Biotest

NEWSLETTER No. 65 2005 December

Comparison of Air Samplers for Environmental Monitoring regarding ISO 14698

by Dr. Jrgen Horn
Environmental monitoring is a must in the pharmaceutical industry. To follow current guidelines and recommendations it is necessary to validate the microbial airborne sampling system as described in the ISO 14698, Biocontamination Control. This worldwide accepted guideline is the only one, which describes in detail, the procedure for an acceptable validation. Regarding this guideline several commercial air sampling systems, such as sedimentation, impaction, slit-to-agar and several sieve samplers, plus filter sampler, centrifugal sampler and impinger are discussed. It can be shown that the centrifugal sampler system, RCS, is the only system which fulfils all requirements of ISO 14698.

Environmental Monitoring is a must in the pharmaceutical industry. The FDA guidelines for Aseptic Processing from September 2004 suggests that the air sampler should be evaluated based on collection efficiency, cleaning ability, ability to be sterilized and disruption of unidirectional air flow. Further devices have to be calibrated and used according to appropriate procedures. The EU GMP Guide, Annex 1, Manufacture of Sterile Medicinal Products makes a direct reference to the applicable EN / ISO Standards. A specific Standard for the purpose of Environmental Monitoring is ISO 14698 Biocontamination control. It is a valid worldwide published standard, and is mandatory in Europe as there is a reference in the EU GMP Guide. Even companies not producing in Europe or not exporting to Europe cover themselves by application of ISO 14698, as this represents the worldwide technical standard in this field. ISO 14698 is a useful checklist on what to do in Environmental Monitoring and how to validate equipment such as air samplers. The same validation procedures for air samplers may, of course, be used for validation of the associated media. An important point is that validation of air sampling is done by using real bioaerosols and real air sampling and not any surrogate testing involving direct inoculation of test strains on media.

There is evidence that direct inoculation may yield erroneous results. Willeke et al in 1995, published that slit to agar and sieve plate samplers recovered 0% of aerosols of gram negative organisms on McConkey, where direct inoculation yields normal growth. Herbig et al published that Hydrogen peroxide gassed polyethylene bags containing gelatine filters allowed Bacillus spores to grow when sprayed onto the filters. This is erroneously taken as evidence that air sampling in Hydrogen peroxide gassed Isolators is possible using gelatine filters. Several publications showed that real air sampling in atmospheres with residual hydrogen peroxide leads to inhibition of growth due to accumulation of hydrogen peroxide in the water phase. Real validations need real conditions as in the technical standard described in ISO 14698. If surrogate tests are correct this may be simply by chance and does not predict a true outcome with the next surrogate testing procedure. Therefore, different air sampling procedures with different air samplers were compared to existing international norms and guidelines. Sedimentation Passive air sampling using sedimentation plates is an adjunct to active air sampling. Settle plates can be used for qualitative or semi-quantitative air monitoring. The data generated by passive air sampling can be useful when considered in combination with results from active air samplers. Sedimentation has been widely used in Europe and is now part of the FDA guidelines. Validation should address desiccation caused by lengthy sampling periods and/or high air flows and, optimally, will include recovery from sedimenting real aerosols after real sampling conditions in aseptic and in uncontrolled environments. Impaction Different types of air samplers use this principle. Physics demands that particles in air have to be accelerated to be deposited from the air on to a surface. The smaller the particle, the higher the requirement for acceleration. On the other hand, high acceleration will yield higher impaction forces with all particles and may thereby destroy the viability of microorganisms on these particles. Therefore, ISO 14698 suggests not only methods for physical efficiency, but also for biological efficiency, where the recovery of vegetative cells of even sensitive microorganisms can be determined. Slit to Agar Sampler Cassella Slit to Agar Sampler, Mattson Garvin Slit to Agar Sampler Limited validation results to ISO like conditions (14698 was a draft in 1995) showing 0% recovery on selective McConkey and variable recovery rates on Tryptic Soy Agar depending on the adjustable impaction speed with the Mattson Garvin. Instruments are not portable and not easy to handle. Need power supply. The entire instruments are not sterilisable. Exhaust may cause air turbulence. Transport of agar plate/agar slides needs special precautions as they are open systems.

Sieve Samplers SAS Super 90, 100, 180 No published data about biological and physical efficiency according to ISO 14698. Published data has shown that physical efficiency at below 2 m is less than 10% of modern centrifugal samplers such as the RCS Plus or High Flow (fig.1 from ). Fungal spores may vary widely in concentration over the year and need to be detected reliably. Spores are mostly in the 1 2 m range. The total count is likely to be incorrect due to the omission of the variable amount of spores under 2 m. The entire instrument cannot be sterilised, only the sieve plate. Heavier than centrifugal samplers. Transport of agar plates needs special precautions as they are open systems. Unused petri-dishes in gastight packages in the isolator have to be discarded after opening. No hydrogen peroxide neutralising Tryptic Soy media available. Only an inflexible, expensive pressure adaptor for compressed gasses is offered. Pictures on the company website show environmental monitoring and images of microorganisms on selective media, such as for example gram negatives on McConkey, that are not supported by biological validation data: The results from Willeke, demonstrated cell wall destruction of gram negative microorganisms on impact and subsequent inhibition on McConkey, as bile salts enter through the cell wall hole. The images therefore, most likely, represent directly inoculated microorganisms and are misleading for air sampling. Yield in air sampling is statistically significantly lower than on other sieve plate samplers or centrifugal samplers. User has to validate the SAS according to ISO 14698. Fig.1 : Physical collection efficiencies for the sieve sampler system SAS Super 90 and centrifugal sampler system RCS Plus. Spores, which are mostly in the 1 2 m range, will be not detected by the sieve sampler system sufficiently. (published data from )

MAS 100 No published data about biological and physical efficiency according to ISO 14698. Exhaust air which leaves the instrument, may disturb air flow which could result in turbulence. Turbulence may aerosolize microorganisms from surfaces resulting in contamination. The entire instrument cannot be sterilised, only the sieve plates. Heavier than centrifugal samplers. Exchange of battery requires a screw driver and is cumbersome. Transport of agar plates needs special precautions as they are open systems. Unused petri-dishes in gastight packages in isolators have to be discarded. No hydrogen peroxide neutralising Tryptic Soy media. Very expensive additional instrumentation for compressed gases is required. User has to validate the MAS according to ISO 14698. M AIR T Although M AIR T uses special agar cassettes as a closed system there is no published data about biological and physical efficiency according to ISO 14698. Acceleration to a higher speed during the sampling process, yielding higher impact, has been seen to be critical for viability as outlined in ISO 14698. Exhaust air leaving the instrument, may disturb air flow resulting in turbulence. Turbulence may aerosolize microorganisms from surfaces resulting in contamination. The entire instrument cannot be sterilised, only the sieve plates. Heavier than centrifugal samplers. Transport of agar plates needs special precautions. Unused agar cassettes in isolators have to be discarded. Special hydrogen peroxide neutralising Tryptic Soy Media (Antiperoxide cassettes) are supplemented with 1% Pyruvate. 1% Pyruvate is documented to be inhibitory and gives lowers yield and is therefore not suitable for sensitive detection of low levels of contamination. Very expensive additional instrumentation for compressed gases is required. User has to validate the M AIR T according to ISO 14698. AIR IDEAL No published data about biological and physical efficiency according to ISO 14698. Plastic heads do not permit NIST traceability of air flow, have no documented serial number and are expensive. Plastic heads discolour and may change dimension after repeat autoclaving. Excessive turbulence as air is discharged to the sides of the instrument. Turbulence may aerosolize microorganisms from surfaces resulting in contamination. Does not have an optional data download capability to meet cGMP requirements for data traceability. Transport of agar plates needs special precautions as they are open systems. Unused petri-dishes in gastight packages in isolators have to be discarded. No hydrogen peroxide neutralising Tryptic Soy media. No possibility for testing compressed gases. Use has to validate AIR IDEAL according to ISO 14698. 4

Centrifugal samplers RCS Plus and High Flow Completely validated against the different draft versions and final version of ISO 14698. Physical sampling efficiency was found to be 100% for 3-4 m particles sizes and the lowest detection limit of 0.4 m, is sufficient to collect all important bioaerosols. Biological collection efficiencies were found to be acceptable and comparable to efficiencies obtained with the all glass impinger (fig. 2). Centrifugal sampling is the only system with an additionally validated media range according to ISO 14698. One special medium using patented hydrogen peroxide and disinfectant neutralising technology on Tryptic Soy is available (TCI-). Only one medium is required to monitor for anaerobes, aerobe bacteria and fungi, and is validated for all three uses (TCI-). Easy and safe transport of unused and used agar strips as each strip is housed in its own closed system. Unused, gassed agar strips in isolator may be saved for second/third gassing working cycle if left unopened. All air exposed head parts of the instrument are autoclaveable. The entire sampler may be sterilised by gassing with Hydrogen peroxide in an Isolator providing a completely sterile instrument. -sterile agar strips may be loaded in to the isolator after venting thereby providing a completely sterile air sampling unit. This completely sterile unit may be taken out of the isolator and used to monitor critical filling operations or media fills. Airflow is directed downwards at the side of the instruments, so this does not disturb unidirectional airflow. Inexpensive flexible adapter solution for compressed gases. Fig. 2: Biological collection efficiencies for centrifugal sampler RCS Plus and RCS High Flow. Biological collection efficiencies was found to be very good and comparable to efficiencies obtainable using the all glass impinger
100
90
78 82
75 70 94
96 80

75

67

67

65

50

48

25

0
RCS High Flow TC RCS High Flow TSM
E. coli ATCC10536

RCS Plus TSM

All Glass Impinger

C. albicans

M. luteus

Biological validation of RCS High Flow and RCS Plus

Filtration Samplers The Sartorius MD8 air sampler uses Gelatine in depth Filters with 50% water content. After sampling the filter is deposited on agar in a petri dish for recovery of microorganisms. While the water content of the gelatine filter is enough to accumulate residual hydrogen peroxide in isolators to toxic levels exceeding 100 ppm it is too low (agar has around 95% water content) to allow recovery of vegetative cells. The ASM Manual of Environmental Microbiology cites three publications which say filtration samplers have the disadvantage of viability loss of vegetative cells due to desiccation. Filtration sampling is used to monitor desiccation resistant forms such as fungal spores or bacterial endospores. Buddemeyer claims gelatine filters with 50% water completely eliminate problems with desiccation with agars without providing any validation data. Hydrogen peroxide gassing validation is a surrogate test only, using gelatine filters gassed in bags, then spraying with Bacillus with no real air sampling which revealed the hydrogen peroxide accumulation to toxic levels. This means no air sampling in isolators with residual peroxide is possible due to accumulation of toxic peroxide which cannot be neutralised in situ in the filter during sampling. For comparable consumable costs you should compare the agar media costs to the costly gelatine filters. The machine is able to samples viruses also, which need PCR techniques or cell culture for detection. The regulations, however, do not require viral monitoring. The instrument cannot be sterilized. Monitoring for compressed gases is unavailable. The user has to validate the MD8 according to ISO 14698 which appears unachievable for vegetative cells after reviewing published handbooks. Whereas filter sampling may be a reference method for sampling spores other methods have to be used in addition if vegetative cells have to be recovered. Impingement An all glass impinger draws air through a suction tube accelerated via a jet at the bottom. The bottom part is filled with a liquid which can be used for solid agar preparation or for filtration. Impingement is an effective method for various sizes of microorganisms. Accelerative speed may go up to nearly sonic speed, microbes can be disrupted or separated during sampling and start to grow in the collection fluid which may increase counts. Impingement is an effective method in the hands of experienced operators who know the system. Parameters have to be set and validated. Due to the variables the manufacturer is unable to provide such validation. In situ neutralisation of hydrogen peroxide and disinfectants, by additives like catalase in the collection fluid, is possible. No solution for compressed gasses is described.

Conclusion In comparison to all known commercial microbial airborne sampling systems, centrifugal sampling with the RCS Plus and RCS High Flow instruments is the only known sampling system which is completely validated according requirements of ISO 14698. It is the only sampling system that can demonstrate unrestricted use for monitoring of all relevant microorganisms in cleanrooms, isolators and compressed gases.

Literature: 1.) Shelby L. Stewart, Sergey A. Grinshpun, Klaus Willeke, Silva Terzieva, Vidmantus Ulevivias, Jean Donnelly Effect of Impact Stress on Microbiology Recovery on an Agar Surface Applied and Environmental Microbiology 1995, 1232-1239 2.) H. J. Bssler, KF Nieth, E. Herbig Tests on the Colony Growth Properties of Sartorias Gelatine Membrane Filters after Exposure to Vapour Phase Hydrogen Peroxide European Journal of Parenteral Sciences 1996, 55-57 Serge Ohresser, Stephanie Griveau, Christian Schann Validation of Microbial Recovery from Hydrogen Peroxide-Sterilized Air PDA Journal of Pharmaceutical Science and Technology 2004, 75-80 J. Horn, M. Backes, E.-C. Schepp Air Monitoring in Isolators Poster Q3, 102nd General Meeting of the American Society of Microbiology May 19th 23rd, 2002, Salt Lake City L. Soltis, J. Horn, M. Backes, E.-C. Schepp Comparison of 5 Airsamplers for General Airsampling and for Hydrogenperoxide Containing Environments 103rd General Meeting of the American Society for Microbiology May 18th 22nd, 2003, Washington R. M. Lee, P. A. Hartmann Optimal Pyruvate Concentration for the Recovery of Coliforms from Food and Water Journal of Ford Protection 1999, Vol. 52, 119-121 Y. Mizunoe, S. N. Wai, A. Takade, S.-I. Yoshida Restoration of Cultureability of Starvation Stressed and Low Temperature Stressed Escherichia Coli 0157 Cells by Using H2O2 Degrading Compounds. Arch. Microbiology 1999, Vo. 172, 63-67. Mark P. Buttner, Klaus Willeke, Sergey A. Grinshpun Sampling and Analysis of Airborne Microorganisms Manual of Environmental Microbiology, American Society for Microbiology 1997, 629640. Julie Buddemeyer Selecting and Active Airsampling Methodology Controlled Environments, July 2005

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