Cleaner monitoring

The inside of an isolator is well protected from microbiological contamination by an air supply with a microbially retentive system (HEPA filtration) and reproducible decontamination. Microbiological contamination may occur with the loss of the integrity of gloves, i.e. with pinhole leaks or tears, mistakes in the transfer of materials or contaminated settle plates. In sterility testing, isolator samples for sterility testing (if unsterile) will develop as an aerosol, when samples are drawn for sterility testing. Because of these risks the EU guidelines for GMP requires microbiological monitoring of the air in isolators. The decontamination of isolators is usually performed by gassing with hydrogen peroxide and sometimes with peracetic acid, ozone or formaldehyde. These antimicrobial substances are all very water-soluble. If 1,000 litres of air (106ml) are drawn over a 18ml petridish this would result in a 56,000 fold accumulation of residues of these gases in the agar. Therefore, even very small amounts of 1ppm or less of these gases after the aeration phase could produce inhibitory concentrations after air sampling. Gelatine disposables with a total weight of 260mg and a 50% water content of 130mg do have a much higher accumulation of 7.7 x 106 fold of residual water soluble antimicrobial gases when 106ml of air is drawn through the gelatine disposable. Fig. 1 shows the concentration of hydrogen peroxide obtained in agar by sampling 1,000 litres of air in a Quality Control Isolator directly after the end of the aeration phase. Even after aeration and extended waiting overnight, residual hydrogen peroxide accumulated to inhibitory levels as shown in Fig. 1. Table 1 shows inhibition of Staphylococci by various small peroxide concentrations in the agar. The accumulation of residual peroxide to inhibitory concentration has been shown by M. Stafford1 at the PDA SE Chapter Isolator interest group in 2001 and by J. Horn2 at the PDA General Meeting in New Orleans 2002. The ISO DIS 14698-1,2 Biocontamination control calls for culture media to be of standard type and non-selective. Appropriate additives should be included to overcome or minimise residual antimicrobial activity. The USP calls for supplementation with additives to overcome or minimise the effects of sanitising agents or antibiotics. The EP calls for repeating the test under conditions without antimicrobial activity. The latter one is difficult to validate given the arithmetically enormous accumulation of residual antimicrobials in the air. The supplementation with additives already assures the neutralisation of antimicrobials during the sampling phase, thereby avoiding all potential damage to the airborne microorganisms. The ideal test would be the aerosolisation of microorganisms into the air with antimicrobial residues followed by air sampling simultaneous of the aerosolised microorganisms and the antimicrobials. As this is difficult to comprehend, air sampling in air with antimicrobial agents was performed by M. Stafford and was followed by immediate inoculation of test strains. In case of gelatine disposables, test organisms have to be sprayed immediately onto the gelatine disposables before placing them onto standard Tryptic Soy Agar. If exposed, gelatine disposables are placed first on agar than the relative high content of antimicrobial in the 0.130ml water phase is readily diluted in the over 17ml water content of a 18ml standard Tryptic Soy petridish resulting in a 130 fold dilution of the possible antimicrobial activity to possibly non-inhibitory levels. After gassing and aeration of an isolator with hydrogen peroxide, peracetic acid, ozone and formaldehyde, residual antimicrobial levels in the air of 1.0; 1.5; 1.0 and 1.25 ppm were obtained for the antimicrobials mentioned above. This was followed by air sampling of 1,000 litres of air with a RCS high flow air sampler using TCI-g Agar Strips able to neutralise high amounts of all of the above antimicrobials. This was compared to a MAS 100 airsampler using standard Tryptic Soy Agar petridishes without neutralising capacity and to a MD8 with non-neutralising gelatine disposables. Test strains were inoculated with a 10 – 100 cfu inoculum onto exposed agars directly after air sampling, and sprayed respectively onto gelatine disposables before placing them onto standard Tryptic Soy Agar. Control experiments sampling 1,000 litres of air under a HEPA filter with unidirectional flow without antimicrobials were performed to demonstrate growth properties of the system without anti microbial challenge. This was also the case with the anaerobe test strain Clostridium sporogenes which due to the supplementation not only showed an inhibited growth in anaerobic incubation, but also showed excellent growth as on the blood agar. However, C. sporogenes on standard Tryptic Soy Agar tends to have variable growth and occasional poor yields. As expected from the results of M. Stafford, the MAS 100 using standard unsupplemented Tryptic Soy Agar and the MD-8 with gelatine disposables, supported growth only with air containing no antimicrobial residues from the HEPA filter but not with air from the isolator with residues of antimicrobials. Growth on TCI-g occurred after exposure to all antimicrobials not only with hydrogen peroxide as shown by M. Stafford. TCI-g and RCS Systems like the RCS High Flow or the RCS Isolator are therefore ideally suited for monitoring any air from isolators or cleanrooms. There is no need to consider any residues of antimicrobials as they are all neutralised by the supplements in the Tryptic Soy Agar of the TCI-g Agar Strips. Owing to increased recovery of anaerobes, the TCI-g medium is also ideally suited for the required periodical screening for anaerobes. This medium fulfils all the requirements of ISO 14698 and USP under all possible conditions in any cleanroom and isolator.

References

1. M. Stafford The Effect of VHP on Testing Devices and Culture Media Used in Sterility Testing Isolators. PDA SE Chapter Isolator Interest Group, Raleigh, N. C., Oct. 2nd, 2001

2. Horn, J., Backes, M., Schepp, E.-C., Wenz, P. Comparative Air Sampling in Isolators using RCS High Flow with TCI-g against M-Air Tester with Anti-Peroxide Cassettes and MD-8 Air Scan with Gelatine filter. PDA Annual Meeting, Dec. 9th – 12th, 2002, New Orleans

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