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Environmental Analysis
How Environmental Monitoring Equipment can Reduce the Likelihood of Contaminations
Environmental monitoring of cleanrooms and isolators has been subject to a significant rise in regulatory requirements in recent years, as contaminations can spoil pharmaceutical products, slow their time to market and even result in recalls. The resulting need for sound and effective environmental monitoring processes has made the selection and handling of monitoring equipment used for monitoring increasingly important. Pharmaceutical companies and their suppliers have therefore been working to assess and improve the products used to test surfaces and ambient air in critical environments.
A key concern of decision-makers in pharmaceutical production is that the instruments and disposables they use are validated according to established standards, in particular those of the FDA. As a matter of course, reputable suppliers that maintain a comprehensive range of environmental monitoring products make sure they fully validate their instruments for all relevant markets the world over, knowing that this gives them a competitive edge by saving their customers considerable time and costs. Many providers of culture media disposables perform studies to support the in-house validation that end-users need to perform.
Tony Ancrum, Associate Director Environmental / RTU / Hardware Service Merck Millipore Merck KGaA
Frankfurter Str. 250 64293 Darmstadt, Germany
Email:
tony.ancrum@merckgroup.com Tel : +49 (0) 6151 72 3740
Disposables which are intended for use in critical environments should be produced under at least equally controlled environmental conditions. Settle plates, contact plates and swabs should thus be filled in cleanrooms that meet high microbiological standards. They are usually double or triple packaged, depending on the class of cleanroom for which they are intended. This allows the outermost bag to be removed on advancing into the next higher class of cleanroom. For class A (ISO 5) cleanrooms and isolators, transparent triple bagging is
perfectly suited. To neutralise a certain amount of H2O2 which may accumulate on the culture media (e.g. as a consequence of active air sampling), Merck Millipore supplements all culture media designed for use in isolators.
Dosing the radiation Anne-Grit Klees, ph.D
(corresponding author) Product Manager
heipha Dr. Müller GmbH Lilienthalstrasse 16
69214 Eppelheim, Germany
Email:
anne-grit.klees@merckgroup.com Tel: + 49 (0) 62 21 -7 59 01 - 9 02
IET November / December 2012
Because the final packaged product cannot be sterilised outright, it is gamma-irradiated. However, the radiation dose must be finely tuned to ensure a contaminant-free product while also guaranteeing that the media does not degenerate and thus affect its capacity to grow microorganisms. In addition, the plastic packaging material must be qualified to withstand the radiation so that it does not become damaged easily when handled or shed particles that would compromise cleanroom safety. Over the years, Merck Millipore has found doses of between 9 and 20 kGy to provide just the right balance. Because aseptic filling is not a terminal sterilisation process, a Sterility Assurance Level (SAL) cannot be defined. However, supplier introduced colonies are only very rarely detected on gamma-irradiated media. When contaminants are indeed encountered these are usually not viable. In 2011, Merck Millipore irradiated some 14 million contact and settle plates. Not a single one was found to be contaminated by viable microorganisms as a consequence of the production process. Even so, as a precautionary measure, such culture media should be visually checked before use.
Minimising moisture loss
Passive air sampling generally involves the use of solid media settle plates. These usually contain Tryptic Soy Agar (TSA), a general purpose
medium, or Sabouraud Dextrose Agar (SDA), which is ideally suited for growing yeasts and molds. The plates are placed at certain locations within the test area. After removal of their lids they are exposed to the air for a pre-determined period of time. Following exposition, the plates are closed and incubated, whereupon any colonies found are counted and the organisms identified.
A critical issue with settle plates is that, while exposed, they lose water due to evaporation, leading to an increasingly dry skin on the agar surface. This can lead to poor growth of certain microbes on the media and thus to an underestimation of the proportion of these organisms in the air. Over a typical four-hour exposure period in a unidirectional airflow cabinet, TSA plates were found to lose up to 16% of their original weight. However, when these plates were inoculated with typical contaminants and incubated, all recovery rates exceeded 70%. 1
To
enable prolonged exposure and incubation periods while ensuring that the plates continue to deliver reliable results, settle plates should be poured to a particularly high filling level. However, the maximum length of exposure must be validated for each production line, taking into consideration air flow, temperature, relative air humidity and turbulences.
Dealing with residues
Surface sampling helps to determine the presence of viable microorganisms on surfaces in critical environments and on personnel. Typical surfaces that require such bioburden testing are laminar airflow workbenches, floors, gloved hands and difficult to reach areas such as the interior of tubing or filling needles.
To sample flat or convex surfaces contact plates are used. These plates are filled so that the solid media, usually TSA or SDA, protrudes. The agar surface is pressed against the surface to be tested, and any media residues are subsequently removed from the sampling site. After incubation the number of resulting colonies is determined.
By coming into contact with test surfaces, contact plates may pick up residues of sanitising agents used for cleaning or disinfecting cleanrooms. These substances, of which hundreds are used in pharmaceutical facilities around the world, may inhibit the growth of contaminants taken up on the same plate. To counteract the growth- inhibiting properties of such sanitisers, so-called neutralisers are added to the culture media. Appropriate combinations of lecithin, polysorbate 80, histidine and sodium thiosulphate can neutralise up to 80% of the sanitisers typically used. However, some of the remaining substances, such as polyhexamethylene biguanides, have proved very tricky. To cover a larger proportion of the sanitisers, Merck Millipore recently introduced a new neutralising mixture, designated Neutraliser A, in TSA contact plates. This has proved successful in neutralising all sanitisers tested so far, with recovery rates all above 50% compared to control plate counts. 2
1Sandle, T. (2011): Microbial recovery on settle plates in unidirectional airflow cabinets. Clean Air and Containment Review 6/2011.
2Hedderich, R. and Klees, A. (2012): Neutralization of Disinfectants by Culture Media used in Environmental Monitoring in Environmental Monitoring – A comprehensive Handbook, Volume 6: 159-180
www.envirotech-online.com
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