Explain the sterility test and what actions would you take when faced with a sterility test failure?
The test is applied to substances, preparations or articles which, according to the Pharmacopoeia, are required to be sterile. However, a satisfactory result only indicates that no contaminating micro-organism has been found in the sample examined in the conditions of the test. The test for sterility is carried out under aseptic conditions. The working conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls.
What media is used for the sterility test?
Media for the test may be prepared as described below, or equivalent commercial media may be used provided that they comply with the growth promotion test. The following culture media have been found to be suitable for the test for sterility. Fluid thioglycollate medium is primarily intended for the culture of anaerobic bacteria; however, it will also detect aerobic bacteria. Soya-bean casein digest medium is suitable for the culture of both fungi and aerobic bacteria.
What microorganisms are used for growth promotion testing?
Aerobic bacteria Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Anaerobic bacterium Clostridium sporogenes, Fungi Candida albicans, Aspergillus niger
What test methods can be used for sterility testing?
The test may be carried out using the technique of membrane filtration or by direct inoculation of the culture media with the product to be examined. Appropriate negative controls are included. The technique of membrane filtration is used whenever the nature of the product permits, that is, for filterable aqueous preparations, for alcoholic or oily preparations and for preparations miscible with or soluble in aqueous or oily solvents provided these solvents do not have an antimicrobial effect in the conditions of the test.
The test may be considered invalid only if one of 4 conditions are fulfilled, what are they?
a) the data of the microbiological monitoring of the sterility testing facility show a fault; b) a review of the testing procedure used during the test in question reveals a fault; c) microbial growth is found in the negative controls; d) after determination of the identity of the micro-organisms isolated from the test, the growth of this species or these species may be ascribed unequivocally to faults with respect to the material and/or the technique used in conducting the sterility test procedure.
What options do you have if your repeat testing fails?
If no evidence of microbial growth is found in the repeat test the product examined complies with the test for sterility. If microbial growth is found in the repeat test the product examined does not comply with the test for sterility. Discuss parametric release with respect to aseptic and terminally sterilised products
In one word describe the sterility test and then justify your description
Sterility testing is designed to demonstrate the presence or absence of extraneous viable contaminating microorganisms in biological parenterals designed for human use. The sterility test is a mandatory product release test. It is, however, statistically poor at detecting anything other than gross contamination
Stability OOS / OOT - representative of 12 months of special study
Obvious changes - excipient, API, process change? Packaging material look at original results and earlier timepoints - 0, 3, 6, 12
Extrapolate data to suggest when may drop OOS
Retest / retain - check increase in impurities
Pull stability forward to support extrapolation
Replenish stock then pull from market - market supply and stability conditions
Sterility test limitations
- The size of sample becomes a limitation above a batch size of 3200 units. Sampling theory suggests that, up to a batch size of 3200 units, the chance of detecting a contaminant rises with the size of the sample, although this effect tails off dramatically with batch sizes over 3200. Therefore, the regulatory authorities, the CFR and Pharmacopeia’s have limited the sample size to no more than 20 units per batch. A batch size of 10,000 units, with 20 sterility samples, has a one in forty chance of detecting a contaminant.
- Volume of samples are found in your monographs. For small volume parenteral, the entire contents should be collected, whereas for large volumes, only a proportion needs to be collected per the monograph. If only a proportion of the contents of a unit is tested, the probability of detection is reduced because of the way in which bacteria divide and potentially stick together.
- Patterns of contamination is a test limitation because contamination is not likely to be random within a batch, rendering random sampling basically useless. In the case of aseptically manufactured product, contamination is likely to have resulted from a “process event”. Therefore, it is usual practice to collect samples filled at the beginning, middle and end of the filling run, as well as after any significant intervention. This way, segregation exists, if needed to justify release of a partial batch.
- Unfortunately, just as in Environmental Monitoring, the Testers can be a source of contamination. It is becoming more common to use isolator technology for sterility testing as the controls are more stringent. Care has to be taken with the sterilization of the outside of the containers so that the test is not contaminated, but also that any contaminating microorganisms in the product are unaffected. The test requires excellent manipulative skills on the part of the operator, as well as superb aseptic technique in order to reduce contamination by the operator.
- Selectivity of the growth media used
- Selectivity of the growth conditions used
- The results are either ‘growth’ or ‘no growth’, as seen by the observer. Sufficient growth for the observer to detect, implies a microbial level of at least ten million bacteria per millilitre, and that the testing solution is sufficiently clear for the growth to be seen. Molds do not grow in this way, and it is hoped that the mycelium is sufficiently large and concentrated enough to be seen.
- If the tester is proven to be the source of contamination, then the test is invalid and may be repeated. If no obvious cause can be assigned to the contamination a repeat is allowed, but even if the second result is negative, the company must have a very strong case that the batch can be released. This is because testing conditions should be so good that contamination by the operator is very rare and so the contamination must have originated in the product.
Sterility Testing Methodology
- Capturing the bacteria - Microorganisms may be captured by membrane filtration or by direct inoculation into liquid media. Membrane filtration is the method of choice as this allows the removal of any anti-microbial effect of the solution and there is no limit on volume tested. It is not generally useful for non-aqueous products or suspensions. Direct inoculation is used for suspensions and other products which don’t lend themselves to filtration; however, problems may occur with direct inoculation due to anti-microbial effects which are difficult to counter
- Selection of correct growth media - Two media are recommended in the pharmacopoeia, tryptic soy broth, or a variant, and fluid thioglycolate broth. This is to ensure that all possible microorganisms are captured. The former is used for yeasts and molds and aerobic bacteria. The latter is used for bacteria and contains a reducing substance to create anaerobic conditions within the testing container.
- Selection of growth (incubation) conditions - The incubation temperature is 30°C for bacteria and 23°C for yeasts and molds. Incubation is for at least 14 days to allow the organisms time to adapt to the media used.
Contamination in the laboratory through people, equipment and environment are all possible sources of a positive sterility test. Because of the economic importance of the test and regulatory expectations of the testing conditions, companies are video recording the testing so as to be able to invalidate tests contaminated due to poor technique more easily.
What are the limitations of sterility testing?
The sterility test has severe limitations as a monitor of the sterility of a batch of product. These limitations are due to:
* Size of samples
* Volume of samples
* Patterns of contamination
* Testers as a source of contamination
* Selectivity of the growth media used
* Selectivity of the growth conditions used
That said; the sterility test is the only test available to the competent authority which may be required to examine a sample for sterility.
When can sterility testing results be deemed to be invalid?
EM data of sterility test facility shows a fault
Review of test procedure reveals a fault
Growth in negative controls
Identity and origin of containment can to attributed to faults in materials or technique used in conducting the sterility test procedure
