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5. Viral subunit vaccines: the influenza vaccines are prepared by treating intact influenza virus
particles from
embryonated hens eggs, infected with influenza virus with surface active agent. The virus particles
are disrupted
and release the two viral subunits, haemagglutinin and neuraminidase that are required in the
vaccine. Another
example of viral subunit vaccine is hepatitis vaccine.
1. Killing:
Killing is the process in which live bacteria in the culture is killed.
Heat or formalin are required to kill the cells of Bordetella pertussis (whooping cough vaccine).
Phenol is
commonly used to kill Vibrio cholerae (cholera vaccine) and Salmonella typhi (typhoid vaccine).
2. Separation:
Separation is the process by which bacterial cells are separated from the fermentation medium.
Centrifugation or precipitation is commonly used for the separation of cells from the culture
fluid.
In the case of vaccines prepared from cells, the fluid is discarded and the cells are
resuspended in a salinesolution. If the vaccines are prepared from a constituent of the fluid, then
the cells are discarded.
3. Fractionation:
The process by which components are extracted from bacterial cells or from the medium is
called fractionation.
The antigens of Pseudomonas aeruginosa are extracted with water.
The polysacchride antigens of Neisseria meningitides are separated from the cells by treatment
with hexadecyltrimethyl ammonium bromide and those of S. pneumonia with ethanol.
The purity can be further improved by resolubilisation and precipitation.
4. Detoxification:
Detoxification is the process by which toxins are converted to harmless toxoids.
Formalin is used to detoxify the toxins of C. diphtheria and C. tetani.
Detoxification is generally performed in the fermenter.
5. Adsorption:
The mineral adjuvants or carriers are used to increase immunogenicity and decrease toxicity.
Eg. Aluminum hydroxide, aluminum phosphate, calcium phosphate etc.
Diphtherai vaccine, tetanus vaccine are prepared as adsorbed vaccines.
6. Conjugation:
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Conjugation is the linking of a vaccine component (poor immune response), with a vaccine
component that induces a good immune response.
The immunogenicity for infants of Hemophilus influenza type B is greatly enhanced by the
conjugation with diphtheria and tetanus toxoids.
Cell fluid provides infected fluids that contain little debris, which can be separated by filtration.
Most viral vaccines are not inactivated because they are made from cultures consisting of live
attenuated virus.
Poliomyelitis vaccine is inactivated with formalin and rabies vaccine with b-propiolactone.
When processing is complete, the bulk materials may be stored (-70oC) until needed for
blending into final vaccine.
Blending
Blending is the process in which various components of a vaccine are mixed to form a final
product.
A single component final bulk is prepared by adding bacterial suspensions or bacterial
components. Eg. BCG vaccine, cholera vaccine, diphtheria vaccine etc.
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Adjuvants such as adsorbents and preservatives may be included in the final bulk.
When viral vaccines are blended, adequate antigenicity and infectivity should be maintained.
Final bulk is added to appropriate containers.
Live attenuated viral vaccines lose potency in the suspension, hence they are stored at low
temperatures with stabilizers. Eg. Poliomyelitis stabilized with magnesium chloride or sucrose.
In-process control
In process quality control is the control exercised over starting materials and intermediates.
The quality control of diphtheria and tetanus vaccines requires that the products are tested for
the presence of free toxins.
Adequate infectivity of the virus from the tissue cultures is an indicator of the adequate virus
content of the starting materials and since infectivity is destroyed in the activation process.
In case of tissue culture substrates, to exclude contamination with infectious agents from the
source animal or in the case of human diploid cells, to exclude abnormal cellular characters.
Monkey kidney cell cultures are tested for simian herpes B virus, simian virus 40 and
mycoplasmas.
product control:
Final product control is the quality control exercised by the monographs of a pharmacopoeia
over products in their final containers.
All vaccines are tested for identity, potency and safety.
Combined vaccines are required to pass tests prescribed for each of the separate components.
Identity tests
Potency tests
• Safety tests, namely; Sterility tests, Free formalin tests, Abnormal toxicity testing, Phenol
concentration, Presence of aluminum and calcium
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Identity tests:
The identities of bacterial vaccines can be checked by precipitation and agglutination reactions.
Inactivated viral vaccines are tested by observation of the specific antibody responses in
vaccinated animals and live viral vaccines by neutralization of their cytopathic effects by specific by
specific antisera.
Potency assay:
Vaccines are tested for potency in which the amount of the vaccine that is required to protect
animals from a defined challenge dose of the pathogen.
Variable doses are injected into a specific groups of animals, and the mortality rate is observed.
Vaccines containing live microorganisms are generally tested for potency by counts of their
viable cells. EG. BCG vaccine.
The potency of live viral vaccines is estimated by using substrates of living cells.
Potency of many viral vaccines are also checked by physicochemical or serological techniques.
Safety assay:
Viral vaccines have some problems for safety testing as compared to bacterial vaccines.
Killed bacterial vaccines must be completely free from living microbes used in the production
process.
The final product must provide an assurance that all microoganisms have been killed.
Incomplete virus inactivation is detected by inoculation of susceptible tissue cultures and of
susceptible animals.
The cultures are examined for cytopathic effects and the animals for symptoms of disease.
Sterility assay:
All vaccines must be bacteriologically and mycologically sterile.
In each batch of a product the number of containers to be tested depends on the batch size
and is the subject of pharmacopoeial regulations.
Membrane filtration method is commonly used for sterility testing of vaccines.
The concentration of free formalin may not exceed 0.02% which is estimated by color
development with Acetylacetone
Phenol concentration
Phenol is used as a preservative in different types of vaccines. Its concentration must not
exceed 0.5w/v%.
Preparation
The strain grown on a suitable culture media, shows around 20 million colonies (seed lot
system).
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After suitable growth, they are separated by filtration in the form of cake.
The cake is homogenized in a grinding flask and suspended in a sterile liquid medium designed
to preserve the antigenicity and the viability of the vaccine (determined by plate count).
The suspension is transferred into a final sterile container and freeze dried under conditions
designed to prevent microbial contamination and finally sealed.
It is available as white pellets or powder, which when reconstituted, yields an opalescent and
homogenous suspension.
BCG tends to clump very badly when grown in conventional liquid media, and for a number of
years the preparation of a homogenous suspension depended on grinding these clumps with steel
balls using a sterile ball mill.
To overcome this problem a non-ionic surfactant (polyoxyethylene ether) is included in the
growth medium.
This causes the organism to grown throughout the medium instead of clumps or pellets, which
may even make the reconstitution difficult.
This also improves the appearance of the product.
Fluffiness is reduced, and reconstitution is assisted by the inclusion of dextran, which is also
used for drying the cells.
The medium also contains glucose which prevents excessive drying and, in the optimum
concentration, allows retention of optimum amount of moisture.
Storage:
BCG vaccine should be stored in sealed light resistant glass containers at a temperature
between 2 – 8oC. The reconstituted vaccine should be used immediately after preparation.
Dose:
Prophylactic, by intracutaneous injection, as a single dose, 0.1ml.
Nowadays, a new needleless technique of inoculation, involving penetration of the skin by a
high pressure jet of the preparation, is often used for mass vaccination.
Percutaneous vaccine is used in this method.
Use:
BCG vaccine is used as an immunizing agent which provides protection against tuberculosis.
Bacterial strains are mixed together to contain 1000 million organisms of Salmonella typhi and
750 million
organisms of each of Salmonella paratyphi A and B.
The suspension is transferred to final sterile containers and freeze dried.
The sterility and abnormal toxicity is evaluated further.
Storage:
Store in well closed containers at a temperature between 2 – 8oC.
Dose:
Prophylactic, 0.5ml (subcutaneous), 2 - 3 injections at 2 – 4 weeks interval.
Booster dose may be given every 1 – 2 years.
uses:
TAB or TABC mixed polyvalent vaccine is used in the prophylaxis of enteric infections.
TAB is also mixed with tetanus vaccine and cholera vaccine.
VIRAL VACCINES
Viral vaccines are prepared by using free living animals, fertile eggs and tissue cultures.
Viruses require a living medium for growth unlike bacteria which can grow on non-living media.
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Viruses are responsible for infectious diseases in man, animals and plants.
Important viral diseases are influenza, common cold, measles, mumps, poliomyelitis, smallpox,
yellow fever,
rabies etc.
For preparation of viral vaccines, viruses are usually grown in the chorioallantoic membrane of
incubated fertilevhen eggs or in whole animals.
POLIOMYELITIS VACCINE
Two types
1. Inactivated or salk-formalin vaccine
2. Live oral or sabin vaccine
Inactivated poliomyelitis vaccine is an aqueous suspension of suitable strains of poliomyelitis
virus, type I, II andvIII, grown in suitable cell cultures and inactivated by a suitable method.
Sabin poliomyelitis vaccine is an aqueous suspension of suitable live, attenuated strains of
poliomyelitis virus,vtype I, II and III, grown in suitable cell cultures. It may contain any one of the
three virus types or a mixture of twovor three of them.
RABIES VACCINE
Rabies vaccine is a suspension of a suitable strain of fixed rabies virus grown in suitable
approved cell cultures and inactivated by suitable method.
The vaccine is prepared immediately before use by reconstitution from the dried vaccine with a
suitable sterile liquid.
Preparation:
Rabies vaccine may be prepared by injecting sheep, rabbits, suckling rats and mice or other
suitable animals intracerebrally with the rabies virus.
Those animals which show typical paralysis are sacrificed and brains are harvested by opening
their skulls in specially arranged sterile rooms.
Brains are emulsified to make 10% suspension in saline solutions.
The suspension is inactivated by using phenol or formaldehyde or b-propiolactone.
Brain emulsion is incubated at 37oC for 24 hours and then diluted with equal quantity of normal
saline.
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The final vaccine may contain a suitable preservative and sufficient neutralizers to adjust the
pH to 7.2.
Rabies vaccine complies with the tests for absence of live virus, sterility and abnormal toxicity.
It should be stored in sealed, light resistant containers at a temperature between 2 – 8oC.
Vaccines containing nervous tissue may cause an allergic response in the brain, leading to
nerve deterioration and paralysis.
These reactions are rare when vaccines are prepared in fertile hen or duck eggs.
Preparation:
The viruses are grown in chick embryo cells or in suitable approved cell cultures.
The virus suspensions are harvested at a time appropriate to the strain of virus being used and
suspensions of the same component are pooled.
The clarified preparations of the individual viral components are mixed and a suitable stabilizer
is added.
The vaccine does not contain any added antimicrobial preservative.
The final product is freeze dried to a moisture content shown to be favorable to the stability of
the dried vaccine.
The vaccine is prepared immediately before use by reconstitution from the dried vaccine with
the liquid stated on the label.
The vaccine is prepared by mixing lyophilized measles 5000 TCID50 of Schwarz strain, mumps
5000 TCID50 and rubella 1000 TCID50 or lyophilized measles 5000 TCID50 of Edmonston Zagreb
strain, mumps 5000 TCID50 and rubella 4000 TCID50 per unit dose (0.5 ml).
A single dose is injected (subcutaneous or intramuscular in children older than 12 months for
protection against these three diseases.)
This reacts with bicarbonate, phosphate and protein impurities in the toxoid to produce a
precipitate containing aluminum hydroxide and phosphate.
Then the precipitate is washed and suspended in saline containing bactericide.
Alum is used to potentiate the effect of antigens (adjuvants).
APT produces more antibodies than FT and TAF.
DPT vaccine is prepared from Diphtheria formol toxoid, tetanus formol toxoid and suspension
of killed Bordetella pertussis.
These three components are mixed in the following proportions to form DPT vaccine.
Merthiolate (0.01%) is commonly added as a preservative immediately before mixing and then
the preparations to form DPT vaccine are mixed.
The antigenic properties of DPT vaccine are adversely affected by exposure to the action of
certain antimicrobial preservatives like phenol and some of the quaternary ammonium compounds.
DIPHTHERIA ANTITOXIN
Diphtheria antitoxin is a sterile, non-pyrogenic solution containing the specific antitoxic
antibodies obtained from the serum of healthy horses and have the power of neutralizing the toxin
formed by Corynebacterium diphtheria.
Preparation
The method of preparation is divided in following steps
1. Preparation of toxin
A pure culture of Corynebacterium diphtheria is grown in a suitable culture media at 37oC for 4
– 5 days.
After incubation, 0.5% phenol is added and the culture media is filtered through bacteria proof
filters.
The filtrate (crude toxin) is converted into a toxoid.
2. Selection of horse
Horses are selected for production of Diphtheria antitoxin because they are easy to handle and
readily produce antitoxins.
Considerable quantity of blood can be withdrawn at a time without any ill effects. The horses
selected must be free from diseases.
Horses are kept in an isolated place for 7 days and then carefully examined for infectious
diseases.
The first dose of toxoid is not more than 5ml which is gradually increased daily or after 1 - 2
days for about 2
months and as much as 600 ml is injected for the final dose.
Storage
Diphtheria antitoxin is stored in containers protected from light at a temperature between 2 –
8oC.
It should not be allowed to freeze.
Dose
Diphtheria antitoxin is administered by subcutaneous or intramuscular injection.
For prophylactic use, the dose is not less than 1500 IU and for therapeutic use, the dose is not
less than 50,000 IU.
Use
Passive immunizing agent for diphtheria.
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TETANUS ANTITOXIN
Tetanus antitoxin is a preparation containing antibodies that have the power of specifically
neutralizing the toxins formed by Clostridium tetani.
It is obtained by fractionation from serum of horses or other mammals that have been
immunized against tetanus toxins.
Method of preparation, storage and dose are similar to diphtheria antitoxin.
ANTIVIRAL SERA
These are used in passive immunization against certain viruses.
It acts differently as viruses are intracellular parasites while antibodies cannot penetrate the
cells.
Inactivation of viruses takes place in body fluids or on surfaces at which invasion occurs.
The main source of antiviral antibodies is human serum.
This is because the horse is not susceptible to many viruses against which protection may be
required. Eg.
Measles, rubella, poliomyelitis etc.
However, official antiviral sera (Eg. Rabies antiserum) is prepared in horses.
Rabies antiserum or Antirabies serum (ARS)
Antirabies serum (equine rabies immunoglobulin, ERIG) is a refined, concentrated and
lyophilized serum from horses hyperimmunized by repeated injections of fixed rabies virus.
Horses are injected with dead virus and when a good level of immunity has developed, are
injected with live viruses.
The method of protein purification used to refine the serum are designated to separate the
gamma-globulin
fraction that contains the antiviral antibodies.
Antirabies serum is indicated promptly after suspected exposure and is give simultaneously
with rabies vaccine to non-immunized individuals.
ANTIVENOMS
The incidence of snakebites in different parts of the world and the recognition of the particular
species of greatest medical importance is fundamental to the appropriate design of monospecific
and polyspecific antivenoms in countries and regions. Up-to-date knowledge is therefore highly
relevant to antivenom manufacturers and regulators, especially for the selection of the most
appropriate venoms, or venom mixtures, to be used in the production and quality control of
antivenoms.
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Venom: The toxic secretion of a specialized venom gland which, in the case of snakes, is delivered
through the fangs and provokes deleterious effects. Venoms usually comprise many different
protein components of variable structure and toxicity.
There are variations in venom composition and antigenicity within the geographical range of a
single taxonomic species as well as other causes of intra-species variation. Therefore, pooled
representative samples of venoms should be prepared from snakes of different geographical
origins and ages. Cross-neutralization of venom outside the range of venoms used for
immunization may extend the range of therapeutic applications of some antivenoms. Results of
preclinical potency testing may be used to identify a potential cross-neutralization capacity of
antivenoms, which should subsequently be confirmed by clinical testing in envenomed patients. In
vitro immunological cross-reactivity should not be used as the single basis for recommending
therapeutic use of an antivenom outside the range of venoms used in its production.
Procedure:
The snake is gently removed from its cage with a hook and either placed on a foam rubber pad
before being pinned behind the head or encouraged to crawl into a transparent plastic tube. For
very dangerous species, the use of short-acting general anaesthesia, or moderate cooling (15°C)
during milking can be considered (e.g. inhaled sevofluorane, halothane or even carbon dioxide) as
it reduces the risk of accidents both to the snake and to the snake-handler. For the collection of
venom, the snake’s head is grasped between index finger and thumb, just behind the angle of the
jaw, while the snake’s body is held between the trunk and the arm of the snake handler. By
applying gentle pressure, the snake's jaws are forced open, the fangs exposed and, in the case of
vipers, erected. In the case of large vipers, the dental sheath is retracted when necessary with
clean forceps
.
The fangs are pushed through a plastic/parafilm membrane hooked over the lip of a glass vessel,
and venom is squeezed out. The use of siliconized containers might be considered to minimize
venom attaching to the container surface. While a brief electrical impulse of moderate intensity can
be applied to stimulate venom secretion, this technique is not used or required by most venom
producers, although it may help in avoiding debris in the venom.
Any venom sample contaminated with blood should be rejected. After venom extraction, the fangs
are carefully withdrawn from the collection vessel, while preventing damage to the mouth and
dentition and avoiding the snake’s impaling itself with its own fangs.
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DIAGNOSTIC AGENTS
Diagnostic preparations are used as in vivo diagnostics, when injected intradermally into the
patients.
Diagnostics tests are based upon hypersensitivity reactions and it shows sensitivity to antigens by
the presence of antibodies.
It is also used to assess immunocompetency in individuals with possible cell mediated
immunodeficiency.
2. Schick control
Schick control is the Schick test toxin that has been heated at a temperature not lower than
70oC and not higher than 85oC for atleast five minutes to destroy the toxin.
It may be used in conjugation with Schick test toxin in order to exclude reactions due to non-
specific substances.
Schick control is prepared from the same batch of Schick test toxin with which it is to be stored
at a temperature of 2 – 8oC.
Left arm Right arm Explanation Inference
(Toxin) (control) Toxin is- Individual is-
Large flushed area No reaction Not neutralized Not immune
No reaction No reaction Neutralized Immune
Large flushed area Small flushed area Not neutralized; Not immune to a
sensitivity pseudo-reactor
to broth constituents
Small flushed area Small flushed area Neutralized; sensitivity Immune and a
to pseudoreactor
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broth constituents
TUBERCULIN TEST
Persons suffering from tuberculosis or who have recovered from an active or latent infection
are extremely
sensitive to a protein constituent of the tubercle bacillus, Mycobacterium tuberculosis.
The tuberculins contain this protein and, therefore, when they are applied to or injected into the
skin of sensitized individuals a local inflammation is set up.
Minute doses are sufficient to cause the reaction.
In general, only people who have never been infected will give a negative result.
The bacterial protein (tuberculin), which may be regarded as a toxin, acts as an antigen which
stimulates the
tissues to produce corresponding antibodies.
These antibodies react strongly with the antigen when they meet it again, causing inflammation,
and even
necrosis, of the cells in the area.
This hypersensitivity reaction, or allergy, appears to be a defense mechanism designed to limit
the spread of
infection.
The mechanism of immunity to tuberculosis is uncertain.
In individuals who are not actually suffering from the disease the presence of tuberculin
sensitivity usually
indicates immunity.
It is the tuberculin negative group, therefore, that can usefully be given BCG vaccine after
which, if the
immunization has been successful the tuberculin reaction should be positive.
OLD TUBERCULIN
Tuberculin was first made by Koch, and as the official preparation is still based on his
preparation it is known as Old tuberculin.
It is the heat-concentrated fluid from a culture of either the human or bovine strain of
Mycobacterium tuberculosis grown in a fluid medium.
Early products contained large amounts of nonspecific, reaction-causing impurities, partly from
the meat broth used for growing the organisms and partly from the metabolism and autolysis of the
latter.
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Unwanted broth constituents are now excluded by the use of a synthetic medium containing 5%
glycerin, dextrose and, as the only sources of nitrogen. asparagine (an amino acid) and ammonium
salts.
After inoculation with the organisms the medium is incubated at 37oC for six weeks or more.
Mycobacterium tuberculosis has a very long generation time of about a day and, therefore,
prolonged incubation is necessary to obtain abundant growth.
The culture is then steamed for an hour.
This kills the organisms and destroys the antigenicity of many of the protein impurities but does
not harm the
tuberculin which is very heat stable.
The preparation is then concentrated to about one-tenth of its original volume, a preservative
bactericide is added, and the solution is sterilized by filtration.
TUBERCULIN TESTS
1. The Mantoux Test
This is the most precise method. Initially, a very low dose (I unit) is injected intracutaneously in
a volume of 0,1 ml. If the result is negative the dose is increased to 10 units and, if there is still no
reaction, to 100 units. Graded dosage is necessary because of the great difference in tuberculin
sensitivity between individuals. It is necessary to start with a low dose since very sensitive persons
may show severe reactions to the higher concentrations. To avoid repeated testing, the WHO has
used, in some of its surveys, a single dose of 5 units, which explains why the Pharmacopoeia
mentions four strengths.
This has become the most popular method in the UK because the method of inoculation is less
painful, simpler, and more reliable than use of a syringe and needle. A spring-release device
makes several intracutaneous punctures of equal depth through a film of tuberculin previously
applied to the skin. PPD is preferred for this lest.