Monitoring isolators and cleanrooms for biocontamination
Recent testing shows new findings from air sampling with antimicrobial agents
By Dr. Juergen Horn
The inside of an isolator is well protected from microbiological contamination by air supply combined with a microbially-retentive system—HEPA filtration—and reproducible decontamination. Microbiological contamination may occur, however, if there is a loss of integrity in the gloves (like pinhole leaks or tears), mistakes in transfer of materials or contaminated settle plates.
In the case of isolator sterility testing, drawn samples will develop an aerosol. Due to these risks, the European Union's (EU) guidelines for good manufacturing practices (GMP) require microbiological monitoring of the air in isolators.
Decontamination of isolators is usually performed by gassing with hydrogen peroxide, sometimes with peracetic acid, ozone or formaldehyde—antimicrobial substances that are all water-soluble. If 1,000 liters of air (106 ml) are drawn over an 18-ml petridish, this would result in a 56,000 fold accumulation of residue of the gases in the agar.
Therefore, even very small amounts—one part per million (ppm) or less—of these gases after the aeration phase could produce inhibitory concentrations after air sampling. Gelatin disposables with a total weight of 260 mg and 50-percent water content of 130 mg have a much higher accumulation of 7,7 x 106 fold of residual water-soluble anti-microbial gases when 106 ml of air is drawn through the gelatin disposable. (Residues in 106 ml of air are accumulated in 0.13 ml water of the gelatin. 106 ml:0.13=7,7 x 106.)
Figure 1 shows the concentration of hydrogen peroxide obtained in the agar by sampling 1,000 liters of air in a quality-control isolator directly after the aeration phase. Even after aeration and extended waiting, overnight residual hydrogen peroxide accumulated to inhibitory levels. Table 1 (page 27) 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 Southeast 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 must be included to overcome or minimize residual antimicrobial activity. The United States Pharmacopeial Convention (USP) calls for supplementation with additives to overcome or minimize the effects of sanitizing agents or antibiotics. The European Pharmacopeial (EP) calls for repeating the test under conditions without antimicrobial activity—which is difficult to validate given the arithmetically enormous accumulation of residual antimicrobials in the air.
The supplementation with additives assures neutralization of antimicrobials during the sampling phase, thereby avoiding all potential damage to airborne microorganisms. The ideal test would be the aerosolization of microorganisms into air with antimicrobial residues, followed by air sampling simultaneous of the aerosolized microorganisms and the antimicrobials.
Air sampling with antimicrobial agents
Since this is difficult to realize, air sampling with antimicrobial agents was performed by Stafford's team, followed by immediate inoculation of test strains.
In the case of gelatin disposables, test organisms must be sprayed immediately onto the disposables before placing them onto standard Tryptic Soy Agar. If exposed, gelatin disposables are placed first on agar; then, the relatively high content of antimicrobial in the 0,130-ml water phase is readily diluted in the over-17 ml water content of a 18-ml standard Tryptic Soy petridish. This results in a 130-fold dilution of the possible antimicrobial activity to possibly non-inhibitory levels.
The published test on colony growth properties of Sartorius gelatin disposables after exposure to vapor phase hydrogen peroxide demonstrated only gassing and aeration of gelatin disposables in polyethylene bags.
Concentrations of 5 to 20 ppm in the air inside the polyethylene bags were reached. Afterwards, the gelatin disposables were sprayed with 300 B. subtilis spores where no growth inhibition occurred. No active air sampling of 1,000 L air—which leads to accumulation of residues of water-soluble inhibitors, such as hydrogen peroxide and formaldehyde—was performed.
By sampling 1,000 L, or 1 million milliliters of air, through a 0.260-ml gelatin disposable with 0.13-ml water content, a considerable concentration of residual growth inhibitors occur in the gelatin's water phase.
The normal procedure for fulfilling USP, EP and FDA regulations would be sampling 1,000 L of air followed by a growth promotion test with 10 to 100 microorganisms. Further uninhibited growth of vegetative cells is expected of more resistant spores, which require more than 10 ppm direct contact of hydrogen peroxide for killing.
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 above antimicrobials. This was followed by air sampling of 1,000 L of air with a RCS High Flow air sampler using TCI-γ Agar Strips able to neutralize high amounts of all of the above antimicrobials.
This was compared to a MAS 100 (Merck) air sampler using standard Tryptic Soy Agar petridishes without neutralizing capacity, and to a MD8 (Sartorius) with non-neutralizing gelatin disposables. Test strains were inoculated with a 10 to 100 cfu inoculum onto exposed agars directly after sampling air, and sprayed onto gelatin disposables before placing them onto standard Tryptic Soy Agar.
Control experiments, sampling 1,000 L of air under a HEPA filter, with unidirectional flow without antimicrobials, were performed to demonstrate growth properties of the system without antimicrobial challenge.
Figure 2 (page 26) shows unconstrained growth of all test strains using TCI-γ under all conditions, with accumulated residues of all antimicrobial agents.
This was even 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 comparable growth on blood agar; whereas C. sporogenes on standard Tryptic Soy Agar tends toward variable growth and sometimes poor yields.
As expected from the results of Stafford and team, the MAS 100 using standard unsupplemented Tryptic Soy Agar and the MD-8 with gelatin 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-γ occurred after exposure to all antimicrobials, not only with hydrogen peroxide, as shown by Stafford. TCI-γ 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 neutralized by the supplements in the Tryptic Soy Agar of the TCI-γ Agar Strips. Due to increased recovery of anaerobes, the TCI-γ 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.
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-γ 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
3. Bässler, H. J., Nieth, K. F., Herbig, E., Tests on the Colony Growth Properties of Sartorius Gelatin Membrane Filters After Exposure to Vapour Phase Hydrogen Peroxide; Europ. J. Parenteral Sciences 1996, 1 (2), ppg 55-57
Dr. Juergen Horn is a senior scientist with Biotest AG. He can be reached at: firstname.lastname@example.org