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BYDr. Suman PattanayakAssociate ProfessorDepartment of Pharma Analysis & QA.

Vijaya Institute of Pharmaceutical Sciences for Women

B. Pharm IV year / II SemDRA, IPR & Patents

A sterility test is essentially a test which assesses whether a sterilized pharmaceutical or medical product is free from contaminating microorganisms by incubation of either the whole or a part of that product with a nutrient medium.

Destructive test and is of questionable suitability for testing large, expensive or delicate products or equipment.

Random sampling should be applied to products that have been processed and filled aseptically. With products sterilized in their final containers, samples should be taken from the potentially coolest

A sterility test is intended to demonstrate that no viable organisms are present, but failure to detect them could simply be a consequence of the use of unsuitable media or inappropriate cultural conditions.

To be certain that no organisms are present it would be necessary to use a universal culture medium suitable for the growth of any possible contaminant and to incubate the sample under an infinite variety of conditions.

Clearly, no such medium or combination of media are available and, in practice , only media capable of supporting non-fastidious bacteria, yeasts and moulds are employed.

The sterility test does have an important application in monitoring the microbiological quality of filter-sterilized, aseptically filled products and does offer a final check on terminally sterilized articles

There are three alternative methods available when conducting sterility tests.

1.The direct inoculation procedure involves introducing test samples directly into nutrient media. (European Pharmacopoeia)

1.Fluid thioglycollate medium or

Fluid mercaptoacetate Medium

2. Soyabean casein digest medium or

Tryptone soya broth

Composition: w/vPancreatic Digest of Casein 15.0 gYeast Extract (water-soluble) 5.0 g Glucose monohydrate/anhydrous 5.5 g/5.0 g Sodium chloride 2.5 g L- Cystine 0.5 g Sodium thioglycollate 0.5 g 0.1% Resazurin Sodium Solution (freshly prepared)

1.0 mL

Granulated Agar (moisture not more than 15%) 0.75 g Purified Water 1000 mL Polysorbate 80 5.0 mL

pH after sterilization (measured at room temperature): 7.1± 0.2 particularly suitable for the cultivation of anaerobic organisms (incubation temperature 30–35°C)

Medium 1 (Fluid Thioglycollate Medium)

Medium 2 (Soybean-Casein Digest Medium)

Composition w/vPancreatic Digest of Casein 17.0 g Pepsin Digest of Soybean Meal 3.0 gGlucose monohydrate/anhydrous 2.5 g /2.3 g Sodium chloride 5.0 g Di potassium hydrogen phosphate 2.5 gPurified Water 1000 mL Polysorbate 80- 5.0 mL

pH after sterilization (measured at room temperature): 7.3±0.2 which will support the growth of both aerobic bacteria (incubation temperature 30–35°C) and fungi (incubation temperature 20–25°C)

Recommended by most pharmacopoeias, the method by which the great majority of products are examined.

membrane filter (pore size 0.45 μm); any microorganism present being retained on the surface of the filter. After washing in situ, the filter is divided aseptically and portions are transferred to suitable culture media which are then incubated at the appropriate temperature for the required period of time.

Water-soluble solids can be dissolved in a suitable diluent and processed in this Way oil-soluble products may be dissolved in a suitable solvent, e.g. isopropyl myristate.

precaution against accidental contamination, product testing must be carried out under conditions of strict asepsis using, for example, a laminar airflow cabinet to provide a suitable environment.

3. A sensitive method for detecting low levels of contamination in intravenous infusion fluids involves the addition of a concentrated culture medium to the fluid in its original container, such that the resultant mixture is equivalent to single strength culture medium. In this way, sampling of the entire volume is achieved.

precaution against accidental contamination, product testing must be carried out under conditions of strict asepsis using, for example, a laminar airflow cabinet to provide a suitable environment.

sampling of air and surfaces and carrying out tests using samples' known’ to be sterile (negative controls).

samples that have been subjected to a very reliable sterilization process, e.g. radiation, or samples that have been subjected to a sterilization procedure more than once

In order to minimize the risk of introducing contaminants from the surroundings or from the operator during the test itself, isolators are often employed which physically separate the operator from the materials under test.

preservative, its activity must be nullified in some way during sterility testing so that an inhibitory action in preventing the growth of any contaminating microorganisms is overcome.

This is achieved by the following methods.Specific inactivationAn appropriate inactivating (neutralizing) agent is incorporated into the culture media. The inactivating agent must be non-toxic to microorganisms

Inhibitory agents Inactivating agentsPhenols, cresols None (dilution)Alcohols None (dilution)Parabens Dilution and TweenMercury compounds -SH compoundsQuaternary ammonium Lecithin + Lubrol W;compounds Lecithin + Tween (Letheen)

Benzylpenicillin † b-Lactamase from Bacillus cereus

Ampicillin

Other antibiotics† None (membrane filtration)Sulphonamides p-Aminobenzoic acid

*Neutralizing agents.

The antimicrobial agent is diluted in the culture medium to a level at which it ceases to have any activity, for example phenols, cresols and alcohols.

Membrane filtration (antibiotic-no specific in activators are available)

Basically, a solution of the product is filtered through a hydrophobic- edged membrane filter that will retain any contaminating microorganisms. The membrane is washed in situ to remove any traces of antibiotic adhering to the membrane and is then transferred to appropriate culture media.

Dilution

Positive controls

small numbers of suitable microorganisms to grow in media in the presence of the sample is measured.

The microorganism used for positive control tests with a product containing an antimicrobial agent must.

indicates a satisfactory inactivationTo be sensitive to that agent

In practice, a positive control (medium with added test sample) and a negative control (medium without it) are inoculated simultaneously-not necessary to do both.

All the controls may be conducted either before,or in parallel with, the test itself, providing that the same batches of media are used for both.

If the controls are carried out in parallel with the tests and one of the controls gives an unexpected result, the test for sterility may be declared invalid, and, when the problem is resolved, the test may be repeated.Specific casesthe sterility testing of parenteral products, ophthalmic and other non-injectable preparations, catgut and surgical sutures will be found in the European Pharmacopoeia. These procedures cannot conveniently be applied to items like surgical dressings and medical devices because they are too big.

In such cases the most convenient approach is to immerse the whole object in culture medium in a sterile flexible bag, but care must be taken to ensure that the liquid penetrates to all parts and surfaces of the materialSamplingA sterility test attempts to infer the state (sterile or non-sterile) of a batch from the results of an examination of part of a batch.p -the proportion of infected containers in a batch.q- the proportion of non-infected containers in a batchEqn……p + q = 1 or q = 1 - p.

Suppose also that a sample of two items is takenfrom a large batch containing 10% infected containers.The probability of a single item taken atrandom being infected is p = 0.1 (10% = 0.1),whereas the probability of such an item being noninfectedis given by q = 1 - p = 0.9. The probabilityof both items being infected is p2 = 0.01, and of bothitems being non-infected, q2 = (1– p)2 = 0.81. Theprobability of obtaining one infected item and onenon-infected item is 1 – (0.01 + 0.81) = 0.18 = 2pq.

In a sterility test involving a sample size of n containers,the probability p of obtaining n consecutive‘steriles’ is given by qn = (1 - p)n. Values for variouslevels of p (i.e. proportion of infected containers in abatch) with a constant sample size are given in

Re-testsUnder certain circumstances a sterility test may be repeated, but the only justification for repeating the test is unequivocal evidence that the first test was invalid; a re-test cannot be viewed as a second opportunity for the batch to pass when it has failed the first time. Circumstances that may justify a re-test would include, for example, failure of the air filtration system in the testing facility which might have permitted airborne contaminants to enter the product or media during testing, non-sterility of the media used for testing, or evidence that contamination arose during testing from the operating personnel or a source other than the sample under test.

The Sample size increases, the probability of the batch being passed as sterile decreases. the correct conclusion to be drawn from a satisfactory test result is that the batch has passed the sterility test not that the batchis sterile.

Tests for Sterility

Tests for sterility are carried out by two methods: (a) Membrane Filtration Method (b) Direct Transfer / Inoculation Method.

The Membrane Filtration Method is used as the method of choice wherever feasible.

Fluid Thioglycollate Medium (Medium 1) and Soybean-Casein Digest Medium (Medium 2) are the two media generally used for tests for sterility.

Media used in Sterility Testing

Method of Preparation: The pancreatic digest of casein, yeast extract, glucose, sodium chloride, L-cystine, agar and water are mixed in the proportions given above and heat until dissolved. Sodium thioglycollate is dissolved in the solution. The specified quantity of Polysorbate 80 is added if this ingredient is to be included. If necessary, 1 M sodium hydroxide or 1 M hydrochloric acid is added so that after the solution is sterilized its pH will be 7.1± 0.2. If the solution is not clear, mixture is heated to boiling and filtered while hot through moistened filter paper. Resazurin sodium solution is added and mix.

Method of Preparation: The ingredients are mixed in the proportions given above with slight warming. The solution is cooled to room temperature. The specified quantity of Polysorbate 80 is added if this ingredient is to be included. If necessary, sufficient 1 M sodium hydroxide or 1M hydrochloric acid so that after the solution is sterilized its pH will be 7.3± 0.2. If the solution is not clear it is filtered through moistened filter paper.

Method of Membrane Filtration Procedure

The filter should be a membrane filter disc of cellulose esters or other suitable plastics, having a nominal average pore diameter not exceeding 0.45 μm.

Cellulose nitrate filters are recommended for aqueous, oily and weakly alcoholic solutions and cellulose acetate filters for strongly alcoholic solutions. The entire unit should be sterilized by appropriate means with the membrane filter and sterile airways in place. The method of sterilization should not be deleterious to the membrane, eg, weaken it or change the nominal average pore diameter. The sterile airways should provide free access to the sterilizing agent. After sterilization, the apparatus should be free of leaks to the atmosphere except through the sterile airways.

Method of Direct Transfer Procedures Liquids and soluble or dispersible solids:Appropriate quantities of the preparation to be examined are added directly into Medium 1 and Medium 2. Approximately equal quantities of the preparation should be added to each vessel of medium. The test vessels of Medium 1 is incubated at 30 - 35°C and the vessels of Medium 2 is incubated at 20- 25°C. The volume of Medium 1 should be such that the air space above the medium in the container is minimized. The volume of Medium 2 should be such that sufficient air space is left above the medium to provide conditions that permit the growth of obligate aerobes. Unless otherwise prescribed, in no case should the volume of material under test be greater than 10% of the volume of the medium alone, i.e, 90% medium and 10% product. If a large volume of product is to be tested it may be preferable to use concentrated media, prepared so as to take the subsequent dilution into account.

Where appropriate the concentrated medium may be added directly to the product in its container. Wherever possible solid articles such as devices should be tested by immersion in or filling with culture media. Immerse all parts of each article in sufficient medium contained in one vessel to completely cover all parts. The volume of Medium 1 should be such that the air space above the medium in the container is minimized. The volume of Medium 2 should be such that sufficient air space is left above the medium to provide conditions that permit the growth of obligate aerobes. Place half the articles into Medium 1 and the remaining half into Medium 2. Incubate the test vessels of Medium 1 at 30 - 35°C and the vessels of Medium 2 at 20 - 25°C.

Ointments and oily preparations: Ointments and oily preparations may be tested by the method of Direct Transfer if testing by the method of Membrane Filtration is not feasible, i.e. when a suitable solvent is not available Incubation and examination of sterility tests: All test vessels of Medium 1 are incubated at 30 - 35°C. The vessels of Medium 2 are incubated at 20 - 25°C. All test and control vessels, other than the subcultured vessels referred to below, must be incubated for at least 14 days unless microbial contamination is detected at an earlier time. If turbidity, precipitate, or other evidence of microbial growth during incubation is seen: the suspected growth is examined microscopically by Gram stain; attempts are made to grow single colonies using appropriate microbiological methods; colonies of each type of micro-organism present are examined for colonial morphology and cellular morphology by Gram stain; attempts are made to identify the isolates, as far as the genus, and preferably species.

Interpretation of the test results: If microbial growth is not evident in any of the vessels inoculated with the product, the sample tested complies with the test for sterility, if microbial growth is evident the product does not comply with the test for sterility unless it can be clearly demonstrated that the test was invalid for causes unrelated to the product being examined. If the test is declared to be invalid it may be repeated with the same number of units as in the original test. If there is no evidence of growth in any vessels inoculated with the product during the repeat test the product passes the test for sterility. This interpretation applies even if growth occurs in negative product control vessels. If there is evidence of growth in the test vessels the product fails the test for sterility. Further testing is not permitted under any circumstances

Evaluation of Sterilization Method Sterile products possess several unique properties, such as freedom from microorganism, pyrogens, particulates and high standards of purity and quality. This ultimate goal in the manufacture of sterile products can be attained by evaluation of sterilization procedure. The sterilization processes are likely to be subjected to the most detailed and complex validation procedures. The judgment of sterility has relied on official sterility test. A validated manufacturing procedure is one which has been proved to do what it purports to do. The proof of evaluation is obtained through the collection and evaluation of data, preferably beginning, from the process development phase and continuing through the production phase. Evaluation of processing includes equipments, process, personnel, material etc.

The principle involve in the evaluation of sterilization process are:

i. To build sterility into product. ii. Perform a maximum level of probability. iii. Establish specification and performance characteristic. iv. To provide greater assurance of support of the result. v. Specific methodology, process and equipment. vi. Final product testing using validated analytical method and vii. Verification, calibration and maintenance of equipments used in the processes.

Evaluation of sterilization methods are done to ensure that the product produce by design process should be of best quality. The process control and finished product testing alone are not sufficient to assure product quality. When testing a specified portion of the total product and if the specified portion passes the test of sterility, it cannot assure that the total product is sterile. Evaluation of sterilization methods provides a high degree of assurance which indicates a specific process will consistently produce a product that will meets it predetermined specifications and quality assurance. So this action proves that any procedure, process, equipments, material activity or system actually leads to the expected result and produce quality product. This concept of evaluation has been expended to encompass a wide range of activities from analytical methods used for quality control of drug substance and drug products.