STERILITY TESTING

PHARMACEUTICAL MICROBIOLOGYSEMESTER V

Dr. Jigar Shah

Examples for which compliance with the test is required

• Ready made injections – including solutions and suspension, both aqueous and oily

• Solids for injection – including a number of materials from biological sources, e.g. heparin, hyaluronidase, and the antibiotics

• Eye drops, eye ointments and eye lotions• Water for injections• Immunological products – vaccines, antisera, and

diagnostic preparations• Implants• Catgut• Absorbable haemostatics

Every batch of final containers must be tested for sterility, except in special circumstances, issues are allowed until tests have been passed

Special circumstances are –

•Preparation required in emergency by medical practitioner and manufacturer has no stock, tests are under process and results take several days to come, so issue is allowed only when –

(a)Bulk from which the containers are filled has been tested and has passed, and

(b)Tests on samples from some of the filled containers are set up, examined daily and, if contamination is detected, the practitioner is notified at once.

•When the substance is so unstable that it loses appreciable activity if held until completion of the test. In this case the bulk test is waived. E.g. Liquid BCG vaccines – freeze dried form

Tests for Sterility

• The tests for sterility are intended for detecting the presence of viable forms of micro-organisms in or on pharmacopoeial preparations.

• The tests must be carried out under conditions designed to avoid accidental contamination of the product during the test.

• The working conditions in which the tests are performed should be monitored regularly by sampling the air and the surfaces of the working area and by carrying out control tests.

Tests for Sterility

• The tests are based upon the principle that if micro-organisms are placed in a medium which provides nutritive material and water, and kept at a favourable temperature, the organisms will grow and their presence can be indicated by a turbidity in the originally clear medium.

• Sterility tests can only show that organisms capable of growing in the test media under the selected conditions are absent from the fraction of the batch that has been tested

To obtain reliable results from sterility tests, it is necessary to –•Take sufficient samples

•To use sensitive culture media

•During testing, need to reduce accidental contamination to a minimum

•Careful supervision of operatives, regular air sampling and full scale runs using N. broth

SAMPLING OF LOTS• The sampling schedules set out the minimum

number of items to be sampled from each batch (lot) and the minimum quantity to be tested from each container, unless otherwise justified and authorised.

• The batch size and conditions of manufacture should be considered when planning a sampling regimen. It is assumed that the product has been manufactured under conditions designed to exclude contamination.

• A batch of product is defined as a homogeneous collection of sealed containers prepared in such a manner that the risk of contamination is the same for each of the units of the batch.

Important aspects of sampling are:1) Stages at which samples should be taken:- For heat sterilized products, the need for tests on the

final containers which is the only stage where they are relevant.

- For aseptically sterilized products, it is advisable to test the bulk from which the final containers will be filled. (i.e. testing at bulk as well final containers)

2) Selection of the samples:- Samples must be representative of the whole of the

bulk material and the lot of final containers.- For the bulk, material must be thoroughly mixed

before the sample is taken- For final containers, the samples must be selected at

random, but-

Organization / Pharmacopoeia

Testing stageBulk Final container

WHO √ √Therap Sub Regulations

X (except in emergency)

√

USP √ √ (IF bulk passed, fewer containers)

EP X √Int Pharm X √

Selection of the samples Continued…

(a) For terminally sterilised products the samples should be made up from units drawn from various sites throughout the steriliser load.

- Some of the units should be taken from that place in the load known to be least accessible to the sterilising agent and less satisfactory sterilizing conditions are believed to exist.

- Samples may be taken representatively from across each load.

Selection of the samples Continued…

b) For aseptically prepared products, the samples should be taken at regular intervals during the filling operations in such a way that every filling point is represented by an approximately equivalent number of samples.

- Further, the first and last items dispensed at each filling point and the first item filled after any machine break-down or change, any non-validated intervention or interruption, should be included amongst the samples.

- Also, an identical group of containers has been filled with the same product from the same bulk lot.

Selection of the samples Continued…

c) For articles sterilized by continuous process, such as radiation sterilization, samples are selected from the total number of similar items subjected to uniform sterilization durian an appropriate period, which the U.S.P. suggests should not exceed one day.

3) Sample Size:

Sampling Schedule - Minimum Number of Items to be tested from each Batch

Minimum Quantity of Product to be tested from each container in the Sample

Culture Media- Sterility test media must initiate and maintain the

vigorous growth of small numbers of –(a) The aerobic and anaerobic bacteria that can be

cultivated on artificial culture media including –(i) Common saprophytes,(ii)Pyogenic cocci and(iii)Spore bearing bacteria pathogenic to man(b) The lower fungi, i.e. yeasts and moulds responsible

for spoilage.- Separate media specifically designed for (i) aerobes,

(ii) anaerobes and (iii) the lower fungi.

Culture Media…

- Some joint media such as broths can support growth of aerobes and anaerobes, aerobes and lower fungi or even all 3 types of organism.

- The WHO report lists a number of media used for bacterial sterility testing and classifies them as follows:

- 1) For aerobes: a) peptone broth, b) glucose peptone broth

- 2) For anaerobes: a) Cooked-meat medium, b) Semi fluid meat medium, c) Liver broth

Culture Media…

- 3) For aerobes and anaerobes: a) Fluid thioglycollate medium, b) Soyabean-casein digest medium.

Culture Media…

- Distribute into suitable vessels, sterilize in an autoclave- Cool to 25º and store at 20º to 30º, avoiding excess of light

- If more than upper one third has acquired a pink color, medium may be restored once by reheating in a water-bath until pink color disappears and cooling rapidly.

- Medium more than 3 weeks old should not be used.- Use fluid thioglycollate medium by incubating it at 30º - 35º under aerobic conditions.

Culture Media…

- Use soyabean-casein digest medium by incubating it at 20º to 25º under aerobic conditions.

- The media used should comply with the following tests carried out before or in parallel with the test on the preparation being examined.

- Sterility: Incubate portions of the medium 1 at 30º - 35º and medium 2 at 20º - 25º for not less than 7 days; no growth of microorganisms occurs.

Culture Media…

- Growth Promotion test: Test each autoclaved load of each lot of the medium for its growth promoting qualities by separately inoculating duplicate test containers of each medium with about 100 viable micro-organisms of each of strains listed in table 1 and incubating according to the conditions specified.

- The test media are satisfactory if clear evidence of growth appears in all inoculated media containers within 7 days.

- If freshly prepared media are not used within 2 days, store them in dark, preferably at 2º to 25º.

Culture Media…

- Tests for bacteriostasis and fingistasis:- Prepare cultures of bacteria and fungi from the

strains of micro-organisms mentioned in table 1.- Inoculate sterility test media with about 100 viable

microorganisms using volumes of medium listed for liquids in table 2.

- Add specified portions of examine preparation to the containers already containing the inoculum and culture medium.

- Incubate the containers under the conditions listed in table 1 for not less than 7 days.

Culture Media…

- If preparation is bacteriostatic and/or fungistatic, use a neutralizing agent.

- Specified amount of preparation and larger volumes of medium used to determine the ratio of preparation to medium in which the growth of organisms is not adversely affected.

- If the specified amount of preparation is bacteriostatic in the medium, decrease amount of preparation to find the maximum amount that does not adversely affect the growth of test organism in medium.

- For liquids and suspensions, if this amount is less than 1 ml, increase the quantity of medium so that 1 ml is sufficiently diluted to prevent inhibition of growth.

Test Procedures- Method A – Membrane Filtration, - Method B – Direct Inoculation.- Method A – for a) an oil, b) an ointment that can be put into solution, c) a non bacteriostatic solid not readily soluble in the

culture medium and d) a soluble powder or a liquid that possesses inherent

bacteristatic and fungistatic properties.- For liquid products where the volume > 100ml or more

Test Procedures…

- Precautions: A laminar sterile airflow cabinet – to avoid accidental contamination.

- Working conditions monitored regularly by sampling the air and surfaces of the working area.

- General Procedures: Antimicrobial agent and needle attached to a syringe barrel filled with non-absorbent cotton.

- Method A: a) Apparatus: A suitable unit consists of a closed reservoir and a receptacle between which a properly supported membrane of appropriate porosity is placed. Nominal pore size not > 0.45µm and diameter of approx. 47mm.

Test Procedures…

- Diluting fluids: - Fluid A: Dissolve 1g of peptic digest of animal

tissue in water to make 1 liter, filter / centrifuge, adjust to pH 7.1±0.2, dispense into flasks in 100-ml quantities and sterilise at 121º for 20 minutes.

- Fluid B: If the test sample contains lecithin or oil, use fluid A to each liter of which has been added 1 ml of polysorbate 80, adjust to pH 7.1±0.2, dispense into flasks in 100-ml quantities and sterilise at 121º for 20 minutes.

Test Procedures…

- Method of test:1) For aqueous solution: - Prepare each membrane by aseptically transferring

fluid A + media + preparation being examined.- Alternatively, first, combined quantities of preparation

+ prescribed in the two media.- Draw the liquid rapidly through filter with aid of

vacuum- If the solution being examined has antimicrobial

properties, wash the membrane by filtering 100ml of sterile fluid A, three times, or quantities should be sufficient to allow growth of small inoculum of organisms.

Test Procedures…

- After filtration, aseptically remove membrane from holder, cut the membrane in half, immerse one half of membrane in 100ml of soyabean-casein digest medium and incubate at 20º to 25º for not < 7days.

- Similar, other membrane in FTM (30º to 35º).2) For liquids immiscible with aqueous vehicles and

suspensions: - Same as above but add sufficient quantity of fluid A

to achieve rapid filtration- Sometimes use sterile enzyme preparations such as

penicillinase or cellulase to aid in dissolving insoluble substances.

- If lecithin is there, use fluid B for diluting.

Test Procedures…

3) For oils and oily solutions: - Low viscous, filter without dilution th’gh dry memb.- Viscous oils – dilute as per needed using sterile

diluent like isopropyl myristate – filter by applying pressure or suction gradually.

- Wash the membrane at least 3 times sterile fluid B (100 ml) or to heat not > than 45º and use warm solutions for washing membrane.

4) For ointments and creams: - Dilute ointments in a fatty base and emulsions of the

w/o type to give a fluid concentration of 1%w/v, by heating (if necessary), not > than 40º with a suitable sterile diluent.

Test Procedures…

5) For soluble solids:- Dissolve substance in a suitable sterile solvent for each

medium and carry out test described under for aqueous solutions.

6) For sterile devices:- Aseptically pass a sufficient volume of fluid B through

each of not less than 20 devices so that not less than 100ml is recovered from each device.

- Collect fluids in sterile containers and filter the entire volume through membrane filter funnel, and follow the test as under for aqueous solutions.

Method B Direct Inoculation- Quantities of the sample to be used

Direct Inoculation…- Method of test1) For aqueous solutions and suspensions:Remove liquid from test containers with a sterile pipette

or with a sterile syringe or a needle

Aseptically transfer specified volume of the material from each container to a vessel of the culture medium

Mix the liquid with the medium but do not aerate excessively

Incubate the inoculated media for not less than 14 days, unless otherwise specified in the monograph.

Direct Inoculation…

- Incubate at 30º to 35º in case of fluid thioglycollate medium and at 20º to 25º in case of soyabean casein digest medium.

- If test sample renders the medium turbid, it is difficult to examine presence or absence of microbial growth by visual examination.

- Transfer suitable portions of the same medium to fresh vessels between the third and seventh days after test is started.

- Continue incubation of transfer vessels for not less than 7 additional days after the transfer and for a total of not less than 14 days.

Direct Inoculation…

2) For oils and oily solutions:- In media, add 0.1% w/v of (4 –tert -octylphenoxy)

polyethoxyethanol, 1% w/v of polysorbate 80 or other suitable emulsifying agent, in an appropriate conc.

- Oil containing cultures should be shaken gently each day.

3) For ointments:- Preparation is diluted ten fold in a sterile diluent such

as fluid B or any other aqueous vehicle capable of dispersing test material homogenously throughout the fluid mixture.

- Mix 10ml of fluid mixture with 80 ml of the medium and proceed same as aqueous solutions & suspension.

Direct Inoculation…

4) For solids:- Mix, and incubate same as 1)

5) For sterile devices:

- For articles of such size and shape as permit complete imersion in not more than 1000ml of culture medium test the intact article, using the appropriate media and incubate same as 1).

Direct Inoculation…

- For transfusion or infusion assemblies or where the size of an item makes immersion impracticable, flush the lumen of each of 20 units with a sufficient quantity of fluid thioglycollate medium and soyabean casein medium to yield a recovery of not less than 15ml of each medium, and incubate with not less than 100 ml of each of two media.

- Where the presence of the specimen being tested, interferes with test because of bacteriostatic action, rinse the article thoroughly with minimum amount of fluid A. Recover the rinsed fluid and test as described for sterile devices under method A.

Observation and Interpretation of Results- At interval during incubation period, and at its

conclusion, examine media for macroscopic evidence of microbial growth.

- If no evidence of growth is found, the preparation being examined passes the test for sterility.

- If evidence of microbial growth is found, reserve the containers, and it is demonstrated that causes not due to preparation, hence the tests for sterility are invalid and may be recommenced.

- Perform a retest using the same number of samples, volumes to be tested and the media – if no evidence of microbial growth is then found, the preparation being examined passes the test for sterility.

Observation and Interpretation of Results…

- If evidence of microbial growth is found, isolate and identify the organisms.

- If they are not readily distinguishable from those growing in the containers reserved in the first test, the preparation being examined fails the test for sterility.

- If they are readily distinguishable from those growing in the containers reserved in the first test, perform a second retest using the twice number of samples.

- If no evidence of microbial growth is found in second retest, the preparation being examined passes the test for sterility.

- If evidence of growth of any micro-organisms is found in second retest, the preparation being examined fails the test for sterility.

CONTROL TESTS• The results of sterility tests relied upon either

negative result (sterility of the sample) or positive result (contamination of the result)

• A negative result could also be due to –1) Inability of the broth to support microbial growth. The

causes of thisa) Inadequate formulationb) Accidental omission of an ingredientc) Overheating during preparation and sterilizationd) In anaerobic media, failure to boil off oxygen from

excessively oxygenated containers

CONTROL TESTS2) Inhibition of Contaminants by a substance added in

the test. This could be-a) The sample itselfb) A neutralising agent destroy the antibacterial effect

of an ingredient of the sample• A positive result could be caused by-1) Lack of sterility of medium2) Accidental contamination during testing

• Therefore, control tests are essential to show that these factors are most unlikely to be the explanation of the result.

NEGATIVE CONTROLS• Here, no growth is expected.(a) A container of medium from each batch used for the test is

incubated at the same time as the test containers• This control serves three purposes – (i) It confirms that the medium is sterile(ii) It shows the oxidation-reduction qualities of indicator

containing anaerobic media are satisfactory.(iii) It serves as a standard with which the corresponding test

container can be compared during and after incubation.(b) Any substance, other than the sample, added to a test tube

should be proved sterile by incubating suitable amounts in appropriate media.

POSITIVE CONTROLS (FERTILITY TESTS)• In this, growth is expected(a) The sensitivity of media must be confirmed to check that it

has not been affected by any error in preparation or storage.• Each type of medium is inoculated with an appropriate

organism and after incubation under suitable conditions is examined for growth.

• Europ Pharm suggests – S. aureus as aerobe- Plectridium sphenoides as anaerobe (spore containing

m.org.)- Candida albicans as the yeast

POSITIVE CONTROLS (FERTILITY TESTS)(b) The medium must be shown capable of supporting the

growth of small numbers of bacteria in the presence of sample.

• Organisms are added to containers in which there are exactly the same quantities of the same inclusions as in the tests

• The EP requires at least two tubes for each type of organism.• The bacteria are incubated at 30 to 32˚C for 7 days and the

yeast at 22 to 25˚C for 2 days.3) Controls to check working conditions and operators’

technique• Used at regular and frequent intervals to confirm that the risk

of accidental contamination is low

POSITIVE CONTROLS (FERTILITY TESTS)

a) General air sampling: High quality of the air supply to the room is being maintained

b) Air sampling at each working space: Settling plates at and near the working place help to detect poor technique and excessive movement

c) Dummy runs: Tests are performed with materials known to be sterile, e.g. ampoules or bottles of Water for Injections or Sodium Chloride have been sterilized for longer times and at higher temperature

• The operators should not be aware that the run is a control because this might encourage the exceptional care.

Modifications in filtration technique

• Davies and Fishburn pointed out several drawbacks in official method of sterility testing like

1) Tremendous efforts required to discover which medicaments inhibitory and to find suitable neutralizing agents

2) Loss of sensitivity of medium due to high dilution is used to overcome inhibitory effects

• Recommended a different technique where sterile bacteria proof asbestos cellulose filter pad was used. It is used to retain organisms on a pad and bactericide and inhibitory medicaments are filtered by washing with sterile solvent.

Modifications in filtration technique• Sykes and Hooper developed a new method in which pad

was replaced by a membrane filter.• Advantages- Membrane are so thin that retention of inhibitory substances

is very small- Quicker filtration- For oil, not need to dissolve them in an organic solvent first.

Oils pass through easily and quickly- The light peroleum may adversely affect the viability of

some organisms.

Advantages of Filtration Technique• Wide application – can be used for solutions, soluble solid,

insoluble solids, oils, ointments, syringes like articles• Very large volume can be tested with one pad• Small volume of broth is required than direct inoculation

into culture media• Apply to substances for which no satisfactory inactivators

are known• Some strong antibacterial agents like mercurials and

quaternary ammonium compounds, inactivated by treatment with appropriate neutralising solution.

• Subculturing is often eliminated

Disadvantages of Filtration Technique

• The possibility of adsorption of sufficient medicament • Highly skilled staff and exceptionally good aseptic

techniques are necessary. • A vertually sterile environment is essential• A room with laminar air flow and conventional asepsis

laboratory is needed.