Sterility testing

DEFINITION: Sterility Testing: It is a procedure carried out to detect and

conform absence of any viable form of microbes in or on pharmacopeia
preparation or product.
PRINCIPLE : Sterility testing only shows that organisms capable of
growing in selected conditions are absent from the fraction of batch that
has been tested. If the microorganism are present in the product can be
indicated by a turbidity in the clear medium.
OBJECTIVE OF STERILITY TESTING:
For validation of sterilization process.
To check presence of microorganisms in preparation which are sterile.
To prevent issue of contaminated product in market.
K.I.P.M. GIDA, GORAKHPUR, U.P.
STEPS INVOLVED IN STERILITY TE TESTING
1) Sampling
2) Selection of the quantity of the product to be used
3) Method of sterility testing
i ) METHOD 1 Membrane filtration method
ii) METHOD 2 Direct inoculation method
4) Observation and interpretation Must be carried out under
aseptic condition.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Sampling
The sample must be representative of the whole of the bulk
material & a lot of final containers.
MAINLY FOLLOWED BY TWO RULES:
A fixed percentage of the final container are selected.
A fixed number of container are taken independent of the lot or
batch size.
K.I.P.M. GIDA, GORAKHPUR, U.P.
According to Indian Pharmacopoeia following guidelines for
determining the minimum number of items are:
Selection of the quantity of the product to be used
Selection of the quantity of the product to be used for sterility
testing depends mainly on the volume or weight in the container.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Method of sterility testing
Membrane filtration method (METHOD 1):
Membrane filtration Appropriate for : (advantage)
Filterable aqueous preparations
Alcoholic preparations
Oily preparations
Preparations miscible with or soluble in aqueous or oily (solvents
with no antimicrobial effect)
All steps of this procedure are performed aseptically in a Class 100
Laminar Flow Hood
K.I.P.M. GIDA, GORAKHPUR, U.P.
Membrane filter 0.45 porosity
Filter the test solution
After filtration remove the filter
Cut the filter in to two halves
First halves (For Bacteria) Second halves (For Fungi)
Transfer in 100 ml culture media
(Fluid Thioglycollate medium)
Incubate at 30-35
0
C for not less then 7 days

Transfer in 100 ml culture media
(Soyabeans-Casein Digest medium)
Incubate at 20-25
0
C for not less then 7 days

Observe the growth in the media

Observe the growth in the media

K.I.P.M. GIDA, GORAKHPUR, U.P.
Suitable for samples with small
volumes
volume of the product is not more
than 10% of the volume of the
medium
suitable method for aqueous
solutions, oily liquids, ointments
an creams
Direct inoculation of the culture
medium suitable quantity of the
preparation to be examined is
transferred directly into the
appropriate culture medium &
incubate for not less than 14 days.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Direct inoculation method (METHOD 2):
Observation and results
Culture media is examined during and after at the end of incubation.
The following observations are possible:
1) No evidence of growth Pass the test for sterility.
2) There is evidence of growth Re-testing is performed same no.
of sample, volume & media as in original test No evidence of
growth Pass the test for sterility.
3) There is evidence of growth isolate & identify the organism.
Re-testing is performed with twice no. of sample if:
No evidence of growth Pass the test for sterility.
There is evidence of growth Fail the test for sterility


K.I.P.M. GIDA, GORAKHPUR, U.P.
Particulate matter monitoring
Particulate matter is defined as unwanted mobile insoluble matter other
than gas bubble present in the product.
If the particle size of foreign matter is larger than the size of R.B.C.. It can
block the blood vessel.
The permit limits of particulate matter as per I.P. are follows:




Source of particulate matter:
1. Intrinsic contamination: The material which are originally present in
the parenteral solution e.g. Barium ions leach in parenteral & react
with sulphur ions in the product to form barium sulphate crystals.
2. Extrinsic contamination: The material which comes from the
environment e.g. Shedding of material from cloth, body, & cotton,
paper, rubber, tissue etc.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Methods for monitoring particulate matter
contamination:
1) Visual method
2) Coulter counter method
3) Filtration method
4) Light blockage method
Identification of particulate matter:
1) Microscopy
2) X-ray powder diffraction
3) Mass spectroscopy
4) Polarized light spectroscopy
5) Scanning electron microscopy (SEM)
K.I.P.M. GIDA, GORAKHPUR, U.P.
Faculty seal packaging / leaking testing
The sealed ampoules are subjected to small cracks which occur due
to rapid temperature changes or due to mechanical shocks.









Vials & bottles are not suitable for this test because the sealing
material used is not rigid.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Filled & sealed ampoules
Dipped in 1% Methylene blue solution
Under negative pressure in vacuum chamber
Vacuum released colored solution enter into the ampoule
Defective sealing
Pyrogen Testing
Pyrogen = Pyro (Greek = Fire) + gen (Greek = beginning).
Fever producing, metabolic by-products of microbial growth and
death.
Bacterial pyrogens are called Endotoxins. Gram negative bacteria
produce more potent endotoxins than gram + bacteria and fungi.
Endotoxins are heat stable lipopolysaccharides (LPS) present in
bacterial cell walls, not present in cell-free bacterial filtrates
Stable to at least 175
o
C; steam sterilization ineffective
Water soluble; monomer unit of LPS can be 10,000 Daltons (1.8 nm)
so endotoxins can easily pass through 0.22m filters
Sources: Water (main), raw materials, equipment, process
environment, people, and protein expression systems if using gram
negative bacteria.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Biological properties of endotoxin
Pyrogen elevate the circulatory levels of inflammatory cytokines
which may be followed by fever, blood coagulation, hypotension
Low doses of Pyrogen: asymptomatic inflammation reaction
Moderate doses: fever & changes in plasma composition
High doses: cardiovascular dysfunction, vasodilation,
vasoconstriction, endothelium dysfunction, multiple organ failure
& finally death.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Sources of pyrogen
1) Equipment
2) Containers (Glass , plastic , metal)
3) Solvent
4) Solute
Elimination of pyrogens
Dry heat sterilization : For glass wares, metal equipments,
powders, waxes, oils, heat stable drugs
650
o
C temp - 1 min
250
o
C temp - 30 min
180
o
C temp - 240 min
Ultra filtration
Reverse osmosis : RO membrane is composed of cellulose
acetate phthalate/ polyamide
Distillation
Adsorption method

K.I.P.M. GIDA, GORAKHPUR, U.P.
Principal:
Rabbits are used to perform this test because their body temp
increases when pyrogen are introduced into their bodies by
parenteral route
3 healthy adult rabbits of either sex, each weighing NLT 1.5 kg are
selected
Do not use any rabbit
having a temp higher than 39.8
o
C
Showing temp variation >0.2
o
C between two successive reading
in the determination of initial temp
Sham test is performed within 7 days of actual test
Animal showing temp increase over 0.6
o
C should be removed from
pyrogen testing
K.I.P.M. GIDA, GORAKHPUR, U.P.
Method :
Dissolve the subs being examined in, or dilute it with a pyrogen free
saline solution
Warm the liquid being examined to approx. 38.5
o
C temp before
injection
The volume of injection is NLT 0.5ml/kg & NMT 10ml/kg of body
weight
Withhold water during test
Clinical thermometer is inserted into the rectum of rabbit to record
body temp
2 normal reading of rectal temp are should be taken prior to the test
injection at an interval of half an hr & its mean is calculated- initial
temp
The solution under test is injected through an ear vein
Record the temp of each rabbit in an interval of 30 min for 3 hrs
The difference between initial temp & maximum temp is recorded-
taken as response
K.I.P.M. GIDA, GORAKHPUR, U.P.
Interpretation of results
K.I.P.M. GIDA, GORAKHPUR, U.P.
Bacterial endotoxin (LAL) test )
To detect or quantify endotoxins of gram-ve bacterial origin
Reagent: amoebocyte lysate from horseshoe crab (Limulus
polyphemus or Tachypleus tridentatus).
The name of the test is also Limulus amebocyte lysate (LAL)
test

K.I.P.M. GIDA, GORAKHPUR, U.P.
Mechanism of LAL Test:

The test is based on the primitive blood-clotting mechanism of
the horseshoe crab
enzymes located with the crab's amebocyte blood cells endotoxin


Initiation of an enzymatic coagulation cascade

proteinaceous gel
Test performance (short)
Avoid endotoxin contamination
Before the test:
interfering factors should not be present
equipment should be depyrogenated the sensitivity of the lysate
should be known
Test:
equal Volume of LAL reagent and test solution (usually 0.1 ml of
each) are mixed in a depyrogenated test-tube
Incubation at 37C, 1 hour
remove the tube - invert at (180) observe the result
pass-fail test
K.I.P.M. GIDA, GORAKHPUR, U.P.
LAL test
Three different techniques:
1. The gel-clot technique - gel formation
2. The turbidimetric technique - the development of Turbidity
after cleavage of an endogenous substrate
3. The chromogenic technique - the development of color after
cleavage of a synthetic peptide- chromogen complex
K.I.P.M. GIDA, GORAKHPUR, U.P.
Advantages of LAL test
Fast - 60 minutes vs. 180 minutes
Greater Sensitivity ,Less Variability
Much Less False, Positives ,Much Less Expensive
Alternative to Animal Model, cheaper,
particularly useful for:
Radiopharmaceuticals and cytotoxic agents
Blood products
Water for injection
Production facilities of
parenterals
The production area where the parenteral preparation are
manufactured can be divided into five sections:
Clean-up area
Preparation area
Aseptic area
Quarantine area
Finishing & packaging area
K.I.P.M. GIDA, GORAKHPUR, U.P.
Clean-up area:
It is not aseptic area.
All the parenteral products must be free from foreign particles &
microorganism.
Clean-up area should be withstand moisture, dust & detergent.
This area should be kept clean so that contaminants may not be carried
out into aseptic area.
Preparation area:
In this area the ingredients of the parenteral preparation are mixed &
preparation is made for filling operation.
It is not essentially aseptic area but strict precautions are required to
prevent any contamination from outside.

K.I.P.M. GIDA, GORAKHPUR, U.P.
Aseptic area:
The parenteral preparations are filtered, filled into final container
& sealed should be in aseptic area.
The entry of personnel into aseptic area should be limited &
through an air lock.
Ceiling, wall & floor of that area should be sealed & painted.
The air in the aseptic area should be free from fibers, dust and
microorganism.
The High efficiency particulate air filters (HEPA) is used for air.
UV lamps are fitted in order to maintain sterility.

K.I.P.M. GIDA, GORAKHPUR, U.P.
Quarantine area:
After filling, sealing & sterilization the parenteral product are
held up in quarantine area.
Randomly samples were kept foe evaluation.
The batch or product pass the evaluation tests are transfer in to
finishing or packaging area.
Finishing & packaging area:
Parenteral products are properly labelled and packed.
Properly packing is essential to provide protection against
physical damage.
The labelled container should be packed in cardboard or plastic
container.
Ampoules should be packed in partitioned boxes


K.I.P.M. GIDA, GORAKHPUR, U.P.
Lyophilization or freeze drying
Lyophilization or freeze drying is a process in which water is
removed from a product after it is frozen and placed under a
vacuum, allowing the ice to change directly from solid to vapor
without passing through a liquid phase.
The process consists of three separate, unique, and interdependent
processes;
Freezing,
Primary drying (sublimation), and
Secondary drying (desorption).

K.I.P.M. GIDA, GORAKHPUR, U.P.
The advantages of Lyophilization include:
Ease of processing a liquid, which simplifies aseptic handling
Enhanced stability of a dry powder
Removal of water without excessive heating of the product
Enhanced product stability in a dry state
Rapid and easy dissolution of reconstituted product
Disadvantages of Lyophilization include:
Increased handling and processing time
Need for sterile diluent upon reconstitution
Cost and complexity of equipment

K.I.P.M. GIDA, GORAKHPUR, U.P.
The Lyophilization process generally includes the following
steps:
Dissolving the drug and excipients in a suitable solvent, generally
water for injection (WFI).
Sterilizing the bulk solution by passing it through a 0.22 micron
bacteria-retentive filter.
Filling into individual sterile containers and partially stoppering
the containers under aseptic conditions.
Transporting the partially stoppered containers to the lyophilizer
and loading into the chamber under aseptic conditions.
Freezing the solution by placing the partially stoppered
containers on cooled shelves in a freeze-drying chamber or pre-
freezing in another chamber.
Applying a vacuum to the chamber and heating the shelves in
order to evaporate the water from the frozen state.
Complete stoppering of the vials usually by hydraulic or screw rod
stoppering mechanisms installed in the lyophilizers.

K.I.P.M. GIDA, GORAKHPUR, U.P.
There are many new parenteral products, including anti-infectives,
biotechnology derived products, and in-vitro diagnostics which are
manufactured as lyophilized products.
Additionally, inspections have disclosed potency, sterility and
stability problems associated with the manufacture and control of
lyophilized products.

K.I.P.M. GIDA, GORAKHPUR, U.P.
K.I.P.M. GIDA, GORAKHPUR, U.P.