, 2 and 3 grown in suitable cell cultures and inactivated by a validated method; a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively.

The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Adsorbed) and Poliomyelitis Vaccine (Inactivated). FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate, separately or together, of suitable quantities of bulk purified diphtheria toxoid and bulk purified tetanus toxoid and admixture of suitable quantities of an inactivated suspension of B. pertussis and purified monovalent harvests of human polioviruses 1, 2 and 3 or a suitable quantity of a trivalent pool of such purified monovalent harvests. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Specific toxicity Use not less than 5 healthy mice each weighing between 14 and 16 g for the vaccine group and for the saline control. Use mice of the same sex or distribute males and females equally between the groups. Allow the animals access to food and water for at least 2 hours before injection and during the test. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing a quantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse of the control group with 0.5 ml of a 0.9 per cent sterile solution of sodium chloride, preferably containing the same amount of antimicrobial preservative as that injected with the vaccine. Weigh the groups of mice immediately before the injection and 72 hours and 7 days after the injection. The vaccine 15

Production
General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for antisera and vaccines, and with the following test for specific toxicity of the diphteria and tetanus components : inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from non-

ADSORBED DIPHTHERIA TETANUS PERTUSSIS AND POLIOMYLITIS (INACTIVATED) VACCINE

IP 2007

complies with the test if: (a) at the end of 72 hours the total mass of the group of vaccinated mice is not less than that preceding the injection; (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60 per cent of that per control mouse; and (c) not more than 5 per cent of the vaccinated mice die during the test. The test may be repeated and the results of the tests combined. Bovine serum albumin. Determine on the poliomyelitis components by a suitable immunochemical method (2.2.14) during preparation of the final bulk vaccine; before addition of the adsorbent, the amount of bovine serum albumin is such that the content in the final vaccine will be not more than 50 ng per single human dose. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for specific toxicity and antimicrobial preservative, and the assays for the diphtheria, tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided that the free formaldehyde content has been determined on the bulk purified antigens, the inactivated B. pertussis suspension and the purified monovalent harvests or the trivalent pool of polioviruses or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Provided that the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine is within the limits approved for the particular preparation.

reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear s u p e r n a t a n t obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The centrifugation residue obtained in identification A may be used. Other suitable methods for separating the bacteria from the adsorbent may also be used. Identify pertussis vaccine by agglutination of the bacteria from the resuspended precipitate by antisera specific to B. pertussis or by the assay of the pertussis component prescribed under Assay. D. The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14) such as the determination of D-antigen by enzyme-linked immunosorbent assay (ELISA).

Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). If the test is carried out in guinea pigs, the lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose; if the test is carried out in mice, the lower confidence limit (P = 0.95) of the estimated potency is not less than 60 IU per single human dose. Pertussis component Carry out the assay as stated under Pertussis Vaccine. The estimated potency is not less than 4 IU per single human 16

Identification
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant is obtained. The clear supernatant

IP 2007

A. D., T., P., POLIOMYLITIS (INACTIVATED) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE

dose and the lower confidence limit (P = 0.95) of the estimated potency is not less than 2 IU per single human dose. Poliomyelitis component D-antigen content As a measure of consistency of production, determine the Dantigen content for human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14) using a reference preparation calibrated in Units of D-antigen. For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits approved for the particular product. Poliomyelitis vaccine (inactivated) reference preparation is calibrated in Units and is intended for use in the assay of D-antigen. The Unit and the IU are equivalent. In vivo test The vaccine complies with the in vivo assay as stated under Poliomyelitis Vaccine (Inactivated). Labelling. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose; (2) the minimum number of International Units of pertussis vaccine per single human dose; (3) the nominal amount of poliovirus of each type (1, 2 and 3), expressed in units of D-antigen per single human dose; (4) the type of cells used for production of the poliomyelitis component; (5) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults; (6) the name and the amount of the adsorbent; (7) that the vaccine must be shaken before use; (8) that the vaccine is not to be frozen.

The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. PRP is a linear copolymer composed of repeated units of 3--D-ribofuranosyl-(11)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. The carrier protein, when conjugated to PRP, is capable of inducing a Tcell-dependent B-cell immune response to the polysaccharide.

Production
General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for antisera and vaccines, and with the following test for specific toxicity of the diphtheria and tetanus components: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal dies in the second test, the vaccine does not comply with the test. During development studies and wherever revalidation is necessary, it shall be demonstrated by tests in animals that the vaccine induces a T-cell dependent B-cell immune response to PRP. As part of consistency studies the assays of the diphtheria, tetanus, pertussis and poliomyelitis components are carried out on a suitable number of batches of vaccine reconstituted for use. For subsequent routine control, the assays of these components may be carried out without mixing with the haemophilus component. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. 17

Adsorbed Diphtheria, Tetanus, Pertussis, Poliomyelitis (Inactivated) and Haemophilus Type b Conjugate Vaccine
Diphtheria, Tetanus, Pertussis, Poliomyelitis (Inactivated) and Haemophilus Type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid; tetanus formol toxoid; an inactivated suspension of Bordetella pertussis; suitable strains of human polioviruses 1, 2 and 3 grown in suitable cell cultures and inactivated by a suitable method; polyribosylribitol phosphate (PRP) covalently bound to a carrier protein; a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. The product is presented with the haemophilus component in a separate container, the contents of which are mixed with the other components immediately before use.

A. D., T., P., POLIOMYLITIS (INACTIVATED) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE

IP 2007

The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Adsorbed), Poliomyelitis Vaccine (Inactivated) and Haemophilus Type b Conjugate Vaccine. FINAL BULK VACCINE The final bulk of the diphtheria, tetanus, pertussis and poliomyelitis components is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, and bulk purified tetanus toxoid onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate and admixture of suitable quantities of an inactivated suspension of B. pertussis and of purified, monovalent harvests of human polioviruses 1, 2 and 3 or a suitable quantity of a trivalent pool of such monovalent harvests. Suitable antimicrobial preservatives may be added. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent. A stabiliser may be added. Only final bulk that complies with the following requirements may be used in the preparation of the final lot. Specific toxicity Use not less than 5 healthy mice each weighing between 14 and 16 g, for the vaccine group and for the saline control. Use mice of the same sex or distribute males and females equally between the groups. Allow the animals access to food and water for at least 2 hours before injection and during the test. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing a quantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse of the control group with 0.5 ml of a 0.9 per cent sterile solution of sodium chloride, preferably containing the same amount of antimicrobial preservative as that injected with the vaccine. Weigh the groups of mice immediately before the injection and 72 hours and 7 days after the injection. The vaccine complies with the test if (a) at the end of 72 hours the total mass of the group of vaccinated mice is not less than that preceding the injection; (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60 per cent of that per control mouse; and (c) not more than 5 per cent of the vaccinated mice die during the test. The test may be repeated and the results of the tests combined. Bovine serum albumin. Determine on the poliomyelitis components by a suitable immunochemical method (2.2.14) during preparation of the final bulk vaccine, before addition 18

of the adsorbent, the amount of bovine serum albumin is such that the content in the final vaccine will not be more than 50 ng per single human dose. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk of the haemophilus component is freeze-dried. Only a final lot that is satisfactory with respect to the test for osmolality shown below and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for specific toxicity and antimicrobial preservative, and the assays for the diphtheria, tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided that the free formaldehyde content has been determined on the bulk purified antigens, the inactivated B. pertussis suspension and the purified monovalent harvests or the trivalent pool of polioviruses or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Provided that the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine, reconstituted where applicable, is within the limits approved for the particular preparation. Free PRP Unbound PRP is determined on the haemophilus component after removal of the conjugate, for example by anion-exchange, size-exclusion or hydrophobic chromatography (2.4.16), ultrafiltration or other validated methods. The amount of free PRP is not greater than that approved for the particular product.

Identification
Identification tests A, B, C and D are carried out using the vial containing the diphtheria, tetanus, pertussis and poliomyelitis components; identification test E is carried out on the vial containing the haemophilus component. A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain

IP 2007

A. D., T., P., POLIOMYLITIS (INACTIVATED) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE

vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear s u p e r n a t a n t obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The centrifugation residue obtained in identification A may be used. Other suitable methods for separating the bacteria from the adsorbent may also be used. Identify pertussis vaccine by agglutination of the bacteria from the resuspended precipitate by antisera specific to B. pertussis or by the assay of the pertussis component prescribed under Assay. D. The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14), such as determination of D-antigen by enzymelinked immunosorbent assay (ELISA). E. The haemophilus component is identified by a suitable immunochemical method (2.2.14) for PRP.

Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject per kg of the rabbits mass a quantity of the vaccine equivalent to 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier; 0.1 mg of PRP for a vaccine with tetanus toxoid as carrier; 0.025 mg of PRP for a vaccine with OMP as carrier. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). If the test is carried out in guinea-pigs, the lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose; if the test is carried out in mice, the lower confidence limit (P = 0.95) of the estimated potency is not less than 60 IU per single human dose. Pertussis component Carry out the assay as stated under Pertussis Vaccine. The estimated potency is not less than 4 IU per single human dose and the lower confidence limit (P = 0.95) of the estimated potency is not less than 2 IU per single human dose. Poliomyelitis component D-antigen content As a measure of consistency of production, determine the Dantigen content for human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14) using a reference preparation calibrated in Units of D-antigen. For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits approved for the particular product. Poliomyelitis vaccine (inactivated) reference preparation is calibrated in Units and intended for use in the assay of D-antigen. The Unit and the IU are equivalent. In vivo test The vaccine complies with the in vivo assay as stated under Poliomyelitis Vaccine (Inactivated). Labelling. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose; (2) the minimum number of International Units of pertussis vaccine per single human dose; (3) the nominal amount of poliovirus of each type (1, 2 and 3), expressed in 19

Tests
The tests for specific toxicity, aluminium, free formaldehyde, antimicrobial preservative and sterility are carried out on the container with diphtheria, tetanus, pertussis and poliomyelitis components; the tests for PRP content, water, sterility and pyrogens are carried out on the container with the haemophilus component. Some tests for the haemophilus component may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. PRP. Minimum 80.0 per cent of the amount of PRP stated on the label. PRP is determined either by assay of ribose (2.7.1) or phosphorus (2.7.1), by an immunochemical method (2.2.14) or by anion-exchange liquid chromatography with pulsedamperometric detection. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Water (2.3.43). Maximum 3.0 per cent for the haemophilus component.

ADSORBED PERTUSSIS VACCINE (ACELLULAR COMPONENT)

IP 2007

Units of D-antigen per single human dose; (4) the type of cells used for production of the poliomyelitis component; (5) the number of micrograms of PRP per single human dose; (6) the type and nominal amount of carrier protein per single human dose; (7) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults; (8) the name and the amount of the adsorbent; (9) that the vaccine must be shaken before use; (9) that the vaccine is not to be frozen.

individual components that comply with the following requirements; after demonstration of consistency, the tests need not be applied routinely to each batch. Adenylate cyclase. Not more than 500 ng in the equivalent of 1 dose of the final vaccine, determined by immunoblot analysis or another suitable method. Tracheal cytotoxin. Not more than 2 pmol in the equivalent of 1 dose of the final vaccine, determined by a suitable method such as a biological assay or liquid chromatography (2.4.14). Absence of residual dermonecrotic toxin. Inject intradermally into each of 3 unweaned mice, in a volume of 0.1 ml, the amount of component or antigenic fraction equivalent to 1 dose of the final vaccine. Observe for 48 hours. No dermonecrotic reaction is demonstrable. Specific properties. The components of the vaccine are analysed by one or more of the methods shown below in order to determine their identity and specific properties (activity per unit amount of protein) in comparison with reference preparations. Pertussis toxin Chinese hamster ovary (CHO) cell-clustering effect and haemagglutination as in vitro methods; lymphocytosispromoting activity, histamine-sensitising activity and insulin secretory activity as in vivo methods. The toxin shows ADPribosyl transferase activity using transducin as the acceptor. Filamentous haemagglutinin Haemagglutination and inhibition by specific antibody. Pertactin, fimbrial-2 and fimbrial-3 antigens. Reactivity with specific antibody. Pertussis toxoid The toxoid induces in animals production of antibodies capable of inhibiting all the properties of pertussis toxin. PURIFIED COMPONENTS Production of each component is based on a seed-lot system. The seed cultures from which toxin is prepared are managed to conserve or where necessary restore toxinogenicity by deliberate selection. None of the media used at any stage contains blood or blood products of human origin. Media used for the preparation of seed lots and inocula may contain blood or blood products of animal origin. Pertussis toxin and, where applicable, filamentous haemagglutinin and pertactin are purified and, after appropriate characterisation, detoxified using suitable chemical reagents, by a method that avoids reversion of the toxoid to toxin, particularly on storage or exposure to heat. Other components such as fimbrial-2 and fimbrial-3 antigens are purified either 20

Adsorbed Pertussis Vaccine (Acellular Component)

Pertussis Vaccine (Acellular Component, Adsorbed) is a preparation of individually prepared and purified antigenic components of Bordetella pertussis adsorbed on a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. The vaccine contains either pertussis toxoid or a pertussis toxin, like protein free from toxic properties, produced by expression of a genetically modified form of the corresponding gene. Pertussis toxoid is prepared from pertussis toxin by a method that renders the latter harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. The vaccine may also contain filamentous haemagglutinin, pertactin (a 69 kDa outer-membrane protein) and other defined components of B. pertussis such as fimbrial2 and fimbrial-3 antigens. The latter 2 antigens may be copurified. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended.

Production
General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. Reference vaccine A batch of vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine is preferably stabilised by a method that has been shown to have no significant effect on the assay procedure when the stabilised and non-stabilised batches are compared. CHARACTERISATION OF COMPONENTS During development of the vaccine, the production process shall be validated to demonstrate that it yields consistently

IP 2007

ADSORBED PERTUSSIS VACCINE (ACELLULAR COMPONENT)

separately or together, characterised and shown to be free from toxic substances. The purification procedure is validated to demonstrate appropriate clearance of substances used during culture or purification. The content of bacterial endotoxins is determined to monitor the purification procedure and to limit the amount in the final vaccine. The limits applied for the individual components are such that the final vaccine contains less than 100 IU per single human dose. Before detoxification, the purity of the components is determined by a suitable method such as polyacrylamide gel electrophoresis (PAGE) or liquid chromatography. SDS-PAGE or immunoblot analysis with specific monoclonal or polyclonal antibodies may be used to characterise subunits. Requirements are established for each individual product. Only purified components that comply with the following requirements may be used in the preparation of the final bulk vaccine. Sterility (2.2.11). Carry out the test for sterility using for each medium a quantity of purified component equivalent to not less than 100 doses. Absence of residual pertussis toxin This test is not necessary for the product obtained by genetic modification. Use a group of not fewer than 5 histaminesensitive mice each weighing between 18 and 26 g. Inject into each mouse the equivalent of 1 human dose intravenously or twice the human dose intraperitoneally, diluted to not more than 0.5 ml with phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin. Inject diluent into a second group of control mice. After 5 days, inject 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 hours. If no animal dies, the preparation complies with the test. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject three-fold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as above; the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. A validated test based on the clustering effect of the toxin for Chinese hamster ovary (CHO) cells may be used instead of the test on mice. Residual detoxifying agents and other reagents The content of residual detoxifying agents and other reagents is determined and shown to be below approved limits unless 21

validation of the process has demonstrated acceptable clearance. Antigen content Determine the antigen content by a suitable immunochemical method (2.2.14) and protein nitrogen by sulphuric acid digestion (2.2.30) or another suitable method. The ratio of antigen content to protein nitrogen is within the limits established for the product. FINAL BULK VACCINE The vaccine is prepared by adsorption of suitable quantities of purified components, separately or together, onto aluminium hydroxide or hydrated aluminium phosphate. A suitable antimicrobial preservative may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for absence of residual pertussis toxin and irreversibility of pertussis toxoid, antimicrobial preservative, free formaldehyde and the assay have been carried out with satisfactory results on the final bulk vaccine, these tests may be omitted on the final lot.

Identification
Subject the vaccine to a suitable desorption procedure such as the following: dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution; maintain at 37 for about 16 h and centrifuge until a clear supernatant liquid is obtained. Examined by a suitable immunochemical method (2.2.14), the clear supernatant liquid reacts with specific antisera to the components stated on the label.

Tests
Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not fewer than 5 histaminesensitive mice. Inject intraperitoneally into the first group twice

IP 2007

the single human dose of the vaccine stored at 2 to 8. Inject intraperitoneally into the second group twice the single human dose of the vaccine incubated at 37 for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 hours. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5.0 per cent of the total number of mice die following histamine challenge. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as above; the strain is suitable if more than 50.0 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Assay The capacity of the vaccine to induce the formation of specific antibodies is compared with the same capacity of a reference preparation examined in parallel; antibodies are determined using suitable immunochemical methods (2.2.14) such as enzyme-linked immunosorbent assay (ELISA). The test on mice shown below uses a three-point model but, after validation, for routine testing a single-dilution method may be used. Requirement The capacity to induce antibodies is not significantly (P = 0.95) less than that of the reference vaccine. The following test model is given as an example of a method that has been found to be satisfactory. 22

Selection and distribution of test animals Use in the test healthy mice (for example, CD1 strain) of the same stock 4 to 8 weeks old. Distribute the animals in 6 groups of a number appropriate to the requirements of the assay. Use 3 dilutions of the vaccine under examination and 3 dilutions of a reference preparation and attribute each dilution to a group of mice. Inject intraperitoneally or subcutaneously into each mouse 0.5 ml of the dilution attributed to its group. Collection of serum samples 4 to 5 weeks after vaccination, bleed the mice individually under anaesthesia. Store the sera at -20 until tested for antibody content. Antibody determination Assay the individual sera for content of specific antibodies to each component using a validated method such as the ELISA test shown below. ELISA Microtitre plates (poly(vinyl chloride) or polystyrene as appropriate for the specific antigen) are coated with the purified antigen at a concentration of 100 ng per well. After washing, unreacted sites are blocked by incubating with a solution of bovine serum albumin and then washed. Two-fold dilutions of sera from mice immunised with test or reference vaccines are made on the plates. After incubation at 22 to 25 for 1 h, the plates are washed. A suitable solution of anti-mouse IgG enzyme conjugate is added to each well and incubated at 22 to 25 for 1 h. After washing, a substrate is added from which the bound enzyme conjugate liberates a chromophore which can be quantified by measurement of absorbance. The test conditions are designed to obtain a linear response for absorbance with respect to antibody content over the range of measurement used and absorbance values within the range 0.1 to 2.0. A reference antiserum of assigned potency is used in the test and serves as the basis for calculation of the antibody levels in test sera. A standardised control serum is also included in the test. The test is not valid if (a) the value found for the control serum differs by more than 2 standard deviations from the assigned value; (b) the confidence interval of the potency estimate is greater than 50.0 per cent to 200.0 per cent. Calculation The antibody titres in the sera of mice immunised with reference and test vaccines are calculated and from the values obtained the potency of the test vaccine in relation to the reference vaccine is calculated by the usual statistical methods. Labelling. The label states (1) the names and amounts of the components present in the vaccine; (2) where applicable, that

IP 2007

ADSORBED PERTUSSIS VACCINE (ACELLULAR CO-PURIFIED)

the vaccine contains a pertussis toxin-like protein produced by genetic modification; (3) the name and amount of the adsorbent; (4) that the vaccine must be shaken before use; (5) that the vaccine is not to be frozen.

Tracheal cytotoxin. Not more than 2 pmol in the equivalent of 1 dose of the final vaccine, determined by a suitable method such as a biological assay or liquid chromatography (2.4.14). Absence of residual dermonecrotic toxin. Inject intradermally into each of 3 unweaned mice, in a volume of 0.1 ml, the amount of antigenic fraction equivalent to 1 dose of the final vaccine. Observe for 48 hours. No dermonecrotic reaction is demonstrable. Specific properties. The antigenic fraction is analyzed by one or more of the methods shown below in order to determine the identity and specific properties (activity per unit amount of protein) of its components in comparison with reference preparations. Pertussis toxin Chinese hamster ovary (CHO) cell-clustering effect and haemagglutination as in vitro methods; lymphocytosispromoting activity, histamine-sensitising activity and insulin secretory activity as in vivo methods. The toxin shows ADP-ribosyl transferase activity using transducin as the acceptor. Filamentous haemagglutinin Haemagglutination and inhibition by specific antibody. Pertactin, fimbrial-2 and fimbrial-3 antigens. Reactivity with specific antibody. Pertussis toxoid The toxoid induces in animals the production of antibodies capable of inhibiting all the properties of pertussis toxin. PURIFIED ANTIGENIC FRACTION Production of the antigenic fraction is based on a seed-lot system. The seed cultures are managed to conserve or, where necessary, restore toxinogenicity by deliberate selection. None of the media used at any stage contains blood or blood products of human origin. Media used for the preparation of seed batches and inocula may contain blood or blood products of animal origin. The antigenic fraction is purified and, after appropriate characterisation, detoxified using suitable reagents by a method that ensures minimal reversion of toxoid to toxin, particularly on or exposure to heat. The purification procedure is validated to demonstrate appropriate clearance of substances used during culture or purification. The content of bacterial endotoxins is determined to monitor the purification procedure and to limit the amount in the final vaccine. The limits applied are such that the final vaccine contains not more than 100 IU per single human dose. Before detoxification, the purity of the antigenic fraction is 23

Adsorbed Pertussis Vaccine (Acellular, Co-Purified)

Pertussis Vaccine (Acellular, Co-Purified, Adsorbed) is a preparation of antigenic components of Bordetella pertussis adsorbed on a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. The vaccine contains an antigenic fraction purified without separation of the individual components. The antigenic fraction is treated by a method that transforms pertussis toxin to toxoid, rendering it harmless while maintaining adequate immunogenic properties of all the components and avoiding reversion to toxin. The antigenic fraction is composed of pertussis toxoid, filamentous haemagglutinin, pertactin (a 69 kDa outermembrane protein) and other defined components of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. It may contain residual pertussis toxin up to a maximum level approved by the competent authority. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended.

Production
General provisions. The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. Reference vaccine. A batch of vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine is preferably stabilised, by a method that has been shown to have no significant effect on the assay procedure when the stabilised and non-stabilised batches are compared. CHARACTERISATION OF COMPONENTS During development of the vaccine, the production process shall be validated to demonstrate that it yields consistently an antigenic fraction that complies with the following requirements; after demonstration of consistency, the tests need not be applied routinely to each batch. Adenylate cyclase. Not more than 500 ng in the equivalent of 1 dose of the final vaccine, determined by immunoblot analysis or another suitable method.

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determined by a suitable method such as polyacrylamide gel electrophoresis (PAGE) (2.4.12) or liquid chromatography (2.4.14). SDS-PAGE or immunoblot analysis with specific monoclonal or polyclonal antibodies may be used to characterise subunits. Requirements are established for each individual product. Only a purified antigenic fraction that complies with the following requirements may be used in the preparation of the final bulk vaccine. Sterility (2.2.11). Carry out the test for sterility using for each medium a quantity of purified antigenic fraction equivalent to not less than 100 doses of the final vaccine. Test for residual pertussis toxin. Use 3 groups of not fewer than 5 histamine-sensitive mice each weighing between 18 and 26 g. Using phosphate-buffered saline containing 0.2 per cent of gelatin, prepare a series of dilutions of the purified antigenic fraction that have been shown to yield a graded response and attribute each dilution to a separate group of mice. Inject intraperitoneally into each mouse the dilution attributed to its group. Inject diluent into a fourth group of control mice. After 5 days, inject intraperitoneally into each mouse 1 mg of histamine base in a volume not exceeding 0.5 ml. Record the number of animals that die within 24 h of histamine challenge. Calculate the weight or volume of a preparation that sensitises 50.0 per cent of the mice injected using a suitable statistical method such as probit analysis. The residual activity of pertussis toxin does not exceed that of batches shown to be safe in clinical studies. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as described above; the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. A validated test based on the clustering effect of the toxin for Chinese hamster ovary (CHO) cells may be used instead of the test on mice. Residual detoxifying agents and other reagents. The content of residual detoxifying agents and other reagents is determined and shown to be below approved limits unless validation of the process has demonstrated acceptable clearance. Antigen content. Determine the complete quantitative antigen composition of the antigenic fraction by suitable immunochemical methods (2.2.14) and protein nitrogen by sulphuric acid digestion or another suitable method. The ratio of total antigen content to protein nitrogen is within the limits established for the product. 24

FINAL BULK VACCINE The vaccine is prepared by adsorption of a suitable quantity of the antigenic fraction onto aluminium hydroxide or hydrated aluminium phosphate. A suitable antimicrobial preservative may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for residual pertussis toxin, reversibility of toxoid, antimicrobial preservative, free formaldehyde and the assay have been carried out with satisfactory results on the final bulk vaccine, these tests may be omitted on the final lot.

Identification
Subject the vaccine to a suitable desorption procedure such as the following: dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution; maintain at 37 for about 16 h and centrifuge until a clear supernatant is obtained. Examine by a suitable immunochemical method (2.2.14), the clear supernatant reacts with specific antisera to the components in the vaccine.

Tests
Test for residual pertussis toxin. Use 3 groups of not fewer than 5 histamine-sensitive mice (see under Production) each weighing between 18 and 26 g. Using phosphate-buffered saline containing 0.2 per cent w/v of gelatin, prepare a series of dilutions of the vaccine under examination that have been shown to yield a graded response and attribute each dilution to a separate group of mice. Inject intraperitoneally into each mouse the dilution attributed to its group. Inject diluent into a fourth group of control mice. After 5 days, inject intraperitoneally into each mouse 1 mg of histamine base in a volume not exceeding 0.5 ml. Note the number of animals that die within 24 h of histamine challenge. Calculate the weight or volume of a preparation that sensitises 50 per cent of the mice injected using a suitable statistical method such as probit analysis. The residual activity of pertussis toxin does not exceed that of batches shown to be safe in clinical studies.

IP 2007

BACILLUS CALMETTE-GUERIN VACCINE (FREEZE-DRIED)

Reversibility of toxoid. Carry out the test for residual pertussis toxin described above using the vaccine incubated at 37 for 4 weeks in parallel with a sample stored at 2 to 8. The degree of reversibility does not exceed that of batches shown to be safe in clinical studies. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Sterility (2.2.11). Complies with the test for sterility. Assay The vaccine complies with the assay as stated under Adsorbed Pertussis Vaccine (Acellular Component). Labelling. The label states (1) the names and amounts of the antigenic components present in the vaccine; (2) the maximum amount of residual pertussis toxin present in the vaccine; (3) the maximum degree of reversion of toxoid to toxin during the period of validity; (4) the name and amount of the adsorbent; (5) that the vaccine must be shaken before use; (6) that the vaccine is not to be frozen.

designed that all cultures and vaccines are protected from direct sunlight and from ultraviolet light at all stages of manufacture, testing and storage. Production of the vaccine is based on a seed-lot system. The production method shall have been shown to yield consistently BCG vaccines that induce adequate sensitivity to tuberculin in man, that have acceptable protective potency in animals and are safe. The vaccine is prepared from cultures which are derived from the master seed lot by as few subcultures as possible and in any case not more than 12 subcultures e.g. If the secondary seed lot is 4 culture passages removed from the primary seed lot, the no. of passages from the secondary seed lot must not exceed 8. The capacity of the working seed lot to induce sensitivity to tuberculin in guinea-pigs is demonstrated. If a bioluminescence test or other biochemical method is used instead of viable count, the method is validated against the viable count for each stage of the process at which it is used. SEED LOT The strain used to establish the master seed lot is chosen for and maintained to preserve its stability, its capacity to sensitise man and guinea-pigs to tuberculin and to protect animals against tuberculosis, and its relative absence of pathogenicity for man and laboratory animals. The strain used shall be identified by historical records that include information on its origin and subsequent manipulation. A suitable batch of vaccine is prepared from the first working seed lot and is reserved for use as the comparison/ in-house reference vaccine. When a new working seed lot is established, a suitable test for delayed hypersensitivity in guinea-pigs is carried out on a batch of vaccine prepared from the new working seed lot; the vaccine is shown to be not significantly different in activity from the comparison vaccine. Only a working seed lot that complies with the following requirements may be used for propagation.

Bacillus Calmette-Guerin Vaccine (Freeze-Dried)

Freeze-dried BCG Vaccine is a preparation of live bacteria derived from a culture of the bacillus of Calmette and Gurin (Mycobacterium bovis BCG) capacity of which to protect against tuberculosis has been established. Vaccine complies with the requirements stated under Vaccines with the following modifications.

Identification
The bacteria in the working seed lot are identified as Mycobacterium bovis BCG using microbiological techniques, which may be supplemented by molecular biology techniques (for example, nucleic acid amplification and restrictionfragment-length polymorphism). Sterility (2.2.11). Complies with the test for sterility, carried out using 10 ml for each medium. The working seed lot complies with the test for sterility except for the presence of mycobacteria. Virulent mycobacteria Examine the working seed lot as prescribed under Tests, using 10 guinea pigs. 25

Production
General provisions The production method is validated to demonstrate that the product, if tested, would comply with the tests for safety and efficacy. BCG vaccine shall be produced by a staff consisting of healthy persons who do not work with other infectious agents; in particular they shall not work with virulent strains of Mycobacterium tuberculosis, during the course of production cycle nor shall they be exposed to a known risk of tuberculosis infection. BCG vaccine is susceptible to sunlight: the procedures for the preparation of the vaccine shall be so

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IP 2007

PROPAGATION AND HARVEST The bacteria are grown in a suitable medium for not more than 21 days by surface or submerged culture. The culture medium shall contain no substances known to cause toxic or allergic reactions in human beings or to cause the bacteria to become virulent for guinea-pigs. The culture is harvested and suspended in a sterile liquid medium that protects the viability of the vaccine as determined by a suitable method of viable count. Test for purity. Purity is checked by acid fast staining. FINAL BULK VACCINE The final bulk vaccine is prepared from a single harvest or by pooling a number of single harvests. A stabiliser may be added; if the stabiliser interferes with the determination of bacterial concentration on the final bulk vaccine, the determination is carried out before addition of the stabiliser. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Virulent mycobacteria. Examine as prescribed under Tests. Sterility (2.2.11). Complies with the test for sterility using 10 ml for each medium except for the presence of mycobacteria. Count of viable units Determine the number of viable units per ml by viable count on solid medium using a method suitable for the vaccine under examination or by determination of adenosine triphosphate by a bioluminescence reaction. Carry out the test in parallel on a reference preparation of the same strain. Bacterial concentration Determine the total bacterial concentration by a suitable method, either directly by determining the mass of the microorganisms, or indirectly by an opacity method that has been calibrated in relation to the mass of the organisms; if the bacterial concentration is determined before addition of a stabiliser, the concentration in the final bulk vaccine is established by calculation. The total bacterial concentration is within the limits approved for the particular product by National Regulatory Authority. The ratio of the count of viable units to the total bacterial concentration is not less than that approved for the particular product by National Regulatory Authority. FINAL LOT The final bulk vaccine is distributed into sterile containers and freeze-dried to a moisture content favourable to the stability of the vaccine; the containers are closed either under vacuum or under a gas that is not deleterious to the vaccine. 26

Except where the filled and closed containers are stored at a temperature of -20 or lower, the expiry date is not later than 4 years from the date of harvest. Only a final lot that complies with the following requirement for count of viable units and with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the test for virulent mycobacteria has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Provided the test for excessive dermal reactivity has been carried out with satisfactory results on the working seed lot and on 5 consecutive final lots produced from it, the test may be omitted on the final lot. Count of viable units Determine the number of viable units per ml of the reconstituted vaccine by viable count on solid medium using a method suitable for the vaccine under examination or by determination of adenosine triphosphate by a bioluminescence reaction. The ratio of the count of viable units after freezedrying to that before is not less than that approved for the particular product.

Identification
BCG vaccine is identified by microscopic examination of the bacilli in stained smears demonstrating their acid-fast property and by the characteristic appearance of colonies grown on solid medium. Alternatively, molecular biology techniques (like nucleic acid amplification) may be used.

Tests
Virulent mycobacteria If this test is satisfactory at final bulk stage it can be omitted at the final lot. Inject subcutaneously or intramuscularly into each of 6 guineapigs, each weighing between 250 and 400 g and having received no treatment likely to interfere with the test, a quantity of vaccine equivalent to at least 50 human doses. Observe the animals for at least 42 days. At the end of this period, kill the guinea-pigs and examine by autopsy for signs of infection with tuberculosis, ignoring any minor reactions at the site of injection. Animals that die during the observation period are also examined for signs of tuberculosis. The vaccine complies with the test if none of the guinea-pigs shows signs of tuberculosis and if not more than one animal dies during the observation period. If 2 animals die during this period and autopsy does not reveal signs of tuberculosis repeat the test on 6 other guinea-pigs. The vaccine complies with the test if not more than one animal dies during the 42 days following the injection and autopsy does not reveal any sign of tuberculosis.

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DIPHTHERIA AND TETANUS VACCINE (ADSORBED)

Sterility (2.2.11). The reconstituted vaccine complies with the test for sterility except for the presence of mycobacteria. Excessive dermal reactivity Use 6 healthy white or pale-coloured guinea-pigs, each weighing not less than 250 g and having received no treatment likely to interfere with the test. Inject intradermally into each guinea-pig, according to a randomised plan, 0.1 ml of the reconstituted vaccine and of 2 tenfold serial dilutions of the vaccine and identical doses of the comparison vaccine. Observe the lesions formed at the site of the injection for 4 weeks. The vaccine complies with the test if the reaction it produces is not markedly different from that produced by the comparison vaccine. Temperature stability Maintain samples of the freeze-dried vaccine at 37 for 4 weeks. Determine the number of viable units in the heated vaccine and in unheated vaccine as described below. The number of viable units in the heated vaccine is not less than 20.0 per cent of that in unheated vaccine. Water (2.3.43). Not more than 3.0 per cent, determined by the semi-micro determination of water. Assay Determine the number of viable units in the reconstituted vaccine by viable count on solid medium or using a suitable validated biochemical method for the vaccine under examination. The number is within the range stated on the label. Determine the number of viable units in the comparison vaccine in parallel. Labelling. The label states (1) the minimum and maximum number of viable units per ml in the reconstituted vaccine; (2) that the vaccine must be protected from direct sunlight; (3) that the vaccine is to be used immediately after broaching the container; (4) the age group for which the vaccine is intended; (5) the dose for each age group; (6) follow instructions as mentioned in the product insert/leaflet.

Production
General provisions Bulk purified diphtheria and tetanus toxoids The bulk purified diphtheria and tetanus toxoids are prepared as described in the monographs on Diphtheria vaccine (adsorbed) and Tetanus vaccine (adsorbed) and comply with the requirements prescribed therein. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Absence of toxin and irreversibility of toxoid Inject subcutaneously into each of 5 guinea-pigs at least 500 Lf of the non-incubated bulk purified toxoid in a volume of 1 ml, using the same buffer solution as for the final vaccine, without adsorbent. Animals that die shall be autopsied and examined for symptoms of diphtheria intoxication (red adrenals). The bulk purified toxoid shall pass the test if no guinea-pig shows symptoms of specific intoxication within six weeks of injection and if at least 80 per cent of the animals survive the test period. The guinea-pigs shall not have been used previously for experimental purposes. Alternatively, a cell-culture test system may be used; in this case, the sensitivity of the test shall have been demonstrated to be not less than that of the guinea-pig test, and the test procedures shall be approved by the National Regulatory Authority. Each bulk purified toxoid shall be tested to ensure that reversion to toxicity cannot take place on storage. The bulk purified toxoid shall be diluted in order to obtain the same concentration and chemical environment as that present in the final bulk vaccine, except for the presence of adjuvant. To determine whether reversion has occurred, diluted toxoids that have been stored at 37 for six weeks shall be tested. The test employed shall be approved by the National Regulatory Authority and should be sufficiently sensitive to detect very small amounts of toxin. No toxicity shall be detected. Intradermal tests in guinea-pigs and cell-culture tests both are considered to be suitable.

Diphtheria and Tetanus Vaccine (Adsorbed)

Diphtheria and Tetanus Vaccine (Adsorbed) is a preparation of diphtheria formol toxoid and tetanus formol toxoid adsorbed on mineral carrier. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively. The specification for individual component used in formulation is referred in the text of individual monograph. 27

Antigenic purity Not less than 1500 Lf per mg of protein nitrogen for diphtheria toxoid and not less than 1000 Lf/mg of protein nitrogen for tetanus toxoid. FINAL BULK VACICNE The final bulk vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid and tetanus toxoid onto mineral carrier such as hydrated aluminium phosphate,

DIPHTHERIA AND TETANUS VACCINE (ADSORBED)

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aluminium hydroxide; the resulting mixture is approximately isotonic with blood. Suitable antimicrobial preservatives may be added. Antimicrobial preservatives of the phenolic type must not be used. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot.

Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity, free formaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.

Identification
A. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Maintain at 37o for about 16 hours and centrifuge. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate. B. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. pH (2.4.24). 6.0 to 7.0. Specific toxicity. Use 5 normal, healthy guinea-pigs weighing between 250 and 350 g which have been maintained for at least 1 week on a uniform, unrestricted diet, and have not been previously treated with any material that will interfere with the test. Weigh the animals separately and record their weights. Inject subcutaneously into each animal 5 times the dose stated on the label. Weigh all the animals at weekly intervals for 6 weeks. None of the animals shows any symptoms of diphtheria or tetanus toxaemia or dies from diphtheria within 42 days or loses weight at the end of the test. If more than one animal dies from non-specific causes or loses weight, repeat the test. If an animal dies or loses weight in the second test, the vaccine fails the test. Assay Diphtheria toxoid Complies with the test as stated under Diphtheria Vaccine (Adsorbed). Tetanus toxoid Complies with the test as stated under Tetanus Vaccine (Adsorbed). Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Free formaldehyde (2.3.20). Maximum 0.2 g/l Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is filled and stored aseptically into sterile containers. The containers are closed so as to prevent contamination. 28

Identification
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). Dissolve in the vaccine under examination by adding sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant liquid is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate or visible floccules. B. Tetanus toxoid is identified by a suitable immuno-chemical method (2.2.14). The clear supernatant liquid obtained during test A reacts with a suitable tetanus antitoxin, giving a precipitate or visible floccules.

Tests
Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity Aluminium (2.3.9.). Not more than 1.25 mg per single human dose when hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent. pH (2.4.24). The pH of the vaccine is within the range approved for the product (6.0 to 7.0). Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the quantity stated on the label. Assay Diphtheria component Carry out one of the described methods for the assay of Diphtheria Vaccine (Adsorbed). Viz a) Intradermal challenge method, b) Lethal challenge method, c) Antibody induction method, d) Validated serological assay in guinea pigs or mice as approved by National Regulatory Authority. Tetanus component Carry out one of the described methods for the assay of Tetanus Vaccine (Adsorbed) Viz a) Antibody induction

IP 2007

DIPHTHERIA AND TETANUS VACCINE (ADSORBED) FOR ADULTS AND ADOLESCENTS

method; b) Challenge method in guinea pigs/mice; c) Validated serological assay in guinea pigs or mice as approved by National Regulatory Authority. Labelling. The label states (1) the human dose; (2) the minimum Lf units per single human dose or the minimum International Units per single human dose if potency test done by challenge method; (3) the name and the amount of the adsorbent and preservative; (4) that the vaccine must be shaken before use; (5) that the vaccine is not to be frozen.

Specific toxicity Inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal dies in the second test, the vaccine does not comply with the test. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable physicochemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for free formaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.

Diphtheria and Tetanus Vaccine (Adsorbed) for Adults and Adolescents

Diphtheria and Tetanus Vaccine (Adsorbed) for Adults and Adolescents is a preparation of diphtheria formol toxoid and tetanus formol toxoid adsorbed on a mineral carrier. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively.

Production
General provisions Bulk purified diphtheria and tetanus toxoids The bulk purified diphtheria and tetanus toxoids are prepared as described in the monographs on Diphtheria vaccine (adsorbed) and Tetanus vaccine (adsorbed) and comply with the requirements prescribed therein. FINAL BULK VACCINE The vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid and tetanus toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide. Suitable antimicrobial preservatives may be added. Certain antimicrobial preservatives, particularly those of the phenolic type, adversely affect the antigenic activity and must not be used. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot.

Identification
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. If a satisfactory result is not obtained with a vaccine adsorbed on aluminium hydroxide, carry out the test as follows. Centrifuge 15 ml of the vaccine under examination and suspend the residue in 5 ml of a freshly prepared mixture of 1 volume of a 56 g/l solution of sodium edetate and 49 volumes of a 90 g/l solution of disodium hydrogen phosphate. Maintain at 37 for not less than 6 hours and centrifuge. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain

Identification
A. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Maintain at 37o for about 16 hours and centrifuge. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate. B. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. pH (2.4.24 ). 6.0 to 7.0. 29

DIPHTHERIA, TETANUS AND PERTUSSIS VACCINE (ADSORBED)

IP 2007

vaccines, is given as an example. The clear supernatant obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate.

respectively. The Bordetella pertussis suspension is prepared by growth of suitable strains in an appropriate medium, under controlled conditions. The specification for individual component used in formulation is referred in the text of individual monograph.

Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable physicochemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. pH (2.4.24). 6.0 to 7.0. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Assay Diphtheria component Carry out the prescribed method for assay of Diphtheria Vaccine by lethal challenge method described in the assay of Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 2 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Labelling. The label states (1) the human dose; (2) the minimum number of International Units of each component per single human dose, if potency determined by challenge method or the minimum Lf units per single human dose if test done by antibody induction method; (3) the name and the amount of the adsorbent; (4) that the vaccine must be shaken before use; (5) that the vaccine is not to be frozen.

Production
General provisions The production method must be validated to demonstrate that the product if tested, would comply with the tests for safety as described under monographs of Diphtheria Vaccine, Tetanus Vaccine (Adsorbed) and Pertussis Vaccine. Bulk purified diphtheria and tetanus toxoids, bulk inactivated B. pertussis suspension The bulk purified diphtheria and tetanus toxoids and inactivated B. pertussis suspension are prepared as described in the monograph on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed) and Pertussis Vaccine respectively and comply with the respective requirements. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid and tetanus toxoid onto hydrated aluminium phosphate or aluminium hydroxide and admixture of an appropriate quantity of a suspension of inactivated B. pertussis. The B. pertussis concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 I.U. per single human dose. If two or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. Suitable antimicrobial preservatives may be added to the bulk vaccine. Antimicrobial preservatives particularly those of phenolic type which affect the antigenic activity must not be used. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 percent and not more than115.0 percent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is filled and stored aseptically into sterile containers. The containers are closed so as to prevent contamination. 30

Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed)

Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed) is a preparation of diphtheria formol toxoid, tetanus formol toxoid adsorbed on mineral carrier and a suspension of killed Bordetella pertussis organisms. The formal toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani,

IP 2007

DIPHTHERIA, TETANUS AND PERTUSSIS VACCINE (ADSORBED)

Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity of diphtheria, tetanus and pertussis components, free formaldehyde, antimicrobial preservative and the Assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.

diphtheria toxemia or tetanus, the vaccine does not comply with the test. If more than one animal dies from non-specific causes, repeat the test once; if more than one animal dies in the second test, the vaccine does not comply with the test. Pertussis component. Use not less than 10 healthy mice each weighing between 14 and 16 g for the vaccine group and for the saline control. Use mice of the same sex or distribute males and females equally between the groups. Allow the animals access to food and water for at least 2 hours before injection and during the test. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing a quantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse of the control group with 0.5 ml of a 0.9 per cent sterile solution of sodium chloride, preferably containing the same amount of antimicrobial preservative as that injected with the vaccine. Weigh the mice groups immediately before the injection and 72 hours and 7 days after the injection. The vaccine complies with the test if: (a) at the end of 72 hours the total mass of the group of vaccinated mice is not less than that preceding the injection; (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60 per cent of that per control mouse; and (c) not more than 5 per cent of vaccinated mice should die during the test. The test may be repeated and the results of the tests combined. Aluminium (2.3.9). Not more than 1.25 mg per single human dose, when hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent. pH (2.4.24). 6.0 to 7.0. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative (2.2.2 ) Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not more than115.0 per cent of the intended amount. Assay Diphtheria component Carry out one of the methods for the assay as stated under Diphtheria Vaccine (Adsorbed). Tetanus component Carry out one of the methods for the assay as stated under Tetanus Vaccine (Adsorbed). Pertussis component Carry out the assay as stated under Pertussis Vaccine. Labelling. The label states (1) in case done by challenge method the minimum number of International Units; if units (as applicable for each component) per single human dose; 31

Identification
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). Dissolve in the vaccine under examination by adding sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant is obtained; reserve the precipitate for identification test C. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immuno-chemical method (2.2.14). The clear supernatant obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis component is identified by agglutination of the bacteria from the resuspended centrifugation residue (see identification test A; other suitable methods for separating the bacteria from the adsorbent may also be used) by antisera specific to B. pertussis or by the assay of the pertussis component.

Tests
Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Each final lot shall be tested for abnormal toxicity by injecting intraperitoneally one human dose , but not more than 0.25 ml into each of the five mice weighing between 17 to 22 g and at least one human dose but not more than 1.0 ml into each of the two guinea pigs weighing between 250 and 350 g. The preparation passes the test if none of the animals dies or shows signs of ill health in 7 days following the injection. If one of the animal dies or shows the signs of ill health, repeat the test. The preparation passes the test if none of the animals in the second group dies or shows signs of ill health in the time interval specified. Specific toxicity Diphtheria and tetanus components. Inject subcutaneously five times the single human dose stated on the label into each of five healthy guinea-pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. The animals should be weighted every week and observations be made. If within 42 days of the injection any of the animals shows signs of or dies from

D., T., P., (WHOLE CELL), HEPATITIS B (rDNA) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE (ADSORBED)

IP 2007

(2) In case done by antibody induction method the minimum number of International Units per single human dose of pertussis component and minimum number of Lf of diphtheria toxoid and tetanus toxoid; (3) the name and the amount of the adsorbent and preservative; (4) that the vaccine must be shaken before use; (5) that the vaccine is not to be frozen; (6) for vaccine contained in single-dose containers where the space is too small to accommodate the full name of the vaccine, the abbreviation DTP may be used in the label and the container provided that the same code is also stated in the label on the package.

The final product contains a suitable antimicrobial preservative. The antigenic properties of the vaccine are adversely affected by the presence of certain antimicrobial preservatives particularly those of the phenolic type and some of the quaternary ammonium type and must not be used.

Production
General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. If the vaccine is presented with the haemophilus component in a separate vial, as part of consistency studies the assays of the diphtheria, tetanus, pertussis and hepatitis B are carried out on a suitable number of batches of vaccine reconstituted as for use. For subsequent routine control, the assays of these components may be carried out without mixing with the haemophilus component. The production method is validated to demonstrate that the product, if tested, would comply with the following test for specific toxicity of the diphtheria and tetanus component: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria, toxemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal shows signs of or dies in the second test, the vaccine does not comply with the test. The stability of the final lot and the relevant intermediates is evaluated using one or more indicator tests. For the haemophilus component, such tests may include determination of molecular size, determination of free PRP in the conjugate and kinetics of depolymerisation. Taking account of the results of the stability testing, release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the period of validity. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production 32

Diphtheria, Tetanus, Pertussis (Whole Cell), Hepatitis B (rDNA) and Haemophilus Type b Conjugate Vaccine (Adsorbed)
Diphtheria, Tetanus, Pertussis (Whole cell), Hepatitis B (rDNA) and Haemophilus Type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of diphtheria formol toxoid containing not less than 1,500 Lf, (2.2.16) per mg of protein nitrogen, purified tetanus formol toxoid containing not less than 1,000 Lf, (2.2.16), per mg of protein nitrogen, hepatitis B surface antigen and haemophilus type b conjugated to suitable protein with a mineral adsorbent to which a suspension of killed Bordetella pertussis has been added. Mineral adsorbent is a suspension of hydrated aluminium hydroxide, aluminium phosphate or calcium phosphate, in saline solution or other appropriate solution isotonic with blood. The formol toxoids are prepared from the toxin produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively, in suitable media. The toxins are converted to toxoids by treatment with formaldehyde solution by methods which avoid reversibility of the toxoids. Hepatitis B surface antigen is a component protein of hepatitis B virus; the antigen is obtained by recombinant DNA technology. The polysaccharide, polyribosyl ribitol phosphate, PRP is a linear copolymer composed of repeated units of 3--Dribofuranosyl-(11)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. The carrier protein, when conjugated to PRP, is capable of inducing a T-cell dependent B-cell immune response to the polysaccharide. The product may be presented with the haemophilus component in a separate container, the contents of which are mixed with the other components immediately before or during use.

IP 2007

D., T., P., (WHOLE CELL), HEPATITIS B (rDNA) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE (ADSORBED)

process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilized by a method that has been shown to have no effect on the assay procedure. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine, Hepatitis B Vaccine (rDNA) and Haemophilus Type b Conjugate Vaccine. FINAL BULK VACCINE Vaccine with all components in the same container The final bulk is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, bulk purified tetanus toxoid, bulk purified hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate, admixture of an appropriate quantity of a suspension of inactivated B. pertussis component and admixture of a suitable quantity of PRP conjugate; the resulting mixture is approximately isotonic with blood. The B. pertussis concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 IU per single human dose. If 2 or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. Suitable antimicrobial preservatives may be added. Vaccine with the haemophilus component in a separate container The final bulk is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, bulk purified tetanus toxoid, bulk purified hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate, admixture of an appropriate quantity of a suspension of inactivated B. pertussis component and admixture of a suitable quantity of PRP conjugate; the resulting mixture is approximately isotonic with blood. The B. pertussis concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 IU per single human dose. If 2 or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. The final bulk is filled separately. Suitable antimicrobial preservatives may be added. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent. A stabilizer may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. 33

Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended content. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity of diphtheria toxoid, tetanus toxoid and pertussis component and antimicrobial preservative and the assays for the diphtheria, tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. If an in vivo assay is used for the hepatitis B component, provided it has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Free PRP Unbound PRP is determined after removal of the conjugate, for example by anion exchange, size exclusion or hydrophobic chromatography (2.4.16), ultrafiltration or other validated methods. The amount of free PRP is not greater than that approved for the particular product. Osmolality (2.4.23). The osmolality of the vaccine is within the limits approved for the particular preparation. pH (2.4.24). 6.0 to 7.0. Description. Whitish turbid liquid in which the mineral carrier tends to settle down slowly on keeping.

Identification
Tests A, B, C, D and E may be omitted if test F is carried out. Test F may be omitted if tests A, B, C, D and E are carried out. A. Diphtheria toxoid. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Maintain at 37o for about 16 hours and centrifuge. Reserve the residue for test C. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate. B. Tetanus toxoid. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. C. Pertussis component. To a suspension of the residue

D., T., P., (WHOLE CELL), HEPATITIS B (rDNA) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE (ADSORBED)

IP 2007

obtained in test A in saline solution add a suitable Bordetella pertussis antiserum; agglutination indicates presence of pertussis component. D. Hepatitis B surface antigen. The suspension of the residue obtained in test A gives a positive reactions when tested by suitable in-vitro assay. E. PRP. The suspension of the residue obtained in the test A gives a positive reaction when tested by a suitable immunochemical method for PRP. F. The vaccine confers an active immunity in mice and guineapigs when administered as directed in the test for Assay.

shows signs of ill health, repeat the test. The preparation passes the test if none of the animals in the second group dies or shows signs of ill health in the time interval specified. Pyrogens (2.2.8). This test is carried out for Haemophilus influenzae type b vaccine only if Haemophilus influenzae type b vaccine is presented as separate lyophilized vial. The vaccine complies with the test for pyrogens. Inject per kg of the rabbits mass a quantity of the vaccine equivalent to: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria toxoid as carrier; 0.1 mg of PRP for a vaccine with tetanus toxoid as carrier protein; 0.025 mg of PRP for vaccine with OMP as carrier. Specific toxicity Diphtheria and tetanus components Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). Pertussis component Complies with the test as stated under Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed). Assay Diphtheria toxoid (adsorbed) Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). Tetanus toxoid (adsorbed) Complies with the test as stated under assay of Tetanus Vaccine (Adsorbed). Pertussis vaccine Complies with the test as stated under Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed). Hepatitis B surface antigen (adsorbed) Complies with the test as stated under Hepatitis B Vaccine (Adsorbed). Storage. When stored under the prescribed conditions the vaccine may be expected to retain potency for not less than 2 years from the date on which the potency test for the pertussis component was started. Labelling. The label states (1) the human dose; (2) Diphtheria and Tetanus components; (a) in case done by challenge method, the minimum number of International Units (as applicable for each component) per single human dose; (b) in case done by antibody induction, the minimum Lf units per single human dose; (3) pertussis component IU or IOU per single human dose; (4) hepatitis B component - mg HBsAg per single human dose; (5) haemophilus conjugate component 34

Tests
If the product is presented with the haemophilus component in a separate container; the tests for specific toxicity of diphtheria toxoid, tetanus toxoid and pertussis component, aluminium, free formaldehyde, antimicrobial preservative and sterility are carried out on the container with the diphtheria, tetanus, pertussis and hepatitis B components; the tests for PRP content, water (where applicable), sterility and pyrogens are carried out on the container with the haemophilus component. If the haemophilus component is freeze-dried, some tests may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. PRP. Not less than 80.0 per cent of the amount of PRP stated on the label. PRP is determined either by assay of ribose (2.7.1), or phosphorus (2.7.1), by an immunochemical method (2.2.14) or by anion exchange liquid chromatography with pulsed amperometric detection. Aluminium (2.3.9). Not more than 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Each final lot shall be tested for abnormal toxicity by injecting intraperitoneally one human dose, but not more than 0.25 ml into each of five mice weighing between 17 and 22 g and at least one human dose but not more than 1.0 ml into each of two guinea pigs weighing between 250 and 350 g. The preparation passes the test if none of the animals dies or shows signs of ill health in seven days following the injection. If one of the animals dies or

IP 2007

DIPHTHERIA TETANUS PERTUSSIS (WHOLE CELL), HEPATITIS B (rDNA) VACCINE (ADSORBED)

- mg PRP per single human dose; (6) the type and nominal amount of carrier protein per single human dose; (7) the name and amount of adsorbent and added preservative; (8) that the vaccine must be shaken before use; (9) that the vaccine is not to be frozen.

from diphtheria, toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal shows signs of or dies in the second test, the vaccine does not comply with the test. The stability of the final lot and the relevant intermediates is evaluated using one or more indicator tests. Taking account of the results of the stability testing, release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the period of validity. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine and Hepatitis B Vaccine (rDNA). FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, tetanus toxoid, pertussis components and hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended content. pH (2.4.24). 6.0 to 7.0. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity of diphtheria toxoid, tetanus toxoid and pertussis component and antimicrobial preservative and the assays for the diphtheria, 35

Diphtheria, Tetanus, Pertussis (Whole Cell) and Hepatitis B (rDNA) Vaccine (Adsorbed)
Diphtheria, Tetanus, Pertussis and Hepatitis B (rDNA) Vaccine (Adsorbed) is a combined vaccine composed of diphtheria formol toxoid containing not less than 1,500 Limes flocculationis (Lf) (2.2.16), per mg of protein nitrogen, purified tetanus formol toxoid containing not less than 1,000 Lf ( 2.2.16), per mg of protein nitrogen and hepatitis B surface antigen with a mineral adsorbent to which a suspension of killed Bordetella pertussis has been added. Mineral adsorbent is a suspension of hydrated aluminium hydroxide, aluminium phosphate or calcium phosphate in saline solution or other appropriate solution isotonic with blood. The formol toxoids are prepared from the toxin produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively, in suitable media. The toxins are converted to toxoids by treatment with formaldehyde solution by methods, which avoid reversibility of the toxoids. Hepatitis B surface antigen is a component protein of hepatitis B virus; the antigen is obtained by recombinant DNA technology. The final product contains a suitable antimicrobial preservative. The antigenic properties of the vaccine are adversely affected by the presence of certain antimicrobial preservatives particularly those of the phenolic type and some of the quaternary ammonium type and must not be used.

Production
General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the following test for specific toxicity of the diphtheria and tetanus component: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies

DIPHTHERIA TETANUS PERTUSSIS (WHOLE CELL), HEPATITIS B (rDNA) VACCINE (ADSORBED)

IP 2007

tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. If an in vivo assay is used for the hepatitis B component, provided it has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine is within the limits approved for the particular preparation. Description. Whitish turbid liquid in which the mineral carrier tends to settle down slowly on keeping.

Abnormal toxicity (2.2.1). Each final lot shall be tested for abnormal toxicity by injecting intraperitoneally one human dose, but not more than 0.25 ml into each of five mice weighing between 17 and 22 g and at least one human dose but not more than 1.0 ml into each of two guinea pigs weighing between 250 and 350 g. The preparation passes the test if none of the animals dies or shows signs of ill health in seven days following the injection. If one of the animals dies or shows signs of ill health, repeat the test. The preparation passes the test if none of the animals in the second group dies or shows signs of ill health in the time interval specified. Specific toxicity Diphtheria and tetanus components Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). Pertussis component Complies with the test as stated under Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed). Assay Diphtheria toxoid (adsorbed) Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). Tetanus toxoid (adsorbed) Complies with the test as stated under assay for Tetanus Vaccine (Adsorbed). Pertussis vaccine Complies with the test as stated under Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed). Hepatitis B surface antigen (adsorbed) Complies with the test as stated under Hepatitis B Vaccine (Adsorbed). Storage. When stored under the prescribed conditions the vaccine may be expected to retain potency for not less than 2 years from the date on which the potency test for the pertussis component was started. Labelling. The label states (1) the human dose (ml); (2) diphtheria and tetanus components, (a) in case done by challenge method, the minimum number of International Units (as applicable for each component) per single human dose and (b) in case done by antibody induction, the minimum Lf units per single human dose; (3) pertussis component - IU or IOU per single human dose; (4) hepatitis B component - mg HBsAg per single human dose; (5) the name and amount of adsorbent and added preservative; (6) that the vaccine must be shaken before use; (7) that the vaccine is not to be frozen. 36

Identification
Tests A, B, C and D may be omitted if test E is carried out. Test E may be omitted if tests A, B C and D are carried out. A. Diphtheria toxoid. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Maintain at 37o for about 16 hours and centrifuge. Reserve the residue for test C. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate. B. Tetanus toxoid. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. C. Pertussis component. To a suspension of the residue obtained in test A in saline solution add a suitable B. pertussis antiserum; agglutination indicates presence of pertussis component. D. Hepatitis B surface antigen. The suspension of the residue obtained in test A gives a positive reactions when tested by suitable in-vitro assay. E. The vaccine confers an active immunity in mice and guineapigs when administered as directed under Assay.

Tests
pH (2.4.24). 6.0 to 7.0. Aluminium (2.3.9). Not more than 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility.

IP 2007

D., T., P., (WHOLE CELL) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE (ADSORBED)

Diphtheria, Tetanus, Pertussis (Whole Cell) and Haemophilus Type b Conjugate Vaccine (Adsorbed)
Diphtheria, Tetanus, Pertussis and Haemophilus type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of diphtheria formol toxoid containing not less than 1,500 Limes flocculationis; (Lf), (2.2.16) per mg of protein nitrogen, purified tetanus formol toxoid containing not less than 1,000 Lf, (2.2.16), per mg of protein nitrogen, and Haemophilus type b conjugated to suitable protein with a mineral adsorbent to which a suspension of killed Bordetella pertussis has been added. The mineral adsorbent is a suspension of hydrated aluminium hydroxide, aluminium phosphate or calcium phosphate, in saline solution or other appropriate solution isotonic with blood. The formol toxoids are prepared from the toxin produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively in suitable media. The toxins are converted to toxoids by treatment with formaldehyde solution by methods which avoid reversibility of the toxoids. The polysaccharide, polyribosyl ribitol phosphate, PRP is a linear copolymer composed of repeated units of 3--D-ribofuranosyl-(11)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. The carrier protein, when conjugated to PRP, is capable of inducing a Tcell dependent B-cell immune response to the polysaccharide. The product may be presented with the haemophilus component in a separate container, the contents of which are mixed with the other components immediately before or during use. The final product contains a suitable antimicrobial preservative. The antigenic properties of the vaccine are adversely affected by the presence of certain antimicrobial preservatives particularly those of the phenolic type and some of the quaternary ammonium type must not be used.

The production method is validated to demonstrate that the product, if tested, would comply with the following test for specific toxicity of the diphtheria and tetanus component: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria, toxemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal shows signs of or dies in the second test, the vaccine does not comply with the test. The stability of the final lot and the relevant intermediates is evaluated using one or more indicator tests. For the haemophilus component, such tests may include determination of molecular size, determination of free PRP in the conjugate and kinetics of depolymerisation. Taking account of the results of the stability testing, release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the period of validity. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilized by a method that has been shown to have no effect on the assay procedure. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Whole Cell) and Haemophilus influenzae Type b Conjugate Vaccine. FINAL BULK VACCINE Vaccine with all components in the same container The final bulk is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, bulk purified tetanus toxoid, onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate, admixture of an appropriate quantity of a suspension of inactivated B. pertussis component and admixture of a suitable 37

Production
General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. If the vaccine is presented with the haemophilus component in a separate vial, as part of consistency studies, the assays of the diphtheria, tetanus and pertussis are carried out on a suitable number of batches of vaccine reconstituted for use. For subsequent routine control, the assays of these components may be carried out without mixing with the haemophilus component.

D., T., P., (WHOLE CELL) AND HAEMOPHILUS TYPE CONJUGATE VACCINE (ADSORBED)

IP 2007

quantity of PRP conjugate; the resulting mixture is approximately isotonic with blood. The B. pertussis concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 IU per single human dose. If 2 or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. Suitable antimicrobial preservatives may be added. Vaccine with the haemophilus component in a separate container The final bulk is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, bulk purified tetanus toxoid onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate, admixture of an appropriate quantity of a suspension of inactivated B. pertussis component and admixture of a suitable quantity of PRP conjugate; the resulting mixture is approximately isotonic with blood. The B. pertussis concentration of the final bulk vaccine does not exceed that corresponding to opacity of 20 IU per single human dose. If 2 or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. The final bulk is filled separately. Suitable antimicrobial preservatives may be added. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent. A stabilizer may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85.0 per cent and not greater than 115.0 percent of the intended content. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Where the haemophilus component is in a separate container, the final bulk of the haemophilus component is freeze-dried. Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests specific toxicity of diphtheria toxoid, tetanus toxoid and pertussis component and antimicrobial preservative and the assays for the diphtheria, tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the 38

final lot. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Free PRP. Unbound PRP is determined after removal of the conjugate, for example by anion exchange, size exclusion or hydrophobic chromatography (2.4.16), ultrafiltration or other validated methods. The amount of free PRP is not greater than approved for the particular product. Osmolality (2.4.23). The osmolality of the vaccine is within the limits approved for the particular preparation. pH (2.4.24). 6.0 to 7.0. Description. Whitish turbid liquid in which the mineral carrier tends to settle down slowly on keeping.

Production Identification
Tests A, B, C and D may be omitted if test E is carried out. Test E may be omitted if tests A, B, C and D are carried out. A. Diphtheria toxoid. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Maintain at 37o for about 16 hours and centrifuge. Reserve the residue for test C. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate. B. Tetanus toxoid. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. C. Pertussis component. To a suspension of the residue obtained in test A in saline solution add a suitable Bordetella pertussis antiserum; agglutination indicates presence of pertussis component. D. PRP. The suspension of the residue obtained in test A gives a positive reaction when tested by a suitable immunochemical method for PRP. E. The vaccine confers an active immunity in mice and guinea pigs when administered as directed in the test for Potency.

Tests
If the product is presented with the haemophilus component in a separate container; the tests for specific toxicity of diphtheria toxoid, tetanus toxoid and pertussis component, aluminium, free formaldehyde, antimicrobial preservative and sterility are carried out on the container with the diphtheria, tetanus and pertussis components; the tests for PRP content, water (where applicable), sterility and pyrogens are carried out on the container with the haemophilus component.

IP 2007

DIPHTHERIA VACCINE (ADSORBED)

If the haemophilus component is freeze-dried, some tests may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. PRP. Not less than 80.0 per cent of the amount of PRP stated on the label. PRP is determined either by assay of ribose (2.7.1) or phosphorus (2.7.1), by an immunochemical method (2.2.14) or by anion exchange liquid chromatography with pulsed amperometric detection. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Each final lot shall be tested for abnormal toxicity by injecting intraperitoneally one human dose , but not more than 0.25 ml into each of the five mice weighing between 17 to 22 g and at least one human dose but not more than 1.0 ml into each of the two guinea pigs weighing between 250 and 350 g. The preparation passes the test if none of the animals dies or shows signs of ill health in 7 days following the injection . If one of the animal dies or shows the signs of ill health, repeat the test . The preparation passes the test if none of the animals in the second group dies or shows signs of ill health in the time interval specified. Pyrogens (2.2.8). This test is carried out for Haemophilus influenzae type b vaccine only if Haemophilus influenzae type b vaccine is presented as separate lyophilized vial. The vaccine complies with the test for pyrogens. Inject per kg of the rabbits mass a quantity of the vaccine equivalent to: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria toxoid as carrier; 0.1 mg of PRP for a vaccine with tetanus toxoid as carrier protein; 0.025 mg of PRP for vaccine with OMP as carrier. Specific toxicity Diphtheria and tetanus components Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). Pertussis component Complies with the test as stated under Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed). Assay Diphtheria toxoid (adsorbed) 39

Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). Tetanus toxoid (adsorbed) Complies with the test as stated under assay of Tetanus Vaccine (Adsorbed). Pertussis vaccine Complies with the test as stated under Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed). Storage. When stored under the prescribed conditions the vaccine may be expected to retain potency for not less than 2 years from the date on which the potency test for the pertussis component was started. Labelling. The label states (1) the human dose (ml); (2) Diphtheria and Tetanus components (a) in case done by challenge method the minimum number of international units (as applicable for each component) per single human dose; (b) in case done by antibody induction method, the minimum Lf units per single human dose; (3) pertussis component IU or IOU per single human dose; (4) haemophilus conjugate component - g PRP per single human dose; (5) the type and nominal amount of carrier protein per single human dose; (6) the name and amount of adsorbent and added preservative; (7) that the vaccine must be shaken before use; (9) that the vaccine is not to be frozen.

Diphtheria Vaccine (Adsorbed)

Diphtheria Vaccine (Adsorbed) is a preparation of diphtheria formol toxoid with a mineral adsorbent. The formol toxoid is prepared from the toxin produced by the growth of Corynebacterium diphtheriae.

Production
General provisions The maximum number of Lf units per single human dose of diphtheria vaccine (adsorbed) is 30. Bulk purified toxoid For the production of diphtheria toxin, from which toxoid is prepared, seed cultures are managed in a defined seed-lot system in which toxinogenicity is conserved and, where necessary, restored by deliberate reselection. A highly toxinogenic strain of Corynebacterium diphtheriae with known origin and history is grown in a suitable liquid medium. At the end of cultivation, the purity of each culture is tested and contaminated cultures are discarded. Toxin-containing culture medium is separated aseptically from the bacterial mass

DIPHTHERIA VACCINE (ADSORBED)

IP 2007

as soon as possible. The toxin content (Lf per ml) is checked to monitor consistency of production. Single harvests may be pooled to prepare the bulk purified toxoid. The toxin is purified to remove components likely to cause adverse reactions in humans. The purified toxin is detoxified with formaldehyde by a method that avoids destruction of the immunogenic potency of the toxoid and reversion of the toxoid to toxin, particularly on exposure to heat. Alternatively, purification may be carried out after detoxification. Only bulk purified toxoid that complies with the following requirements may be used in the preparation of the final bulk vaccine. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Absence of toxin and irreversibility of toxoid Inject subcutaneously into each of 5 guinea-pigs at least 500 Lf of the non-incubated bulk purified toxoid in a volume of 1 ml, using the same buffer solution as for the final vaccine, without adsorbent. Animals that die shall be autopsied and examined for symptoms of diphtheria intoxication (red adrenals). The bulk purified toxoid shall pass the test if no guinea-pig shows symptoms of specific intoxication within six weeks of injection and if at least 80 per cent of the animals survive the test period. The guinea-pigs shall not have been used previously for experimental purposes. Alternatively, a cell-culture test system may be used; in this case, the sensitivity of the test shall have been demonstrated to be not less than that of the guinea-pig test, and the test procedures shall be approved by the National Regulatory Authority. Each bulk purified toxoid shall be tested to ensure that reversion to toxicity cannot take place on storage. The bulk purified toxoid shall be diluted in order to obtain the same concentration and chemical environment as those present in the final bulk vaccine, except for the presence of adjuvant. To determine whether reversion has occurred, diluted toxoids that have been stored at 37 for six weeks shall be tested. The test employed shall be approved by the National Regulatory Authority and should be sufficiently sensitive to detect very small amounts of toxin. No toxicity shall be detected. Intradermal tests in guinea-pigs and cell-culture tests both are considered to be suitable. Cell culture method Using the same buffer solution as for the final vaccine, without adsorbent, prepare a solution of bulk purified toxoid at 100 Lf per ml. Divide the solution into 2 equal parts. Maintain 1 part at 5 3 and the other at 37 for 6 weeks. Carry out a test in Vero cells for active diphtheria toxin using 50 l per well of both samples. The sample should not contain antimicrobial 40

preservatives and detoxifying agents should be determined to be below the concentration toxic to Vero cells. Non-specific toxicity may be eliminated by dialysis. Use freshly trypsinised Vero cells at a suitable concentration, for example 2.5 105 per ml and a reference diphtheria toxin diluted in 100 Lf per ml diphtheria toxoid. A suitable reference diphtheria toxin will contain either not less than 100 LD50/ml or 67 to 133 lr/100 in 1 Lf and 25,000 to 50,000 minimal reacting doses for guinea-pig skin in 1 Lf (diphtheria toxin RP is suitable for use as the reference toxin). Dilute the toxin in 100 Lf/ml diphtheria toxoid to a suitable concentration, for example 2 10-4 Lf per ml. Prepare serial twofold dilutions of the diluted diphtheria toxin and use undiluted test samples (50 l per well). Distribute them in the wells of a sterile tissue culture plate containing a medium suitable for Vero cells. To ascertain that any cytotoxic effect noted is specific to diphtheria toxin, prepare in parallel dilutions where the toxin is neutralised by a suitable concentration of diphtheria antitoxin, for example 100 IU/ml. Include control wells without toxoid or toxin and with non-toxic toxoid at 100 Lf per ml on each plate to verify normal cell growth. Add cell suspension to each well, seal the plates and incubate at 37 for 5 to 6 days. Cytotoxic effect is judged to be present where there is complete metabolic inhibition of the Vero cells, indicated by the pH indicator of the medium. Confirm cytopathic effect by microscopic examination or suitable staining such as MTT dye. The test is invalid if 5 10-5 Lf per ml of reference diphtheria toxin in 100 Lf per ml toxoid has no cytotoxic effect on Vero cells or if the cytotoxic effect of this amount of toxin is not neutralised in the wells containing diphtheria antitoxin. The bulk purified toxoid complies with the test if no toxicity neutralisable by antitoxin is found in either sample. Antigenic purity. Not less than 1,500 Lf per mg of protein nitrogen. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption of a suitable quantity of bulk purified toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide; the resulting mixture is approximately isotonic with blood. Suitable antimicrobial preservatives may be added. Certain antimicrobial preservatives, particularly those of the phenolic type, adversely affect the antigenic activity and must not be used. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot.

Identification
Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Maintain at 37o for about 16 hours and centrifuge. The clear supernatant

IP 2007

reacts with a suitable diphtheria antitoxin and yields a precipitate. pH (2.4.24). 6.0 to 7.0. Specific toxicity. Use 5 normal, healthy guinea-pigs weighing between 250 and 350 g, which have been maintained for at least 1 week on a uniform, unrestricted diet, and have not been previously treated with any material that will interfere with the test. Weigh the animals separately and record their weights. Inject subcutaneously into each animal 5 times the dose stated on the label. Weigh all the animals at weekly intervals for 6 weeks. None of the animals shows any symptoms of diphtheria or tetanus toxaemia or dies from diphtheria within 42 days or loses weight at the end of the test. If more than one animal dies from non-specific causes or loses weight, repeat the test. If an animal dies or loses weight in the second test, the vaccine fails the test. Potency. Determine by any of the methods of biological assay of Adsorbed Diphtheria Vaccine described. Biological assay of adsorbed diphtheria vaccine (a) Intradermal challenge method The potency of adsorbed diphtheria vaccine is determined by comparing the dose necessary to protect guinea-pigs against the erythrogenic effects of a range of intradermal injections of diphtheria toxin with the dose of the Standard preparation of adsorbed diphtheria toxoid necessary to give the same protection. For this comparison, the Standard preparation of adsorbed diphtheria toxoid and a suitable preparation of diphtheria toxin, for use as a challenge toxin, are required. Standard preparation The Standard preparation is International standard of Diphtheria toxoid, adsorbed, or another suitable preparation the potency of which has been determined in relation to the International Standard. Suggested method Test animals. Use white guinea-pigs, weighing between 250 and 350 g, from the same stock. Distribute the guinea-pigs into no fewer than six equal groups; use groups containing a number of animals sufficient to obtain results that fulfill the requirements for a valid Assay prescribed below. The guineapigs are all of the same sex or the males and females are distributed equally among the groups. If the challenge toxin to be used has not been shown to be stable or has not been adequately standardized, include five guinea-pigs as unvaccinated controls. Selection of the challenge toxin. Select a preparation of diphtheria toxin containing 67 to 133 Limes reactionis/100 (Lr/ 100) in Limes flocculationis (Lf) and 25,000 to 50,000 minimal reacting doses for guinea-pig skin in 1 Lf. If the challenge 41

toxin preparation has been shown to be stable, it is not necessary to verify the activity for every assay. Preparation of the challenge toxin solutions. Immediately prior to use, dilute the challenge toxin with a suitable diluent to obtain a challenge toxin solution containing about 512x10-4 Lf in 0.2 ml. Dilute a portion of this challenge toxin solution to give a series of five 4-fold dilutions. Determination of potency of the vaccine. Prepare in saline solution dilutions of the vaccine under examination and of the Standard preparation such that, for each, the dilutions form a series differing by not more than 2.5 fold steps and in which the dilutions of intermediate concentration, when injected subcutaneously in 1.0 ml volumes into guinea-pigs, result in an intradermal score of approximately three when the animals are challenged. Allocate the dilutions, one to each of the groups of guinea-pigs, and inject subcutaneously 1.0 ml of each dilution into each guinea-pig in the group to which that dilution is allocated. After 28 days shave both flanks of each guinea-pig and inject each animal intradermally with 0.2 ml of the challenge toxin solution and with 0.2 ml of each of the five dilutions thereof in such a way as to minimize interference between adjacent sites. If necessary, inject the unvaccinated control guinea-pigs with dilutions containing 8x10-5, 4x10-5, 2x10-5, 1x10-5 and 5x10-6 Lf of the challenge toxin. Examine all the injection sites 48 hours after injection of the challenge toxin and record the incidence of specific diphtheria erythema. Record also the number of sites free from such reactions as the intradermal challenge score. Tabulate the intradermal challenge scores for all the animals receiving the same dilution of vaccine and use those data with a suitable transformation, such as (score)2 or arcsin [(score/6)2], to obtain an estimate of the relative potency for each of the test preparations by parallel-line quantitative analysis. The test is not valid unless (a) for both the preparation under examination and the Standard preparation, the mean score obtained at the lowest dose level is more than three; (b) if applicable, the toxin dilution that contains 4x10-5 Lf gives a positive erythema in at least 80.0 per cent of the control guineapigs and the dilution that contains 2x10-5 Lf gives no reaction in at least 80 per cent of the guinea-pigs (if these criteria are not met a different toxin has to be selected); (c) the fiducial limits of the assay fall between 50.0 and 200.0 per cent of the estimated potency; (d) the statistical analysis shows no deviation from linearity and parallelism. The test may be repeated but when more than one test is performed the results of all valid tests must be combined in the estimate of potency. The lower fiducial limit of error of the estimated potency is not less than 30 Units per dose. (b) Lethal challenge method Test animals. Use healthy, white or light-coloured guinea-

DIPHTHERIA VACCINE (ADSORBED)

IP 2007

pigs from the same stock, weighing between 250 and 350 g. Distribute them into six groups of sixteen; and four groups of four. The guinea-pigs should all be of the same sex or the males and females should be distributed equally between the six groups of sixteen. Challenge toxin. Select a preparation of diphtheria toxin containing not less than 100 LD50 in 1.0 ml. Preparation of the challenge toxin solutions. Immediately prior to use, prepare from the challenge toxin by dilution in phosphate buffered saline pH 7.4, or normal saline a challenge toxin containing approximately 100 LD50 in 1.0 ml. Dilute portions of this challenge toxin solution to 2LD50, 1 LD50 and LD50 in the same solution Determination of potency of the vaccine. Prepare in saline solution three dilutions of the vaccine under examination and three dilutions of the Standard preparation such that for each, the dilutions form a series differing by not more than 2.5 fold steps and in which the dilutions of intermediate concentration, when injected subcutaneously in 1.0 ml volumes into guineapigs, protect approximately 50 per cent of the animals from the lethal effects of the subcutaneous injection of the quantity of diphtheria toxin prescribed for this test. Allocate the six dilutions, one to each of the six groups of sixteen guinea-pigs, and inject subcutaneously 1.0 ml of each dilution into each guinea-pig in the groups to which that dilution is allocated. After 28 days inject subcutaneously into each animal in the six groups of sixteen, 1.0 ml of the challenge toxin solution. Allocate the challenge toxin solution and the three dilutions made from it, one to each of the four groups of four guineapigs and inject subcutaneously 1.0 ml of each toxin solution into each guinea-pig in the group to which that solution is allocated. Examine the guinea pigs twice in a day, remove dead animals and kill the animals showing definite signs of diphtheria. Count the number of surviving animals 5 days later and calculate the potency of the vaccine under examination relative to the potency of the Standard preparation on the basis of the number of animals that survive in each of the six groups of sixteen, using appropriate statistical methods. The test is not valid unless (a) for the vaccine under examination and the Standard preparation the 50.0 per cent protective doses lie between the largest and smallest doses of the preparations given to the guinea-pigs; (b) the survivors among the four groups of guinea-pigs injected with the challenge toxin and its dilutions indicate that the challenge was approximately 100 LD50 and; (c) statistical analysis shows parallelism, linearity and a significant slope of the doseresponse lines. The test may be repeated any number of times but when more than one test is performed the results of all valid tests must be combined in the estimate of potency. If the lower limit of 95.0 per cent confidence interval of estimated potency is less than 30 IU per single human dose then the 42

limits of the 95.0 per cent confidence interval should be within 50 to 200 of the estimated potency. (c) Antibody induction method Inject subcutaneously on each of two occasions separated by an interval of not more than 4 weeks, one-fiftieth of the stated human dose diluted to 1 ml with saline solution, into each of 10 normal, healthy guinea-pigs weighing between 250 and 350 g. Not earlier than 2 weeks and not later than 3 weeks after the second injection, collect the serum from each animal and determine the antitoxin content of the serum of each animal, as described under Diphtheria Antitoxin or any other method approved by National Regulatory Authority. The geometric mean of the antitoxin contents shall be not less than 2.0 Units per ml with reference to the Diphtheria antitoxin standard. (d) Any other validated serological assay in guinea pigs or mice as approved by National Regulatory Authority Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under, Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity, free formaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.

Identification
Diphtheria toxoid is identified by a suitable immunochemical method. The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/ v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant liquid is obtained. The clear supernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate.

Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the absorbent.

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HAEMOPHILUS TYPE B CONJUGATE

Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. pH (2.4.24). 6.0 to 7.0. Assay Carry out one of the prescribed methods for the assay of Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Labelling. The label states (1) the human dose; (2) the minimum Lf units per single human dose or the minimum International Units per single human dose if potency test done by challenge method; (3) the name and the amount of the adsorbent and preservative; (4) that the vaccine must be shaken before use; (5) that the vaccine is not to be frozen.

immunogenicity test on mice. Taking account of the results of the stability testing, release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the period of validity. SEED LOT The strain of H. influenzae type b used in preparing Haemophilus type b conjugate vaccine shall be identified by a record of its history, including the source from which it was obtained and the tests made to determine the characteristics of the strain. The strain shall have been shown to be capable of producing Type b polysaccharide. The production of PRP and of the carrier protein is based on defined seed lot systems. Master seed lot and working seed lot shall be properly characterized and defined. Cultures derived from the working seed shall have the same characteristics as of the master seed lot. The sample of culture of single harvests taken before killing shall be tested for contamination by examination of Gram-stained smears and by inoculation on suitable media. H. Influenzae Type b Polysaccharide (PRP) H. influenzae Type b is grown in a liquid medium that does not contain high-molecular-weight polysaccharides; if any ingredient of the medium contains blood-group substances, the process shall be validated to demonstrate that after the purification step they are no longer detectable. The culture may be inactivated. PRP is separated from the culture liquid and purified by a suitable method. Volatile matter, including water, In the purified polysaccharide is determined by methods such as thermogravimetry, Karl Fischer or any other suitable method. All chemical analysis shall be based on the dry weight of the polysaccharide, in its salt form. Only those pools of PRP that comply with the following requirements may be used in the preparation of the conjugate. The partially purified PRP shall be stored frozen at or below -20.

Haemophilus Type b Conjugate Vaccine

Haemophilus Type b Conjugate Vaccine is a liquid or freezedried preparation of a polysaccharide, derived from a suitable strain of Haemophilus influenzae type b, covalently bound to a carrier protein. The polysaccharide, polyribosylribitol phosphate, referred to as PRP, is a linear copolymer composed of repeated units of 3--D-ribofuronosyl-(11)-ribitol-5phosphate [(C10H19O12P)n], with a defined molecular size. The carrier protein, when conjugated to PRP, is capable of inducing a T-cell-dependent B-cell immune response to the polysaccharide.

Identification
The PRP is identified by an immunochemical method (2.2.14) or other suitable method (e.g. 1H or 13C NMR spectroscopy). Molecular size. The percentage of PRP eluted before a given K0 value or within a range of K0 values, is determined by gel filtration or high performance size-exclusion chromatography (HPSEC) (2.4.16), either alone or in combination with light scattering and refractive index detectors (e.g. multiple angle laser light scattering i.e. MALLS) or any other suitable method. An acceptable value is established for the particular product and each batch of PRP must be shown to comply with this limit. Limits for currently approved products, using the indicated stationary phases, are shown for information in 43

Production
General provisions The production method shall have been shown to yield consistently H. influenzae type b conjugate vaccines of adequate safety and immunogenicity in humans. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy of Vaccines. The stability of the final lot and relevant intermediates is evaluated using one or more indicator tests. Such tests may include determination of molecular size, determination of free PRP in the conjugate and the

HAEMOPHILUS TYPE B CONJUGATE

IP 2007

Table 1 - Specifications for different components of Haemophilus Type b Conjugate Vaccine. Polysaccharide Type of PRP Nominal amount per dose Type Carrier Protein Purity Nominal amount per dose 18 g Coupling method Cyanogen bromide activation of PRP Carbodiimidemediated coupling Reductive amination one(step method) or N-hydroxysuccinimide activation Thioether bond Conjugation Procedure

Polysaccharide 25 g (size reduced) K0 60.0 per cent: 0.6-0.7 Polysaccharide: 10 g PRP 50.0 per cent K0: 0.30 Polysaccharide 10 g (size reduced) Dp=15-35 or 10-35

Diphtheria toxoid

>1500 Lf per mg of protein nitrogen

Activated diphtheria toxoid (D-AH+), cyanogen bromide activated PRP ADH-activated PRP (PRP-cov.-AH) + tetanus toxoid + EDAC Direct coupling of PRP to CRM 197 (cyanoboro-hydride activated)

Tetanus toxoid

>1500 Lf per mg of protein nitrogen

20 g

CRM 197 diphtheria protein

>90.0 per cent diphtheria protein

25g

Polysaccharide (size-reduced) K0 0.3 0.6

15 g

Meningococcal group B outer membrane protein (OMP)

Outer membrane 125 or protein vesicles 250 g <8.0 per cent of lipopolysaccharide

PRP activation by CDI PRP-IM + BuA2 + BrAc = PRP-BuA2-BrAc + thioactivated OMP

Abbreviations: ADH = BrAc = BuA2 = CDl =

adipic acid dihydrazide bromoacetyl chloride butane-1, 4-diamide carbonyldi-imidazole

Dp EDAC IM Mw

= = = =

degree of polymerization l-ethyl-3-(3-dimethylaminopropyl) carbodimide imidazolium weight-average molecular weight.

Table 2 - Requirements on bulk conjugate Test Specifications Free Polysaccharide (PRP) Unbound protein Protein Carrier Tetanus toxoid CRM 197 <20.0 per cent <1.0 per cent, where applicable <25.0 per cent <1.0 per cent or <2.0 per cent, depending on the coupling method 0.3-0.7 50.0 per cent 0.3-0.6

Diphtheria toxoid <37.0 per cent <5.0 per cent, where applicable

OMP <15.0 per cent Not applicable

PRP to protein ratio Molecular size (K0): Crosslinked agarose for chromatography R Cross-linked agarose for chromatography R1

1.25-1.75 95.0 per cent <0.75

0.30-0.55 60.0 per cent <0.2

0.05-0.1 85.0 per cent < 0.25

0.6-0.7

85.0 per cent <0.5

44

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HAEMOPHILUS TYPE B CONJUGATE

Tables 1 and 2. Where applicable, the molecular-size distribution is also determined after chemical modification of the polysaccharide. A validated determination of the degree of polymerization or of the weight-average molecular weight and the dispersion of molecular masses may be used instead of the determination of molecular size distribution. Ribose (2.7.1). Not less than 32.0 per cent, calculated with reference to the dried substance, as estimated by Bial reaction for pentose, using D-ribose as a standard or any other suitable assay. Phosphorus (2.7.1). 6.8 per cent to 9.0 per cent, calculated with reference to the dried substance. Protein (2.3.49). Not more than 1.0 per cent, calculated with reference to the dried substance. Use sufficient PRP to allow detection of 1 per cent of protein (e.g. a minimum of 1 mg of PRP). Nucleic acid (2.7.1). Not more than 1.0 per cent, calculated with reference to the dried substance by spectroscopy or any other suitable method. Bacterial endotoxins (2.2.3 ). Not more than 25 IU of endotoxin per microgram of PRP. Residual reagents. Where applicable, tests are carried out to determine residues of reagents used during inactivation and purification. An acceptable value for each reagent is established for the particular product and each batch of PRP must be shown to comply with this limit. Where validation studies have demonstrated removal of a residual reagent, the test on PRP may be omitted. Carrier protein The carrier protein is chosen in a way so that when the PRP is conjugated it is able to induce a T-cell-dependent immune response. Currently approved carrier proteins and coupling methods are listed for information in Table 1. The carrier proteins are produced by culture of suitable microorganisms; the bacterial purity of the culture is verified; the culture may be inactivated; the carrier protein is purified by a suitable method. Only a carrier protein that complies with the following requirements may be used in preparation of the conjugate.

Diphtheria toxoid. Diphtheria toxoid is produced as stated under Diphtheria Vaccine (Adsorbed) and complies with the requirements prescribed there for bulk purified toxoid. Tetanus toxoid. Tetanus toxoid is produced as stated under Tetanus Vaccine (Adsorbed) and complies with the requirements prescribed there for bulk purified toxoid except that the antigenic purity is not less than 1500 Lf per mg of protein nitrogen. Diphtheria protein CRM 197. Suitable tests are carried out, for validation or routinely, to demonstrate that the product is non-toxic. The protein obtained contains not less than 90.0 per cent of diphtheria CRM 197 protein, when prepared by liquid chromatography (2.4.14) or any other suitable method. The carrier protein shall be characterized by a suitable chemical or physicochemical method like SDS-PAGE, HPLC, isoelectric focusing, amino acid sequencing, circular dichroism, fluorescence spectroscopy, peptide mapping or mass spectroscopy, as appropriate. OMP (Meningococcal group B outer membrane protein complex) OMP complex of Neisseria meningitidis complies with the following requirements for lipopolysaccharide and pyrogens. Lipopolysaccharide. Not more than 8.0 per cent of lipopolysaccharide, determined by a suitable method. Pyrogens (2.2.8). Inject into each rabbit 0.25 g of OMP per kg body weight, for determining the pyrogenic effect. Bulk conjugate PRP is chemically modified to enable conjugation; it is usually partly depolymerised either before or during this procedure. Reactive functional groups or spacers may be introduced into the carrier protein or PRP prior to conjugation. The conjugate is obtained by the covalent binding of PRP and carrier protein. Where applicable, unreacted but potentially reactogenic functional groups are made unreactive by means of capping agents; the conjugate is purified to remove reagents. Where validation studies have demonstrated removal of a residual reagent (eg. CN, Br etc.), the test on bulk conjugate may be omitted. Only a bulk conjugate that complies with the following requirements may be used in preparation of the final bulk vaccine. For each test and for each particular product, limits of acceptance are established and each batch of conjugate must be shown to comply with these limits. Limits applied to currently approved products for some of these tests are listed for information in Table 2. PRP. The PRP content is determined by assay of phosphorus (2.7.1) or by assay of ribose (2.7.1) or by an immunochemical method (2.2.14) or by any suitable method. 45

Identification
The carrier protein is identified by a suitable immunochemical method (2.2.14). Sterility (2.2.11). Carry out the test for sterility using for each medium 10 ml or the equivalent of one hundred doses, whichever is less.

HEPATITIS A (INACTIVATED) AND HEPATITIS B (rDNA) VACCINE (ADSORBED)

IP 2007

Protein (2.7.1). The protein content is determined by a suitable chemical method. PRP to protein ratio. Determine the ratio by calculation. Molecular size. Molecular-size distribution is determined by gel filtration or size-exclusion chromatography (2.4.16), using a gel matrix, appropriate to the expected size of the conjugate. Free PRP. Unbound PRP is determined after removal of the conjugate, for example by size-exclusion or hydrophobic chromatography (2.4.16), ultra-filtration or other validated methods. Free carrier protein. Free carrier protein is determined by a suitable method (which may include deriving the content by calculation from the results of other tests). The amount is within the limits approved for the particular product. Unreacted functional groups. No unreacted functional groups are detectable in the bulk conjugate unless process validation has shown that unreacted functional groups detectable at this stage are removed during the subsequent manufacturing process (for example, owing to short half-life). Residual reagents. Removal of residual reagents such as cyanide, EDAC (ethyldimethylaminopropylcarbodimide) and phenol is confirmed by suitable tests or by validation of the purification process. Sterility (2.2.11). Carry out the tests for sterility using for each medium 10 ml or the equivalent of one hundred doses, whichever is less. FINAL BULK VACCINE An adjuvant, an antimicrobial preservative and a stabilizer may be added to the bulk conjugate before dilution to the final concentration with a suitable diluent. Only a final bulk vaccine that complies with the following requirements may be used in preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final lots are filled in suitable containers, under stringent aseptic conditions. Only a final lot that is satisfactory with respect to each of the requirements given under Identification, Tests and Assay may be released for use. Provided the test for antimicrobial preservative has been carried out on the final bulk vaccine, it may be omitted on the final lot. 46

Identification
The vaccine is identified by a suitable immunochemical method (2.2.14) for PRP or the assay serves also to identify the vaccine.

Tests
Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject per kg of the rabbits mass a quantity of the vaccine equivalent to 1 g of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier; 0.1 g of PRP for a vaccine with tetanus toxoid as carrier; 0.025 g of PRP for a vaccine with OMP as carrier. pH (2.4.24). The pH of the vaccine, reconstituted if necessary, is within the range approved for the product (6.5 to 7.5). Aluminium (2.3.9). When aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent, Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Water (2.3.43). Not more than 3.0 per cent. PRP. Not less than 80.0 per cent of the amount of PRP stated on the label as determined by ribose assay (2.7.1) or by phosphorus assay (2.7.1) or by an immunochemical method (2.2.14) or by any other suitable method like colorimetery or by anion exchange liquid chromatography (2.4.14) with pulsed amperometric detection. Free PRP. Unbound PRP is determined after removal of the conjugate for example by size-exclusion or hydrophobic chromatography (2.4.16), ultra filtration or other validated methods. Labelling. The label states (1) the number of micrograms of PRP per human dose; (2) the type and nominal amount of carrier protein per single human dose; (3) for vaccine contained in single-dose containers where the space is too small to accommodate the full name of the vaccine the abbreviation Hib may be used in the label on the container provided that the same code is also stated in the label on the package.

Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine (Adsorbed)

Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine (Adsorbed) is a suspension consisting of a suitable strain of hepatitis A virus, grown in cell cultures and inactivated by a

IP 2007

HEPATITIS B (rDNA) VACCINE

validated method, and of hepatitis B surface antigen (HBsAg), a component protein of hepatitis B virus obtained by recombinant DNA technology; the antigens are adsorbed on a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate.

carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot.

Identification
The vaccine is shown to contain hepatitis A virus antigen and hepatitis B surface antigen by suitable immunochemical methods (2.2.14), using specific antibodies or by the mouse immunogenicity tests described under assay.

Production
General provisions The two components are prepared as described in the monographs on Hepatitis A Vaccine (Inactivated, Adsorbed) and Hepatitis B Vaccine (rDNA) and comply with the requirements prescribed therein. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for antisera and vaccines. Reference preparation The reference preparation is part of a representative batch shown to be at least as immunogenic in animals as a batch that, in clinical studies in young, healthy adults, produces not less than 95.0 per cent seroconversion, corresponding to a level of neutralizing antibody recognized to be protective, after a full-course primary immunization. For hepatitis A, an antibody level not less than 20 mIU/ml determined by enzymelinked immunosorbent assay is recognized as being protective. For hepatitis B, antibody level not less than 10 mIU/ml against HBsAg is recognized as being protective. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more inactivated harvests of hepatitis A virus and one or more batches of purified antigen of Hepatitis B (rDNA). Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde (where applicable) and antimicrobial preservative content (where applicable) have been carried out on the final bulk vaccine with satisfactory results, they may be omitted on the final lot. If the assay of the hepatitis A and/or the hepatitis B component is carried out in vivo, then provided it has been 47

Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Bacterial endotoxins (2.2.3). Less than 2 IU per human dose.

Assay
Hepatitis A component Complies with the assay as stated under Inactivated Hepatitis A Vaccine (Adsorbed). Hepatitis B component Complies with the assay as stated under Hepatitis B Vaccine (rDNA). Labelling. The label states (1) the amount of hepatitis A virus antigen and hepatitis B surface antigen per container; (2) the type of cells used for production of the vaccine; (3) the name and amount of the adsorbent used; (4) that the vaccine must be shaken before use; (5) that the vaccine must not be frozen.

Hepatitis B Vaccine (rDNA)

Hepatitis B Vaccine (rDNA) is a non-infectious preparation containing the purified major surface antigen of Hepatitis B virus (HBsAg). This preparation is white or almost white translucent liquid in which the mineral carrier tends to settle down slowly on keeping but is free from foreign particles/ floccules.

Production
General provisions The antigen is manufactured by recombinant DNA technology by culturing genetically engineered yeast cells or other suitable

HEPATITIS B (rDNA) VACCINE

IP 2007

cell lines which carry the gene that codes for major surface antigen of the Hepatitis-B virus as approved by the competent authority. Several physico-chemical steps are employed to purify the Hepatitis-B surface antigen (HBsAg). The vaccine may contain the product of the S gene (major protein), a combination of the S gene and pre-S2 gene products (middle protein) or a combination of S gene, the pre-S2 gene, and preS1 gene products (large protein). The purity of the antigen is determined by comparison with a reference preparation using liquid chromatography or other suitable methods such as SDSPAGE with any suitable staining method. The purified antigen is finally adsorbed on aluminium hydroxide or aluminium phosphate. The method used for production of the vaccine must have been shown to yield a product consistently complying with the requirements for immunogenicity and safety. It must also have been shown to induce specific, protective antibodies in human beings. The production method must be validated to demonstrate that the product if tested, would comply with the tests for safety and efficacy. Reference preparation. A part of a batch shown to be at least as immunogenic as a batch that was used in clinical studies and approved by National Regulatory Authority and determined by any suitable method. For hepatitis B, antibody level not less than 10 mIU/ml against HBsAg is recognized as being protective.. Characterisation of the substance Development studies are carried out to characterize the antigen. The complete protein, lipid and carbohydrate structure of the antigen is established. The morphological characteristics of the antigen particles are established by electron microscopy. The buoyant density of the antigen particles is determined by a physico-chemical method, for example gradient centrifugation. The antigenic epitopes are characterized. The protein fraction of the antigen is characterized in terms of the primary structure (for example, by determination of the aminoacid composition, by partial amino-acid sequence analysis and by peptide mapping). PROPAGATION AND HARVEST Identity, microbial purity, plasmid retention and consistency of yield are determined at suitable production stages. If mammalian cells are used, tests for extraneous agents and mycoplasmas are performed in accordance with tests for extraneous agents in viral vaccines for human use. PURIFIED ANTIGEN Only a purified antigen that complies with the following requirements may be used in the preparation of the final bulk. 48

Total protein (2.3.49). The total protein is determined by a validated method. The content is within the limits approved for the specific product. Antigen content and identification. The quantity and specificity of HBsAg is determined in comparison with the International standard for HBsAg subtype ad or an in-house reference, by a suitable immunochemical method such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunoblot (preferably using a monoclonal antibody directed against a protective epitope) or single radial diffusion. The antigen/protein ratio is within the limits approved for the specific product. The molecular weight of the major band in a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced conditions corresponds to the value expected from the gene sequence and possible glycosylation. Antigenic purity. The purity of the antigen is determined by comparison with a reference preparation using liquid chromatography or other suitable methods such as SDS-PAGE with staining. A suitable method is sensitive enough to detect a potential contaminant at a concentration of 1.0 per cent of total protein. Not less than 95.0 per cent of the total protein consists of hepatitis B surface antigen. Composition. The content of proteins, lipids, nucleic acids and carbohydrates is determined. Host-cell and vector-derived DNA (2.2.15). If mammalian cells are used for production, not more than 10 pg of DNA in the quantity of purified antigen equivalent to a single human dose of vaccine. Caesium. If a caesium salt is used during production, a test for residual caesium is carried out on the purified antigen. The content is within the limits approved for the specific product. Sterility (2.2.11). The purified antigen complies with the test for sterility, carried out using 10 ml for each medium. Additional tests on the purified antigen may be required depending on the production method used: for example, a test for residual animal serum where mammalian cells are used for production or tests for residual chemicals used during extraction and purification. FINAL BULK VACCINE An antimicrobial preservative and an adjuvant may be included in the vaccine. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount.

IP 2007

HEPATITIS B (rDNA) VACCINE

Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde, antimicrobial preservative content and the assay in animals, where applicable, have been carried out on the final bulk vaccine with satisfactory results, they may be omitted on the final lot.

the test. Use animals of the same sex in the test. Inject similar groups of animals with the reference preparation of HepatitisB vaccine (r DNA). One group of control animals remains unvaccinated but is injected intraperitoneally with the same volume of the diluent alone. Anaesthetize and bleed the animals 28 to 42 days later, keeping the individual sera separate. Assay the individual sera for specific HBsAg antibody concentration by a suitable immunochemical method such as ELISA or RIA. Calculate the result of the assay by standard statistical methods (5.7). From the distribution of reaction levels measured on all the sera in the unvaccinated (control group), the maximum reaction level that can be expected to occur in an unvaccinated animal for that particular assay is determined. Any response in the vaccinated animals that exceeds this level is by definition seroconversion. The percentage of animals showing seroconversion in each group is transformed (for example, probit transformation) and a parallel line model, using the log dose response curve, is applied to the data. The potency of the preparation under examination relative to the reference preparation is thus established. The test is not valid unless (a) for both the test and reference vaccine, the ED50 lies between the smallest and the largest doses given to animals ; (b) the statistical analysis shows no deviation from linearity or parallelism; (c) the fiducial limits of the estimated relative potency fall between 33.0 and 300.0 per cent of the estimated potency. Method B (In vitro) The potency of the vaccine under examination is determined by an in vitro method that has been validated against the biological test. Enzyme Linked Immunosorbent Assay (ELISA) using monoclonal antibodies specific for protection inducing epitopes of HBsAg have been shown to be suitable. Adequate number of dilutions and replicates of the vaccine under examination and the reference standard are employed in the assay. The data obtained is analyzed by a parallel-line model and may be suitably transformed for statistical evaluation. Commercially available kits for measuring HBsAg in vitro may be used provided they are validated to produce equally precise and accurate results. The test is not valid unless (a) the statistical analysis shows no deviation from linearity or parallelism; (b) the fiducial limits of the estimated relative potency fall between 80.0 and 125.0 per cent of the estimated potency. The acceptance criteria are approved for a given reference preparation by the National Regulatory Authority in the light of the validation data. Labelling. The label states (a) the amount of HBsAg per dose; (b) the type of cells used for production of the vaccine; (c) the 49

Identification
The assay or, where applicable, the electrophoretic profile, also serves to identify the vaccine.

Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbant. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject the equivalent of one human dose into each rabbit. A validated test for bacterial endotoxins may be used instead of the test for pyrogens. Assay. The vaccine complies with the assay of Hepatitis-B Vaccine (rDNA) described below. Potency. The upper fiducial limit (P = 0.95) of the estimated relative potency is not less than 1.0. Determine the potency either in animals (Method A) or by a validated in vitro procedure (Method B) described below: Method A (Biological) The potency of the vaccine under examination is determined in animals by comparing in given conditions its capacity to induce specific anti-HBsAg antibodies in mice or guinea-pigs with the same capacity as with the reference standard. Inject intraperitoneally not less than three doses of suitable dilutions of the vaccine under examination diluted with adjuvant used in the vaccine into groups of a suitable strain of mice, weighing between 15 and 20 g (about 5 weeks old), of either sex distributed randomly into several groups of mice. Healthy guinea pigs weighing between 300 and 350g (about 7 weeks old) that have not been previously treated with any material that will interfere with the test will also be suitable for

INACTIVATED HEPATITIS A VACCINE (ADSORBED)

IP 2007

name and amount of the adjuvant; (d) that the vaccine must be shaken before use; (e) that the vaccine must not be frozen.

Virus concentration. The virus concentration of each master and working seed lot is determined to monitor consistency of production. Extraneous agents (2.7.3). The master and working seed lots comply with the requirements for seed lots for virus vaccines. In addition, if primary monkey cells have been used for isolation of the strain, measures are taken to ensure that the strain is not contaminated with simian viruses such as simian immunodeficiency virus and filoviruses. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are being handled. Animal serum (but not human serum) may be used in the cell culture media. Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from extraneous agents. The cell culture media may contain a pH indicator, such as phenol red and approved antibiotics at the lowest effective concentration. Not less than 500 ml of the cell cultures employed for vaccine production is set aside as uninfected cell cultures (control cells). Multiple harvests from the same production cell culture may be pooled and considered as a single harvest. Only a single harvest that complies with the following requirements may be used in the preparation of the vaccine. When the determination of the ratio of virus concentration to antigen content has been carried out on a suitable number of single harvests to demonstrate consistency, it may subsequently be omitted as a routine test.

Inactivated Hepatitis A Vaccine (Adsorbed)

Hepatitis A Vaccine (Inactivated, Adsorbed) is a liquid preparation of a suitable strain of hepatitis A virus grown in cell cultures, inactivated by a validated method and adsorbed on a mineral carrier. The vaccine is an opalescent suspension. The vaccine complies with the monograph on Vaccines.

Production
Production of the vaccine is based on a virus seed-lot system and a cell-bank system. The production method shall have been shown to yield consistently vaccines that comply with the requirements for immunogenicity, safety and stability. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Unless otherwise justified and authorised, the virus in the final vaccine shall not have undergone more passages from the master seed lot than were used to prepare the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. Reference preparation. A part of a batch shown to be at least as immunogenic as a batch that, in clinical studies in young healthy adults, produced not less than 95.0 per cent seroconversion, corresponding to a level of neutralising antibody accepted to be protective, after a full-course of primary immunisation is used as a reference preparation. An antibody level of 20 mIU /ml determined by enzyme-linked immunosorbent assay is recognised as being protective. Substrate for virus propagation The virus is propagated in a human diploid cell line or in a continuous cell line approved by the competent authority. SEED LOT The strain of hepatitis A virus used to prepare the master seed lot shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. Only a seed lot that complies with the following requirements may be used for virus propagation. Description. A clear, colourless or light coloured liquid.

Identification
The test for antigen content also serves to identify the single harvest. Sterility (2.2.11). The single harvest complies with the test for sterility, carried out using 10 ml for each medium. Mycoplasmas (2.7.4). The single harvest complies with the test for mycoplasmas carried out using 1ml for each medium. Control cells. The control cells of the production cell culture comply with a test for identity and the requirements for extraneous agents. Antigen content. Determine the hepatitis A antigen content by a suitable immunochemical method (2.2.14) to monitor production consistency; the content is within the limits approved for the particular product. Ratio of virus concentration to antigen content. The consistency of the ratio of the concentration of infectious virus, as determined by a suitable cell culture method, to antigen content is established by validation on a suitable number of single harvests. 50

Identification
Each master and working seed lot is identified as hepatitis A virus using specific antibodies.

IP 2007

INACTIVATED HEPATITIS A VACCINE (ADSORBED)

PURIFICATION AND PURIFIED HARVEST The harvest, which may be a pool of several single harvests, is purified by validated methods. If continuous cell lines are used for production, the purification process shall have been shown to reduce consistently the level of host-cell DNA. Only a purified harvest that complies with the following requirements may be used in the preparation of the inactivated harvest. Virus concentration. The concentration of infective virus in the purified harvest is determined by a suitable cell culture method to monitor production consistency and as a starting point for monitoring the inactivation curve. Antigen:total protein ratio. Determine the hepatitis A virus antigen content by a suitable immunochemical method (2.2.14). Determine the total protein by a validated method. The ratio of hepatitis A virus antigen content to total protein content is within the limits approved for the particular product. Bovine serum albumin. Not more than 50 ng in the equivalent of a single human dose, determined by a suitable immunochemical method (2.2.14). Where appropriate in view of the manufacturing process, other suitable protein markers may be used to demonstrate effective purification. Residual host-cell DNA (2.2.15). If a continuous cell line is used for virus propagation, the content of residual host-cell DNA, determined using a suitable method is not greater than 100 pg in the equivalent of a single human dose. Residual chemicals. If chemical substances are used during the purification process, tests for these substances are carried out on the purified harvest (or on the inactivated harvest), unless validation of the process has demonstrated total clearance. The concentration must not exceed the limits approved for the particular product. INACTIVATION AND INACTIVATED HARVEST Several purified harvests may be pooled before inactivation. In order to avoid interference with the inactivation process, virus aggregation must be prevented or aggregates must be removed immediately before and/or during the inactivation process. The virus suspension is inactivated by a validated method; the method shall have been shown to be consistently capable of inactivating hepatitis A virus without destroying the antigenic and immunogenic activity; as part of the validation studies, an inactivation curve is plotted representing residual live virus concentration measured on at least three occasions (for example, on days 0, 1 and 2 of the inactivation process). If formaldehyde is used for inactivation, the presence of excess free formaldehyde is verified at the end of the inactivation process. Only an inactivated harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. 51

Inactivation. Carry out an amplification test for residual infectious hepatitis A virus by inoculating a quantity of the inactivated harvest equivalent to 5 per cent of the batch or if the harvest contains the equivalent of 30,000 doses or more, not less than 1,500 doses of vaccine into cell cultures of the same type as those used for production of the vaccine and incubating the cells for at least 28 days. Make two passages and at the end of incubation carry out a test of suitable sensitivity for residual infectious virus. No evidence of hepatitis A virus multiplication is found in the samples taken at the end of the inactivation process. Use infective virus inocula concurrently as positive controls to demonstrate cellular susceptibility and absence of interference. Sterility (2.2.11). The inactivated viral harvest complies with the test for sterility, carried out using 10 ml for each medium. Bacterial endotoxins (2.2.3). Not more than 2 IU of endotoxin in the equivalent of a single human dose. Antigen content. Determine the hepatitis A virus antigen content by a suitable immunochemical method (2.2.14). Residual chemicals. As stated under Purification and Purified Harvest. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more inactivated harvests. Approved adjuvants, stabilisers and antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers. The containers are then closed so as to avoid contamination. Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde (where applicable) and antimicrobial preservative content (where applicable) and the assay have been carried out on the final bulk vaccine with satisfactory results, these tests may be omitted on the final lot. If the assay is carried out using mice or other animals, then provided it has been carried with satisfactory results on the final bulk vaccine, it may be omitted on the final lot.

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IP 2007

Identification
The vaccine is shown to contain hepatitis A virus antigen by a suitable immunochemical method using specific antibodies or by the mouse immunogenicity test described under Assay.

antibodies against hepatitis A virus by a suitable immunochemical method (2.2.14). Calculations. Carry out the calculations by the usual statistical methods (5.7) for an assay with a quantal response. From the distribution of reaction levels measured on all the sera in the unvaccinated group, determine the maximum reaction level that can be expected to occur in an unvaccinated animal for that particular assay. Any response in vaccinated animals that exceeds this level is by definition a seroconversion. Make a suitable transformation of the percentage of animals showing seroconversion in each group (for example, a probit transformation) and analyse the data according to a parallelline log dose-response model. Determine the potency of the test preparation relative to the reference preparation. Validity conditions. The test is not valid unless (a) for both the test and the reference vaccine, the ED50 lies between the smallest and the largest doses given to the animals; (b) the statistical analysis shows no significant deviation from linearity or parallelism; (c) the fiducial limits of the estimated relative potency fall between 33.0 and 300.0 per cent of the estimated potency. Potency. The upper fiducial limit (P = 0.95) of the estimated relative potency is not less than 1.0. In vitro assay Carry out an immunochemical determination of antigen content (2.2.14) with acceptance criteria validated against the in vivo test. The acceptance criteria are approved for a given reference preparation by the National Regulatory Authority in the light of the validation data. Labelling. The label states (1) the biological origin of the cells and; (2) the adjuvant used for the preparation of the vaccine.

Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent. Free formaldehyde (2.3.20). When formaldehyde has been used for inactivation, the vaccine complies with the test prescribed in Vaccines. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Assay The vaccine complies with the assay of Hepatitis A vaccine. The assay is carried out either in vivo, by comparing in given condition the capacity to induce specific antibodies in mice with the same capacity of a reference preparation or in vitro by an immunochemical determination of antigen content (2.2.14). In vivo assay The test on mice shown below is given as an example of a method that has been found suitable for a given vaccine; other validated methods may also be used. Selection and distribution of the test animals. Healthy mice from the same stock, about 5 weeks old and from a strain shown to be suitable should be used in the test. Use animals of the same sex. Distribute the animals in at least seven equal groups of a number suitable for the requirements of the assay. Determination of potency of the vaccine under examination. Using a 0.9 per cent w/v solution of sodium chloride containing the aluminium adjuvant used for the vaccine, prepare at least three dilutions of the vaccine under examination and matching dilutions of the reference preparation. Allocate the dilutions one to each of the groups of animals and inject subcutaneously not more than 0.5 ml of each dilution into each animal in the group to which that dilution is allocated. Maintain a group of unvaccinated controls, injected subcutaneously with the same volume of diluent. After 28 to 32 days, anaesthetise and bleed all animals, keeping the individual sera separate. Assay the individual sera for specific 52

Inactivated Hepatitis B Vaccine

Inactivated Hepatitis B Vaccine is a non-infectious inactivated liquid preparation derived from the surface antigen of Hepatitis B virus (HbsAg). This preparation is white or almost white translucent liquid in which the mineral carrier tends to settle down slowly on keeping but is free from foreign particles / floccules.

Production
The antigen is harvested and purified from the plasma of human carriers of Hepatitis B virus. The surface antigen contains all the three antigen species (S, Pre-S1, Pre-S2). The individual donor plasma is shown by sensitive tests to be seronegative for HIV-I and HIV-2 and for HCV. The plasma pool is tested for freedom from adventitious viruses and blood borne

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INACTIVATED HEPATITIS B VACCINE

transmissible pathogens by appropriate methods. The purified antigen is further inactivated by a validated method, usually with formalin or any other inactivating agent, to render the hepatitis B virus harmless. The preparation is also tested for the residual HBV DNA using a sensitive test approved by the competent authority and the level is shown to be less than 1 pg HBV DNA per 50 doses. The method used for production of the vaccine must have been shown to yield a product consistently complying with the requirements of immunogenicity, safety and stability. The production method must also be validated to demonstrate that the product, if tested, would comply with the tests for safety and efficacy. Reference preparation. A part of a batch shown to be at least as immunogenic as a batch that produced in clinical studies in young healthy adults not less than 95.0 per cent seroconversion, corresponding to a level of neutralizing antibody accepted to be protective (HbsAg antibody titre not less than 10 mIU/ml after a full course of primary immunization determined by enzyme-linked immunosorbent assay (ELISA) is used as a reference preparation. Characterisation of the substance Development studies are carried out to characterize the antigen. The complete protein, lipid and carbohydrate structure of the antigen is established. The morphological characteristics of the antigen particles are established by electron microscopy. The buoyant density of the antigen particles is determined by a physico-chemical method (2.4.29), for example gradient centrifugation. The antigenic epitopes are characterized. The protein fraction of the antigen is characterized in terms of the primary structure (for example, by determination of the aminoacid composition, by partial amino-acid sequence analysis and by peptide mapping). PROPAGATION AND HARVEST Identity, microbial purity, plasmid retention and consistency of yield are determined at suitable production stages. PURIFIED ANTIGEN Only a purified antigen that complies with the following requirements may be used in the preparation of the final bulk. Total protein (2.3.49). The total protein is determined by a validated method. The content is within the limits approved for the specific product. Antigen content and identification. The quantity and specificity of HBsAg is determined in comparison with the International standard for HBsAg subtype ad or an in-house reference, by a suitable immunochemical method such as radio immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunoblot (preferably using a monoclonal antibody directed against a protective epitope) or single radial diffusion. 53

The antigen/protein ratio is within the limits approved for the specific product. The molecular weight of the major band in a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced conditions corresponds to the value expected from the gene sequence and possible glycosylation. Antigenic purity. The purity of the antigen is determined by comparison with a reference preparation using liquid chromatography or other suitable methods such as SDS-PAGE with staining. A suitable method is sensitive enough to detect a potential contaminant at a concentration of 1.0 per cent of total protein. Not less than 95.0 per cent of the total protein consists of hepatitis B surface antigen. Composition. The content of proteins, lipids, nucleic acids and carbohydrates is determined. Sterility (2.2.11). The purified antigen complies with the test for sterility carried out using 10 ml for each medium. Additional tests on the purified antigen may be required depending on the production method used. FINAL BULK VACCINE An antimicrobial preservative and an adjuvant may be included in the vaccine. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical or physico-chemical method. The amount is not less than the 85.0 per cent and not greater than 115.0 per cent of that stated on the label. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde, antimicrobial preservative content and the assay in animals, where applicable, have been carried out on the final bulk vaccine with satisfactory results, they may be omitted on the final lot.

Identification
The assay or, where applicable, the electrophoretic profile, also serves to identify the vaccine.

Tests
Aluminium (2.3.9). When hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent, the vaccine

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IP 2007

complies with the test prescribed in the monograph on Vaccines. Test for inactivating agent. The concentration of any inactivating agent remaining in the final vaccine shall be determined by methods approved by the competent authority. The concentration shall not exceed a specified upper limit. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject the equivalent of one human dose into each rabbit. A validated test for bacterial endotoxins (2.2.3) may be used instead of the test for pyrogens. Assay The upper fiducial limit (P = 0.95) of the estimated relative potency is not less than 1.0. Determine the potency by method A (Biological) as described under Hepatitis-B vaccine (rDNA). Labelling. The label states (1) the amount of HBsAg per dose; (2) the name and amount of inactivating agent; (3) the name and amount of the adjuvant; (4) that the vaccine must be shaken before use; (5) that the vaccine must not be frozen.

recommends new strains corresponding to prevailing epidemiological evidence. The origin and passage history of virus strains shall be approved by the National Regulatory Authority. Substrate for virus propagation Influenza virus seed to be used in the production of vaccine is propagated in fertilised eggs from chicken flocks free from specified pathogens or in suitable cell cultures, such as chickembryo fibroblasts or chick kidney cells obtained from chicken flocks free from specified pathogens. For production, the virus of each strain is grown in the allantoic cavity of eggs derived from specific pathogen free flocks. SEED LOT The production of vaccine is based on a seed-lot system. Working seed lots represent not more than fifteen passages from the approved reassorted virus or the approved virus isolate. The final vaccine represents one passage from the working seed lot. The haemagglutinin and neuraminidase antigens of each seed lot are identified as originating from the correct strain of influenza virus by suitable methods. Only a working virus seed lot that complies with the following requirements may be used in the preparation of the monovalent pooled harvest. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. Mycoplasmas (2.7.4). Carry out the test for mycoplasmas using 10 ml. PROPAGATION AND HARVEST An antimicrobial agent may be added to the inoculum. After incubation at a controlled temperature, the allantoic fluids are harvested and combined to form a monovalent pooled harvest. An antimicrobial agent may be added at the time of harvest. At no stage in the production, penicillin or streptomycin is used. MONOVALENT POOLED HARVEST To limit the possibility of contamination, inactivation is initiated as soon as possible after preparation. The virus is inactivated by a method that has been demonstrated on three consecutive batches to be consistently effective for the manufacturer. The inactivation process shall have been shown to be capable of inactivating the influenza virus without destroying its antigenicity; the process should cause minimum alteration of the haemagglutinin and neuraminidase antigens. The inactivation process shall also have been shown to be capable of inactivating avian leucosis viruses and mycoplasmas. If the monovalent pooled harvest is stored after inactivation, it 54

Inactivated Influenza Vaccine (Split Virion)

Influenza Vaccine (Split Virion, Inactivated) is a sterile, aqueous suspension of a strain or strains of influenza virus, type A or B, or a mixture of strains of the two types grown individually in eggs derived from specific pathogen free flock or cell cultures, inactivated and treated so that the integrity of the virus particles has been disrupted without diminishing the antigenic properties of the haemagglutinin and neuraminidase antigens. The stated amount of haemagglutinin antigen for each strain present in the vaccine is 15 g per dose, unless clinical evidence supports the use of a different amount.

Production
The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Choice of vaccine strain The World Health Organisation reviews the world epidemiological situation annually and if necessary

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INACTIVATED INFLUENZA VACCINE (SPLIT VIRION)

is held at a temperature of 53. If formaldehyde solution is used, the concentration does not exceed 0.2 g/l of formaldehyde at any time during inactivation; if betapropiolactone is used, the concentration does not exceed 0.1 per cent v/v at any time during inactivation. Before or after the inactivation procedure, the monovalent pooled harvest is concentrated and purified by high-speed centrifugation or other suitable method and the virus particles are disrupted into component subunits by the use of approved procedures. For each new strain, a validation test is carried out to show that the monovalent bulk consists predominantly of disrupted virus particles. Only a monovalent pooled harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. Haemagglutinin antigen. Determine the content of haemagglutinin antigen by an immunodiffusion test (2.2.14), by comparison with a haemagglutinin antigen reference preparation or with an antigen preparation calibrated against it. Carry out the test at 20 to 25. For some vaccines, the physical form of the haemagglutinin particles prevents quantitative determination by immunodiffusion after inactivation of the virus. For these vaccines, a determination of haemagglutinin antigen is made on the monovalent pooled harvest before inactivation. The production process is validated to demonstrate suitable conservation of haemagglutinin antigen and a suitable tracer is used for formulation, for example, protein content. Neuraminidase antigen. The presence and type of neuraminidase antigen are confirmed by suitable enzymatic or immunological methods (2.2.14) on the first three monovalent pooled harvests from each working seed lot. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. Viral inactivation. Carry out as described below under Tests. Chemicals used for disruption. Tests are carried out on the monovalent pooled harvest for the chemicals used for disruption, the limits being approved by the National Regulatory Authority. FINAL BULK VACCINE Appropriate quantities of the monovalent pooled harvests are blended to make the final bulk vaccine. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. 55

Sterility (2.2.11). Carry out the test for sterility using 10 ml for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under Tests and Assay may be released for use. Provided that the test for viral inactivation has been performed with satisfactory results on each monovalent pooled harvest and that the tests for free formaldehyde, ovalbumin and total protein have been performed with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Description. The vaccine is a slightly opalescent liquid.

Identification
The assay serves to confirm the antigenic specificity of the vaccine.

Tests
Viral inactivation. Inoculate 0.2 ml of the vaccine into the allantoic cavity of each of ten fertilised eggs and incubate at 33 to 37 for 3 days. The test is not valid unless at least eight of the ten embryos survive. Harvest 0.5 ml of the allantoic fluid from each surviving embryo and pool the fluids. Inoculate 0.2 ml of the pooled fluid into a further ten fertilised eggs and incubate at 33 to 37 for 3 days. The test is not valid unless at least eight of the ten embryoes survive. Harvest about 0.1 ml of the allantoic fluid from each surviving embryo and examine each individual harvest for live virus by a haemagglutination test. If haemagglutination is found for any of the fluids, carry out for that fluid a further passage in eggs and test for haemagglutination; no haemagglutination occurs. Total protein (2.3.49). Not more than six times the total haemagglutinin content of the vaccine as determined in the assay, but in any case, not more than 100 g of protein per virus strain per human dose and not more than a total of 300 g of protein per human dose. Ovalbumin. Not more than 1 g of ovalbumin per human dose, determined by a suitable technique using a suitable reference preparation of ovalbumin. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity.

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IP 2007

Bacterial endotoxins (2.2.3). Not more than 100 IU of endotoxin per human dose. Assay Determine the content of haemagglutinin antigen by an immunodiffusion test (2.2.14), by comparison with an appropriate haemagglutinin antigen reference preparation. Carry out the test at 20 to 25. The confidence interval (P = 0.95) of the assay is not greater than 80.0 per cent to 125.0 per cent of the estimated content. The lower confidence limit (P = 0.95) of the estimate of haemagglutinin antigen content is not less than 80.0 per cent of the amount stated on the label for each strain. For some vaccines, quantitative determination of haemagglutinin antigen with respect to available reference preparations is not possible. An immunological identification of the haemagglutinin antigen and a semi-quantitative determination are carried out instead by suitable methods. Labelling. The label complies with the requirements stated under Vaccines and also states (a) that the vaccine has been prepared on eggs; (b) the strain or strains of influenza virus used to prepare the vaccine; (c) the method of inactivation; (d) the haemagglutinin content in g per virus strain per dose; (e) the season during which the vaccine is intended to protect.

approved by the competent authority. Substrate for virus propagation Influenza virus seed to be used in the production of vaccine is propagated in fertilised eggs from chicken flocks free from specified pathogens or in suitable cell cultures, such as chickembryo fibroblasts or chick kidney cells obtained from chicken flocks free from specified pathogens. For production, the virus of each strain is grown in the allantoic cavity of eggs derived from SPF flocks. SEED LOT The production of vaccine is based on a seed-lot system. Working seed lots represent not more than fifteen passages from the approved reassorted virus or the approved virus isolate. The final vaccine represents one passage from the working seed lot. The haemagglutinin and neuraminidase antigens of each seed lot are identified as originating from the correct strain of influenza virus by suitable methods. Only a working virus seed lot that complies with the following requirements may be used in the preparation of the monovalent pooled harvest. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. Mycoplasmas (2.7.4). Carry out the test for mycoplasmas using 10 ml.

Inactivated Influenza Vaccine (Surface Antigen)

Influenza Vaccine (Surface Antigen, Inactivated) is a sterile, aqueous suspension of a strain or strains of influenza virus, type A or B, or a mixture of strains of the two types grown individually in eggs derived from specific pathogen free flocks or cell cultures, inactivated and treated so that the preparation consists predominantly of haemagglutinin and neuraminidase antigens, without diminishing the antigenic properties of these antigens. The stated amount of haemagglutinin antigen for each strain present in the vaccine is 15 g per dose, unless clinical evidence supports the use of a different amount.

PROPAGATION AND HARVEST An antimicrobial agent may be added to the inoculum. After incubation at a controlled temperature, the allantoic fluids are harvested and combined to form a monovalent pooled harvest. An antimicrobial agent may be added at the time of harvest. At no stage in the production penicillin or streptomycin is used. MONOVALENT POOLED HARVEST To limit the possibility of contamination, inactivation is initiated as soon as possible after preparation. The virus is inactivated by a method that has been demonstrated on three consecutive batches to be consistently effective for the manufacturer. The inactivation process shall have been shown to be capable of inactivating the influenza virus without destroying its antigenicity; the process should cause minimum alteration of the haemagglutinin and neuraminidase antigens. The inactivation process shall also have been shown to be capable of inactivating avian leucosis viruses and mycoplasmas. If the monovalent pooled harvest is stored after inactivation, it is held at a temperature of 53. If formaldehyde solution is used, the concentration does not exceed 0.2 g/l of formaldehyde at any time during inactivation; if betapropiolactone is used, the concentration does not exceed 56

Production
The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Choice of vaccine strain The World Health Organisation reviews the world epidemiological situation annually and if necessary recommends new strains corresponding to prevailing epidemiological evidence. The origin and passage history of virus strains shall be

IP 2007

INACTIVATED INFLUENZA VACCINE (SURFACE ANTIGEN)

0.1 per cent v/v at any time during inactivation. Before or after the inactivation process, the monovalent pooled harvest is concentrated and purified by high-speed centrifugation or other suitable method. Virus particles are disrupted into component subunits by approved procedures and further purified so that the monovalent bulk consists mainly of haemagglutinin and neuraminidase antigens. Only a monovalent pooled harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. Haemagglutinin antigen. Determine the content of haemagglutinin antigen by an immunodiffusion test (2.2.14), by comparison with a haemagglutinin antigen reference preparation or with an antigen preparation calibrated against it. Carry out the test at 20 to 25. Neuraminidase antigen. The presence and type of neuraminidase antigen are confirmed by suitable enzymatic or immunological methods (2.2.14) on the first three monovalent pooled harvests from each working seed lot. Sterility (2.2.11). Carry out the test for sterility, using 10 ml for each medium. Viral inactivation. Carry out the test described below under Tests. Purity. The purity of the monovalent pooled harvest is examined by polyacrylamide gel electrophoresis or by other approved techniques. Mainly haemagglutinin and neuraminidase antigens shall be present. Chemicals used for disruption and purification. Tests are carried out on the monovalent pooled harvest for the chemicals used for disruption and purification, the limits being approved by the competent authority. FINAL BULK VACCINE Appropriate quantities of the monovalent pooled harvests are blended to make the final bulk vaccine. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. 57

Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the test for viral inactivation has been performed with satisfactory results on each monovalent pooled harvest and that the tests for free formaldehyde, ovalbumin and total protein have been performed with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Description. The vaccine is a clear liquid.

Identification
The assay serves to confirm the antigenic specificity of the vaccine.

Tests
Viral inactivation. Inoculate 0.2 ml of the vaccine into the allantoic cavity of each of ten fertilised eggs and incubate at 33 to 37 for 3 days. The test is not valid unless at least eight of the ten embryos survive. Harvest 0.5 ml of the allantoic fluid from each surviving embryo and pool the fluids. Inoculate 0.2 ml of the pooled fluid into a further ten fertilised eggs and incubate at 33 to 37 for 3 days. The test is not valid unless at least eight of the ten embryos survive. Harvest about 0.1 ml of the allantoic fluid from each surviving embryo and examine each individual harvest for live virus by a haemagglutination test. If haemagglutination is found for any of the fluids, carry out for that fluid a further passage in eggs and test for haemagglutination; no haemagglutination occurs. Total protein (2.3.49). Not more than 40 g of protein other than haemagglutinin per virus strain per human dose and not more than a total of 120 g of protein other than haemagglutinin per human dose. Ovalbumin. Not more than 1 g of ovalbumin per human dose, determined by a suitable technique using a suitable reference preparation of ovalbumin. Free formaldehyde (2.3.20). Complies with the test for free formaldehyde as stated under General Requirements for Vaccines for Human Use. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Sterility (2.2.11). Complies with the test for sterility. Bacterial endotoxins (2.2.3). Not more than 100 IU of endotoxin per human dose. Assay Determine the content haemagglutinin antigen by an

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IP 2007

immunodiffusion test (2.2.14), by comparison with an appropriate haemagglutinin antigen reference preparation. Carry out the test at 20 to 25. The confidence interval (P = 0.95) of the assay is not greater than 80.0 per cent to 125.0 per cent of the estimated content. The lower confidence limit (P = 0.95) of the estimate of haemagglutinin antigen content is not less than 80.0 per cent of the amount stated on the label for each strain. Labelling. The label states (1) that the vaccine has been prepared on eggs; (2) the strain or strains of influenza virus used to prepare the vaccine; (3) the method of inactivation; (4) the haemagglutinin content in micrograms per virus strain per dose; (5) the season during which the vaccine is intended to protect.

SEED LOT The production of vaccine is based on a seed-lot system. Working seed lots represent not more than fifteen passages from the approved reassorted virus or the approved virus isolate. The final vaccine represents one passage from the working seed lot. The haemagglutinin and neuraminidase antigens of each seed lot are identified as originating from the correct strain of influenza virus by suitable methods. Only a working virus seed lot that complies with the following requirements may be used in the preparation of the monovalent pooled harvest. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. Mycoplasmas (2.7.4). Carry out the test for mycoplasmas using 10 ml.

Inactivated Influenza Vaccine (Whole Virion)

Influenza Vaccine (Whole Virion, Inactivated) is a sterile, aqueous suspension of a strain or strains of influenza virus, type A or B, or a mixture of strains of the two types grown individually in eggs derived from specific pathogen free flocks or cell culture and inactivated in such a manner that their antigenic properties are retained. The stated amount of haemagglutinin antigen for each strain present in the vaccine is 15 g per dose, unless clinical evidence supports the use of a different amount.

PROPAGATION AND HARVEST An antimicrobial agent may be added to the inoculum. After incubation at a controlled temperature, the allantoic fluids are harvested and combined to form a monovalent pooled harvest. An antimicrobial agent may be added at the time of harvest. At no stage in the production is penicillin or streptomycin used. MONOVALENT POOLED HARVEST To limit the possibility of contamination, inactivation is initiated as soon as possible after preparation. The virus is inactivated by a method that has been demonstrated on three consecutive batches to be consistently effective for the manufacturer. The inactivation process shall have been shown to be capable of inactivating the influenza virus without destroying its antigenicity; the process should cause minimum alteration of the haemagglutinin and neuraminidase antigens. The inactivation process shall also have been shown to be capable of inactivating avian leucosis viruses and mycoplasmas. If the monovalent pooled harvest is stored after inactivation, it is held at a temperature of 53. If formaldehyde solution is used, the concentration does not exceed 0.2 g/l of formaldehyde at any time during inactivation; if betapropiolactone is used, the concentration does not exceed 0.1 per cent v/v at any time during inactivation. Before or after the inactivation process, the monovalent pooled harvest is concentrated and purified by high-speed centrifugation or other suitable method. Only a monovalent pooled harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. Haemagglutinin antigen. Determine the content of haemagglutinin antigen by an immunodiffusion test (2.2.14), by comparison with a haemagglutinin antigen reference 58

Production
The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Choice of vaccine strain The World Health Organisation reviews the world epidemiological situation annually and, if necessary, recommends new strains corresponding to prevailing epidemiological evidence. The origin and passage history of virus strains shall be approved by the competent authority. Substrate for virus propagation Influenza virus seed to be used in the production of vaccine is propagated in fertilised eggs from chicken flocks free from specified pathogens or in suitable cell cultures, such as chickembryo fibroblasts or chick kidney cells obtained from chicken flocks free from specified pathogens .For production, the virus of each strain is grown in the allantoic cavity of eggs derived from specific pathogen free flocks.

IP 2007

JAPANESE ENCEPHALITIS VACCINE (HUMAN)

preparation or with an antigen preparation calibrated against it. Carry out the test at 20 to 25. Neuraminidase antigen. The presence and type of neuraminidase antigen are confirmed by suitable enzymatic or immunological methods (2.2.14) on the first three monovalent pooled harvests from each working seed lot. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. Viral inactivation. Carry out the test described below under Tests. FINAL BULK VACCINE Appropriate quantities of the monovalent pooled harvests are blended to make the final bulk vaccine. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under Tests and Assay may be released for use. Provided that the test for viral inactivation has been performed with satisfactory results on each monovalent pooled harvest and that the tests for free formaldehyde, ovalbumin and total protein have been performed with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Description. The vaccine is a slightly opalescent liquid.

the allantoic fluid from each surviving embryo and examine each individual harvest for live virus by a haemagglutination test. If haemagglutination is found for any of the fluids, carry out for that fluid a further passage in eggs and test for haemagglutination; no haemagglutination occurs. Total protein (2.3.49). Not more than six times the total haemagglutinin content of the vaccine as determined in the assay, but in any case, not more than 100 g of protein per virus strain per human dose and not more than a total of 300 g of protein per human dose. Ovalbumin. Not more than 1 g of ovalbumin per human dose, determined by a suitable technique using a suitable reference preparation of ovalbumin. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum amount shown to be effective and is not greater than 115.0 per cent of the quantity stated on the label. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Sterility (2.2.11). Complies with the test for sterility. Bacterial endotoxins (2.2.3). Not more than 100 IU of endotoxin per human dose. Assay Determine the content of haemagglutinin antigen by an immunodiffusion test (2.2.14), by comparison with an appropriate haemagglutinin antigen reference preparation. Carry out the test at 20 to 25. The confidence interval (P = 0.95) of the assay is not greater than 80.0 per cent to 125.0 per cent of the estimated content. The lower confidence limit (P = 0.95) of the estimate of haemagglutinin antigen content is not less than 80.0 per cent of the amount stated on the label for each strain. Labelling. The label states (1) that the vaccine has been prepared on eggs; (2) the strain or strains of influenza virus used to prepare the vaccine; (3) the method of inactivation; (4) the haemagglutinin content in micrograms per virus strain per dose; (5) the season during which the vaccine is intended to protect.

Identification
The assay serves to confirm the antigenic specificity of the vaccine.

Tests
Viral inactivation. Inoculate 0.2 ml of the vaccine into the allantoic cavity of each of ten fertilised eggs and incubate at 33 to 37 for 3 days. The test is not valid unless at least eight of the ten embryos survive. Harvest 0.5 ml of the allantoic fluid from each surviving embryo and pool the fluids. Inoculate 0.2 ml of the pooled fluid into a further ten fertilised eggs and incubate at 33 to 37 for 3 days. The test is not valid unless at least eight of the ten embryos survive. Harvest about 0.1 ml of 59

Japanese Encephalitis Vaccine (Human)

Japanese Encephalitis Vaccine for human use is a liquid or freeze dried preparation of Japanese encephalitis virus grown in approved substrate and inactivated by a validated method.

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IP 2007

Production
General provisions The vaccine is produced on the basis of virus seed lot system The production method shall have been shown to yield consistently the vaccines that comply with the tests for immunogenicity, safety and stability. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Substrate for virus propagation The virus is propagated in an approved cell substrate like a Vero cell line. SEED LOT The strain of Japanese encephalitis virus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. The National Regulatory Authority shall determine the acceptable number of passages from the master virus seed lot to produce working virus seed lots. Only a working seed lot that complies with the following tests may be used for virus propagation.

if present are reduced to an acceptable level by suitable method of purification. Serum and trypsin used in the preparation of cell suspension and media are shown to be free from infectious extraneous agents. The cell culture media may contain a pH indicator such as phenol red. Not less than 500 ml of the cell cultures employed for vaccine production are set aside as uninfected cell cultures (control cells). The virus suspension is harvested on one or more occasions during incubation. Multiple harvests from the same production cell culture may be pooled and considered as a single harvest. Virus harvests that comply with the tests given under Identification and Virus concentration are pooled in the preparation of the inactivated viral harvest. Control cells. The control cells of the production cell culture from which the single harvest is derived should comply with the test for identification and with the tests for extraneous agents (2.7.3). Purification and inactivation The virus harvest may be concentrated and/or purified by suitable methods; the virus harvest is inactivated by a validated method at a fixed, well defined stage of the process which may be before, during or after any concentration or purification. The method shall have been shown to be capable of inactivating Japanese encephalitis virus without destruction of the immunogenic activity. If formalin is used, the concentration shall at no time exceed 1:2000. Only an inactivated viral suspension that complies with the following tests may be used in the preparation of the final bulk vaccine. Inactivation. Inactivation is confirmed by carrying out an amplification test for residual infectious Japanese encephalitis virus. Inoculate a quantity of inactivated viral suspension equivalent to not less than 25 doses into cell cultures of the same type as those used for production of the vaccine. Make a passage after 7 days. Maintain the cultures for a further period of 14 days and then examine the cell cultures for Japanese encephalitis virus using an immunofluorescence test. No Japanese encephalitis virus is detected. Alternatively, 5 ml of each culture fluid is pooled on day 14 and 21 and 0.03 ml is inoculated intracerebrally into each of the 10 mice weighing between 12 and 15 g. The mice are observed for 14 days for symptoms caused by Japanese encephalitis virus, and mice showing symptoms of Japanese encephalitis virus are sacrificed and virus presence is confirmed by immunofluorescence test. No Japanese encephalitis virus shall be detected. Residual host-cell DNA (2.2.15). The content of residual hostcell DNA, determined using a suitable method, should not be greater than 10 ng per single human dose if cells are used in the production. 60

Identification
Each working seed lot is identified as Japanese encephalitis virus using specific antibodies by an approved method. Virus concentration. The virus concentration of each working seed lot is determined by a cell culture method using immunofluoresence or any other approved method. Extraneous agents (2.7.3). The working seed lot complies with the tests for the virus seed lots. PROPAGATION AND HARVEST a) Mouse brain vaccine The vaccine is prepared by using a seed-lot system. An approved strain of virus is grown by inoculating intracerebrally into healthy mice. Virus harvests are pooled, concentrated and inactivated by addition of formalin or any other suitable inactivating agent. It may contain a suitable preservative. The vaccine may be issued in single or multidose containers. b) Cell culture vaccine All processing of the cell bank and subsequent cell cultures are done under aseptic conditions in an area where no other cells are handled. Approved animal (but not human) serum may be used in the media, but the final medium for maintaining cell growth during virus multiplication does not contain animal serum; the media may contain human albumin. Serum proteins,

IP 2007

JAPANESE ENCEPHALITIS VACCINE (HUMAN)

FINAL BULK VACCINE The final bulk vaccine is prepared from one or more inactivated viral suspensions. An approved stabilizer may be added to maintain the activity of the product. Thiomersal can be used as preservative. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Formaldehyde (2.3.20). Not more than 0.01 per cent w/v. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers. The containers are then sealed so as to prevent contamination. Only a final lot that complies with each of the tests given under Identification, Tests and Assay may be released for use. Provided that the test for inactivation has been carried out with satisfactory results on the inactivated virus suspension and the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, these tests may be omitted on the final lot.

Antimicrobial preservative. Determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum amount shown to be effective and is not greater than 115.0 per cent of that stated on the label. Biological assay Potency of Japanese encephalitis virus vaccine is determined by titrating the neutralizing antibodies produced in the immunized mice by plaque reduction method or serum neutralization test (SNT) using appropriate cell culture. Standard preparation The Standard preparation is a freeze-dried Japanese encephalitis virus vaccine the potency of which has been determined in relation to the Japanese encephalitis reference vaccine obtained from the National Institute of Health, Tokyo, Japan. Suggested method Preparation of challenge virus suspension The approved challenge virus strain is stored in freeze-dried form or in liquid form stored below -70 . Prepare a working pool of the challenge virus strain by inoculating intracerebrally 0.03 ml of 100 fold dilution of the standard strain in Hanks balanced salt solution containing 5 per cent calf serum into a suitable number of 2-day old suckling mice. Sacrifice the animals after they show characteristic symptoms of encephalitis and become moribund. Harvest their brains aseptically, wash them in chilled sterile saline solution to remove blood clots. Homogenize the brains with Hanks balanced salt solution containing 5 per cent calf serum to make a 10 per cent emulsion. Centrifuge the emulsion at 2000 g for 30 minutes. Dilute the supernatant with Hanks balanced salt solution containing 5 per cent calf serum so as to contain about 200 Plaque-Forming Units (PFU) of the virus per 0.4 ml. Determination of potency Prepare appropriate dilutions of the vaccine under examination and of the Standard Preparation in a suitable medium. Inject intraperitoneally in two doses of 0.5 ml each at 7-day interval into at least 20 mice of 4 weeks of age. Bleed each mouse 7 days after the second injection, pool the separated serum from each group and inactivate the sera by heating at 56 for 30 minutes. The inactivated sera may be stored at -20, if necessary. Dilute the sera appropriately, e.g. 1:40. 1:160, 1:640 etc., mix with an equal volume of the challenge virus suspension and incubate at 37 for 90 minutes for neutralization. Inoculate the mixture into cell cultures and overlay the infected cells with 1 per cent agar. After incubation for an appropriate time (about 61

Identification
The vaccine is shown to contain Japanese encephalitis virus antigen by a suitable immunochemical method using specific antibodies, alternatively, the Assay also serves to identify the vaccine.

Tests
Complies with the test for Inactivation under final Purification and Inactivation. Sterility (2.2.11). Complies with the test for sterility. Bacterial endotoxins (2.2.3). Less than 25 IU per single human dose. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin (for cell culture vaccine). Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Residual host-cell DNA (for continuous cell line vaccines) (2.2.15). Not more than 10 ng per single human dose. Free formaldehyde (2.3.20). Not more than 0.01 per cent w/v.

MEASLES AND RUBELLA VACCINE (LIVE)

IP 2007

48 hours), stain the cells and count the number of plaques formed on the cultures to obtain the plaque reduction rates for the vaccine under examination and the Standard preparation. Calculate the neutralizing antibody titres for each group using standard statistical methods (5.7). The test is not valid unless (a) the mean number of plaques obtained with the Standard preparation is between 100 and 150 per dish and (b) the potency of the vaccine under examination is not less than that of the Standard preparation. Labelling. The label states (1) the biological origin of the cells used for the preparation of the vaccine; (2) the strain of virus used.

following test for thermal stability and with each of the tests given below under Identification and Tests and Assay may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37 for 7 days. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37 for 7 days and for vaccine stored at 2 to 8. For each component, the virus concentration of the heated vaccine is not more than 1.0 log10 lower than that of the unheated vaccine.

Identification

Measles and Rubella Vaccine (Live)

Measles and Rubella Vaccine (Live) is a freeze-dried preparation of suitable attenuated strains of measles virus and rubella virus grown in suitable cell cultures. The vaccine is reconstituted immediately before use to give a clear liquid that may be coloured owing to the presence of a pH indicator.

When the vaccine reconstituted as stated on the label is mixed with antibodies specific for measles virus and rubella virus, it is no longer able to infect cell cultures susceptible to these viruses. When the vaccine reconstituted as stated on the label is mixed with quantities of specific antibodies sufficient to neutralize any one viral components, the second viral component infects susceptible cell cultures.

Tests
Water (2.3.43). Not more than 3.0 per cent, determined by Karl Fischer, semi-micro determination of water or by any suitable validated method. Sterility (2.2.11). The reconstituted vaccine complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Assay A. Mix the vaccine with a sufficient quantity of antibodies specific for rubella virus.Titrate the vaccine for infective measles virus at least in triplicate, using at least eight cell cultures for each dilution 0.5 log10 step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated measles virus concentration is not less than that stated on the label; the minimum measles virus concentration stated on the label is not less than 1x103 CCID50 per single human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration are greater than 0.3. Measles vaccine (Live) RS is suitable for use as a reference preparation. B. Titrate the vaccine for infective rubella virus at least in triplicate, using at least eight cell cultures for each 0.5 log10 62

Production
General provisions The two components are prepared as described in the monographs on Measles vaccine (live) and Rubella vaccine (live) and comply with the tests prescribed therein. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. FINAL BULK VACCINE Virus harvests for each component are pooled and clarified to remove cells. A suitable stabilizer may be added and the pooled harvests diluted as appropriate. Suitable quantities of the pooled harvest for each component are mixed. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT For each component, a minimum virus concentration for release of the product is established such as to ensure, in the light of stability data, that the minimum concentration stated on the label will be present at the end of the period of validity. Only a final lot that complies with the tests for minimum virus concentration of each component for release, with the

IP 2007

MEASLES VACCINE (LIVE)

dilution step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated rubella virus concentration is not less than that stated on the label; the minimum rubella virus concentration stated on the label are not less than 1x103 CCID50 per single human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration are greater than 0.3. Rubella vaccine (Live) RS is suitable for use as a reference preparation. Labelling. The label states (1) the strains of virus used in the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) the minimum virus concentration for each component of the vaccine; (4) the time within which the vaccine must be used after reconstitution; (5) that the vaccine must not be given to a pregnant woman and that a woman should not become pregnant within two months after having the vaccine.

information on the origin of the strain and its subsequent manipulation. Virus seed lots are prepared in large quantities and stored at temperatures below -20 if freeze-dried, or below -60 if not freeze-dried. Only a seed lot that complies with the following tests may be used for virus propagation.

Identification
The master and working seed lots are identified as measles virus by serum neutralization in cell culture, using specific antibodies. Virus concentration. The virus concentration of the master and working seed lots is determined to monitor consistency of production. Extraneous agents (2.7.3). The working seed lot complies with the tests for seed lots. Neurovirulence (2.7.5). The master/working seed lot complies with the test for neurovirulence of live virus vaccines. Macaca and Cercopithecus monkeys susceptible to measles virus are suitable for the test. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are handled. Suitable animal (but not human) serum may be used in the growth medium, but the final medium for maintaining cell growth during virus multiplication does not contain animal serum. Serum and trypsin used in the preparation of cell suspensions and culture media are shown to be free from extraneous agents. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration. It is preferable to have a substrate free from antibiotics during production. Not less than 500 ml of the production cell culture is set aside as uninfected cell cultures (control cells). The viral suspensions are harvested at a time appropriate to the strain of virus being used. Only a single harvest that complies with the following tests may be used in the preparation of the final bulk vaccine.

Measles Vaccine (Live)

Measles Vaccine (Live) is a freeze-dried preparation of a suitable attenuated strain of measles virus. The vaccine is reconstituted immediately before use, as stated on the label, to give a clear liquid that may be coloured owing to the presence of a pH indicator.

Production
General provisions The production of vaccine is based on a virus seed-lot system and, if the virus is propagated in human diploid cells, a cellbank system. Unless otherwise justified and authorized, the virus in the final vaccine shall have undergone no more passages from the master seed lot than were used to prepare the vaccine shown in clinical studies to be satisfactory with respect to abnormal toxicity and efficacy; even with authorized exceptions, the number of passages beyond the level used for clinical studies shall not exceed five. The production method is validated to demonstrate that the product, if tested, would comply with the tests for abnormal toxicity and efficacy. Substrate for virus propagation The virus is propagated in human diploid cells or in cultures of chick embryo cells derived from a chicken flock free from specified pathogens. SEED LOT The strain of measles virus used in the production of measles vaccine shall be identified by historical records that include 63

Identification
The single harvest contains virus that is identified as measles virus by serum neutralisation in cell culture, using specific antibody. Virus concentration. The virus concentration in the single harvest is determined as prescribed under Assay to monitor consistency of production and to determine the dilution to be used for the final bulk vaccine. Extraneous agents (2.7.3). Complies with the test for extraneous agents.

MEASLES, MUMPS AND RUBELLA VACCINE (LIVE)

IP 2007

Control cells. If human diploid cells are used for production, the control cells comply with the test for identification and extraneous agents. FINAL BULK VACCINE Virus harvests that comply with the above tests are pooled and clarified to remove cells. A suitable stabilizer may be added and the pooled harvests diluted as appropriate. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). The final bulk vaccine complies with the test for sterility carried out using 10 ml for each medium. FINAL LOT A minimum virus concentration for release of the product is established so as to ensure, in the light of stability data, that the minimum concentration stated on the label will be present at the end of the period of validity. Only a final lot that complies with the tests for minimum virus concentration for release, with the following requirement for thermal stability and with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37 for 7 days. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37 for 7 days and for vaccine stored at 2 to 8. The virus concentration of the heated vaccine is not more than 1.0 log10 lower than that of the unheated vaccine.

Assay Titrate the vaccine for infective virus at least in triplicate, using at least five cell cultures for each 0.5 log10 dilution step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated virus concentration is not less than that stated on the label; the minimum virus concentration stated on the label is not less than 1 103 CCID50 per human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration is greater than 0.3. Measles vaccine (Live) RS is suitable for use as a reference preparation. Labelling. The label states (1) the strain of virus used for the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) the minimum virus concentration; (4) the time within which the vaccine must be used after reconstitution.

Measles, Mumps and Rubella Vaccine (Live)

Measles, Mumps and Rubella Vaccine (Live) is a freeze-dried preparation of suitable attenuated strains of measles virus, mumps virus and rubella virus grown in suitable cell cultures. The vaccine is reconstituted immediately before use to give a clear liquid that may be coloured owing to the presence of a pH indicator.

Production
General provisions The three components are prepared as described in the monographs on Measles Vaccine (Live), Mumps Vaccine (Live) and Rubella Vaccine (Live) and comply with the tests prescribed therein. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. FINAL BULK VACCINE Virus harvests for each component are pooled and clarified to remove cells. A suitable stabilizer may be added and the pooled harvests diluted as appropriate. Suitable quantities of the pooled harvest for each component are mixed. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. 64

Identification
When the vaccine reconstituted as stated on the label is mixed with specific measles antibodies, it is no longer able to infect susceptible cell cultures.

Tests
Water (2.3.43). Not more than 3.0 per cent, determined by Karl Fischer, semi-micro determination of water or by any suitable validated method. Sterility ( 2.2.11). The reconstituted vaccine complies with the test for sterility. Abnormal toxicity ( 2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14).

IP 2007

MENINGOCOCCAL POLYSACCHARIDE VACCINE

FINAL LOT For each component, a minimum virus concentration for release of the product is established such as to ensure, in the light of stability data, that the minimum concentration stated on the label will be present at the end of the period of validity. Only a final lot that complies with the tests for minimum virus concentration of each component for release, with the following requirement for thermal stability and with each of the requirements given below under Identification and Tests may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37 for 7 days. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37 for 7 days and for vaccine stored at 2 to 8. The virus concentration of the heated vaccine is not more than 1.0 log10 lower than that of the unheated vaccine.

is not less than 1x103 CCID50 per single human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration are greater than 0.3. Measles vaccine (Live) RS is suitable for use as a reference preparation. B. Mix the vaccine with a sufficient quantity of antibodies specific for measles virus and rubella virus. Titrate the vaccine for infective mumps virus at least in triplicate, using at least eight cell cultures for each dilution 0.5 log10 step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated mumps virus concentration is not less than that stated on the label; the minimum mumps virus concentration stated on the label is not less than 5 x 103 CCID50 per single human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration are greater than 0.3. Mumps vaccine (Live) RS is suitable for use as a reference preparation. C. Mix the vaccine with a sufficient quantity of antibodies specific for mumps virus. Titrate the vaccine for infective rubella virus at least in triplicate, using at least eight cell cultures for each dilution 0.5 log10 step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated rubella virus concentration is not less than that stated on the label; the minimum rubella virus concentration stated on the label is not less than 1x103 CCID50 per single human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration are greater than 0.3. Rubella vaccine (Live) RS is suitable for use as a reference preparation. Labelling. The label states (1) the strains of virus used in the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) the minimum virus concentration for each component of the vaccine; (4) the time within which the vaccine must be used after reconstitution; (5) that the vaccine must not be given to a pregnant woman and that a woman should not become pregnant within two months after having the vaccine.

Identification
When the vaccine reconstituted as stated on the label is mixed with antibodies specific for measles virus, mumps virus and rubella virus, it is no longer able to infect cell cultures susceptible to these viruses. When the vaccine reconstituted as stated on the label is mixed with quantities of specific antibodies sufficient to neutralize any two viral components, the third viral component infects susceptible cell cultures.

Tests
Water (2.3.43). Not more than 3.0 per cent, determined by Karl Fischer, semi-micro determination of water or by any suitable validated method. Sterility (2.2.11). The reconstituted vaccine complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Assay A. Mix the vaccine with a sufficient quantity of antibodies specific for mumps virus and rubella virus.Titrate the vaccine for infective measles virus at least in triplicate, using at least eight cell cultures for each dilution 0.5 log10 step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated measles virus concentration is not less than that stated on the label; the minimum measles virus concentration stated on the label 65

Meningococcal Polysaccharide Vaccine

Meningococcal Polysaccharide Vaccine is a freeze-dried preparation of one or more purified capsular polysaccharides obtained from one or more suitable strains of Neisseria meningitidis group A, group C, group Y and group W135 that are capable of consistently producing polysaccharides known to be safe and effective in man.

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IP 2007

N. meningitidis group A polysaccharide consists of partly O-acetylated repeating units of N-acetylmannosamine, linked with 16 phosphodiester bonds. N. meningitidis group C polysaccharide consists of partly O-acetylated repeating units of sialic acid, linked with 29 glycosidic bonds. N. meningitidis group Y polysaccharide consists of partly O-acetylated alternating units of sialic acid and D-glucose, linked with 26 and 14 glycosidic bonds. N. meningitidis group W135 polysaccharide consists of partly O-acetylated alternating units of sialic acid and D-galactose, linked with 26 and 14 glycosidic bonds.

medium. The liquid media used and the final medium are semisynthetic and free from substances precipitated by cetrimonium bromide (hexadecyltrimethylammonium bromide) and do not contain blood-group substances or highmolecular-mass polysaccharides. The bacterial purity of the culture is verified by microscopic examination of Gram-stained smears and by inoculation into appropriate media. The cultures are centrifuged and the polysaccharides precipitated from the supernatant by addition of cetrimonium bromide. The precipitate obtained is harvested and may be stored at or below -20 awaiting further purification. PURIFIED POLYSACCHARIDES The polysaccharides are purified, after dissociation of the complex of polysaccharide and cetrimonium bromide, using suitable procedures to remove successively nucleic acids, proteins and lipopolysaccharides. The purification step consists of ethanol precipitation of the polysaccharides or purification with chloroform and n-butanol or by cold phenol treatment, which are then dried and stored at or below -20. The loss on drying is determined by thermogravimetry, Karl Fischer or any other suitable method and the value is used to calculate the results of the other chemical tests with reference to the dried substance. Only purified polysaccharides that tested comply with the following requirements may be used in the preparation of the final bulk vaccine. Protein (2.7.1). Not more than 10 mg of protein per gram of purified polysaccharidefor group A and C organisms and less than 50 mg of protein per gram of polysaccharide for group Y and W135 calculated using bovine plasma albumin as a reference or other methods approved by National Regulatory Authority. Nucleic acids (2.7.1). Not more than 10 mg of nucleic acids per gram of purified polysaccharide, calculated with reference to the dried substance. O-Acetyl groups (2.7.1). Not less than 2 mmol of O-acetyl groups per gram of purified polysaccharide for group A, not less than 1.5 mmol per gram of polysaccharide for group C, not less than 0.3 mmol per gram of polysaccharide for groups Y and W135, all calculated with reference to the dried substance. Phosphorus (2.7.1). Not less than 80 mg of phosphorus per gram of group A purified polysaccharide, calculated with reference to the dried substance. Sialic acid (2.7.1). Not less than 800 mg of sialic acid per gram of group C polysaccharide and not less than 560 mg of sialic acid per gram of purified polysaccharide for groups Y and W135, all calculated with reference to the dried substance. Use the following reference solutions: 66

Production
General provisions Production of the meningococcal polysaccharides is based on a well defined seed-lot system. The method of production shall have been shown to yield consistently meningococcal polysaccharide vaccines of satisfactory immunogenicity and safety for man. The production method is validated to demonstrate that the product, if tested, would comply with the test of abnormal toxicity for antisera and vaccines. SEED LOT The strains of N. meningitidis used for the master seed lots shall be identified by historical records that include information on their origin and by their biochemical, serological, physicochemical or molecular characteristics. Cultures from the working seed lot shall have the same characteristics as the strain that was used to prepare the master seed lot. The strains have the following characteristics: a) Colonies obtained from a culture are round, uniform in shape and smooth with a mucous, opalescent, greyish appearance.

b) Gram staining reveals characteristic Gram-negative diplococci in coffee-bean arrangement. c) e) The oxidase test is positive. Suspensions of the culture agglutinate with specific antisera of known titre.

d) The culture utilizes glucose and maltose.

PROPAGATION AND HARVEST The working seed lots are cultured on solid media that do not contain blood-group substances or ingredients of mammalian origin. The inoculum may undergo one or more subcultures in liquid medium before being used for inoculating the final

IP 2007

MENINGOCOCCAL POLYSACCHARIDE VACCINE

Group C polysaccharide. A 150 mg/l solution of Nacetylneuraminic acid. Group Y polysaccharide. A solution containing 95 mg/l of Nacetylneuraminic acid and 55 mg/l of glucose. Group W135 polysaccharide. A solution containing 95 mg/l of N-acetylneuraminic acid and 55 mg/l of galactose. Calcium. If a calcium salt is used during purification, determination of calcium is carried out on the purified polysaccharide by a suitable method; the content is within the limits approved for the product. Molecular size. Examine by gel filtration or high performance size-exclusion chromatography (HPSEC) (2.4.16), using agarose for chromatography or cross-linked agarose for chromatography either alone or in combination with light scattering and refractive index detector (e.g. multiple angle LASER light scattering, MALLS) or any other suitable method. Use a column 0.9 m x 15 mm equilibrated with a solvent having an ionic strength of 0.2 mol/kg and a pH of 7.0 to 7.5. Apply to the column about 2.5 mg of polysaccharide in a volume of about 1.5 ml and elute at about 20 ml/h. Collect fractions of about 2.5 ml and determine the content of polysaccharide by a suitable method. At least 65.0 per cent of group A polysaccharide, 75.0 per cent of group C polysaccharide, 80.0 per cent of group Y polysaccharide and 80.0 per cent of group W135 polysaccharide is eluted before a distribution coefficient (K0) of 0.50 is reached. In addition, the percentages eluted before this distribution coefficient are within the limits approved for the particular product.

Only a final bulk vaccine that complies with the following requirement may be used in the preparation of the final lot. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers. The containers are then closed so as to avoid contamination. Only a final lot that is satisfactory with respect to each of the requirements prescribed below under Identification, Tests and Assay may be released for use.

Identification
Carry out an identification test for each polysaccharide present in the vaccine by a suitable immunochemical method (2.2.14).

Tests
Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject each of the rabbit with 1ml per kg body weight of solution containing a) c) 0.025 g of polysaccharide for a monovalent vaccine, 0.075 g of polysaccharide for a trivalent vaccine,

b) 0.050 g of polysaccharide for a bivalent vaccine, d) 0.100 g of polysaccharide for a tetravalent vaccine. Water (2.3.43). Not more than 3.0 per cent, of moisture content by thermogravimetery, Karl Fischer or any other suitable method. Molecular size. Examine by gel filtration or size-exclusion chromatography (2.4.16). Use a column about 0.9 m long and 16 mm in internal diameter equilibrated with a solvent having an ionic strength of 0.2 mol/kg and a pH of 7.0 to 7.5. Apply to the column about 2.5 mg of each polysaccharide in a volume of about 1.5 ml and elute at about 20 ml/h. Collect fractions of about 2.5 ml and determine the content of polysaccharide by a suitable method. Use cross-linked agarose for chromatography and apply a suitable immunochemical method (2.2.14) to establish the elution pattern of the different polysaccharide(s). The vaccine complies with the test if: a) 65.0 per cent of Group A polysaccharide is eluted before K0 of 0.50,

Identification and serological specificity

The identity and serological specificity are determined by a suitable immunochemical method (2.2.14). Identity and purity of each polysaccharide shall be confirmed; it shall be shown that there is not more than 1.0 per cent m/m of groupheterologous N. meningitidis polysaccharide. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject each of the rabbit with 1ml per kg body weight of solution containing a) c) 0.025 g of polysaccharide for a monovalent vaccine, 0.075 g of polysaccharide for a trivalent vaccine,

b) 0.050 g of polysaccharide for a bivalent vaccine, d) 0.100 g of polysaccharide for a tetravalent vaccine. FINAL BULK VACCINE One or more purified polysaccharides of one or more N. meningitidis groups are dissolved in a suitable solvent that may contain a stabilizer. 67

b) 75.0 per cent of Group C polysaccharide is eluted before K0 of 0.50, c) 80.0 per cent of Group Y & W135 polysaccharide is eluted before K0 of 0.50.

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IP 2007

For a tetravalent vaccine (group A + group C + group Y and group W135), use cross linked agarose for chromatography R1 and apply a suitable immunochemical method (2.2.14) to establish the elution pattern of the different polysaccharides. The vaccine complies with the test if K0 for the principal peak is a) not greater than 0.70 for group A and group C polysaccharide, b) not greater than 0.57 for group Y polysaccharide, c) not greater than 0.68 for group W135 polysaccharide. Assay Carry out an assay as stated under each polysaccharide present in the vaccine. For a divalent vaccine (group A + group C), use measurement of phosphorus (2.7.1) to determine the content of polysaccharide A and measurement of sialic acid (2.7.1) to determine the content of polysaccharide C. To determine sialic acid, use as reference solution a 150 mg/l solution of Nacetylneuraminic acid. For a tetravalent vaccine (group A + group C + group Y + group W135) a suitable immunochemical method (2.2.14) is used with a reference preparation of purified polysaccharide for each group. The vaccine contains not less than 70.0 per cent and not more than 130.0 per cent of the quantity of each polysaccharide stated on the label. Labelling. The label states (1) the group or groups of polysaccharides (A, C, Y or W135) present in the vaccine; (2) the number of g of polysaccharide per human dose.

shown in clinical studies to be satisfactory with respect to safety and efficacy even with authorized exceptions, the number of passages beyond the level used for clinical studies shall not exceed five. The production method is validated to demonstrate that the product, if tested, would comply with the tests for safety and efficacy. Substrate for virus propagation The virus is propagated in human diploid cells or in primary cultures of chick embryo cells derived from a chicken flock free from specified pathogens. SEED LOT The strain of mumps virus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. To avoid unnecessary use of monkeys in the test for neurovirulence, Virus seed lots are prepared in large quantities and stored at temperatures below -20 if freeze-dried, or below -60 if not freeze-dried. Only a seed lot that complies with the following tests may be used for virus propagation.

Identification
The master and working seed lots are identified as mumps virus by serum neutralisation in cell culture, using specific antibodies. Virus concentration. The virus concentration of the master and working seed lots is determined to ensure consistency of production. Extraneous agents (2.7.3). The working seed lot complies with the tests for seed lots. Neurovirulence (2.7.5). The master/working seed lot complies with the test for neurovirulence of live virus vaccines. Macaca and Cercopithecus monkeys are suitable for the test. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are handled. Suitable animal (but not human) serum may be used in the growth media. Serum and trypsin used in the preparation of cell suspensions and culture media are shown to be free from extraneous agents. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration. It is preferable to have a substrate free from antibiotics during production. Not less than 500 ml of the production cell culture is set aside as uninfected cell culture (control cells). The viral suspensions are harvested at a time appropriate to the strain of virus being used. 68

Mumps Vaccine (Live)

Mumps Vaccine (Live) is a freeze-dried preparation of a suitable attenuated strain of mumps virus. The vaccine is reconstituted immediately before use to give a clear liquid that may be coloured owing to the presence of a pH indicator.

Production
General provisions The production of vaccine is based on a virus seed-lot system and, if the virus is propagated in human diploid cells, a cellbank system. The production method shall have been shown to yield consistently live mumps vaccines of adequate immunogenicity and safety in man. Unless otherwise justified and authorised, the virus in the final vaccine shall have undergone no more passages from the master seed lot than were used to prepare the vaccine

IP 2007

PERTUSSIS VACCINE

Only a single harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine.

Tests
Water (2.3.43). Not more than 3.0 per cent, determined by Karl Fischer, semi-micro determination of water or by any suitable validated method. Sterility ( 2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Assay Titrate the vaccine for infective virus at least in triplicate, using at least five cell cultures for each 0.5 log10 dilution step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated virus concentration is not less than that stated on the label; the minimum virus concentration stated on the label is not less than 5 x 103 CCID50 per human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration is greater than 0.3. Mumps vaccine (Live) RS is suitable for use as a reference preparation. Labelling. The label states (1) the strain of virus used for the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) the minimum virus concentration and; (4) the time within which the vaccine must be used after reconstitution.

Identification
The single harvest contains virus that is identified as mumps virus by serum neutralization in cell culture, using specific antibodies. Virus concentration. The virus concentration in the single harvest is determined as prescribed under Assay to monitor consistency of production and to determine the dilution to be used for the final bulk vaccine. Sterility (2.2.11). Single harvest complies with sterility test should be processed further. Control cells. The control cells comply with a test for extraneous agents (2.7.3). FINAL BULK VACCINE Single harvests that comply with the above tests are pooled and clarified to remove cells. A suitable stabiliser may be added and the pooled harvests diluted as appropriate. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). The final bulk vaccine complies with the test for sterility, carried out using 10 ml for each medium. FINAL LOT A minimum virus concentration for release of the product is established such as to ensure, in the light of stability data, that the minimum concentration stated on the label will be present at the end of the period of validity. Only a final lot that complies with the tests for minimum virus concentration for release, with the following requirement for thermal stability and with each of the requirements given below under Identification and Tests and Assay may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37 for 7 days. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37 for 7 days and for vaccine stored at 2 to 8. The virus concentration of the vaccine exposed to 37 for 7 days is not more than 1.0 log10 lower than that of the unheated vaccine.

Pertussis Vaccine
Pertussis Vaccine is a sterile saline suspension of inactivated whole cells of one or more strains of Bordetella pertussis.

Production
General provisions Inactivated B. pertussis suspension Production is based on a seed-lot system. One or more strains of B. pertussis with known origin and history are used. Strains, culture medium and cultivation method are chosen in such a way that agglutinogens 1, 2 and 3 are present in the final vaccine. Each strain is grown for 24 to 72 hours in a liquid medium or on a solid medium; the liquid medium used in the final cultivation stage does not contain blood or blood products. Human blood or blood products are not used in any culture media. The bacteria are harvested, washed to remove substances derived from the medium and suspended in a 0.9 per cent w/v solution of sodium chloride or other suitable 69

Identification
When the vaccine is reconstituted as stated on the label is mixed with specific mumps antibodies, it is no longer able to infect susceptible cell cultures.

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IP 2007

isotonic solution. The opacity of the suspension is determined not later than 2 weeks after harvest by comparison with the reference preparation of Opacity and used as the basis of calculation for subsequent stages in vaccine preparation. Single harvests are not used for the final bulk vaccine unless they have been shown to contain B. pertussis cells with the same characteristics with regard to growth and agglutinogens, as the parent strain and to be free from contaminating bacteria and fungi. The bacteria are killed and detoxified in controlled conditions by means of a suitable chemical agent or by heating or by a combination of these methods. Freedom from live B. pertussis is tested using a suitable culture medium. The suspension is maintained at 5 + 3 for a suitable period to diminish its toxicity. FINAL BULK VACCINE Suitable quantities of the inactivated single harvests are pooled to prepare the final bulk vaccine. Suitable antimicrobial preservatives may be added. The bacterial concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 IU per single human dose. If 2 or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity, free formaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.

to 16 g for the vaccine group and for the saline control. Use mice of the same sex or distribute males and females equally between the groups. Allow the animals access to food and water for at least 2 hours before injection and during the test. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing a quantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse of the control group with 0.5 ml of a 0.9 per cent w/v sterile solution of sodium chloride, preferably containing the same amount of antimicrobial preservative as that injected with the vaccine. Weigh the groups of mice immediately before the injection, 72 hours and 7 days after the injection. The vaccine complies with the test if (a) at the end of 72 h the total mass of the group of vaccinated mice is not less than that preceding the injection; (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60.0 per cent of that per control mouse; and (c) not more than 5.0 per cent of the vaccinated mice die during the test. The test may be repeated and the results of the tests combined. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not more than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Assay Carry out the assay of Pertussis Vaccine as described below : Biological assay of pertussis vaccine The potency of PertussisVaccine is determined by comparison of the dose necessary to protect mice against the effects of a lethal dose of Bordetella pertussis challenge culture, administered intracerebrally, with the dose of a reference preparation, calibrated in International Units, required to give the same level of protection. For this comparison, the Standard preparation of Pertussis vaccine & a suitable strain of B. pertussis (e.g.18323, to be used as challenge strain), are required. Reference preparation The reference preparation is an International standard of Pertussis vaccine, consisting of a freeze dried vaccine or another suitable preparation, calibrated in comparison to International standard, from time to time. The International Unit is the activity contained in a stated amount of the International standard, which consists of a quantity of dried pertussis vaccine. The equivalence in International Units of the International Standard is stated by the World Health Organization. Use healthy mice of a suitable strain, weighing between 13 and 16 g from the same stock. Distribute the mice randomly, in 70

Identification
Identify pertussis vaccine by agglutination of the bacteria in the vaccine by antisera specific to B. pertussis.

Tests
Specific toxicity Use not less than 10 healthy mice each weighing between 14

IP 2007

PNEUMOCOCCAL POLYSACCHARIDE VACCINE

six to eight groups of not less than 16 and not more than 24 and four groups of 10. The mice should all be of the same sex or the males and females should be distributed equally between the groups. Half of the groups of 16 to 24 should receive the reference preparation and the other half should receive the vaccine under examination. The four groups of 10 each should be used for the LD50 titration of challenge suspension. Use at least, three dilutions of the reference vaccine and similar dilutions of the vaccine under examination. In each case the dilutions are so selected that the dilution protecting 50 per cent of the mice (ED50) is as near as possible to middle of the dilution range. For example, suggested dilutions are (1/8, 1/40 and 1/200 of the human dose of the vaccine under examination) and (0.5 IU, 0.1 IU and 0.02 IU or any other suitable standardized dilutions, of the reference preparation), each dose being contained in a volume, not exceeding 0.5 ml. For each dilution use 16 to 24 mice and inject intraperitoneally, into each mouse one dose of the dilution. Select a suitable strain of B. pertussis (e.g. 18323), capable of causing the death of mice within 14 days of intracerebral injection. Make two subcultures after reviving the strain on a suitable medium (e.g. B.G. medium) and suspend the harvested growth in a solution containing 1 per cent w/v of casein hydrolysate (e.g. casamino acid) and 0.85 per cent w/v of sodium chloride and having a pH of 7.0 to 7.2 or in another suitable solution. Determine the opacity of the suspension by using 5th International reference preparation for opacity (10 OU) and/or spectophotometerically. Alternatively, aliquots of challenge suspension frozen in liquid nitrogen with a suitable preservative like 10 per cent DMSO may be used, to avoid heterogenicity. After 14 to 17 days of immunization, inject intracerebrally, a dose of 0.02 to 0.03 ml of the challenge dilution randomly, into each immunized mouse. The challenge should contain, approximately 1,00,000 organisms and 100 to 1000 LD50 per dose, in a volume of not more than 0.03 ml. In the same way, inject 4 groups of 10 control mice each, for LD50 titration of challenge preparation, prepared by a series of dilutions, from the dilution selected for challenge. The challenge should be completed within 2 to 2.5 hours of preparation. Exclude any mouse from consideration, that dies within 3 days of challenge. Count the number of mice surviving in each of the groups, after 14 days. On the basis of the numbers of animals surviving in each of the groups of 16 to 24 mice, calculate the potency of vaccine under examination, against the potency of reference preparation. Seed a suitable highest dilution of the challenge suspension, into each of two B.G medium plates, before and after challenge. Incubate the plates at 37 for 48 to 72 hours and calculate the number of colony forming units (CFUs). Calculate the potency of the vaccine by Probit analysis and LD 50 of the challenge suspension by Reed and Munch Method. 71

The test is not valid unless: a) for both the vaccine under examination and the reference preparation, the ED50 (protective dose), lies between the largest and the smallest doses given to the mice; b) the number of animals, which die in the four groups of 10 injected with the challenge suspension and its dilutions indicate that the challenge dose contains 100 to 1000 LD50 and 1 LD50 contains not more than 300 colony forming units; c) and the statistical analysis shows no deviation from linearity or parallelism, in terms of significance of the scope of dose response curve; d) the vaccine passes the requirements for potency, if the test results of a statistically valid assay show that the estimated potency of the vaccine is not less than 4.0 IU per single human dose and the lower fiducial limit (P = 0.95) of estimated potency is not less than 2.0 IU. The test may be repeated once, but when more than one test is performed the results of all valid tests must be combined, in the estimate of potency. Labelling. The label states (1) the minimum number of International Units per single human dose; (2) that the vaccine must be shaken before use; (3) that the vaccine is not to be frozen.

Pneumococcal Polysaccharide Vaccine

Pneumococcal Polysaccharide Vaccine consists of a mixture of equal parts of purified capsular polysaccharide of various serotype antigens prepared from suitable pathogenic strains of Streptococcus pneumoniae in different desired combinations whose capsules have been shown to be made up of polysaccharides that are capable of inducing satisfactory levels of specific antibodies in man. It contains upto 23 immunochemically different capsular polysaccharides listed in the Table 1.

Production
General provisions Production of the vaccine is based on a well defined seed-lot system for each type. The production method shall have been shown to yield consistently pneumococcal polysaccharide vaccines of acceptable immunogenicity and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the tests for abnormal toxicity of vaccines for human use, modified as follows for the tesonn guinea-pig; inject 10 human dose into each guineapig and observe for 12 days. Monovalent bulk polysaccharides The bacteria are grown in a suitable liquid medium that does not contain blood-group substances or high-molecular-mass polysaccharides. The bacterial purity of the culture is verified

PNEUMOCOCCAL POLYSACCHARIDE VACCINE

IP 2007

Table 1- Specifications on monovalent bulk polysaccharides (per cent contents): Molecular Type* 1 2 3 4 5 6B 7F 8 9N 9V 10A 11A 12F 14 15B 17A or 17F 18C 19A 19F 20 22F 23F 33F Proteins Nucleic acids <2 <2 <5 <3 < 7.5 <2 <5 <2 <2 <2 <7 <3 <3 <5 <3 <2 <3 <2 <3 <2 <2 <2 < 2.5 <2 <2 <2 <2 <2 <2 <2 <2 <1 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 Total 3.5-6 0-1 0-1 4-6 2.5-6.0 0-2 1.5-4.0 0-1 2.2-4 0.5-3 0.5-3.5 0-2.5 3-5 1.5-4 1-3 0-1.5 0-1 0.6-3.5 1.4-3.5 0.5-2.5 0-2 0-1 0-2 Phosphorus Molecular size K0 Nitrogen CL-4B** CL-2B*** 0-1.5 0-1.0 0-1.0 0-1.5 <2 2.5-5.0 0-1.0 0-1.0 0-1.0 0-1.0 1.5-3.5 2.0-5.0 0-1.0 0-1.0 2.0-4.5 0-3.5 2.4-4.9 3.0-7.0 3.0-5.5 1.5-4.0 0-1.0 3.0-4.5 0-1.0 < 0.15 < 0.15 < 0.15 < 0.15 < 0.60 < 0.50 < 0.20 < 0.15 < 0.20 < 0.45 < 0.65 < 0.40 < 0.25 < 0.30 < 0.55 < 0.45 < 0.15 < 0.45 < 0.20 < 0.60 < 0.55 < 0.15 < 0.50 15 Uronic acids 45 15 40 12 40 20 15 13 25 20 15 28 13 12 9 25 20 15 20 14 20 20 25 37 HexoMethylsamines pentoses 38 O-acetyl Groups 1.8

12 12.5 12

* The different types are indicated using the Danish nomenclature ** Cross linked agarose for chromatography R *** Cross linked agarose for chromatography R1

and the culture is inactivated with phenol. Impurities are removed by such techniques as fractional precipitation, enzymatic digestion and ultrafiltration. The polysaccharide is obtained by fractional precipitation, washed, and dried in a vacuum to a residual moisture content shown to be favourable to the stability of the polysaccharide. The residual moisture content is determined by drying under reduced pressure over diphosphorus pentoxide or by thermogravimetric analysis and the value obtained is used to calculate the results of the tests shown below with reference to the dried substance. The monovalent bulk polysaccharide is stored at a suitable temperature in conditions that avoid the uptake of moisture. Only a monovalent bulk polysaccharide that complies with the following requirements may be used in the preparation of the final bulk vaccine. Percentage contents of components, 72

determined by the methods prescribed below, are shown in the Table 1. The purified polysaccharides comply with the following tests as applicable: Protein (2.7.1). Comply with the test for protein. Nucleic acids (2.7.1). Comply with the test for nucleic acids. Total nitrogen (2.3.30). Comply with the test for total nitrogen. Phosphorus (2.7.1). Comply with the test for phosphorus. Uronic acids (2.7.1). Comply with the test for uronic acids. Hexosamine (2.7.1). Comply with the test for hexosamine. Methylpentoses (2.7.1). Comply with the test for methylpentoses.

IP 2007

POLYSACCHARIDE VACCINE (INACTIVATED)

O-Acetyl groups (2.7.1). Comply with the test for O-acetyl groups. Sterility (2.2.11). Comply with the test for sterility. Molecular size. Molecular size is determined by gel filtration or high performance size-exclusion chromatography (HPSEC) (2.4.16) using cross linked Agarose for chromatography R or chromatography Agarose for chromatograph R1, either alone or Multiple angle light laser scattering (MALLS) or any other suitable method.

on each monovalent bulk polysaccharide used in the preparation of the final lot.

Identification
The assay also serves to identify the vaccine.

Tests
Sterility (2.2.11). Complies with the test for sterility. Abnormal Toxicity (2.2.1). Complies with the test for abnormal toxicity with the following modifications. Inject 10 human doses each in two guinea pigs weighing between 250 and 350 g by intraperitoneal route and observe for 12 days, Pyrogens (2.2.8). Complies with the test for pyrogens. Inject each of the rabbit with 1 ml of a dilution of the vaccine containing 2.5 g/ml of each polysaccharide. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Phenol (2.3.36). Not more than 2.5 g/l. pH (2.4.24). 4.5 to 7.4. Assay Determine the content of each polysaccharide by a suitable biochemical, physicochemical or immunochemical method (2.2.14), using antisera specific for each polysaccharide contained in the vaccine, including factor sera for types within groups, and purified polysaccharides of each type as standards. The vaccine contains not less than 70.0 per cent and not more than 130.0 per cent of the quantity stated on the label for each polysaccharide. The confidence interval (P = 0.95) of the assay is not less than 80.0 and not more than 120.0 per cent of the estimated content. Labelling. The label states (1) the number of g of each polysaccharide per human dose; (2) the total amount of polysaccharide in the container.

Identification
Confirm the identity of the monovalent bulk polysaccharide by immunochemical method (2.2.14)(except for polysaccharides 7F, 14 and 33F). Specificity For establishing the specificity, no reaction should occur, when the antigens are tested against all the antisera specific for the other polysaccharides of the vaccine, including factor sera for distinguishing types within groups. The polysaccharides are tested at a concentration of 50 g/ml using a method capable of detecting 0.5 g/ml. FINAL BULK VACCINE The final bulk vaccine is obtained by aseptically mixing the different polysaccharide powders. The uniform mixture is aseptically dissolved in a suitable isotonic solution so that one human dose of 0.5 ml contains 25 g of each polysaccharide. An antimicrobial preservative may be added. The solution is sterilized by filtration through a bacteriaretentive filter. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed and filled aseptically into sterile containers (vials or ampoules). Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for phenol and for antimicrobial preservative have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. When consistency of production has been established on a suitable number of consecutive batches, the assay may be replaced by a qualitative test that identifies each polysaccharide, provided that an assay has been performed 73

Poliomyelitis Vaccine (Inactivated)

Poliomyelitis Vaccine (Inactivated) is a liquid preparation of suitable strains of human polioviruses 1, 2 and 3 grown in suitable cell cultures and inactivated by a validated method.

Production
General provisions The production method should consistently yield vaccines of acceptable safety and immunogenicity in man.

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IP 2007

Production of the vaccine is based on a virus seed-lot system. Cell lines are used according to a cell-bank system. If primary, secondary or tertiary monkey kidney cells are used, production complies with the requirements indicated below. Unless otherwise justified and authorised, the virus in the final vaccine shall not have undergone more passages from the master seed lot than was used to prepare the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Substrate for virus propagation The virus is propagated in a human diploid cell line (2.7.2), in a continuous cell line (2.7.2) or in primary, secondary or tertiary monkey kidney cells. Primary, secondary or tertiary monkey kidney cells. The following special requirements for the substrate for virus propagation apply to primary, secondary or tertiary monkey kidney cells. Monkeys used in the preparation of kidney cell cultures for production and control of the vaccine. The animals used are of a species approved by the competent authority, in good health and, unless otherwise justified and authorised, have not been previously employed for experimental purposes. Kidney cells used for vaccine production and control are derived from monitored, closed colonies of monkeys bred in captivity, not from animals caught in the wild; a previously approved seed lot prepared using virus passaged in cells from wild monkeys may, subject to approval by the competent authority, be used for vaccine production if historical data on safety justify this. Monitored, closed colonies of monkeys. The monkeys are kept in groups in cages. Freedom from extraneous agents is achieved by the use of animals maintained in closed colonies that are subject to continuous and systematic veterinary and laboratory monitoring for the presence of infectious agents. The supplier of animals is certified by the competent authority. Each monkey is tested serologically at regular intervals during a quarantine period of not less than 6 weeks imposed before entering the colony and then during its stay in the colony. The monkeys used are shown to be tuberculin-negative and free from antibodies to simian virus 40 (SV40) and simian immunodeficiency virus. If Macaca spp. monkeys are used for production, the monkeys are also shown to be free from antibodies to herpesvirus B (Cercopithecine herpesvirus 1) infection. Human herpesvirus 1 has been used as an indicator for freedom from herpesvirus B antibodies on account of the danger of handling herpesvirus B 74

(Cercopithecine herpesvirus 1). Monkeys from which kidneys are to be removed are thoroughly examined, particularly for evidence of tuberculosis and herpesvirus B (Cercopithecine herpesvirus 1) infection. If a monkey shows any pathological lesion relevant to the use of its kidneys in the preparation of a seed lot or vaccine, it is not to be used nor are any of the remaining monkeys of the group concerned unless it is evident that their use will not impair the safety of the product. All the operations described in this section are conducted outside the area where the vaccine is produced. Monkey cell cultures for vaccine production. Kidneys that show no pathological signs are used for preparing cell cultures. Each group of cell cultures derived from a single monkey forms a separate production cell culture giving rise to a separate single harvest. The primary monkey kidney cell suspension complies with the test for mycobacteria ; disrupt the cells before carrying out the test. If secondary or tertiary cells are used, it shall be demonstrated by suitable validation tests that cell cultures beyond the passage level used for production are free from tumorigenicity. SEED LOT Each of the three strains of poliovirus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. Only a working seed lot that complies with the following requirements may be used for virus propagation.

Identification
Each working seed lot is identified as human poliovirus 1, 2 or 3 by virus neutralisation in cell culture using specific antibodies. Virus concentration. The virus concentration of each working seed lot is determined to define the quantity of virus to be used for inoculation of production cell cultures. Extraneous agents (2.7.3). The working seed lot complies with the requirements for seed lots for virus vaccines. In addition, if primary, secondary or tertiary monkey kidney cells have been used for isolation of the strain, measures are taken to ensure that the strain is not contaminated with simian viruses such as simian immunodeficiency virus, simian virus 40, filoviruses and herpesvirus B (Cercopithecine herpesvirus 1). A working seed lot produced in primary, secondary or tertiary monkey kidney cells complies with the requirements given below under Virus Propagation and Harvest for single harvests produced in such cells.

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POLYSACCHARIDE VACCINE (INACTIVATED)

PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells or viruses are being handled. Approved animal serum (but not human serum) may be used in the cell culture media. Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from extraneous agents. The cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. Not less than 500 ml of the cell cultures employed for vaccine production is set aside as uninfected cell cultures (control cells); where continuous cell lines in a fermenter are used for production, 200 106 cells are set aside to prepare control cells; where primary, secondary or tertiary monkey kidney cells are used for production, a cell sample equivalent to at least 500 ml of the cell suspension, at the concentration employed for vaccine production, is taken to prepare control cell cultures. Only a single harvest that complies with the following requirements may be used in the preparation of the vaccine. The tests for Identification and Sterility may be carried out instead on the purified, pooled monovalent harvest. After demonstration of consistency of production at the stage of the single harvest, the test for virus concentration may be carried out instead on the purified, pooled monovalent harvest. Control cells. The control cells of the production cell culture comply with a test for Identification (if a cell-bank system is used for production) and with the requirements for extraneous agents, where primary, secondary or tertiary monkey kidney cells are used, the tests in cell cultures are carried out as shown below under Test in Rabbit Kidney Cell Cultures and Test in Cercopithecus Kidney Cell Cultures). Test in rabbit kidney cell cultures. Test a sample of at least 10 ml of the pooled supernatant fluid from the control cultures for the absence of herpesvirus B (Cercopithecine herpesvirus 1) and other viruses in rabbit kidney cell cultures. The dilution of supernatant in the nutrient medium is not greater than 1:4 and the area of the cell layer is at least 3 cm2 per ml of inoculum. Set aside one or more containers of each batch of cells with the same medium as non-inoculated control cells. Incubate the cultures at 37 and observe for at least 2 weeks. The test is not valid if more than 20 per cent of the control cells are discarded for non-specific, accidental reasons. Test in Cercopithecus kidney cell cultures. Test a sample of at least 10 ml of the pooled supernatant fluid from the control cultures for the absence of SV40 virus and other extraneous agents by inoculation onto cell cultures prepared from the kidneys of cercopithecus monkeys, or other cells shown to be at least as sensitive for SV40, by the method described under Test in Rabbit Kidney Cell Cultures. The test is not valid if more than 20 per cent of the control cell cultures are discarded 75

for non-specific, accidental reasons.

Identification
The single harvest is identified as containing human poliovirus 1, 2 or 3 by virus neutralisation in cell cultures using specific antibodies. Virus concentration. The virus concentration of each single harvest is determined by titration of infectious virus in cell cultures. Sterility (2.2.11). The single harvest complies with the test for sterility, carried out using 10 ml for each medium. Mycoplasmas (2.7.4). The single harvest complies with the test for mycoplasmas, carried out using 10 ml. Test in rabbit kidney cell cultures. Where primary, secondary or tertiary monkey kidney cells are used for production, test a sample of at least 10 ml of the single harvest for the absence of herpesvirus B (Cercopithecine herpesvirus 1) and other viruses in rabbit kidney cell cultures as described for the control cells. Test in Cercopithecus kidney cell cultures. Where primary, secondary or tertiary monkey kidney cells are used for production, test a sample of at least 10 ml of the single harvest for the absence of SV40 virus and other extraneous agents. Neutralise the sample by a high-titre antiserum against the specific type of poliovirus. Test the sample in primary cercopithecus kidney cell cultures or cells that have been demonstrated to be at least as susceptible for SV40. Incubate the cultures at 37 and observe for 14 days. At the end of this period, make at least one subculture of fluid in the same cell culture system and observe both primary cultures and subcultures for an additional 14 days. PURIFICATION AND PURIFIED MONOVALENT HARVEST Several single harvests of the same type may be pooled and may be concentrated. The monovalent harvest or pooled monovalent harvest is purified by validated methods. If continuous cell lines are used for production, the purification process shall have been shown to reduce consistently the content of substrate-cell DNA to not more than 500 pg per single human dose. Only a purified monovalent harvest that complies with the following requirements may be used for the preparation of the inactivated monovalent harvest.

Identification
The virus is identified by virus neutralisation in cell cultures using specific antibodies or by determination of D-antigen. Virus concentration. The virus concentration is determined by titration of infectious virus.

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Specific activity. The ratio of the virus concentration or the Dantigen content, determined by a suitable immunochemical method (2.2.14). to the total protein content (specific activity) of the purified monovalent harvest is within the limits approved for the particular product. INACTIVATION AND INACTIVATED MONOVALENT HARVEST Several purified monovalent harvests of the same type may be mixed before inactivation. To avoid failures in inactivation caused by the presence of virus aggregates, filtration is carried out before and during inactivation; inactivation is started within a suitable period, preferably not more than 24 h and in any case not more than 72 h, of the prior filtration. The virus suspension is inactivated by a validated method that has been shown to inactivate poliovirus without destruction of immunogenicity; during validation studies, an inactivation curve with at least four points (for example, time 0, 24, 48, and 96 h) is established showing the decrease in concentration of live virus with time. If formaldehyde is used for inactivation, the presence of an excess of formaldehyde at the end of the inactivation period is verified. Only an inactivated monovalent harvest that complies with the following requirements may be used in the preparation of a trivalent pool of inactivated monovalent harvests or a final bulk vaccine. Test for effective inactivation. After neutralisation of the formaldehyde with sodium bisulphite (where applicable), verify the absence of residual live poliovirus by inoculation on suitable cell cultures of two samples of each inactivated monovalent harvest, corresponding to at least 1500 human doses. Take one sample not later than three-quarters of the way through the inactivation period and the other at the end. Inoculate the samples in cell cultures such that the dilution of vaccine in the nutrient medium is not greater then and the area of the cell layer is at least 3 cm2 per ml of inoculum. Set aside one or more containers with the same medium as noninoculated control cells. Observe the cell cultures for at least 3 weeks. Make not fewer than two passages from each container, one at the end of the observation period and the other 1 week before; for the passages, use cell culture supernatant and inoculate as for the initial sample. Observe the subcultures for at least 2 weeks. No sign of poliovirus multiplication is present in the cell cultures. At the end of the observation period, test the susceptibility of the cell culture used by inoculation of live poliovirus of the same type as that present in the inactivated monovalent harvest. Sterility (2.2.11). The inactivated monovalent harvest complies with the test for sterility, carried out using 10 ml for each medium. D-antigen content. The content of D-antigen determined by a 76

suitable immunochemical method (2.2.14) is within the limits approved for the particular preparation. FINAL BULK VACCINE The final bulk vaccine is prepared directly from the inactivated monovalent harvests of human polioviruses 1, 2 and 3 or from a trivalent pool of inactivated monovalent harvests. If a trivalent pool of inactivated monovalent harvests is used, a test for effective inactivation is carried out on this pool instead of on the final bulk vaccine. A stabiliser and an antimicrobial preservative may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Inactivation. Before addition of any antimicrobial preservative, a sample of at least 1500 ml or, for a purified and concentrated vaccine, the equivalent of 1500 doses is tested for residual live poliovirus in cell cultures, as described for the inactivated monovalent harvest. If the final bulk vaccine is prepared from a trivalent pool of inactivated monovalent harvests, the test for inactivation is carried out on that pool rather than on the final bulk vaccine. FINAL LOT Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde and antimicrobial preservative and the in vivo assay have been performed with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided that the test for bovine serum albumin has been performed with satisfactory results on the trivalent pool of inactivated monovalent harvests or on the final bulk vaccine, it may be omitted on the final lot.

Identification
The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemical method such as the determination of D-antigen by enzyme-linked immunosorbent assay (ELISA).

Tests
Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical

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method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Protein content (2.3.49). Not more than 10 g of protein nitrogen per human dose. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Sterility (2.2.11). Complies with the test for sterility. Bacterial endotoxins ( 2.2.3). Not more than 5 IU per human dose. Assay D-antigen content. As a measure of consistency of production, determine the D-antigen content for human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14) using an appropriate reference preparation calibrated in D-antigen units. For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits approved for the particular product. In vivo test. The capacity of the vaccine to induce the formation of neutralizing antibodies is determined in-vivo by one of the following methods: Test in chicks or guinea-pigs. Prepare a suitable series of at least three dilutions of the vaccine under examination using a suitable buffered saline solution. Inject 0.5 ml of the dilutions intramuscularly into groups of ten 3-week-old chickens or groups of ten guinea-pigs, each weighing between 250 and 350 g, using a separate group for each dilution of vaccine. Bleed the animals on the fifth or sixth day after the injection and separate the sera. Examine the sera for the presence of neutralising antibody, at a dilution of 1 in 4, to each of the human polioviruses 1, 2 and 3. Mix 100 CCID50 of virus with the dilution of serum and incubate at 37 for 4 h 30 min to 6 h. Keep at 5 3 for 12 to 18 h. Inoculate the mixtures into cell cultures for the detection of unneutralised virus and read the results up to 7 days after inoculation. For each group of animals, note the number of sera which have neutralising antibody and calculate the dilution of the vaccine giving an antibody response in 50.0 per cent of the animals. Carry out in parallel a control test using a suitable reference preparation. The vaccine complies with the test if a dilution of 1 in 100 or more produces an antibody response for each of the three types of virus in 50.0 per cent of the animals. Test on rats. A suitable in vivo assay method consists of intramuscular injection into the hind limb(s) of not fewer than 3 dilutions of the vaccine under examination and a reference vaccine, using for each dilution a group of 10 specific pathogen-free rats of the suitable strain. Use of 4 dilutions is often necessary to obtain valid results for all 3 serotypes. The 77

number of animals per group must be sufficient to obtain results that meet the validity criteria; groups of 10 rats are usually sufficient although valid results may be obtained with fewer animals per group. The weight of individual animal must not vary by more than 10.0 per cent from the group mean. An inoculum of 0.5 ml per rat is used. The dose range is chosen such that a dose response to all 3 poliovirus types is obtained. Bleed the animals after 20 to 22 days. Neutralising titres against all 3 polivirus types are measured separately using 100 CCID50 of the Sabin strains as challenge viruses. Vero or Hep2 as indicator cells, and neutralization conditions of 3 h at 35 to 37 followed by 18 h at 2 to 8. Results are read following fixation and staining after 7 days of incubation at 35. For a valid antibody assay, the titre of each challenge virus must be shown to be within the range of 10 to 1000 CCID50 and the neutralizing antibody titre of a control serum must be within 2 twofold dilutions of the geometric mean titre of the serum. The potency is calculated by comparison of the preparation of responders for the vaccine under examination and the reference vaccine by the probit method or, after validation, using a parallel-line model. For the probit method it is necessary to establish a cut-off neutralising antibody titre for each poliovirus type to define a responder. Due to interlaboratory variation, it is not possible to define cut-off values that could be applied by all laboratories. Rather, the cut-off values are determined for each laboratory based on a minimum series of 3 tests with the reference vaccine. The midpoint on a log 2 scale of the minimum and maximum geometric mean titres of the series of 3 or more tests is used as the cutoff value. For each of the 3 poliovirus types, the potency of the vaccine is not significantly less than that of the reference preparation. The test is not valid unless (1) for both the test and reference vaccines the ED50 lies between the smallest and the largest doses given to the animals; (2) the statistical analysis shows no significant deviation from linearity or parallelism; (3) the fiducial limits of the estimated relative potency fall between 25.0 per cent and 400.0 per cent of the estimated potency. Labelling. The label states (1) the types of poliovirus contained in the vaccine; (2) the nominal amount of virus of each type (1, 2 and 3), expressed in units of D-antigen per single human dose; (3) the cell substrate used to prepare the vaccine.

Poliomyelitis Vaccine, Live (Oral)

Oral Poliomyelitis Vaccine is a preparation of approved strains of live attenuated poliovirus type 1, 2 or 3 grown in in vitro cultures of approved cells, containing any one type or any combination of the three types of Sabin strains, prepared in a form suitable for oral administration.

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Production
General provisions The vaccine strains and the production method should consistently yield vaccines that are both immunogenic and safe in man. The production of vaccine is based on a virus seed-lot system. Cell lines are used according to a cell-bank system. If primary monkey kidney cells are used, production complies with the requirements indicated below. Unless otherwise justified and authorised, the virus in the final vaccine shall not have undergone more than two passages from the master seed lot. Substrate for virus propagation The virus is propagated in human diploid cells (2.7.2) or in continuous cell lines (2.7.2) or in primary monkey kidney cells (including serially passaged cells from primary monkey kidney cells). Continuous cell lines are approved by the competent authority. Primary monkey cells. The following special requirements for the substrate for virus propagation apply to primary monkey cells. Monkeys used for preparation of kidney cell cultures and for testing of virus. If the vaccine is prepared in monkey kidney cell cultures, animals of a species approved by the competent authority, in good health, and not previously employed for experimental purposes shall be used. The monkeys shall be kept in well-constructed and adequately ventilated animal rooms in cages spaced as far apart as possible. Adequate precautions shall be taken to prevent cross-infection between cages. Not more than two monkeys shall be housed per cage and cage-mates shall not be interchanged. The monkeys shall be kept in the country of manufacture of the vaccine in quarantine groups for a period of not less than 6 weeks before use. A quarantine group is a colony of selected, healthy monkeys kept in one room, with separate feeding and cleaning facilities, and having no contact with other monkeys during the quarantine period. If at any time during the quarantine period the overall death rate of a shipment consisting of one or more groups reaches 5 per cent (excluding deaths from accidents or where the cause was specifically determined not to be an infectious disease), monkeys from that entire shipment shall continue in quarantine from that time for a minimum of 6 weeks. The groups shall be kept continuously in isolation, as in quarantine, even after completion of the quarantine period, until the monkeys are used. After the last monkey of a group has been taken, the room that housed the group shall be thoroughly cleaned and decontaminated before being used for a fresh group. If kidneys from near-term monkeys are used, the mother is quarantined for the term of pregnancy. 78

Monkeys from which kidneys are to be removed shall be anaesthetised and thoroughly examined, particularly for evidence of tuberculosis and cercopithecid herpesvirus 1 (B virus) infection. If a monkey shows any pathological lesion relevant to the use of its kidneys in the preparation of a seed lot or vaccine, it shall not be used, nor shall any of the remaining monkeys of the quarantine group concerned be used unless it is evident that their use will not impair the safety of the product. All the operations described in this section shall be conducted outside the areas where the vaccine is produced. The monkeys used shall be shown to be free from antibodies to simian virus 40 (SV40) and simian immunodeficiency virus. If Macaca spp. are used for production, the monkeys shall also be shown to be free from antibodies to cercopithecid herpesvirus 1 (B virus). Human herpesvirus has been used as an indicator for freedom from B virus antibodies on account of the danger of handling cercopithecid herpesvirus 1 (B virus). Monkey kidney cell cultures for vaccine production. Kidneys that show no pathological signs are used for preparing cell cultures. If the monkeys are from a colony maintained for vaccine production, serially passaged monkey kidney cell cultures from primary monkey kidney cells may be used for virus propagation, otherwise the monkey kidney cells are not propagated in series. Virus for the preparation of vaccine is grown by aseptic methods in such cultures. If animal serum is used in the propagation of the cells, the maintenance medium after virus inoculation shall contain no added serum. Each group of cell cultures derived from a single monkey or from fetuses from no more than ten near-term monkeys is prepared and tested as an individual group. SEED LOT The strains of poliovirus used shall be identified by historical records that include information on the origin and subsequent manipulation of the strains. Working seed lots are prepared by a single passage from a master seed lot and at an approved passage level from the original Sabin virus. Virus seed lots are prepared in large quantities and stored at a temperature below -60. Only a virus seed lot that complies with the following requirements may be used for virus propagation.

Identification
Each working seed lot is identified as poliovirus of the given type, using specific antibodies. Virus concentration. Determined by the method described below, the virus concentration is the basis for the quantity of virus used in the neurovirulence test.

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Extraneous agents (2.7.3). If the working seed lot is produced in human diploid cells (2.7.2) or in continuous cell lines (2.7.2) it complies with the requirements for seed lots for virus vaccines. If the working seed lot is produced in primary monkey cells, it complies with the requirements given below under Propagation and Harvest and Monovalent Pooled Harvest and with the tests in adult mice, suckling mice and guinea-pigs given under Tests for extraneous agents in viral vaccines for human use. Working seed lot shall be free from detectable DNA sequences from simian virus 40 (SV40) Neurovirulence (2.7.6). Each master and working seed lot complies with the test for neurovirulence of poliomyelitis vaccine (oral) in monkeys. Furthermore, the seed lot shall cease to be used in vaccine production if the frequency of failure of the monovalent pooled harvests produced from it is greater than predicted statistically. This statistical prediction is calculated after each test on the basis of all the monovalent pooled harvests tested; it is equal to the probability of false rejection on the occasion of a first test (i.e. 1 per cent), the probability of false rejection on retest being negligible. If the test is carried out only by the manufacturer, the test slides are provided to the control authority for assessment. Genetic markers. Each working seed lot is tested for its replicating properties at temperatures ranging from 36 to 40 as described under Monovalent Pooled Harvest. PROPAGATION AND HARVEST All processing of the cell-banks and subsequent cell-cultures is done under aseptic conditions in an area where no other cells are handled. Approved animal (but not human) serum may be used in the media, but the final medium for maintaining cell growth during virus multiplication does not contain animal serum. Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from live extraneous agents. The cell-culture medium may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. It is preferable to have a substrate free from antibiotics during production. Not less than 5 per cent and not more than 1000 ml of the cell cultures employed for vaccine production are set aside as uninfected cell cultures (control cells); special requirements, given below, apply to control cells when the vaccine is produced in primary monkey cells The virus suspension is harvested not later than 4 days after virus inoculation. After inoculation of the production cell culture with the virus working seed lot, inoculated cells are maintained at a fixed temperature, shown to be suitable, within the range 33 to 35; the temperature is maintained constant to 0.5; control cell cultures are maintained at 33 to 35 for the relevant incubation periods. Only a single virus harvest that complies with the following requirements may be used in the preparation of the monovalent 79

pooled harvest. Virus concentration. The virus concentration of virus harvests is determined as prescribed under Assay to monitor consistency of production and to determine the dilution to be used for the final bulk vaccine. Extraneous agents ( 2.7.3 ). Complies with tests for extraneous agents. Control cells. The control cells of the production cell culture from which the virus harvest is derived comply with a test for identity and with the requirements for extraneous agents or, where primary monkey cells are used, as shown below. Primary monkey cells. The following special requirements apply to virus propagation and harvest in primary monkey cells. Cell cultures. On the day of inoculation with virus seed, each cell culture is examined for degeneration caused by an infective agent. If, in this examination, evidence is found of the presence in a cell culture of any extraneous agent, the entire group of cultures concerned shall be rejected. On the day of inoculation with the virus working seed lot, a sample of at least 30 ml of the pooled fluid removed from the cell cultures of the kidneys of each single monkey or from fetuses from not more than ten near-term monkeys is divided into two equal portions. One portion of the pooled fluid is tested in monkey kidney cell cultures prepared from the same species, but not the same animal, as that used for vaccine production. The other portion of the pooled fluid is, where necessary, tested in monkey kidney cell cultures from another species so that tests on the pooled fluids are done in cell cultures from at least one species known to be sensitive to SV40. The pooled fluid is inoculated into bottles of these cell cultures in such a way that the dilution of the pooled fluid in the nutrient medium does not exceed 1 in 4. The area of the cell sheet is at least 3 cm2 per ml of pooled fluid. At least one bottle of each kind of cell culture remains uninoculated to serve as a control. If the monkey species used for vaccine production is known to be sensitive to SV40, a test in a second species is not required. Animal serum may be used in the propagation of the cells, provided that it does not contain SV40 antibody, but the maintenance medium after inoculation of test material contains no added serum except as described below. The cultures are incubated at a temperature of 35 to 37 and are observed for a total period of at least 4 weeks. During this observation period and after not less than 2 weeks incubation, at least one subculture of fluid is made from each of these cultures in the same cell culture system. The subcultures are also observed for at least 2 weeks. Serum may be added to the original culture at the time of subculturing, provided that the serum does not contain SV40 antibody.

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Fluorescent-antibody techniques may be useful for detecting SV40 virus and other viruses in the cells. A further sample of at least 10 ml of the pooled fluid is tested for cercopithecid herpesvirus 1 (B virus) and other viruses in rabbit kidney cell cultures. Serum used in the nutrient medium of these cultures shall have been shown to be free from inhibitors of B virus. Human herpesvirus has been used as an indicator for freedom from B virus inhibitors on account of the danger of handling cercopithecid herpesvirus 1 (B virus). The sample is inoculated into bottles of these cell cultures in such a way that the dilution of the pooled fluid in the nutrient medium does not exceed 1 in 4. The area of the cell sheet is at least 3 cm2 per ml of pooled fluid. At least one bottle of the cell cultures remains uninoculated to serve as a control. The cultures are incubated at a temperature of 35 to 37 and observed for at least 2 weeks. A further sample of 10 ml of the pooled fluid removed from the cell cultures on the day of inoculation with the seed lot virus is tested for the presence of extraneous agents by inoculation into human cell cultures sensitive to measles virus. The tests are not valid if more than 20 per cent of the culture vessels have been discarded for non-specific accidental reasons by the end of the respective test periods. If, in these tests, evidence is found of the presence of an extraneous agent, the single harvest from the whole group of cell cultures concerned is rejected. If the presence of cercopithecid herpesvirus 1 (B virus) is demonstrated, the manufacture of oral poliomyelitis vaccine shall be discontinued and the competent authority shall be informed. Manufacturing shall not be resumed until a thorough investigation has been completed and precautions have been taken against any reappearance of the infection, and then only with the approval of the competent authority. If these tests are not done immediately, the samples of pooled cell-culture fluid shall be kept at a temperature of -60 or below, with the exception of the sample for the test for B virus, which may be held at 4, provided that the test is done not more than 7 days after it has been taken. Control cell cultures. On the day of inoculation with the virus working seed lot 25 per cent (but not more than 2500 ml) of the cell suspension obtained from the kidneys of each single monkey or from not more than ten near-term monkeys is taken to prepare uninoculated control cell cultures. These control cell cultures are incubated in the same conditions as the inoculated cultures for at least 2 weeks and are examined during this period for evidence of cytopathic changes. The tests are not valid if more than 20 per cent of the control cell cultures have been discarded for non-specific, accidental reasons. At the end of the observation period, the control cell cultures are 80

examined for degeneration caused by an infectious agent. If this examination or any of the tests required in this section shows evidence of the presence in a control culture of any extraneous agent, the poliovirus grown in the corresponding inoculated cultures from the same group shall be rejected. Tests for haemadsorbing viruses. At the time of harvest or within 4 days of inoculation of the production cultures with the virus working seed lot, a sample of 4 per cent of the control cell cultures is taken and tested for haemadsorbing viruses. At the end of the observation period, the remaining control cell cultures are similarly tested. The tests are made as described in (2.7.3), Tests for extraneous agents in viral vaccines for human use. Tests for other extraneous agents. At the time of harvest, or within 7 days of the day of inoculation of the production cultures with the working seed lot, a sample of at least 20 ml of the pooled fluid from each group of control cultures is taken and tested in two kinds of monkey kidney cell culture, as described above. At the end of the observation period for the original control cell cultures, similar samples of the pooled fluid are taken and the tests referred to in this section in the two kinds of monkey kidney cell culture and in the rabbit cell cultures are repeated, as described above under Cell cultures. If the presence of Cercopithecid herpesvirus 1 (B virus) is demonstrated, the production cell cultures shall not be used and the measures concerning vaccine production described above must be undertaken. The fluids collected from the control cell cultures at the time of virus harvest and at the end of the observation period may be pooled before testing for extraneous agents. A sample of 2 per cent of the pooled fluid is tested in each of the cell culture systems specified. Single harvests Tests for neutralised single harvests in monkey kidney cell cultures. A sample of at least 10 ml of each single harvest is neutralised by a type-specific poliomyelitis antiserum prepared in animals other than monkeys. In preparing antisera for this purpose, the immunising antigens used shall be prepared in non-simian cells. Half of the neutralised suspension (corresponding to at least 5 ml of single harvest) is tested in monkey kidney cell cultures prepared from the same species, but not the same animal, as that used for vaccine production. The other half of the neutralised suspension is tested, if necessary, in monkey kidney cell cultures from another species so that the tests on the neutralised suspension are done in cell cultures from at least one species known to be sensitive to SV40.

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POLIOMYELITIS VACCINE, LIVE (ORAL)

The neutralised suspensions are inoculated into bottles of these cell cultures in such a way that the dilution of the suspension in the nutrient medium does not exceed 1 in 4. The area of the cell sheet is at least 3 cm2 per ml of neutralised suspension. At least one bottle of each type of cell culture remains uninoculated to serve as a control and is maintained by nutrient medium containing the same concentration of the specific antiserum used for neutralisation. Animal serum may be used in the propagation of the cells, provided that it does not contain SV40 antibody, but the maintenance medium, after the inoculation of the test material, contains no added serum other than the poliovirus neutralising antiserum, except as described below. The cultures are incubated at a temperature of 35 to 37 and observed for a total period of at least 4 weeks. During this observation period and after not less than 2 weeks incubation, at least one subculture of fluid is made from each of these cultures in the same cell-culture system. The subcultures are also observed for at least 2 weeks. Serum may be added to the original cultures at the time of subculturing, provided that the serum does not contain SV40 antibody. Additional tests are made for extraneous agents on a further sample of the neutralised single harvests by inoculation of 10 ml into human cell cultures sensitive to measles virus. Fluorescent-antibody techniques may be useful for detecting SV40 virus and other viruses in the cells. The tests are not valid if more than 20 per cent of the culture vessels have been discarded for non-specific accidental reasons by the end of the respective test periods. If any cytopathic changes occur in any of the cultures, the causes of these change are investigated. If the cytopathic changes are shown to be due to unneutralised poliovirus, the test is repeated. If there is evidence of the presence of SV40 or other extraneous agents attributable to the single harvest, that single harvest is rejected. MONOVALENT POOLED HARVEST Monovalent pooled harvests are prepared by pooling a number of satisfactory single harvests of the same virus type. Monovalent pooled harvests from continuous cell lines may be purified. Each monovalent pooled harvest is filtered through a bacteria-retentive filter. Only a monovalent pooled harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine.

Virus concentration The virus concentration is determined by the method described below and serves as the basis for calculating the dilutions for preparation of the final bulk, for the quantity of virus used in the neurovirulence test and to establish and monitor production consistency. Genetic markers. A ratio of the replication capacities of the virus in the monovalent pooled harvest is obtained over a temperature range between 36 and 40 in comparison with the seed lot or a reference preparation for the marker tests and with appropriate rct/40- and rct/40+ strains of poliovirus of the same type. The incubation temperatures used in this test are controlled to within 0.1. The monovalent pooled harvest passes the test if, for both the virus in the harvest and the appropriate reference material, the titre determined at 36 is at least 5.0 log greater than that determined at 40. If growth at 40 is so low that a valid comparison cannot be established, a temperature in the region of 39.0 to 39.5 is used, at which temperature the reduction in titre of the reference material must be in the range 3.0 to 5.0 log of its value at 36; the acceptable minimum reduction is determined for each virus strain at a given temperature. If the titres obtained for one or more of the reference viruses are not concordant with the expected values, the test must be repeated. Neurovirulence (2.7.6). Each monovalent pooled harvest complies with the test for neurovirulence of poliomyelitis vaccine (oral). If the test is carried out only by the manufacturer, the test slides are provided to the competent authority for assessment. The TgPVR21 transgenic mouse model provides a suitable alternative to the monkey neurovirulence test for neurovirulence testing of types 1, 2 or 3 vaccines once a laboratory qualifies as being competent to perform the test and the experience gained is to the satisfaction of the competent authority. The test is carried out using a standard operating procedure approved by the competent authority. A suitable procedure (Neurovirulence test of type 1, 2 or 3 live poliomyelitis vaccines (oral) in transgenic mice susceptible to poliovirus) is available from WHO, Quality and Safety of Biologicals, Geneva. Primary monkey cells. The following special requirements apply to monovalent pooled harvests derived from primary monkey cells. Retroviruses. The monovalent pooled harvest is examined using a reverse transcriptase assay. No indication of the presence of retrovirus is found. Test on rabbits. A sample of the monovalent pooled harvest is tested for cercopithecid herpesvirus 1 (B virus) and other viruses by injection of not less than 100 ml into not fewer than 10 healthy rabbits each weighing between 1.5 and 2.5 kg. Each rabbit receives not less than 10 ml and not more than 20 ml, of 81

Identification
Each monovalent pooled harvest is identified as poliovirus of the given type, using specific antiserum.

POLIOMYELITIS VACCINE, LIVE (ORAL)

IP 2007

which 1 ml is given intradermally at multiple sites, and the remainder subcutaneously. The rabbits are observed for at least 3 weeks for death or signs of illness. All rabbits that die after the first 24 h of the test and those showing signs of illness are examined by autopsy, and the brain and organs removed for detailed examination to establish the cause of death. The test is not valid if more than 20.0 per cent of the inoculated rabbits show signs of intercurrent infection during the observation period. The monovalent pooled harvest passes the test if none of the rabbits shows evidence of infection with B virus or with other extraneous agents or lesions of any kind attributable to the bulk suspension. If the presence of B virus is demonstrated, the measures concerning vaccine production described above under Cell cultures are taken. Test on guinea-pigs. Administer to not less than five guineapigs, each weighing between 350 and 450 g, 0.1 ml of the monovalent pooled harvest by intracerebral injection and 0.5 ml by intraperitoneal injection. Measure the rectal temperature of each animal on each working day for 6 weeks. At the end of the observation period carry out autopsy on each animal. In addition, administer to not fewer than five guinea-pigs 0.5 ml by intraperitoneal injection and observe as described above for 2 to 3 weeks. At the end of the observation period, carry out a passage from these animals to not fewer than five guineapigs using blood and a suspension of liver or spleen tissue. Measure the rectal temperature of the latter guinea-pigs for 2 to 3 weeks. Examine by autopsy all animals that, after the first day of the test, die or are killed because they show disease or show for three consecutive days a body temperature higher than 39; carry out histological examination to detect infection with Marburg virus; in addition, inject a suspension of liver or spleen tissue or of blood intraperitoneally into not fewer than three guinea-pigs. If any signs of infection with Marburg virus are noted, confirmatory serological tests are carried out on the blood of the affected animals. The monovalent pooled harvest complies with the test if not fewer than 80.0 per cent of the guinea-pigs survive to the end of the observation period and remain in good health and no animal shows signs of infection with filoviruses virus. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more satisfactory monovalent pooled harvests and may contain more than one virus type. Suitable flavouring substances and stabilisers may be added. Only a final bulk vaccine that complies with the following requirement may be used in the preparation of the final lot. Sterility (2.2.11). Complies with the test for sterility. 82

FINAL LOT Only a final lot that complies with the following requirement for thermal stability and is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Thermal stability. Expose samples of the final lot at 37 for 48 hours. Determine the total virus concentration as described under Assay in parallel for the heated vaccine and for unheated vaccine. The estimated difference between the total virus concentration of the unheated and heated vaccines is not greater than 0.5 log10 infectious virus units (CCID50) per single human dose.

Identification
The vaccine is shown to contain poliovirus of each type stated on the label, using specific antibodies.

Tests
Sterility (2.2.11). Complies with the test for sterility. Assay Titrate for infectious virus at least in triplicate using the method described below. Use an appropriate virus reference preparation to validate each assay. If the vaccine contains more than one poliovirus type, titrate each type separately, using appropriate type-specific antiserum (or preferably a monoclonal antibody) to neutralise each of the other types present. For a trivalent vaccine, the estimated mean virus titres must be: not less than 1 106.0 infectious virus units (CCID50) per single human dose for type 1; not less than 1 105.0 infectious virus units (CCID50) for type 2; and not less than 1 105.8 infectious virus units (CCID50) for type 3. For monovalent or divalent vaccine, the minimum virus titres are decided by the competent authority. Method. Groups of eight to twelve flat-bottomed wells in a microtitre plate are inoculated with 0.05 ml of each of the selected dilutions of virus followed by a suitable cell suspension of the Hep-2 (Cincinnati) line. The plates are incubated at a suitable temperature. Examine the cultures on days 7 to 9. The assay is not valid if (a) the confidence interval (P = 0.95) of the logarithm of the virus concentration is greater than 0.3; (b) the virus concentration of the reference preparation differs by more than 0.5 log CCID50 from the assigned value. Labelling. The label states (1) the types of poliovirus contained in the vaccine; (2) the minimum amount of virus of each type contained in one single human dose; (3) the cell substrate used for the preparation of the vaccine; (4) that the vaccine is not to be injected.

IP 2007

RABIES VACCINE, HUMAN

Rabies Vaccine, Human

Rabies Vaccine for Human use is a freeze-dried or liquid (adsorbed) preparation of a suitable approved, strain of fixed rabies virus grown in an approved cell culture/ embryos of duck/chicken and inactivated by a validated method. The freeze-dried vaccine is reconstituted immediately before use as stated on the label to give a clear or slightly opalescent solution/suspension. It may be coloured owing to the presence of a pH indicator. The vaccine complies with the General Requirements of Vaccines for Human Use.

immunofluoresence or any other approved method. Extraneous agents (2.7.3). The working seed lot complies with the requirements for the virus seed lots. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures are done under aseptic conditions in an area where no other cells are handled. Approved animal (but not human) serum may be used in the media, but the final medium for maintaining cell growth during virus multiplication does not contain animal serum; the media may contain human albumin. Serum proteins, if present are reduced to an acceptable level by a suitable method of purification. Serum and trypsin used in the preparation of cell suspension and media are shown to be free from infectious extraneous agents. The cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. Not less than 500 ml of the cell cultures employed for vaccine production are set aside as uninfected cell cultures (control cells). The virus suspension is harvested on one or more occasions during incubation. Multiple harvests from the same production cell culture may be pooled and considered as a single harvest. When vaccine is prepared in embryonated eggs, the egg proteins are minimized by an appropriate method of purification. The eggs are inoculated with virus seed by the yolk sac route. The infected sterile living embryos are harvested, minced and emulsified in suitable diluent, and stabilizer with aseptic precautions. Emulsions are centrifuged, supernatants are collected and stored as raw virus harvest at a suitable temperature. Viral harvests that comply with the following requirements are pooled in the preparation of the inactivated viral harvest.

Production
General provisions The vaccine is produced on the basis of virus seed lot system and if a cell line is used for virus propagation, a cell-bank system shall be followed. The production method shall have been shown to yield consistently vaccines that comply with the requirements for immunogenicity, safety and stability. Unless otherwise justified and authorized, the virus in the final vaccine shall not have undergone more passages from the master seed lot than was used to prepare the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Substrate for virus propagation The virus is propagated in any suitable approved cell substrate like a human diploid cell line (2.7.2), a continuous cell line, or in duck embroys or in cultures of chicken embryos derived from a flock certified as free from specified pathogens(2.7.7). SEED LOT The strain of rabies virus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. Working seed lots are prepared by not more than five passages from the master seed lot. Only a working seed lot that complies with the following requirements may be used for virus propagation.

Identification
The single harvest contains virus that is identified as rabies virus using specific antibodies by an approved method. Virus concentration. Titrate for infective virus in cell cultures or by any other approved method. The titre is used to monitor consistency of production. Control cells. The control cells of the production cell culture from which the single harvest is derived should comply with a test for identification and with the requirements for extraneous agents (2.7.3). Control eggs. Control eggs shall be tested for freedom from haemagglutinating agents, and other extraneous agents. PURIFICATION AND INACTIVATION The virus harvests may be concentrated and/or purified by suitable methods; the virus harvest is inactivated by a validated method at a fixed, well defined stage of the process which may 83

Identification
Each working seed lot is identified as rabies virus using specific antibodies by an approved method. Virus concentration. The virus concentration of each working seed lot is determined by a cell culture method using

RABIES VACCINE, HUMAN

IP 2007

be before, during or after any concentration or purification. The method shall have been shown to be capable of inactivating rabies virus without destruction of the immunogenic activity. If betapropiolactone is used, the concentration shall at no time exceed 1:3500. Cell culture vaccines Only an inactivated viral suspension that complies with the following requirements may be used in the preparation of the final bulk vaccine. Inactivation. Inactivation is confirmed by carrying out an amplification test for residual infectious rabies virus, not more than 4 days after inactivation or on the sample frozen after inactivation and stored at -70. Inoculate a quantity of inactivated viral suspension equivalent to not less than 25 vaccine doses into cell cultures of the same type as those used for production of the vaccine. Make a passage after 7 days. Maintain the cultures for a further period of 14 days and then examine the cell cultures for rabies virus using an immune fluorescence test. No rabies virus is detected. Alternatively, 5 ml of each culture fluid is pooled on days 14 and 21 and 0.03 ml is inoculated intracerebrally into each of the 10 mice weighing 12 to 15 g. The mice are observed for 14 days for symptoms caused by rabies virus, and mice showing symptoms of rabies are sacrificed and virus presence is confirmed by immunoflurescence test or tested for live virus in cell culture by immunofluorescence test or method of equal sensitivity. No rabies virus should be detected. Residual host-cell DNA (2.2.15). If a continuous cell line is used for virus propagation, the content of residual host-cell DNA, determined using a suitable method, should not be greater than 10 ng per single human dose. Embryonated egg vaccine Only concentrated viral suspensions that comply with the test for sterility, antigen content and endotoxin requirements may be used for preparation of bulk for inactivation. Inactivation is confirmed by carrying out Mice Inoculation Test for residual infectious rabies virus, not more than four days after inactivation or on the sample frozen 4 days after inactivation are stored at 70. Inoculate intracerebrally 0.03 ml into each of 20 mice weighing between 12 and 15 g. The mice are observed for 14 days for symptoms caused by rabies virus and mice showing symptoms of rabies are sacrificed and virus presence is confirmed by an immunoflurescence test or tested for live virus in cell culture by immunofluorescence test or method of equal sensitivity. No rabies virus should be detected. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more inactivated viral suspensions. An approved stabilizer may be added to 84

maintain the activity of the product during and after freezedrying. Thiomersal can be used as preservative. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antigen content. Determine the antigen content by a suitable approved in vitro or in vivo method. The content should be within the limits approved for the particular product. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers and can be freeze-dried in case of lyophilized products. The containers are then sealed so as to prevent contamination and the introduction of moisture. Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the test for inactivation has been carried out with satisfactory results on the inactivated virus suspension and the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, these tests may be omitted on the final lot.

Identification
The vaccine is shown to contain rabies virus antigen by a suitable immunochemical method using specific antibodies, alternatively, the Assay also serves to identify the vaccine.

Tests
Inactivation. Inoculate a quantity equivalent to not less than 25 human doses of vaccine into cell cultures of the same type as those used for production of the vaccine. Make a passage after 7 days. Maintain the culture for a further 14 days and then examine the cell culture for rabies virus using an immunofluorescence test. No rabies virus is detected. Alternatively, inject 0.03ml of the vaccine intracerebrally into each of the 10 mice weighing between 12 and 15g. Neither symptoms of disease in the central nervous system nor death occurs in any of the animal within 14 days. If the inactivation test is already performed on inactivated virus used for final lot, it may be omitted from the test on final lot. Sterility (2.2.11). Complies with the test for sterility. Bacterial endotoxins (2.2.3). Less than 25 IU per single human dose. Pyrogens (2.2.8). Complies with the test for pyrogens. Unless otherwise justified and authorized, inject into each rabbit a single human dose of the vaccine diluted to ten times its volume. Water (2.3.43). Not more than 3.0 per cent determined by an approved method.

IP 2007

RABIES VACCINE, HUMAN

Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Accelerated degradation. The potency determined by method described under Assay of a sample of the preparation under examination after storage at 37 for 4 weeks is not less than 2.5 units per single human dose. This may not be mandatory for lot release, once the consistency of the product is approved by National Regulatory Authority. Bovine serum albumin (for cell culture vaccine). Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Aluminium content (for gel absorbed vaccine) (2.3.9). Not more than 1.25 mg per single human dose. Ovalbumin (for egg based vaccines). Not more than 1 g of ovalbumin per human dose, determined by a suitable technique using a suitable reference preparation of ovalbumin. Residual host-cell DNA (for continuous cell line vaccines) (2.2.15). Should not be greater than 10 ng per single human dose. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Assay The Standard preparation is the international standard or another suitable preparation, the potency of which has been determined in relation to the International standard. The potency of rabies vaccine is determined by comparing the dose necessary to protect mice against a lethal intracerebral dose of rabies vaccine necessary to provide the same protection. For this comparison, a reference preparation of rabies vaccine, calibrated in international units, and a suitable preparation of rabies virus for use as the challenge preparation are necessary. The international unit is the activity contained in a stated quantity of the international standard. The equivalence in international units of the international standard is stated by the World Health Organization. The test described below uses a parallel-line model with at least three points for the vaccine under examination and the reference preparation. Test animals. Use mice of a suitable strain, drawn from a uniform stock three to four weeks old, weighing between 11 and 15 g. Distribute the mice into six groups of at least 16 mice each and four groups of 10 mice each and must be of the same sex or the sexes must be equally distributed among the groups. Throughout the test all mice that die before the fifth day after challenge are excluded from the test and all mice that die with 85

signs of rabies between the fifth and fourteenth day after challenge are counted as failing to resist the challenge. The strain of mice suitable for the test is such that when 0.03 ml containing 5 to 50 LD50 of the challenge virus suspension is injected intracerebrally per mouse there is 100 per cent mortality. Standard challenge virus suspension. A working pool of the challenge virus strain is prepared by injecting intracerebrally 0.03ml of a 10 fold dilution of the CVS strain of rabies virus in 2 per cent v/v sterile inactivated normal horse serum in water for injection or another suitable diluent approved by the competent authority into a suitable number of test animals. The animals when moribund after showing characteristic signs of rabies are sacrificed and their brains harvested aseptically. They are then washed in chilled saline solution to remove blood clots. A 10 per cent suspension of the brains is prepared in a suitable diluent approved by the competent authority and thoroughly homogenised. After centrifuging lightly, the supernatant liquid is distributed into sterile vials and freeze dried. The sealed and freeze-dried supernatant liquid containing vials are stored at -20. When stored under prescribed conditions the virus titre of the freeze-dried preparation may be expected to be maintained for not less than 3 years. Alternatively, the washed brains are homogenised in a suitable diluent approved by the competent authority to give 10 per cent suspension. It is then centrifuged lightly, distributed into sterile ampoules or sterile plastic vials and sealed. The sealed ampoules or plastic vials can be stored at 60 or below. When stored under prescribed conditions, the virus titre may be expected to be maintained for not less than one year. Storage time needs to be validated by the manufacturer. Virus titre of the challenge virus. Prepare ten fold serial dilutions of the standard challenge virus suspension. Using the four groups of 10 mice each, inject 0.03 ml of the virus suspension intracerebrally into each mouse, using a different group for each suspension. Observe the mice for 14 days. Calculate the virus titre of the standard challenge virus suspension in LD50 per dose of 0.03 ml by standard statistical methods. Determination of potency of the vaccine. Reconstitute the standard preparation with a suitable diluent. Prepare at least three 5-fold serial dilutions of the solution of the standard preparation and three 5-fold serial dilutions of the vaccine under examination. For both, the standard preparation and the preparation under examination, the serial dilutions should be prepared in such a way that the lowest dilution protects more than 50 per cent of the injected mice. Allocate one dilution to each of the six groups of 16 mice each. Inject intraperitoneally each mouse in each group with dilutions of the vaccine and reference preparation and repeat the injections. After 7 days, prepare identical dilutions of the vaccine and

RUBELLA VACCINE (LIVE)

IP 2007

reference preparation and repeat the injections. After a further 7 days, inject each vaccinated mouse intracerebrally with 0.03 ml of the standard challenge virus suspension such that on the basis of preliminary titration, 0.03 ml contains between 5 to 50 LD50. Observe the mice for 14 days and record the number of mice surviving the challenge in each group. Calculate the potency of the preparation under examination by standard statistical methods. The vaccine complies with the test if the estimated potency is not less than 2.5 IU per single human dose. The test is not valid unless (a) for both the preparation under examination and the standard preparation, the 50 per cent protective dose lies between the largest and smallest doses given to the mice; (b) there is not deviation from linearity or parallelism of the dose response lines, the confidence limit (P = 0.95) are not less than 25.0 per cent and not more than 400 per cent of the estimated potency; (c) the titre of the challenge virus suspension lies between 5 to 50 LD50. Labelling. The label states the biological origin of the cells used for the preparation of the vaccine.

information on the origin of the strain and its subsequent manipulation. To avoid unnecessary use of monkeys in the test for neurovirulence virus seed lots are prepared in large quantities and stored at temperatures below -20 if freeze-dried, or below -60 if not freeze-dried. Only a seed lot that complies with the following tests may be used for virus propagation.

Identification
The master and working seed lots are identified as rubella virus by serum neutralisation in cell culture, using specific antibodies. Virus concentration. The virus concentration of the master and working seed lots is determined to ensure consistency of production. Extraneous agents (2.7.3). The working seed lot complies with the tests for seed lots. Neurovirulence (2.7.5). The master/working seed lot complies with the test for neurovirulence of live virus vaccines. Macaca and Cercopithecus monkeys are suitable for the test. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are handled. Suitable animal (but not human) serum may be used in the growth medium, but the final medium for maintaining cell growth during virus multiplication does not contain animal serum. Serum and trypsin used in the preparation of cell suspensions and culture media are shown to be free from extraneous agents. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration. It is preferable to have a substrate free from antibiotics during production. Not less than 500 ml of the production cell culture is set aside as uninfected cell culture (control cells). The temperature of incubation is controlled during the growth of the virus. The virus suspension is harvested, on one or more occasions, within 28 days of inoculation. Multiple harvests from the same production cell culture may be pooled and considered as a single harvest. Only a single harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine.

Rubella Vaccine (Live)

Rubella Vaccine (Live) is a freeze-dried preparation of a suitable attenuated strain of rubella virus. The vaccine is reconstituted immediately before use to give a clear liquid or may be coloured owing to the presence of a pH indicator.

Production
General provisions The production of vaccine is based on a virus seed-lot system and a cell-bank system. The production method shall have been shown to yield consistently live rubella vaccines of adequate immunogenicity and safety in man. Unless otherwise justified and authorised, the virus in the final vaccine shall have undergone no more passages from the master seed lot than were used to prepare the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Substrate for virus propagation The virus is propagated in human diploid cells (2.7.2). SEED LOT The strain of rubella virus used in the production of rubella vaccine shall be identified by historical records that include 86

Identification
The single harvest contains virus that is identified as rubella virus by serum neutralization in cell culture, using specific antibodies. Virus concentration. The virus concentration in the single harvest is determined as prescribed under Assay to monitor

IP 2007

TETANUS VACCINE (ADSORBED)

consistency of production and to determine the dilution to be used for the final bulk vaccine. Extraneous agents (2.7.3). The single harvest complies with the tests for extraneous agents. Control cells. The control cells comply with a test for identification and with the tests for extraneous agents (2.7.3). FINAL BULK VACCINE Single harvests that comply with the above tests are pooled and clarified to remove cells. A suitable stabilizer may be added and the pooled harvests diluted as appropriate. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT A minimum virus concentration for release of the product is established such as to ensure, in the light of stability data, that the minimum concentration stated on the label will be present at the end of the period of validity. Only a final lot that complies with the tests for minimum virus concentration for release, with the following requirement for thermal stability and with each of the requirements given below under Identification and Tests may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37 for 7 days. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37 for 7 days and for vaccine stored at 2 to 8. The virus concentration of the heated vaccine is not more than 1.0 log10 lower than that of the unheated vaccine.

Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Assay Titrate the vaccine for infective virus at least in triplicate, using at least five cell cultures for each 0.5 log10 dilution step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated virus concentration is not less than that stated on the label; the minimum virus concentration stated on the label is not less than 1 103 CCID50 per human dose. The assay is not valid if the confidence limits (P=0.95) of the logarithm of the virus concentration is greater than 0.3. Rubella vaccine (Live) RS is suitable for use as a reference preparation. Labelling. The label states (1) the strain of virus used for the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) the minimum virus concentration; (4) the time within which the vaccine must be used after reconstitution; (5) that the vaccine must not be given to a pregnant woman and that a woman must not become pregnant within two months.

Tetanus Vaccine (Adsorbed)

Tetanus Vaccine (Adsorbed) is a preparation of tetanus formol toxoid adsorbed on mineral carrier. The formol toxoid is prepared from the toxin produced by the growth of Clostridium tetani.

Production
General provisions The maximum number of Lf per single human dose of tetanus vaccine is 25. The production method is validated to demonstrate that the product, if tested, would comply with the following test. BULK PURIFIED TOXOID For the production of tetanus toxin, from which toxoid is prepared, seed cultures are managed in a defined seed-lot system in which toxinogenicity is conserved and, where necessary, restored by deliberate reselection. A highly toxinogenic strain of Clostridium tetani with known origin and history is grown in a suitable liquid medium. At the end of cultivation, the purity of each culture is tested and contaminated cultures are discarded. Toxin-containing culture medium is collected aseptically. The toxin content (Lf per ml) is checked to monitor consistency of production. Single harvests may be pooled to prepare the bulk purified toxoid. 87

Identification
When the vaccine reconstituted as stated on the label is mixed with specific rubella antibodies, it is no longer able to infect susceptible cell cultures.

Tests
Sterility (2.2.11). The reconstituted vaccine complies with the test for sterility. Water (2.3.43). Not more than 3.0 per cent, determined by Karl Fischer, semi-micro determination of water or by any suitable validated method. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14).

TETANUS VACCINE (ADSORBED)

IP 2007

The toxin is purified to remove components likely to cause adverse reactions in humans. The purified toxin is detoxified with formaldehyde by a method that avoids destruction of the immunogenic potency of the toxoid and reversion of toxoid to toxin, particularly on exposure to heat. Alternatively, purification may be carried out after detoxification. Only bulk purified toxoid that complies with the following requirements may be used in the preparation of the final bulk vaccine. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Absence of tetanus toxin. Inject subcutaneously at least 500 Lf of purified toxoid in a volume of 1 ml into each of five healthy guinea-pigs, each weighing 250 to 350 g, that have not previously been treated with any material that will interfere with the test. If within 21 days of the injection any of the animals shows signs of or dies from tetanus, the toxoid does not comply with the test. If more than one animal dies from non-specific causes, repeat the test once; if more than one animal dies in the second test, the toxoid does not comply with the test. Irreversibility of toxoid. Using the buffer for the final vaccine, prepare a dilution of the bulk purified toxoid containing the same toxoid concentration as the final vaccine. Divide the dilution into two equal parts. Keep one of them at 2 to 8 and the other at 37 for 6 weeks. Test both dilutions by a suitable sensitive assay for active tetanus toxin, such as inoculation into mice or guinea-pigs. The toxoid complies with the test if neither sample produces any sign of a toxic reaction attributable to tetanus toxin. Antigenic purity. Not less than 1,000 Lf per mg of protein nitrogen. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption of a suitable quantity of bulk purified toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide; the resulting mixture is approximately isotonic with blood. Suitable antimicrobial preservatives may be added. Certain antimicrobial preservatives, particularly those of the phenolic type, adversely affect the antigenic activity and must not be used. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Specific toxicity. Inject five times the dose stated on the label subcutaneously or intraperitoneally into each of five guineapigs. None of the guinea-pigs shows any symptoms of, or dies from, tetanus within 21 days. If more than one animal dies from non-specific causes within this period repeat the test. None of the second group of animals shows symptoms of

tetanus or dies from tetanus or any other cause within 21 days. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Free formaldehyde (2.3.20). Maximum 0.2 g/l. pH (2.4.24). 6.0 and 7.0 Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity for antisera and vaccine. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.

Identification
Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant liquid is obtained. The clear supernatant liquid reacts with a suitable tetanus antitoxin, giving a precipitate.

Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity for antisera and vaccine.

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Potency of tetanus component Assay. Determine by either of the following methods. (1) Inject subcutaneously on each of two occasions separated by an interval of not more than 4 weeks, one-tenth of the stated human dose diluted to 1 ml with saline solution into each of 9 normal, healthy guinea-pigs weighing between 250 and 350 g. Not more than 2 weeks after the second injection, collect the serum from each animal and carry out the biological test for tetanus antitoxin, described under Tetanus antitoxin or any other method approved by National Regulatory Authority. Sera of at least 6 guinea-pigs out of 9 should contain not less than 0.5 Unit of tetanus antitoxin per ml. (2) Carry out the biological assay of adsorbed Tetanus Vaccine (Adsorbed) described below. If the lower limit of the 95.0 per cent confidence interval of estimated potency is less than 40 IU per single human dose then the limits of the 95.0 per cent confidence interval of the estimater of potency shall be within 50 to 200 per cent of the estimated potency unless the lower limit of the 95.0 per cent confidence interval of the estimated potency is greater than 40 IU per single human dose. Biological assay of adsorbed tetanus vaccine The potency of adsorbed tetanus vaccine is determined by comparing the dose of the vaccine required to protect guineapigs or mice from the paralytic effects of a subcutaneous injection of tetanus toxin with the dose of the Standard preparation needed to give the same protection. For this comparison, the Standard preparation of adsorbed tetanus toxoid and a suitable preparation of tetanus toxin, for use as a challenge toxin, are necessary. Standard preparation The Standard preparation is International standard for Tetanus toxoid, adsorbed or another suitable preparation, the potency of which has been determined in relation to the International standard. Suggested method (a) Test on guinea-pigs Test animals. Use healthy guinea-pigs from the same stock and weighing between 250 and 350 g. Distribute them into six groups of sixteen each. The guinea-pigs should all be of the same sex or the males and females should be distributed equally between the groups. If the challenge toxin to be used has not been shown to be stable or has not been adequately standardised, include four further groups of five guinea-pigs to serve as unvaccinated controls. Challenge toxin. Select a preparation of tetanus toxin containing not less than 50 times the 50 per cent paralytic 89

dose per ml. If the challenge toxin preparation has been shown to be stable, it is not necessary to verify the paralytic dose for every assay. Preparation of the challenge toxin solution. Immediately prior to use, prepare from the challenge toxin by dilution with phosphate buffered saline pH 7.4 a challenge toxin solution containing fifty times the 50 per cent paralytic dose per ml. If necessary, dilute portions of this challenge toxin solution 16-, 50- and 160-fold with the same buffer solution. Determination of potency. Prepare in saline solution three dilutions of the vaccine under examination and three dilutions of a solution of the Standard preparation such that, for each, the dilutions form a series differing by not more than 2.5-fold steps and in which the dilutions of intermediate concentration, when injected subcutaneously in 1-ml volumes into guineapigs, protect approximately 50 per cent of the animals from the paralytic effects of the subcutaneous injection of the quantity of tetanus toxin prescribed for this test. Allocate the six dilutions one to each of the six groups of sixteen guinea-pigs and inject subcutaneously 1.0 ml of each dilution into each guinea-pig in the group to which that dilution is allocated. After 28 days inject each animal subcutaneously with 1.0 ml of the challenge toxin solution containing fifty times the 50 per cent paralytic dose. If necessary, allocate the challenge toxin solution and the three dilutions made from it one to each of the four groups of five guinea-pigs and inject subcutaneously 1.0 ml of each toxin solution into each guineapig in the group to which that toxin solution is allocated. Examine the guinea-pigs twice daily, remove and kill all animals showing definite signs of tetanus paralysis. Count the number of guinea-pigs without paralysis 5 days after injection of the challenge toxin and calculate the potency of the vaccine under examination relative to the potency of the Standard preparation on the basis of the number of animals without paralysis in each of the six groups of sixteen, using standard statistical methods. The test is not valid unless (a) for both the vaccine under examination and the Standard preparation, the 50 per cent protective doses lie between the largest and smallest doses of the preparations given to the guinea-pigs; (b) if applicable, the number of paralysed animals among the four groups of five injected with the challenge toxin solution and its dilutions indicate that the challenge was approximately 50 times the 50 per cent paralytic dose; (c) the fiducial limits of assay lie between 50.0 per cent and 200.0 per cent of the estimated potency; (d) the statistical analysis shows no deviations from linearity or parallelism. The test may be repeated any number of times but when more than one test is performed the results of all valid tests must be combined in the estimate of potency. (b) Test on mice Test animals. Use healthy mice from the same stock, weighing

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between 14 and 20 g. Distribute them into six groups of sixteen each. If the challenge toxin to be used has not been shown to be stable or has not been adequately standardised, include four further groups of six mice to serve as unvaccinated controls. The mice should all be of the same sex or the males and females should be distributed equally among the groups. Challenge toxin. Select a preparation of tetanus toxin containing not less than 100 times the 50 per cent paralytic dose per ml. Preparation of the challenge toxin solutions. Immediately prior to use prepare from the challenge toxin by dilution with phosphate buffered saline pH 7.4 a challenge toxin solution containing fifty times the 50 per cent paralytic dose in each 0.5 ml. If necessary, dilute portions of this challenge toxin solution 16-, 50- and 160-fold with the same buffer solution. Determination of potency. Prepare in saline solution three dilutions of the vaccine under examination and three dilutions of a solution of the Standard preparation such that, for each, the dilutions form a series differing by not more than 2.5-fold steps and in which the dilutions of intermediate concentration, when injected subcutaneously in 0.5 ml volumes into mice, protect approximately 50 per cent of the animals from the paralytic effects of the subcutaneous injection of the quantity of tetanus toxin prescribed for this test. Allocate the six dilutions one to each of the six groups of sixteen mice and inject subcutaneously 0.5 ml of each dilution into each mouse in the group to which the dilution is allocated. After 28 days inject each animal subcutaneously with 0.5 ml of the challenge toxin solution containing fifty times the 50 per cent paralytic dose. If necessary, allocate the challenge toxin solution and the three dilutions made from it one to each of the four groups of six mice and inject subcutaneously 0.5 ml of each toxin solution into each mouse in the group to which that toxin solution is allocated. Count the number of mice without paralysis 4 days after injection of the challenge toxin and calculate the potency of the vaccine under examination relative to the potency of the Standard preparation on the basis of the numbers of animals without paralysis in each of the six groups of sixteen, using standard statistical methods. The test is not valid unless (a) for both the vaccine under examination and the Standard preparation, the 50 per cent protective doses lie between the largest and smallest doses of the preparations given to the mice; (b) if applicable, the number of paralysed animals among the four groups of six injected with the challenge toxin solution and its dilutions indicate that the challenge was approximately 50 times the 50 per cent paralytic dose; (c) the fiducial limits of the assay lie between 50.0 per cent and 200.0 per cent of the estimated potency; (d) the statistical analysis shows no deviation from linearity or parallelism. The test may be repeated any number of times but when more than one test is performed the results of all valid 90

tests must be combined in the estimate of potency. (c) Determination of antibodies in guinea-pigs Preparation of serum samples. For preparation of serum samples, the following technique has been found suitable. Invert the tubes containing blood samples 6 times and allow to stand at 37 for 2 h, then at 4 for 2 hours, centrifuge at room temperature at 800 g for 20 min. transfer the serum to sterile tubes and store at a temperature below -20. At least 40.0 per cent yield of serum is obtained by this procedure. Determination of antibody titre. The ELISA and ToBI tests shown below are given as examples of immunochemical methods that have been found suitable for the determination of antibody tire. Determination of antibody titre in guinea-pig serum by enzyme-linked immunosorbent assay (ELISA). Dilutions of test and reference sera are made on ELISA plates coated with tetanus toxoid. A positive guinea-pig serum control and a negative guinea-pig serum control are included on each plate to monitor the assay performance. Peroxidase-conjugated rabbit or goat antibody directed against guinea-pig-IgG is added followed by a peroxidase substrate. Optical density is measured and the relative antibody titre is calculated using the usual statistical methods. Reagents and equipment ELISA plates: 96 wells, columns 1-12, rows A-H. Clostridium tetani guinea-pig antiserum (for vaccines-human use) reference preparation (positive control serum). Peroxidase conjugate. Peroxidase-conjugated rabbit or goat antibody directed against guinea-pig IgG. Tetanus toxoid. Carbonate coating buffer pH 9.6. Dissolve 1.59 g of anhydrous sodium carbonate and 2.93 g of sodium hydrogen carbonate in 1000 ml of water. Distribute into 150 ml bottles and sterilise by autoclaving at 121 for 15 min. Phosphate buffered saline pH 7.4 (PBS). Dissolve with stirring 80.0 g of sodium chloride, 2.0 g of potassium dihydrogen phosphate, 14.3 g of disodium hydrogen phosphate dihydrate and 2.0 g of potassium chloride in 1000 ml of water. Store at room temperature to prevent crystallisation. Dilute to 10 times its volume with water before use. Citric acid solution. Dissolve 10.51 g of citric acid in 1000 ml of water and adjust the solution to pH 4.0 with a 400 g/l solution of sodium hydroxide. Washing buffer. PBS containing 0.5 g/l of polysorbate 20. Diluent block buffer. PBS containing 0.5 g/l of polysorbate 20 and 25 g/l of dried skimmed milk. Peroxidase substrate. Shortly before use, dissolve 10 mg of

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TETANUS VACCINE (ADSORBED)

diammonium 2,2'-azinobis(3-ethylbenzothiazoline-6sulphonate) (ABTS) in 20 ml of citric acid solution. Immediately before use add 5 l of strong hydrogen peroxide solution. Method The description below is given as an example of a suitable plate lay-out but others may be used. Wells 1A-H are for negative control serum and wells 2A-H and 3A-H are for positive control serum for assay monitoring. Wells 4-12A-H are for test samples. Coat each well of the ELISA plates with 100 l of tetanus toxoid solution (0.5 Lf/ml in carbonate coating buffer). Allow to stand overnight at 4 in a humid atmosphere. To avoid interference from temperature gradient, do not stack more than 4 plates high. On the following day, wash the plates thoroughly with washing buffer. Block the plates by addition of 100 l of diluent block buffer to each well. Incubate in a humid atmosphere at 37 for 1 h. Wash the plates thoroughly with washing buffer. Place 100 I of diluent block buffer in each well of the plates, except those of row A. Prepare suitable dilutions of negative control serum, positive control serum (from about 0.01 IU/ml) and test sera. Allocate the negative control serum to column 1, positive control serum to columns 2 and 3 and test sera to columns 4-12 and add 100 I of each serum to the first 2 wells of the column to which it is allocated. Using a multi channel micropipette, make twofold serial dilutions from row B down the plate to row H by transferring 100 l to the following well. Discard 100 l from the last row so that all wells contain 100 I. Incubate at 37 for 2 h. Wash thoroughly with washing buffer. Prepare a suitable dilution (a 1 in 2000 dilution has been found suitable) of peroxidase conjugate in diluent block buffer and add 100 I to each well. Incubate at 37 in a humid atmosphere for 1 h. Wash the plates thoroughly with washing buffer. Add 100 I of peroxidase substrate to each well. Allow to stand at room temperature, protected from light, for 30 min. Read the plates at 405 nm in the same order as addition of substrate was made. Determination of antibody titre in guinea-pig serum by toxinor toxoid-binding inhibition (ToBI). Tetanus toxin or toxoid is added to serial dilutions of test and reference sera; the serum/ antigen mixtures are incubated overnight. To determine unbound toxin or toxoid, the mixtures are transferred to an ELISA plate coated with tetanus antitoxin. Peroxidaseconjugated equine anti-tetanus IgG is added followed by a peroxidase substrate. Optical density is measured and the antibody titre is calculated using the usual statistical methods. A positive control serum and a negative control serum are included on each plate to monitor assay performance. Reagents and equipment Round-bottomed, rigid polystyrene microtitre plates. 91

Flat-bottomed ELISA plates. Tetanus toxin or tetanus toxoid. Clostridium tetani guinea-pig antiserum (for vaccines-human use) reference preparation. Equine anti-tetanus IgG. Peroxidase-conjugated equine anti-tetanus IgG. Carbonate buffer pH 9.6. Dissolve 1.5 g of anhydrous sodium carbonate, 2.39 g of sodium hydrogen carbonate and 0.2 g of sodium azide in 1000 ml of water, adjust to pH 9.6 and autoclave at 121 for 20 min. Sodium acetate buffer pH 5.5. Dissolve 90.2 g of anhydrous sodium acetate in 900 ml of water, adjust to pH 5.5 using a saturated solution of citric acid monohydrate and dilute to 1000 ml with water. Phosphate buffered saline pH 7.2 (PBS). Dissolve 135.0 g of sodium chloride, 20.55 g of disodium hydrogen phosphate dihydrate and 4.80 g of sodium dihydrogen phosphate monohydrate in water and dilute to 15 litres with the same solvent. Autoclave at 100 for 60 min. Diluent buffer. PBS containing 5 g/l of bovine albumin and 0.5 g/l of polysorbate 80. Block buffer. PBS containing 5 g/l of bovine albumin. Tetramethylbenzidine solution. 6 g/l solution of tetramethylbenzidine in alcohol. The substance dissolves within 30-40 min at room temperature. Peroxidase substrate. Mix 90 ml of water, 10 ml of sodium acetate buffer pH 5.5, 1.67 ml of tetramethylbenzidine solution and 20 I of strong hydrogen peroxide solution. Washing solution. Tap water containing 0.5 g/l of polysorbate 80. Method Block the round-bottomed polystyrene microtitre plates by placing in each well 150 I of block buffer. Cover the plates with a lid or sealer. Incubate in a humid atmosphere at 37 for 1 h. Wash the plates thoroughly with washing solution. Place 100 I of PBS in each well. Place 100 l of reference guineapig tetanus antitoxin in the first well of a row. Place 100 l of undiluted test sera in the first well of the required number of rows. Using a multichannel micropipette, make it two fold serial dilutions across the plate (up to column 10), by transfer of 100 I to the following well. Discard 100 l from the last column so that all wells contain 100 l. Prepare 0.1 Lf/ml solution of tetanus toxin or toxoid using PBS a diluent. Add 40 l of this solution to all wells except those column 12. The wells of row 11 are a positive control. Add 40 l of PBS to the wells of column 12 (negative control). Shake the plates gently and cover them with lids. Coat the ELISA plates: immediately before use make

TICK-BORNE ENCEPHALITIS VACCINE (INACTIVATED)

IP 2007

a suitable dilution of equine anti-tetanus IgG in carbonate buffer pH 9.6 and add 100 l to all wells. Incubate the 2 series of plates overnight in a humid atmosphere at 37. To avoid temperature gradient effects, do not stack more than 4 plate high. Cover the plates with lids. On the following day, wash the ELISA plates thoroughly with washing solution. Block the plates by placing in each well 125 l of block buffer. Incubate at 37 in a humid atmosphere for 1 h. Wash the plates thoroughly with washing solution. Transfer 100 l of the preincubation mixture from the polystyrene plates to the corresponding wells of the ELISA plates, starting with column 12 and then from 1 to 11. Cover the plates with a lid. Incubate at 37 in a humid atmosphere for 2 h. Wash the ELISA plates thoroughly with washing solution. Make a suitable dilution (a 1 in 4000 dilution has been found suitable) of the peroxidaseconjugated equine anti-tetanus IgG in diluent buffer. Add 100 l of the dilution to each well and cover the plates with a lid. Incubate at 37 in a humid atmosphere for 1.5 h. Wash the ELISA plates thoroughly with washing solution. Add 100 l of peroxidase substrate to each well. A blue colour develops. Incubate the plates at room temperature. Stop the reaction at a given time (within 10 min) by the addition of 100 l of 2 M sulphuric acid to each well in the same order as the addition of substrate. The colour changes from blue to yellow. Measure the absorbance (2.4.7) at 450 nm immediately after addition of the sulphuric acid or maintain the plates in the dark until reading. (d) Any other validated serological assay in guinea-pigs/mice approved by National Regulatory Authority. Labelling. The label states (1) the human dose (ml); (2) the minimum units per single human dose or the minimum International Units per single human dose if potency test done by challenge method or both; (3) the name and the amount of the adsorbent and preservative; (4) that the vaccine must be shaken before use; (5) that the vaccine is not to be frozen.

consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The virus in the final vaccine shall not have undergone more passages from the master seed lot than the virus in the vaccine used in clinical trials. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity and immunogenicity. Substrate for virus propagation The virus is propagated in chick embryo cells prepared from eggs derived from a chicken flock free from specified pathogens (2.7.7) or in other suitable cell cultures. SEED LOT The strain of virus used is identified by historical records that include information on the origin of the strain and its subsequent manipulation. Virus seed lots are stored at or below -60. Only a seed lot that complies with the following requirements may be used for virus propagation.

Identification
Each seed lot is identified as containing the vaccine strain of tick-borne encephalitis virus by a suitable immunochemical method, preferably using monoclonal antibodies. Virus concentration. The virus concentration of each seed lot is determined by titration in suitable cell cultures to monitor consistency of production. Extraneous agents (2.7.3). Each seed lot complies with the requirements for extraneous agents in viral vaccines for human use; the tests in cell cultures are carried out in human and simian cells only. PROPAGATION AND HARVEST All processing of the cell cultures if performed under aseptic conditions in an area where no other cells are being handled. Serum and trypsin used in the preparation of cell suspensions and media used must be shown to be free from extraneous agents. The cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. At least 500 ml of the cell cultures employed for vaccine production is set aside as uninfected cell cultures (control cells). Only a single harvest that complies with the following requirements may be used in the preparation of the inactivated harvest.

Tick-borne Encephalitis Vaccine (Inactivated)

Tick-borne Encephalitis Vaccine (Inactivated) is a liquid preparation of a suitable strain of tick-borne encephalitis virus grown in cultures of chick-embryo cells or other suitable cell cultures and inactivated by a suitable, validated method.

Production
General provisions The vaccine complies with the General Requirements of Vaccines for Human Use. Production of the vaccine is based on a virus seed lot system. The production method shall have been shown to yield 92

Identification
The single harvest is shown to contain tick-borne encephalitis virus by a suitable immunochemical method, preferably using

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monoclonal antibodies, or by virus neutralization in cell cultures. Sterility (2.2.11). Complies with the test for sterility carried out using 10 ml for each medium. Mycoplasma (2.7.4). Complies with the test for mycoplasmas carried out using 1 ml for each medium. Control cells. The control cells comply with the tests for extraneous agents (2.7.3). If the vaccine is produced using a cell-bank system, the control cells comply with a test for identification. Virus concentration. Determine the virus concentration by titration in suitable cell culture to monitor consistency of production. Inactivation To avoid interference, viral aggregates are removed by filtration immediately before the inactivation process. The virus suspension is inactivated by a validated method; the method shall have been shown to be consistently capable of inactivating tick-borne encephalitis virus without destroying the antigenic and immunogenic activity; as part of the validation studies, an inactivation curve is plotted representing residual live virus concentration measured on not fewer than three occasions. If formaldehyde is used for inactivation, the presence of an excess of free formaldehyde is verified at the end of the inactivation process. Only an inactivated harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. Residual infective virus. Inoculate a quantity of the inactivated harvest equivalent to not less than ten human doses of vaccine in the final lot into primary chicken fibroblast cell cultures, or other cells shown to be at least as sensitive to tick-borne encephalitis virus with not less than 3 cm2 of cell sheet per ml of inoculum. Incubate at 37 1 for 14 days. No cytopathic effect is detected at the end of the incubation period. Collect the culture fluid and inoculate 0.03 ml intracerebrally into each of not fewer than ten mice about 4 weeks old. Observe the mice for 14 days. They show no evidence of tick-borne encephalitis virus infection. Purification Several inactivated single harvests may be pooled before concentration and purification by suitable methods, preferably by continuous-flow, sucrose density-gradient centrifugation. Only a purified, inactivated harvest that complies with the following requirements way be used in the preparation of final bulk vaccine. Sterility ( 2.2.11). Complies with the test for sterility, carried out using 10 ml for each medium. 93

Specific activity. Determine the antigen content of the purified, inactivated harvest by a suitable immunochemical method (2.2.14). Determine the total protein content by a suitable method. The specific activity, calculated as the antigen content per unit mass of protein, is within the limits approved for the specific product. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more purified, inactivated harvests. Only a final bulk vaccine that complies with the following requirement may be used in the preparation of the final lot. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde, bovine serum albumin (where applicable) and pyrogens and the assay have been carried out satisfactory results on the final bulk vaccine, they may be omitted on the final lot.

Identification
The vaccine is shown to contain tick-borne encephalitis virus antigen by a suitable immunochemical method using specific antibodies or by the mouse immunogenicity test described under Assay.

Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.1 g/l. Bovine serum albumin. If bovine serum albumin has been used during production, the vaccine contains not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject into each rabbit, per kg of body mass, one dose of vaccine. Assay The potency is determined by comparing the dose necessary to protect a given proportion of mice against the effects of a lethal dose of tick-borne encephalitis virus, administered intraperitoneally, with the quantity of a reference preparation

TUBERCULIN PURIFIED PROTEIN DERIVATIVE

IP 2007

of tick-borne encephalitis vaccine necessary to provide the same protection. For this comparison an approved reference preparation and a suitable preparation of tick-borne encephalitis virus from an approved strain for use as the challenge preparation are necessary. The following is cited as an example of a method that has been found suitable for a given vaccine. Selection and distribution of test animals. Use healthy mice weighing between 11 and 17 g derived from the same stock. Distribute the mice into not less than six groups of a suitable size to meet the requirements for validity of the test; for titration of the challenge suspension, use not fewer than four groups of ten mice. Use mice of the same sex or distribute males and females equally between groups. Determination of potency of the vaccine. Prepare not less than three suitable dilutions of the vaccine under examination and of the reference preparation; in order to comply with validity criteria four to five dilutions will usually be necessary. Prepare dilutions such that the most concentrated suspension is expected to protect more than 50 per cent of the animals and the least concentrated suspension less than 50.0 per cent. Allocate each dilution to a different group of mice and inject subcutaneously into each mouse 0.2 ml of the dilution allocated to its group. Seven days later make a second injection using the same dilution scale. 14 days after the second injection prepare a suspension of the challenge virus containing not less than 100 LD50 in 0.2 ml. Inject 0.2 ml of this virus suspension intraperitoneally into each vaccinated mouse. To verify the challenge dose, prepare a series of not fewer than three dilutions of the challenge virus suspension at not greater than one-hundredfold intervals. Allocate the challenge suspension and the four dilutions, one to each of the five groups of ten mice, and inject intraperitoneally into each mouse 0.2 ml of the challenge suspension or the dilution allocated to its group. Observe the animals for 21 days after the challenge and record the number of mice that die in the period between 7 and 21 days after the challenge. Calculations. Calculate the results by the usual statistical methods for an assay with quantal responses (5.7). Validity criteria. The test is not valid unless (1) the concentration of the challenge virus is not less than 100 LD50; (2) for both the vaccine under examination and the reference preparation the 50.0 per cent protective dose (PD50) lies between the largest and smallest doses given to the mice; (3) the statistical analysis shows a significant slope and no significant deviation from linearity and parallelism of the doseresponse lines; (4) the fiducial limits (P = 0.95) are not less than 33.0 per cent and not more than 300.0 per cent of the estimated potency. Potency requirement. Include all valid tests to estimate the 94

mean potency and the fiducial limits (P = 0.95) for the mean potency; compute weighed means with the inverse of the squared standard error as weights. The vaccine complies with the test if the estimated potency is not less than that approved by the competent authority, based on data from clinical efficacy trials. Labelling. The label states (1) the strain of virus used in preparation; (2) the type of cells used for production of the vaccine.

Tuberculin Purified Protein Derivative

Tuberculin PPD; Tuberculin Purified Protein Derivative for Human Use Tuberculin Purified Protein Derivative is a preparation made from the heat-treated products of growth and lysis of one or more strains of Mycobacterium tuberculosis that reveal delayed hypersensitivity in animals sensitised by a microorganism of the same species.

Production
It is prepared from the water-soluble fraction obtained by heating in free-flowing steam or in an autoclave and subsequently filtering cultures of the mycobacteria grown in a suitable liquid medium. The active fraction in the filtrate, which is predominantly protein, is separated by precipitation, washed and redissolved. The preparation is free from mycobacteria. An antimicrobial preservative that does not give rise to false positive reactions, such as 0.5 per cent w/v of phenol, and a suitable stabiliser may be added. Phenol is not added to preparations that are to be freeze-dried. The final sterile product is distributed into sterile glass containers which are then sealed so as to prevent microbial contamination or alternatively it is freeze-dried and the containers subsequently sealed.*
* To ensure availability of a preparation of uniform potency, Tuberculin Purified Protein Derivative is produced and issued by the Statens Serum Institute ,Denmark as a powder to be reconstituted as stated on the label.

The preparation may be issued either as a sterile liquid or as a freeze-dried product. If issued as a liquid, it is in a ready-touse form and 0.1 ml constitutes one intradermal dose containing appropriate number of Units. If issued as a freezedried product, it should yield a ready-to-use preparation when reconstituted as per manufacturers instructions. Description. A colourless or pale, straw-coloured liquid, or dry cream-coloured powder, or pellet. The preparation, reconstituted if necessary as stated on the label, complies with the following requirements.

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TUBERCULIN PURIFIED PROTEIN DERIVATIVE

Identification
A. When progressively increasing doses are injected intradermally into specifically sensitised guinea-pigs, reactions occur at the points of injection, varying from erythema to necrosis. When similar injections are administered to nonsensitised guinea-pigs no such reactions occur. B. The potency test described below serves as a test for identity if it is performed on material from the final containers.

derivative with the dose of the appropriate Standard Preparation necessary to give the same effect. The estimated potency is not less 80 per cent and not more than 125 per cent of the stated potency. The fiducial limits of error are not less than 64 per cent and not more than 156 per cent of the stated potency. Standard Preparation The Standard Preparation is the 1st International Standard for Tuberculin, purified protein derivative (PPD), mammalian, established in 1951, consisting of PPD derived from cultures of M. tuberculosis (supplied in ampoules containing 5,00,000 Units) or another suitable preparation the potency of which has been determined in relation to the International Standard. Method Sensitise 12 guinea-pigs each weighing not less than 400 g by the intramuscular injection of a total of about 0.5 ml of a suspension in a suitable mineral oil with or without emulsifier and containing 0.1 mg per ml of heat-inactivated, dried mycobacteria of the same type as that used in the preparation of tuberculin. Use phosphate-buffered saline pH 7.4 containing 0.005 per cent w/v of polysorbate 80 to prepare the dilutions of the Standard preparation and of the preparation under examination and to reconstitute freeze-dried preparations containing no stabiliser. Not less than 1 month and not more than 6 months later carry out the following test : Shave the flanks to provide space for at least three reactions on each side, but not more than a total of 12 injection sites per animal. Use at least three doses of the Standard preparation and at least three doses of the preparation under examination, the highest dose being about 10 times as strong as the lowest. Dilute the preparations so that the lesions produced are 8 to 25 mm in diameter. Allocate the doses to the available sites in a random manner, in a Latin square design. Inject each dose intradermally in the same volume (0.1 or 0.2 ml) at the sites to which it has been allocated. Measure the diameters of the lesions after 24 to 48 hours and calculate the result of the test using standard statistical methods on the basis that the lesion diameters are directly proportional to the logarithm of the concentration of the tuberculin. Storage. Store in light-resistant containers at a temperature between 2 and 8. It should not be allowed to freeze. Labelling. The label states (1) the number of Units per dose of 0.1 ml or per ml or per mg; (2) the total volume in the container (for liquid preparation); (3) the nature and quantity of the reconstituting liquid (for the freeze-dried preparation); (4) the name and proportion of any added substances; (5) the species or strain used; (6) the storage conditions; (7) the date after which the contents are not intended to be used; (8) that care 95

Tests
pH (2.4.24). 6.5 to 7.5. Phenol (if present) (2.3.36). Not more than 0.5 per cent w/v. Sterility (2.2.11). Complies with the tests for sterility. Potency. Carry out the biological assay of tuberculin purified protein derivative described below. Tuberculin Purified Protein Derivative in freeze-dried form complies with the following additional requirements. Live mycobacteria A. Inject 5.0 ml intraperitoneally or subcutaneously into each of two guinea-pigs weighing between 300 and 400 g. Observe the animals for not less than 42 days. Kill the animals and carry out an autopsy. No guinea-pig shows signs of infection with mycobacteria. B. Carry out tests for live mycobacteria in the preparation under examination using suitable culture media. No growth of mycobacteria should occur. Sensitising effect. Inject intradermally into each of three guinea-pigs three times, at intervals of 5 days, a dose of the preparation under examination containing about 500 Units in a volume of 0.1 ml. Two to three weeks after the third injection inoculate the same dose intradermally into these animals and into a control group of three guinea-pigs of the same weight but that have not had any previous injections of tuberculin. The reactions of the two groups are not significantly different after 48 to 72 hours. Toxicity. Inject subcutaneously into 2 healthy guinea-pigs, weighing not less than 250 g and which have not previously been treated with any material which will interfere with the test, 0.5 ml of a solution containing 1,00,000 Units per ml. No harmful effects are produced within 7 days. Biological assay The potency of tuberculin purified protein derivative is determined by comparing the dose necessary to reveal delayed hypersensitivity in guinea-pigs or other animals hypersensitised with mycobacteria of the same type as that used in the preparation of the tuberculin purified protein

TYPHOID (STRAIN TY 21A) VACCINE, LIVE (ORAL)

IP 2007

should be taken to avoid inhaling the powder (for the freezedried preparation).

the growth conditions, strain Ty 21a does not agglutinate to Vi antiserum. Strain Ty 21a agglutinates to H:d flagellar antiserum. Biochemical markers Strian Ty 21a does not produce hydrogen sulphide on Kligler iron agar. This property serves to distinguish Ty 21a from other galactose-epimerase-negative S. typhi strains. Cell growth Strain Ty 21a cells lyse when grown in the presence of 1.0 per cent of galactose. PROPAGATION AND HARVEST The bacteria from the working seed lot are multiplied in a preculture, subcultured once and are then grown in a suitable medium containing 0.001 per cent of galactose at 30 for 13 to 15 hours. The bacteria are harvested. The harvest must be free from contaminating micro-organisms. Only a single harvest that complies with the following requirements may be used for the preparation of the freezedried harvest. pH (2.4.24). 6.8 to 7.5. Optical density The optical density of the culture, measured at 546 nm, is 6.5 to 11.0. Before carrying out the measurement, dilute the culture so that a reading in the range 0.1 to 0.5 is obtained and correct the reading to take account of the dilution.

Typhoid (Strain Ty 21a) Vaccine, Live (Oral)

Typhoid Vaccine (Live, Oral, strain Ty 21a) is a freeze-dried preparation of the live Salmonella typhi strain Ty 21a grown in a suitable medium. When presented in capsules, the vaccine complies with the tests stated under Capsules.

Production
Choice of vaccine strain The main characteristic of the strains is the defect of the enzyme uridine diphosphate-galactose-4 epimerase. The activities of galactopermease, galactokinase and galactose-1phosphate uridyl-transferase are reduced by 50 to 90 per cent. Whatever the growth conditions, the strain does not contain Vi antigen. The strain agglutinates to anti-O:9 antiserum only if grown in medium containing galactose. It contains the flagellar H:d antigen and does not produce hydrogen sulphide on Kligler iron agar. The strain is nonvirulent for mice. Cells of strain Ty 21a lyse if grown in the presence of 1.0 per cent of galactose. SEED LOT The vaccine is prepared using a seed-lot system. The working seed lots represent not more than one subculture from the master seed lot. The final vaccine represents not more than four subcultures from the original vaccine on which were made the laboratory and clinical tests showing the strain to be suitable. Only a master seed lot that complies with the following requirements may be used in the preparation of working seed lots. Galactose metabolism In a spectrophotometric assay, no activity of the enzyme uridine diphosphate-galactose-4-epimerase is found in the cytoplasm of strain Ty 21a compared to strain Ty 2. Biosynthesis of lipopolysaccharide Lipopolysaccharides are extracted by the hot-phenol method and examined by size-exclusion chromatography (2.4.16). Strain Ty 21a grown in medium free of galactose shows only the rough (R) type of lipopolysaccharide. Serological characteristics Strain Ty 21a grown in a synthetic medium without galactose does not agglutinate to specific anti-O:9 antiserum. Whatever 96

Identification
Culture bacteria on an agar medium containing 1.0 per cent of galactose and bromothymol blue. Light blue, concave colonies, transparent due to lysis of cells, should be found. No yellow colonies (galactose-fermenting) should be found. FREEZE-DRIED HARVEST The harvest is mixed with a suitable stabilizer and freeze-dried by a process that ensures the survival of at least 10.0 per cent of the bacteria and to a water content shown to be favourable to the stability of the vaccine. No antimicrobial preservative is added to the vaccine. Only a freeze-dried harvest that complies with the following tests may be used for the preparation of the final bulk.

Identification
Culture bacteria are examined on an agar medium containing 1.0 per cent of galactose and bromoethymol blue. Light blue, concave colonies, transparent due to lysis of cells, should be found. No yellow colonies (galactose-fermenting) should be found.

IP 2007

TYPHOID POLYSACCHARIDE VACCINE

Number of live bacteria Not less than 1 x 10 live S. typhi strain Ty 21a per gram. Water (2.4.43). 1.5 to 4.0 per cent, determined by the semimicro determination of water or any other validated method. FINAL BULK VACCINE The final bulk vaccine is prepared by aseptically mixing one or more freeze-dried harvests with a suitable sterile excipient. Only a final bulk that complies with the following requirement may be used in the preparation of the final lot. Number of live bacteria. Not less than 40 109 live S. typhi strain Ty 21a per gram. FINAL LOT The final bulk vaccine is distributed under aseptic conditions into capsules with a gastro-resistant shell or into suitable containers. Only a final lot that is satisfactory with respect to Identification, Tests and Number of live bacteria will be released for use, except that in the determination of number of live bacteria each dosage unit must contain not less than 4 x 109 live bacteria.
11

Labelling. The label states (1) the minimum number of live bacteria per dosage unit; (2) that the vaccine is for oral use only.

Typhoid Polysaccharide Vaccine

Typhoid Polysaccharide Vaccine is a preparation of purified Vi capsular polysaccharide obtained from Salmonella typhi Ty2 strain or some other suitable strain of known origin and history that has the capacity to produce Vi polysaccharide, and which have been characterized by suitable biochemical, physicochemical, serological or molecular methods. Capsular polysaccharide is a partly 3-O-acetylated repeated units of 2-acetylamino-2-deoxy-D-galactopyranuronic acid with -(1?4)-linkages.

Production
General provisions The production of Vi polysaccharide is based on a defined seed-lot system. The method of production shall have been shown to yield consistently Vi polysaccharide typhoid vaccines of adequate immunogenicity and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the tests for abnormal toxicity. SEED LOT The strain of S. typhi used for the master seed lot shall be identified by historical records that include information on its origin and by its biochemical and serological characteristics. Cultures from the working seed lot shall have the same characteristics as the strain that was used to prepare the master seed lot, shall be demonstrated along with adequate documentation. Only a strain that has the following characteristics may be used in the preparation of the vaccine: (a) stained smears from a culture are typical of S. typhi; (b) the culture utilises glucose without production of gas; (c) colonies on agar are oxidasenegative; (d) a suspension of the culture agglutinates specifically with an appropriate Vi antiserum or colonies form haloes on an agar plate containing a suitable Vi antiserum. PROPAGATION AND HARVEST The working seed lot is cultured on a solid medium, which may contain blood-group substances, or a liquid medium; the inoculum obtained is transferred to a liquid medium, which is used to inoculate the final medium. The liquid medium used and the final medium are semi-synthetic, free from substances that are precipitated by cetrimonium bromide and do not contain blood-group substances or high-molecular-mass 97

Identification
Culture bacteria from the vaccine under examination on an agar medium containing 1.0 per cent of galactose and bromothymol blue. Light blue, concave colonies transparent due to lysis of cells, should be found. No yellow colonies (galactose-fermenting) should be found.

Tests
Microbial contamination (2.2.9). Carry out the test using suitable selective media. Determine the total viable count using the plate-count method. The number of contaminating microorganisms per dosage unit is not greater than 102 bacteria and 20 fungi. No pathogenic bacterium, particularly Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and no Salmonella other than strain Ty 21a are found. Water (2.4.43). 1.5 to 4.0 per cent. Number of live bacteria Carry out the test using not less than five dosage units. Homogenise the contents of the dosage units in a 0.9 per cent w/v solution of sodium chloride at 4 using a mixer in a cold room with sufficient glass beads to emerge from the liquid. Immediately after homogenization prepare a suitable dilution of the suspension using cooled diluent and inoculate brain heart infusion agar, incubate at 36 1 for 20 to 36 hours. The vaccine contains not less than 2 x 109 live S. typhi Ty 21a bacteria per dosage unit.

TYPHOID POLYSACCHARIDE VACCINE

IP 2007

polysaccharides, unless it has been demonstrated that they are removed by the purification process. The bacterial purity of the culture is verified by microscopic examination of Gramstained smears and by inoculation into appropriate media. The culture is then inactivated at the beginning of the stationary phase by the addition of formaldehyde. Bacterial cells are eliminated by centrifugation; the polysaccharide is precipitated from the culture medium by addition of hexadecyltrimethylammonium bromide (cetrimonium bromide). The precipitate is harvested and may be stored at or below -20 before purification. Purified Vi Polysaccharide The polysaccharide is purified, after dissociation of the polysaccharide/cetrimonium bromide complex, using suitable procedures to eliminate successively nucleic acids, proteins and lipopolysaccharides. The polysaccharide is precipitated in its salt form and dried at 5+3; the powder obtained constitutes the purified Vi polysaccharide. The loss on drying is determined by thermogravimetry, Karl Fischer or any other suitable method and is used to calculate the results of the chemical tests shown below with reference to the dried substance. Only those pools of purified Vi polysaccharide that comply with the following requirements may be used in the preparation of the final bulk: Protein (2.7.1). Not more than 10 mg per gram of polysaccharide, calculated with reference to the dried substance. Nucleic acids (2.7.1). Not more than 20 mg per gram of polysaccharide, calculated with reference to the dried substance. O-Acetyl groups (2.7.1). Not less than 2 mmol per gram of polysaccharide, calculated with reference to the dried substance. Molecular size. Examine by gel filtration or size-exclusion chromatography (2.4.16) using cross-linked agarose for chromatography. Use a column 0.9 m long and 16 mm in internal diameter equilibrated with a solvent having an ionic strength of 0.2 mol per kg and a pH of 7.0 to 7.5. Apply about 5 mg of polysaccharide in a volume of 1 ml to the column and elute at about 20 ml/h. Collect fractions of about 2.5 ml. Determine the point corresponding to K0 = 0.25 and make two pools consisting of fractions eluted before and after this point. Determine O-acetyl groups on the two pools. Not less than 50 per cent of the polysaccharide is found in the pool containing fractions eluted before K0 = 0.25.

Bacterial endotoxins (2.2.3). Determine by a suitable method, the content should not be more than 150 IU per mg of polysaccharide. FINAL BULK VACCINE One or more batches of purified Vi polysaccharide are dissolved in a suitable solvent, which may contain an antimicrobial preservative, so that the volume corresponding to one dose contains 25 g of polysaccharide and the solution is isotonic with blood (250 mosm/kg to 350 mosm/kg). Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot; Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. If phenol has been used in the preparation, the content is not more than 2.5 g/l. FINAL LOT The final bulk vaccine is distributed and filled aseptically into sterile containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements prescribed below under Identification, Tests and Assay and with the requirements for Bacterial endotoxins may be released for use. Provided the tests for free formaldehyde and antimicrobial preservative have been carried out on the final bulk vaccine, they may be omitted on the final lot. The vaccine contains minimum of 25 g purified ViPolysaccharide per dose of 0.5 ml.

Identification
Carry out an identification test using a suitable immunochemical method (2.2.14).

Tests
Sterility (2.2.11). Complies with the tests for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. pH (2.4.24). 6.5 to 7.5. O-Acetyl groups (2.7.1). 0.085 ( 25 per cent) mol per dose (25 g of polysaccharide). Test solution. Place 3 ml of the vaccine in each of three tubes (two reaction solutions and one correction solution). 98

Identification
Carry out an identification test using a suitable immunochemical method (2.2.14).

IP 2007

TYPHOID VACCINE (FREEZE DRIED)

Reference solutions. Dissolve 0.150 g of acetylcholine chloride in 10 ml of water (stock solution containing 15 g/l of acetylcholine chloride). Immediately before use, dilute 0.5 ml of the stock solution to 50 ml with water (working dilution containing 150 g/ml of acetylcholine chloride). In ten tubes, place in duplicate (reaction and correction solutions) 0.1 ml, 0.2 ml, 0.5 ml, 1.0 ml and 1.5 ml of the working dilution. Prepare a blank using 3 ml of purified water. Make up the volume in each tube to 3 ml with water. Add 0.5 ml of a mixture of 1 volume of water and 2 volumes of dilute hydrochloric acid to each of the correction tubes and to the blank. Add 1.0 ml of alkaline hydroxylamine solution to each tube. Allow the reaction to proceed for exactly 2 min and add 0.5 ml of a mixture of 1 volume of water and 2 volumes of dilute hydrochloric acid to each of the reaction tubes. Add 0.5 ml of a 20 per cent w/vl solution of ferric chloride in 0.2M hydrochloric acid to each tube, stopper the tubes and shake vigorously to remove bubbles. Measure the absorbance of each solution at 540 nm using the blank as the compensation liquid. For each reaction solution, subtract the absorbance of the corresponding correction solution. Draw a calibration curve from the corrected absorbance for the five reference solutions and the corresponding content of acetylcholine chloride and read from the curve the content of acetylcholine chloride in the test solution for each volume tested. Calculate the mean of the two values. 1 mol of acetylcholine chloride (181.7 g) is equivalent to 1 mol of O-acetyl (43.05 g). Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. If phenol has been used in the preparation, the content is not more than 2.5 g/l. Assay Determine Vi polysaccharide content by a suitable immunochemical method (2.2.14) using a reference purified polysaccharide. The estimated amount of polysaccharide per dose is 80.0 per cent to 120.0 per cent of the content stated on the label. The fiducial limits of error (P = 0.95) of the estimated amount of polysaccharide are not less than 80.0 per cent and not more than 120.0 per cent. Labelling. The label states (1) the number of micrograms of polysaccharide per human dose (25 g); (2) the total quantity of polysaccharide in the container. 99

Typhoid Vaccine
Typhoid Vaccine is a sterile suspension of inactivated Salmonella typhi containing not less than 5 x 108 and not more than 1 x 109 bacteria per human dose. The human dose does not exceed 1.0 ml.

Production
The vaccine is prepared using a seed-lot system from a suitable strain of S. typhi such as, Ty 2. The final vaccine represents not more than 3 subcultures from the strain on which were made the laboratory and clinical tests that showed it to be suitable. The bacteria are inactivated by acetone, by formaldehyde, by phenol or by heating or by a combination of the last two methods. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity (2.2.1) modified to the extent that 0.5 ml of the vaccine is injected subcutaneously or intramuscularly or intraperitoneally into each mouse and 1.0 ml into each guineapig.

Identification
It is identified by specific agglutination. Phenol (2.3.36). If phenol has been used in the preparation, the concentration is not more than 0.5 per cent w/v. Antigenic power. When injected into susceptible laboratory animals, it elicits anti-O, anti-H and, to a lesser extent, anti-Vi agglutinins. Sterility (2.2.11). Complies with the test for sterility. Labelling. The label states (1) the method used to inactivate the bacteria; (2) the number of bacteria per human dose.

Typhoid Vaccine (Freeze Dried)

Freeze Dried Typhoid Vaccine is a freeze-dried preparation of inactivated Salmonella typhi. The vaccine is reconstituted as stated on the label to give a uniform suspension containing not less than 5 x 108 and not more than 1 x 109 bacteria per human dose. The human dose does not exceed 1.0 ml of the reconstituted vaccine.

Production
The vaccine is prepared from a seed-lot system from a suitable strain of S. typhi, such as Ty 2. The final vaccine represents not more than 3 subcultures from the strain on which were made the laboratory and clinical tests that showed it to be suitable. The bacteria are inactivated either by acetone or by

VARICELLA VACCINE, LIVE

IP 2007

formaldehyde or by heat. Phenol is not used in the preparation. The vaccine is distributed into sterile containers and freezedried to a moisture content favourable to the stability of the vaccine. The production method is validated to demonstrate that the product, if tested, would comply with test for abnormal toxicity (2.2.1), modified to the extent that 0.5 ml of the vaccine is injected subcutaneously or intramuscularly or intraperitoneally into each mouse and 1.0 ml into each guineapig.

origin of the strain and its subsequent manipulation. The virus shall at no time have been passaged in continuous cell lines. Seed lots are prepared in the same kind of cells as those used for the production of the final vaccine. To avoid the unnecessary use of monkeys in the test for neurovirulence, virus seed lots are prepared in large quantities and stored at temperatures below -20, if freeze-dried, or below -60, if not freeze-dried. Only a virus seed lot that complies with the following requirements may be used for virus propagation.

Identification
The vaccine reconstituted as stated on the label is identified by specific agglutination. Antigenic power. When injected into susceptible laboratory animals, the reconstituted vaccine elicits anti-O, anti-H and, to a lesser extent, anti-Vi agglutinins. Sterility (2.2.11). The reconstituted vaccine complies with the tests for sterility. Water. Not more than 5.0 percent. Labelling. The label states (1) the method used to inactivate the bacteria; (2) the number of bacteria per human dose; (3) that the vaccine should be used within 8 hours of reconstitution.

Identification
The master and working seed lots are identified as varicella virus by serum neutralisation in cell culture, using specific antibodies. Virus concentration. The virus concentration of the master and working seed lots is determined as prescribed under Assay to monitor consistency of production. Extraneous agents (2.7.3). Complies with the requirements for seed lots for live virus vaccines; a sample of 50 ml is taken for the test in cell cultures. Neurovirulence (2.7.5). Complies with the test for neurovirulence of live virus vaccines. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are handled. Approved animal (but not human) serum may be used in the media. Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from extraneous agents. The cell culture medium may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. It is preferable to have a substrate free from antibiotics during production. 5.0 per cent, but not less than 50.0 ml, of the cell cultures employed for vaccine production is set aside as uninfected cell cultures (control cells). The infected cells constituting a single harvest are washed, released from the support surface and pooled. The cell suspension is disrupted by sonication. Only a virus harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine.

Varicella Vaccine, Live

Varicella Vaccine (Live) is a freeze-dried preparation of a suitable attenuated strain of Herpesvirus varicellae.

Production
General provisions The production of vaccine is based on a virus seed-lot system and a cell-bank system. The production method shall have been shown to yield consistently live varicella vaccines of adequate immunogenicity and safety in man. The virus in the final vaccine shall not have been passaged in cell cultures beyond the 38th passage from the original isolated virus. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Substrate for virus propagation The virus is propagated in human diploid cells (2.7.2). SEED LOT The strain of varicella virus shall be identified as being suitable by historical records which shall include information on the

Identification
The virus harvest contains virus that is identified as varicella virus by serum neutralisation in cell culture, using specific antibodies. Virus concentration. The concentration of infective virus in virus harvests is determined as prescribed under assay to monitor consistency of production and to determine the

100

IP 2007

VIPER VENOM

dilution to be used for the final bulk vaccine. Extraneous agents (2.7.3). Use 50 ml for the test in cell cultures. Control cells. The control cells of the production cell culture from which the single harvest is derived comply with a test for identity and with the requirements for extraneous agents (2.7.3). FINAL BULK VACCINE Virus harvests that comply with the above tests are pooled and clarified to remove cells. A suitable stabiliser may be added and the pooled harvests diluted as appropriate. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers and freeze-dried to a moisture content shown to be favourable to the stability of the vaccine. The containers are then closed so as to prevent contamination and the introduction of moisture. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot.

Labelling. The label states (1) the strain of virus used for the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) that contact with disinfectants is to be avoided; (4) the minimum virus concentration; (5) that the vaccine is not to be administered to pregnant women; (6) the time within which the vaccine must be used after reconstitution.

Viper Venom
Daboia Venom
Viper Venom is the dried secretion obtained from the poison glands of Viperae russelli and other species of Viperae (Fam. Viperidae). Viper Venom contains not less than 50 Mouse Units per mg.

Production
Immediately after extraction, the poisonous secretion is dried from the frozen state. The dried venom is pooled, mixed, dissolved in ice-cold water for injection and then filtered through a bacteria-proof filter to give a stock solution. Further dilutions of the stock solution are made with water for injection under aseptic conditions to give solutions with the required number of Mouse Units per ml. These solutions are then distributed in single dose sterile glass containers, dried from the frozen state and sealed at a pressure not exceeding 2.75 kPa. Description. An almost white or very light yellow, dry powder which when mixed with water yields a clear solution with some insoluble residue.

Identification
When the vaccine reconstituted as stated on the label is mixed with specific Herpesvirus varicellae antibodies, it is no longer able to infect susceptible cell cultures.

Identification
A. Produces almost immediate coagulation of blood and citrated human plasma. B. Mix the soluble fraction from at least 0.6 mg with 1 ml of polyvalent antisnake venom serum and incubate the mixture at 37o for 30 minutes. Inject 0.5 ml of the mixture intravenously into a group of mice weighing between 18 and 20 g. Observe the animals for 24 hours; no animal dies.

Tests
Sterility (2.2.11) Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin. Not more than 0.5 g per human dose, determined by a suitable immunochemical method (2.2.14). Water (2.3.43). Not more than 3.0 per cent, determined by the semi-micro determination of water. Assay Titrate for infective virus, using at least ten cell cultures for each fourfold dilution or by a technique of equal precision. Use a suitable virus reference preparation to validate each assay. The virus concentration is not less than the minimum stated on the label.

Tests
Sterility (2.2.11). Complies with tests for sterility. Assay. Carry out the biological assay of snake venom described below: Biological assay of snake venom Dissolve a quantity of the freeze-dried venom equivalent to 50 Mouse Units in 25 ml of saline solution. Inject 0.5 ml

101

YELLOW FEVER VACCINE (LIVE)

IP 2007

intravenously into each of 10 mice weighing between 18 and 20 g and observe the animals for 24 hours. Not less than 3 and not more than 8 of the mice die in 2 to 24 hours. If the number of deaths is not within this range, change the dilution of the venom suitably. Express the result in terms of number of Mouse Units per mg. NOTE The quantity in mg of the venom which will kill in 2 to 24 hours not less than 3 and not more than 8 mice represents one Mouse Unit. Storage. Store in single dose, light-resistant containers. Labelling. The label states (1) the number of Mouse Units per container; (2) the volume of water for injection to be used for reconstitution.

be only one passage from a master seed lot. A working seed lot shall be used without intervening passage as the inoculum for infecting the tissues used in the production of a vaccine lot, so that no vaccine virus is more than one passage from a seed lot that has passed all the safety tests. Only a virus seed lot that complies with the following requirements may be used for virus propagation.

Identification
The master and working seed lots are identified as containing yellow fever virus by serum neutralisation in cell culture, using specific antibodies. Extraneous agents (2.7.3). Each working seed lot complies with the test for extraneous agents. PROPAGATION AND HARVEST

Yellow Fever Vaccine (Live)

Yellow Fever Vaccine (Live) is a freeze-dried preparation of the 17D strain of yellow fever virus grown in fertilised hen eggs.

Production
General provisions The production of vaccine is based on a virus seed-lot system. The production method shall have been shown to yield consistently the yellow fever vaccine (live) of acceptable immunogenicity and safety for man. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Reference preparation. In the test for neurotropism, a suitable batch of vaccine known to have satisfactory properties in man is used as the reference preparation. Substrate for virus propagation Virus for the preparation of master and working seed lots and for all vaccine batches is grown in the tissues of chick embryos from a flock free from specified pathogens (2.7.7). SEED LOT The 17D strain shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. Virus seed lots are prepared in large quantities and stored at a temperature below -60. Master and working seed lots shall not contain any human protein or added serum. Unless otherwise justified and authorised, the virus in the final vaccine shall be between passage levels 204 and 239 from the original isolate of strain 17D. A working seed lot shall

All processing of the fertilised eggs is done under aseptic conditions in an area where no other infectious agents or cells are handled at the same time. Two per cent but not less than twenty and not more than fifty eggs are set aside as uninfected control eggs. After inoculation and incubation at a controlled temperature, only living and typical chick embryos are harvested. The age of the embryo at the time of virus harvest is reckoned from the initial introduction of the egg into the incubator and shall not be more than 12 days. After homogenisation and clarification by centrifugation, the extract of embryonic pulp is tested as described below and kept at -70 or colder until further processing. Virus harvests that comply with the prescribed tests may be pooled. No human protein is added to the virus suspension at any stage during production. If stabilisers are added, they shall have been shown to have no antigenic or sensitising properties for man. Only a single harvest that complies with the following tests may be used in the preparation of the final bulk vaccine.

Identification
The single harvest contains virus that is identified as yellow fever virus by serum neutralisation in cell culture, using specific antibodies. Extraneous agents (2.7.3). Complies with the tests for extraneous agents. Control eggs. Complies with the tests for extraneous agents (2.7.3). Virus concentration. In order to calculate the dilution for formulation of the final bulk, each single harvest is titrated as described under Assay. FINAL BULK VACCINE Single harvests that comply with the tests prescribed above are pooled and clarified again. A test for protein nitrogen

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YELLOW FEVER VACCINE (LIVE)

content is carried out. A suitable stabiliser may be added and the pooled harvests diluted as appropriate. Only a final bulk vaccine that complies with the following tests may be used in the preparation of the final lot. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Protein nitrogen content (2.3.30). The protein nitrogen content, before the addition of any stabiliser, is not more than 0.25 mg per human dose. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers and freeze-dried to a moisture content shown to be favourable to the stability of the vaccine. The containers are then closed so as to prevent contamination and the introduction of moisture. Only a final lot that is satisfactory with respect to thermal stability and each of the tests given under Identification, Tests and Assay may be released for use. Provided that the test for ovalbumin has been performed with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37 for 14 days. Determine the virus concentration as described under Assay in parallel for the heated vaccine and for unheated vaccine. The difference in the virus concentration between unheated and heated vaccine does not exceed 1.0 log10, and the virus concentration of the heated vaccine is not less than the number of TCID50 or plaque-forming units (PFU) equivalent to 1 103 mouse LD50 per human dose.

The virus concentration is not less than the equivalent in TCID50 or PFU of 1 103 mouse LD50 per human dose. The relationship between mouse LD50 and TCID50 or PFU is established by each laboratory and approved by the competent authority. The method shown below, or another suitable technique, may be used to determine the mouse LD50. Mouse LD50. The statistically calculated quantity of virus suspension that is expected to produce fatal specific encephalitis in 50 per cent of mice of a highly susceptible strain, 4 to 6 weeks of age, after intracerebral inoculation. Appropriate serial dilutions of the reconstituted vaccine are made in diluent for yellow fever virus (0.75 per cent solution of bovine albumin in phosphate-buffered saline pH 7.4, or any other diluent that has been shown to be equivalent for maintaining the infectivity of the virus). Mice of a highly susceptible strain, 4 to 6 weeks of age, are injected intracerebrally under anaesthesia with 0.03 ml of the vaccine dilution. Groups of not less than eight mice are used for each dilution; the series of dilutions is chosen so as to cover the range 0 to 100.0 per cent mortality of the mice. Injection of the mice is performed immediately after the dilutions have been made. The mice are observed for 21 days and all deaths are recorded. Only survivors and deaths caused by typical yellow fever infections are counted in the computations. Mice paralysed on the twenty-first day of observation are counted as survivors. Tests in monkeys for Yellow Fever Vaccine Each master and working seed lot complies with the following tests in monkeys for viraemia (viscerotropism), immunogenicity and neurotropism. The monkeys shall be Macaca spp. susceptible to yellow fever virus and shall have been shown to be non-immune to yellow fever at the time of injecting the seed virus. They shall be healthy and shall not have received previously intracerebral or intraspinal inoculation. Furthermore, they shall not have been inoculated by other routes with neurotropic viruses or with antigens related to yellow fever virus. Not fewer than ten monkeys are used for each test. Use a test dose of 0.25 ml containing the equivalent of not less than 5000 mouse LD50 and not more than 50,000 mouse LD50, determined by a titration for infectious virus and using the established equivalence between virus concentration and mouse LD50 (see under Assay). Inject the test dose into one frontal lobe of each monkey under anaesthesia and observe the monkeys for not less than 30 days. Viraemia (Viscerotropism). Viscerotropism is indicated by the amount of virus present in serum. Take blood from each of the test monkeys on the second, fourth and sixth days after

Identification
When the vaccine reconstituted as stated on the label is mixed with specific yellow fever virus antibodies, there is a significant reduction in its ability to infect susceptible cell cultures.

Tests
Sterility (2.2.11). Complies with the test for sterility. Ovalbumin. Not more than 5 g of ovalbumin per human dose, determined by a suitable immunochemical method (2.2.14). Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Bacterial endotoxins (2.2.3). Not more than 5 IU of bacterial endotoxin per human dose. Water (2.3.43). Not more than 3.0 per cent, determined by the semi-micro determination of water. Assay Titrate for infective virus in cell cultures. Use an appropriate virus reference preparation to validate each assay.

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inoculation and prepare serum from each sample. Prepare 1:10, 1:100 and 1:1000 dilutions from each serum and inoculate each dilution into a group of at least six cell culture vessels used for the determination of the virus concentration. The seed lot complies with the test if none of the sera contains more than the equivalent of 500 mouse LD50 in 0.03 ml and at most one serum contains more than the equivalent of 100 mouse LD50 in 0.03 ml. Immunogenicity. Take blood from each monkey 30 days after the injection of the test dose and prepare serum from each sample. The seed lot complies with the test if at least 90.0 per cent of the test monkeys are shown to be immune, as determined by examining their sera in the test for neutralisation of yellow fever virus described below. It has been shown that a low dilution of serum (for example, 1:10) may contain non-specific inhibitors that influence this test; such serum shall be treated to remove inhibitors. Mix dilutions of at least 1: 10, 1:40 and 1: 160 of serum from each monkey with an equal volume of 17D vaccine virus at a dilution that will yield an optimum number of plaques with the titration method used. Incubate the serum-virus mixtures in a waterbath at 37 for 1 h and then cool in iced water; add 0.2 ml of each serum-virus mixture to each of four cell-culture plates and proceed as for the determination of virus concentration. Inoculate similarly ten plates with the same amount of virus plus an equal volume of a 1:10 dilution of monkey serum known to contain no neutralising antibodies to yellow fever virus. At the end of the observation period, compare the mean number of plaques in the plates receiving virus plus non-immune serum with the mean number of plaques in the plates receiving virus plus dilutions of each monkey serum. Not more than 10 per cent of the test monkeys have serum that fails to reduce the number of plaques by 50.0 per cent at the 1:10 dilution. Neurotropism. Neurotropism is assessed from clinical evidence of encephalitis, from incidence of clinical manifestations and by evaluation of histological lesions, in comparison with ten monkeys injected with the reference preparation. The seed lot is not acceptable if either the onset and duration of the febrile reaction or the clinical signs of encephalitis and pathological findings are such as to indicate a change in the properties of the virus. Clinical evaluation. The monkeys are examined daily for 30 days by personnel familiar with clinical signs of encephalitis in primates (if necessary, the monkeys are removed from their cage and examined for signs of motor weakness or spasticity). The seed lot is not acceptable if in the monkeys injected with it the incidence of severe signs of encephalitis, such as paralysis or inability to stand when stimulated, or mortality is greater than for the reference vaccine. These and other signs of encephalitis, such as paresis, in-coordination, lethargy, tremors or spasticity are assigned numerical values for the severity of symptoms by a grading method. Each day each

monkey in the test is given a score based on the scale: Grade 1 rough coat, not eating, high-pitched voice, inactive, slow moving, shaky, tremors, unco-ordinated, limb weakness, inability to stand, limb paralysis or death (a dead monkey receives a daily score of 4 from the day of death until day 30). Grade 2 Grade 3 Grade 4

A clinical score for a particular monkey is the average of its daily scores; the clinical score for the seed lot is the mean of the individual monkey scores. The seed lot is not acceptable if the mean of the clinical severity scores for the group of monkeys inoculated with it is significantly greater (P = 0.95) than the mean for the group of monkeys injected with the reference preparation. In addition, special consideration is given to any animal showing unusually severe signs when deciding on the acceptability of the seed lot. Histological evaluation. Five levels of the brain are examined including : Block 1 Block II the corpus striatum at the level of the optic chiasma, the thalamus at the level of the mamillary bodies,

Block III the mesencephalon at the level of the superior colliculi, Block IV the pons and cerebellum at the level of the superior olives, Block V the medulla oblongata and cerebellum at the level of the mid-inferior olivary nuclei.

Cervical and lumbar enlargements of the spinal cord are each divided equally into six blocks; 15 m sections are cut from the tissue blocks embedded in paraffin wax and stained with gallocyanin. Numerical scores are given to each hemisection of the cord and to structures in each hemisection of the brain as listed below. Lesions are scored as follows: Grade 1. Minimal: 1 to 3 small focal inflammatory infiltrates. Degeneration or loss of a few neurons. Grade 2. Moderate: 4 or more focal inflammatory infiltrates. Degeneration or loss of neurons affecting not more than one third of cells. Grade 3. Severe: moderate focal or diffuse inflammatory infiltration. Degeneration or loss of up to two third of the neurons. Grade 4. Overwhelming: variable but often severe inflammatory reaction. Degeneration or loss of more than 90.0 per cent of neurons. It has been found that inoculation of yellow fever vaccine into the monkey brain causes histological lesions in different

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anatomical formations of the central nervous system with varying frequency and severity (I. S. Levenbook et al., Journal of Biological Standardization, 1987, 15, 305-313). Based on these two indicators, the anatomical structures can be divided into target, spared and discriminator areas. Target areas are those which show more severe specific lesions in a majority of monkeys irrespective of the degree of neurovirulence of the seed lot. Spared areas are those which show only minimal specific lesions and in a minority of monkeys. Discriminator areas are those where there is a significant increase in the frequency of more severe specific lesions with seed lots having a higher degree of neurovirulence. Discriminator and target areas for Macaca cynomolgus and Macaca rhesus monkeys are shown in the table below: Table 1. The discriminator and target areas for monkey. Type of monkey Discriminator areas Target areas substantia nigra

Macaca cynomolgus globus pallidus putamen anterior/ median thalamic nucleus lateral thalamic nucleus Macaca rhesus

caudate nucleus substantia nigra globus pallidus cervical putamen anterior/ lumbar median thalamic enlargement nucleus lateral thalamic nucleus cervical enlargement lumbar enlargement enlargement

Scores for discriminator and target areas are used for the final evaluation of the seed lot. The individual monkey score is calculated from the sum of individual target area scores in each hemisection divided by the number of areas examined. A separate score is calculated similarly for the discriminator areas. Mean scores for the test group are calculated in two ways: (1) by dividing the sum of the individual monkey discriminator scores by the number of monkeys and (2) by dividing the sum of the individual monkey target and discriminator scores by the number of monkeys. These two mean scores are taken into account when deciding on the acceptability of the seed lot. The seed lot is not acceptable if either of the mean lesion scores is significantly greater (P = 0.95) than for the reference preparation. Labelling. The label states (1) the strain of virus used in preparation; (2) that the vaccine has been prepared in chick embryos; (3) the minimum virus concentration; (4) that contact

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