QUALITY CONTROL AND QUALITY ASSURANCE

OF IMMUNOLOGICALS
ONE MONTH TRAINING REPORT
SUBMITTED AT:
INSTITUTE OF INTEGRATED AND HONOURS STUDIES
(KURUKSHETRA UNIVERSITY, KURUKSHETRA)
DEPARTMEINT OF BIOTECHNOLOGY

SUBMITTED BY:
KHILENDER SHARMA
ROLL NO:201802
EXAM ROLL NO: 201181603
MASTER OF SCIENCE(BIO-TECHNOLOGY) 5YEAR
SEMESTER(VIII)

TRAINING AT:
CENTRAL RESEARCH INSTITUTE, KASAULI
SUPERVISION BY: Dr. Romica Latawa
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DECLARATION

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 ACKNOWLEDGEMENT

Primarily I would thank Almighty for being able to complete this training session with
success. The internship opportunity I had with CRI, Kasauli was a great chance for
learning and professional development. Therefore, I consider myself as a very lucky
individual as I was provided with an opportunity to be a part of it. I am also greatful for
having a chance to meet so many wonderful people and professionals who led me through
this training period. I express my deepest thanks to Dr. Romica Latawa(overall-in-charge)
and Mr. Ashok Gauri for taking part in useful decision & giving me necessary advices and
guidance and arranged all facilities to make my work easier. I choose this moment to
acknowledge their contribution greatfully. I am thankful to all the teachers of IIHS,KUK
and all the staff members of CRI, Kasauli as without their support and blessings, this
report would not have been possible and I am also thankful to my parents, my elders and
my friends who support me always.

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 TOPIC

STERILITY TESTING
OF
PHARMACEUTICAL PRODUCTS

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 INTRODUCTION

 Sterility testing is a GMP(Goods Manufacturing Practices) microbiology testing

requirement used to confirm sterile products do not contain any viable
microorganisms before release and patient administration.
 Sterility testing must be accurate as possible, due to their importance for medical
devices, pharmaceutical products, formulation and other injectables that claim to be
sterile or free from viable microorganism,
 Test for sterility is the qualitative test to be performed on product who gone through
sterilization process to ensure that product are actually sterile and free from viable
microorganism.
 Pharmaceutical products need to be sterilize like Vaccine, injection, syringes,
ophthalmic preparations and surgical instrument etc.
 It is the process of validating the sterilization process is done properly or not while
making sterile preparation.
 Sterility testing must be carried out in aseptic area where chance of unwanted
contamination is zero.

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 TEST OF STERILITY
1. The test of sterility is based on the principle that if microorganism are placed in a
medium which provides nutritive material and water and kept at a favourable
temperature, the microorganism will grow and their presence can be indicated by
turbidity in the originally clear medium.

2. The probability of detecting viable microorganism in the tests for sterility ,

increases with the number present in the given amount of preparations being
examined and according to the species of microorganism present.

3. The external surface of ampoules and closure of vials and bottles should be cleaned
with a suitable antimicrobial agent. If the contents are packed in container under
vaccum, sterile air should be admitted by using suitable sterile devices.

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 No. of items recommended to be tested in the batch as(as per I.P)
No. Preparations No. of containers in a batch Minimum no. of
containers are
recommended to be
tested
1. Parenteral preparations (a). Not more than 100 containers (a) 10% or 4
containers(whichever is
(b). More than 100 but not more than 500 containers greater)
(b). 10 containers
(c). More than 500 containers (c). 2% or 20
container(whichever is less)
(d). For large volume parenterals
(d). 2% or 10
containers(whichever is
less or specified).
2. Ophthalmic and other non- (a) Not more than 200 containers (a) 5% or 2
parenteral preparations containers(whichever is
greater)

(b). More than 200 containers (b) 10 containers

3. Surgical dressings and (a). Catgut, surgical sutures and other sterile medical (a) 2% or 5
Devices devices for use packages(whichever is
greater, max. 20 pack)
(b).Not more than 100 packages (b). 10% or 4
packages(whichever is
(c).More than 100 but not more than 500 packages greater)
(c). 10 packages
(d). More than 500 packages (d). 2% or 20
packages(whichever is less)

TABLE: 1.0

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 CULTURE MEDIA

Media used for sterility test :

1. Fluid thioglycolate medium(FTM):
It is used with clear fluid products. FTM is primarily intended
for used for the culture of anaerobic bacteria; however it will also detect aerobic bacteria.
If more than the upper one-third of the medium has acquired a pink color, the medium
may be restored once by reheating on a water bath or in free flowing steam until the pink
color disappears and cooling rapidly, taking care to prevent the introduction of non-sterile
air into the container. When ready for use, not more than the upper one-tenth of the
medium should have pink color.

2. Alternative thioglycolate medium(ATM):

It is used with turbid or viscid products and for devices having
tubes with small lumine. ATM is incubated in such way that, to assure anaerobic
condition.

3. Soyabean casein digest medium(SCDM):

SCDM is suitable for the culture of both fungi and aerobic.

Note: Medium 1 and 2 are adjusted at pH 7.1, Medium 3 is adjusted at pH 7.3 and
sterilized by autoclaving at 121 Degree(c) for 20 minutes. Anaerobic conditions are
required for growth of some microbial species.

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Table(1.1) Composition of media used for sterility testing:
Components FTM ATM SCDM
L-cystine 0.5 gm 0.5 gm -
Sodium chloride 2.5gm 2.5gm 5.0 gm
Dextrose monohydrated/anhydrous 5.5gm/5.0gm 5.5gm/5.0gm 2.5gm/2.3gm
Yeast extract 5.0gm 5.0gm -
(water insoluble )
Pancreatic digest of casein 15.0gm 15.0gm 17.0gm
Sodium thioglycolate/thioglycolic 0.5gm/0.3ml 0.5gm/0.3ml -
acid
Papaic digest of soyabean meal - - 3.0 gm
Dipotassium hydrogen phosphate - - 2.5gm
Resazurin sodium soluble(0.1%) 1.0 ml - -
Distilled water to make 1000 ml 1000 ml 1000 ml
pH of the medium after 7.1 7.1 7.3
sterilization

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 Suitability of media

The media used for sterility testing should comply the following test:

(1). Sterility of media:

Incubate the portions of Fluid thioglycolate medium/Alternative
thioglycolate medium at 30-35°C and Soyabean casein digest media at 20-25°C for about
14 days and observe the presence of the growth of microorganisms in a sterile media.
(2). Growth promotion test(GPT):
Test each autoclaved load of media for its growth promoting qualities by
separately inoculating duplicate test containers of each medium with about 100 viable
microorganisms of each of the strains. The media is suitable if it promote the growth of
microorganism in all inoculated media containers within specified time. The sterility test
is considered invalid if the test medium shows inadequate growth response.

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  Table(1.2) List of test microorganism for growth promotion test:

MEDIUM TEST TEMPERATURE DURATION CONDITION

MICROORGNISM (°C) (DAYS)
Fluid 1.Clostridium 30 to 35 3 Anaerobic
thioglycolate sporogenes
2.Staphylococcus 30 to 35 3 Aerobic
aureus
3.Pseudomonas 30 to 35 3 aerobic
aeruginosa
Alternative 1.Bacteroides 30 to 35 3 Anaerobic
thioglycolate vulgatus
2.C.sporogenes 30 to 35 3 Anaerobic
3.Bacillus subtilis 30 to 35 3 Anaerobic
Soyabean- 1.Aspergillus 20 to 25 5 Aerobic
casein digest brasiliensis
2.Candida albicans 20 to 25 5 Aerobic
3.Bacillus subtilis 30 to 35 3 Aerobic

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METHODS OF STERILITY TESTING:
1. Method A: Membrane filtration
2. Method B: Direct inoculation

1.Method A: Membrane filtration:

This method is preferred when the substances being examined is (a)
an oil,(b) ointment that can be put into a solution (c) a non-bacteriostatic solid not readily
soluble the culture medium, and (d) a soluble powder or a liquid that possesses inhere
bacteriostatic and fungistatic properties. For liquid products where the volume in the
container is 100ml or more, only Method ‘A’ should be employed.

 This methods need good skills and special knowledge.

 It also calls for routine use of positive and negative controls. A positive control is
small number not more than 100 CFU of microorganism specified in separate
portion of each medium.
Apparatus:
 Reservoir and Container to collect apparatus
 Membrane filters: Pore size 0.45µm, Diameter:50mm, Flow rate: 55-75ml of
water/minute at pressure of 70 mm of mercury.
 Cellulose nitrate filters are used for aqueous, oily and weak alcoholic solutions.
 Cellulose acetate filters are used for strongly alcoholic solutions.
Note: Complete unit should be free from microorganisms including membranes.

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Dilutions fluid:
1. Fluid A: Dissolve 1 gm of peptic digest of animal tissue (bacteriological peptone)
in 1 litre of water. Filter and adjust the pH to 7.1±0.2. Dispense fluid into
flasks(100ml) and sterilize at 121°C for 20 minutes.
2. Fluid B: If the test sample contains lecithin or oil, use Fluid A, to each litre of
which has been added 1 ml of polysorbate 80. Adjust the pH 7.1±0.2 and sterilize
at 121°C for 20 minutes in autoclave.

Quantities of sample to be used:

1. For parenteral preparations:

Whenever possible use the whole contents of the container but nit less then the
quantities, if necessary , dilute to about 100 ml with suitable diluent(e.g fluid A).

2. For ophthalmic and non-injectable preparations:

Take an amount within the range prescribed, if necessary, using the contents of
more than one container and mix thoroughly. For each medium use the amount
specified, taken from the mixed sample.

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Table(1.4) Quantities of injectable preparations used for sterility testing

Preparation Quantity in each container of Minimum quantity to be used for

injectable preparation each culture medium
(a)Liquids Less than 1 ml. Total contents of a container.
Half the contents of a container
1-40 ml. but not less than 1 ml.
20 ml

More than 40 ml but not 10% of the contents of a

more than 100 ml. container but not less than 20 ml
More than 100 ml

(b) Antibiotic liquid - 1 ml.

(c) Other soluble preparations - The whole contents of each

container to provide not less than
(d) Insoluble preparations, 200 mg.
creams and ointments. - The whole contents of each
container to provide not less than
200 mg.
(e) Solids Less than 50 mg Total contents.
50 mg or more but less Half contents but not less than
than 300 mg 50 mg
300 mg to 5g 150 mg
More than 5g 500 mg

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 Table(1.5) Quantities of ophthalmic and non-injectable preparations for sterility
testing

S.No. Formulations Quantity to be Quantity to be

mixed used for each
culture medium
1. Ophthalmic solutions, other non- 10 to 100 ml 5 to 10 ml
injectable liquid preparations.

2. Insoluble preparations to be 1 to 10 gm 0.5 to 1 gm

suspended or emulsified(ointments
and creams), preparations soluble
in water and solvents.

3. Absorbent cotton - Not less than 1 gm

Method of test:
(a) For aqueous solutions:
Transfer aseptically the quantities of the preparation being examined
prescribed in the two media onto the membrane. Draw the liquid rapidly through the filter
with the aid of vacuum. The membrane is removed aseptically, cut into two part, one part
is then immersed in 100 ml of soyabean casein digest media and incubated to 20 to 25°C
for atleast 14 days. Similarly, other part immersed in 100 ml of liquid thiglycollate
medium and incubated at 30 to 35°C for atleast 14 days.
(b) For liquid immiscible with aqueous suspensions:
Carried out the test described under, for aqueous solutions but add a
sufficient quantity of fluid ‘A’ to the pooled sample to achieved rapid filtration.
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(c) For oils and oily solutions:
Filter oils or oily solutions pf sufficiently low viscocity without
dilutions through a dry membrane. Dilute viscous oils are necessary with suitable diluent.
(d) For ointments and creams:
Dilute ointments in fatty base and emulsions of the water-in-oil type to
give a fluid concentration of 1%w/v by heating, if necessary, to not more than 40°C with a
suitable sterile diluent such as isopropyl myristate. If ointments and oils are insoluble in
isopropyl myristate then use method of direct inoculation.
(e) For antibiotic solids, bulks, and blends:
Aseptically remove a sufficient quantity solids from the appropriate
amount of containers prescribed in, mix obtain a composite sample, equivalent to about 6
g of solid, and transfer to sterile flask. Dissolve in about 200 ml of fluid A, and mix.

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