Growth Support and Sterility Controls

The first responsibility in any microbiological exercise that is expected to produce "no growth" results, and for which no growth is the favorable condition, is to ensure that the medium is capable of supporting growth. Maintenance of aseptic clean rooms must ensure that only materials that can be safely presumed to be sterile should be permitted entry.

Growth supportiveness of the media should be verified before use. It should also be checked after it has been in contact with the filling equipment and the product containers. This is to ensure that traces of product, antibiotic, detergent, disinfectant, etc. in antimicrobial concentrations have not been passed into the media from any one of these or other production-related sources.

Sterility of media is best verified by preincubation. This is best done outside of aseptic areas. The prospect of having, for example, 50 litres of microbiological media becoming heavily contaminated through each hour of preincubation within an aseptic filling room should be avoided.

When prepared media are autoclaved for media fills a sample is usually aseptically withdrawn for growth support checks. These can then be simultaneously conducted with preincubation of the media in a laboratory incubator to verify its sterility. Both results are obtainable before the media need be taken into the clean room.

If the only sterilization of media is on-line filtration, an aseptic sample may be taken from the sterile holding vessel for growth support checks. The media should properly be held until these results are obtained, but the risk of contaminating the filling room by preincubation therein is something some companies prefer to avoid. In such a case a risk may have to be taken to fill media for which there is no prior supportive evidence of either sterility or of its ability to support growth. There may be additional risks of contaminating the medium by moving it out of the aseptic filling room for preincubation and back in again for filling. These may have to be tolerated.

A second growth support check should be done on filled units. In principle these may be taken and tested at the beginning or end of the incubation of the media fill. In terms of managing and scheduling it would be best to take them at the beginning. This eliminates taking the whole of the media fill incubation period plus some days before ascertaining that the media was satisfactory. However, in response to a well-known but informal regulatory view that this practice may result in taking the very units that might be contaminated out of the trial, growth support on filled vials is most usually done at the end of the incubation period of the complete set of filled units.

The medium that is universally used for media fills is TSB, because it is used in the pharmacopoeial test for sterility. This is the usual point of reference for the microorganisms and the conditions that should be applied to growth support checks.

Table 3.2 shows the current United States Pharmacopeia (USP) and European Pharmacopoeia (PhEur) requirements for TSB medium growth support when used for the test for sterility; however, the control cultures applying to TSB are cited only for the 20-25°°C incubation condition. Media fills may be incubated at two temperatures, 20-25°C and 30-35°C. It is therefore good sense to replicate the growth support test across the two temperature ranges. All pharmacopoeially recommended microorganisms listed in Table 3.2 should grow profusely in both temperature ranges with seven days' incubation from an initial inoculum of 10 to 100 cfu. Separate media samples should be inoculated with each culture.

In addition to the pharmacopoeial media growth support control cultures, many regulatory agencies insist on at least one isolate from the manufacturing environment being used as a media control.

The logic is that if the TSB is intended to recover microorganisms inhabiting the manufacturing environment, it should be shown to have the ability to support the

Table 3.2 Microorganisms Required for Sterility Test (Media Growth Support Checks in USP XXVI [2003] and PhEur 4th edition [2002] (ATCC Numbers Only are Shown for Convenience)

Medium USP XXVI (2003) PhEur 4th edition (2002)

TSB at 20-25°C Bacillus subtilis Bacillus subtilis

Candida albicans Candida albicans

(ATCC 10231) (ATCC 2091)

Staphylococcus aureus (ATCC 6538P)

growth of those environmental microorganisms. Local microorganisms could be frail, injured, disinfectant-damaged, etc. and therefore could be more difficult to recover in TSB than the pampered, well-nourished subcultures from the culture collection.

Conversely, the local environmental isolates used for media controls will have most likely been maintained in a local culture collection for several months at least, and will probably have recovered from any physiological damage associated with stressful local conditions. It may seem cynical, but it is probably true to state that few microbiologists would choose a local environmental isolate for media fill control because it is difficult to grow or is slow growing in laboratory culture.

Irrespective of these doubts and compromises, local environmental isolates are recommended for media control. The chosen isolate should be changed periodically so that it can be related to the current rather than the historical microflora of the manufacturing environment.

Where antibiotic filling processes are simulated, ensure that at least one of the growth support control cultures is sensitive to the antibiotic, to provide the most sensitive information on the success of the clean-up process.

The preparation of control cultures should be clearly specified in laboratory documentation, and records of subculturing maintained. The FDA prefers that working control cultures be separated by no more than five generations from their national or international culture collection origins. This limits the potential for mutation.

Low inocula must be used in media control, because the intention is to recover microorganisms when they are present only in low numbers. The pharmacopoeias interpret low numbers to mean between 10 and 100 cfu per inoculum. The reference condition for this is surface culture on Tryptone Soy Agar incubated at 30-35°C for at least 48 hours.

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