Monitoring isolator air

By definition, the inside of an isolator is well protected from microbiological contamination.

As a PDA technical report states: "An isolator is sealed or is supplied with air through a microbially retentive filtration system (HEPA minimum) and may be reproducibly decontaminated. When closed, it uses only decontaminated (where necessary) interfaces or Rapid Transfer Ports (RTPs) for material transfer. When open, it allows for the ingress and/or egress of materials through defined openings that have been designed and validated to preclude the transfer of contamination. It can be used for aseptic processing activities, for containment of potent compounds, or simultaneously for both asepsis and containment."¹ Why, then, is it mandatory to carry out micro-organism monitoring in isolators? Even though the air supply, the transfer of objects into and out of the cleanroom area as well as sterilisation are sufficiently controlled, the system-related risk remains that micro-organisms inadvertently carried into the clean area can propagate uncontrollably, thus endangering the product. The PIC/S Recommendation on "Isolators used for Aseptic Processing and Sterility Testing", dated June 2002² lists three major causes of microbiological contamination based on past experience – loss of integrity of gloves, mistakes in transfer of materials into the system and contaminated settle plates – whereas the USP 25, Chapter 1116, gives pinhole leaks or tears in gloves as major contamination risk for isolators.³ Because these risks of contamination cannot be eliminated, the EU Guidelines for GMP⁴ and the British Isolator Guidelines⁵ require microbiological monitoring of the air in isolators.

Monitoring methods According to Thorogood,⁶ for active airborne micro-organism monitoring, methods should be used which avoid disturbance of the air flow pattern and the introduction of growth media into the critical work zone. Furthermore, it is recommended to withdraw an air sample isokinetically from the interior of the isolator. Impaction or filtration methods should then be applied from outside the isolator. The PDA states in its Technical Report No. 341 that active air sampling should always be done in a manner that does not disrupt air flow and requires minimal handling steps to avoid the risk of false positive findings. Both cases emphasise active air sampling, but what are the benefits of this method of monitoring? Compared to passive monitoring with, for example, settle plates, active microbiological sampling has the following advantages: • The sampling head can be positioned in the vicinity of the critical zone – for example, near the point of fill • A large, defined and calibratable volume of air can be sampled • Sampling can be carried out at the same rate as the laminar air flow in an isolator • This method does not require leaving nutrient medium exposed to air flow inside the isolator for any length of time The USP 25 gives a list in Chapter 1116 of some of the air samplers available on the market, including the "Gelatin Filter Sampler". This section of USP 25 stresses that the selection, appropriateness and adequacy of using any particular sampler is the responsibility of the user. It is the user who has to decide which system, according to their opinion, fulfils all the requirements for a GMP-compliant air monitoring system. The gelatine filter sampler mentioned in the USP 25 is well known worldwide as the air sampler MD8 airscan, (Fig. 1). This system basically consists of an air sampler (pump and flow meter), a filter holder that is connected by a flexible hose to the air sampler and a disposable gelatin filter that can be quickly and easily attached to the filter holder. The water-soluble filter material has a moisture content of 46-49% relative humidity (rh) and a pore size of 3µm. The gelatin filters are supplied pre-sterilised by gamma irradiation. They are characterised by validatable and extremely high retention rates for microorganisms and viruses, for example, 99.9995% for Bacillus subtilis niger spores at an inlet velocity of 0.25m/s and 99.94% for T3 coliphages at 80% rh. Their conditions for use are 30°C and 85% rh maximum. During the sampling procedure, the air sampler draws a defined air volume through the gelatin filter (flow rate selectable between 2 and 8m³/h). Any micro-organisms contained in the sampled air are retained on the gelatin filter. After sampling, the filter is placed directly on a medium in a standard 90mm petri dish and incubated. The colonies that develop are counted and recorded as colony forming units per cubic meter (cfu/m³).

Monitoring inside the isolator Having described the equipment requirements for air samplers, we can now turn to the practical user requirements for an airborne microorganism system designed for isolators: • No interference with the normal usage of the isolator • No contamination of the isolator as a result of the transfer of materials – for example, air sampler, or consumables, such as petri dish media, agar strips or filters • Maintenance of the barrier function between the different clean room classes – no contamination of the environment, the isolator or its interior • The method should be user-friendly • Monitoring must give accurate and reliable results • The method should be generally recognised With the MD8 airscan only the disposable gelatin filter and the holder are located inside the isolator. The sampling system has only minimal space requirements and can be positioned in such a way that it does not interfere with routine work (Fig. 2), for example, in isokinetic sampling. The air sampler placed outside the isolator (Fig. 3) is not exposed to sterilants used inside the isolator, such as hydrogen peroxide or peracetic acid, which increases the in-service life of the air sampler. Other air samplers have the disadvantage that the motor for suctioning the air sample is located inside the isolator, which is especially critical when gas is used to decontaminate it. In addition, this type of air sampler poses a potential risk of secondary contamination when it is transferred after sterilisation from outside into the interior of the isolator.⁷ With the MD8 airscan, calibration and adjustment of the air sampling rate, which are prerequisites for ensuring exact measurements and validation of the air sampling system, can be performed outside the isolator, without posing an added risk of contaminating the isolator (Fig. 4). Protecting employees and products makes it necessary to maintain the barrier function between the individual cleanroom classes. For this purpose, HEPA filters or a PTFE membrane filter capsule can be installed between the isolator and the air sampler, thus completely isolating the critical work area inside the isolator (Fig. 5). The installation of filter systems is required by the British Isolator Guidelines, according to which unfiltered air may not exit or enter the isolator. The gelatin filter unit is easy to exchange on the sampling head, enhancing the user-friendliness of the MD8 airscan. The operator needs to use only one hand in the isolator glove to change the filter quickly and reliably (Fig. 6). The MD8 airscan's precision sensor with optimal control characteristics guarantees a constant air sampling rate and allows quantitative evaluation of cfu/m³. The user can calibrate and adjust the air sampler on-site, enabling validatable predictions to be made about the air sampling volume. The "absolute" retention rating of the gelatin filters and the MD8 airscan's fast and efficient sampling of 1m³ of air allow the lowest concentration (1cfu/m³) of microbes to be detected, making the gelatin membrane filter method suitable for meeting the current requirements. The high retention rating and residual moisture of the gelatin filters and their compatibility with H₂O₂ gas sterilisation⁸ decisively contribute toward preventing false negative results.

Further applications The system is "state of the art" for simple, clean and reliable monitoring of airborne micro-organisms in isolators. Moreover, there are several further applications for the gelatin membrane filter method which are rapidly attracting attention. These include microbiological monitoring in cleanrooms and blow-fill-seal systems, measurement of high concentrations of airborne microbes within the scope of occupational health, and safety and quantitative sampling of airborne viruses. Interest in using the method in combination with rapid-test systems or genetic engineering methods, such as those for anthrax sampling, is also increasing.