Environmental monitoring of cleanrooms and isolators has been subject to a significant rise in regulatory requirements recently, as contaminations can harm product, slow the product’s time to market, and even result in recalls. This critical need for effective environmental monitoring processes has made the equipment used for microbial 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 pharmaceutical environments.
The objective of surface monitoring is to determine the presence of viable microorganisms on a variety of critical surfaces, including laminar airflow workbenches, floors, laboratory personnel and difficult-to-reach areas such as the interior of tubing and filling needles.
Contact plates are the equipment of choice for sampling flat or convex surfaces. These 50mm diameter plates are filled so the solid media protrudes, usually Tryptic Soy Agar (TSA), a general purpose medium, or Sabouraud Dextrose Agar (SDA), ideally suited for the growth of yeasts and molds. The agar surface is then pressed against the surface to be tested and incubated to determine the number of colonies, if any, present on the surface.
Several recent advances have made the use of contact plates safer and more effective. First, contact plate media containing only non-animal origin substances has been developed. This minimizes the potential to transmit animal spongiform encephalopathy (TSE/BSE), a risk associated with the use of the animal-derived TSA. The growth-promoting properties of a solid vegetable contact medium were tested for various strains originating from culture collections (see Figure 1). The results indicate culturability roughly comparable to that of TSA.
medium and TSA (test in duplicate)
Also, when coming into contact with test surfaces, contact plates tend to pick up residues of the sanitizing agents used for cleaning or disinfecting cleanrooms. Hundreds of different sanitizers are used in pharmaceutical facilities around the world. In fact, many regulatory agencies actually expect to see two disinfectants used in rotation, with each possessing a different mode of activity. These different sanitizing substances can inhibit the growth of contaminants on the contact plate.
To counteract the growth-inhibiting properties of these sanitizers, neutralizers have been added to the culture media. Appropriate combinations of lecithin, polysorbate 80, histidine and sodium thiosulfate are believed to neutralize as much as 80% of the sanitizers typically used. However, some of the remaining substances, such as polyhexamethylene biguanides, have proved very tricky. To cover a larger proportion of the sanitizers, a new neutralizing mixture was recently introduced by EMD Millipore called Neutralizer A. It has proved successful in neutralizing all sanitizers tested so far, with recovery rates all above 50% compared to control plate counts1.
In addition, residues of antibiotics previously produced in the pharmaceutical facility, such as ß-lactam derivatives, are also able to inhibit microbial growth on contact plates. However, a combination of a new cephalosporinase and penicillinase provides inactivation of a broad spectrum of ß-lactam antibiotics, including cephalosporins of the third and fourth generation.
Figure 2 shows the efficacy of cephalosporinase/penicillinase in neutralizing 13 ß-lactam antibiotics against the more sensitive microorganism tested, Kocuria rhizophila. Only in the case of cefoxitin was neutralization incomplete. When Staphylococcus aureus was the microorganism selected, even cefoxitin was neutralized completely.
For irregular surfaces such as equipment recesses, nooks, crevices, tubing and filling needles, contact plates are impractical. Instead, pre-moistened swabs may be used to validate cleaning and sanitation procedures and to verify that a required hygiene level has been reached.
The swab head is the most critical component of the swab. It needs to be lint-free and extremely low in non-volatile residues and particulates. Swab heads can be made of various materials, including polyester, polyurethane, nylon and cotton, and these materials can be structured or knitted in different ways. The type of material determines the extent to which abrasion may occur, and some materials may be degraded by aggressive solvents and subsequently leave residues.
The swab’s shaft also requires consideration. It needs to be flexible and long enough to reach difficult-to-reach locations, such as tubing interiors.
For maximum recovery, the swab is carefully removed from its tube and rubbed across a surface area using a twisting motion. Conventional swabs are then re-suspended in a specified amount of rinse solution and agitated to transfer any microorganisms present on the swab head into the solution. The collection medium is then tested, usually by direct plating or membrane filtration and incubation of filters on culture media.
A drawback of using conventional swabs is that the recommended procedure involves a number of steps, each of which carries the potential for handling errors and contaminations. To avoid having to open the tube several times, an all-in-one swab system can be used; this system requires only one opening because it already carries a reservoir containing the culture broth. After returning the swab to the tube, the reservoir at the top is snapped and squeezed until the broth fully saturates the swab head. After incubation, the broth is monitored for turbidity as an indicator of contamination.
This ICR swab is gamma-sterilized in its final packaging at a relatively high dose of 25-35 kGy. Despite this, both the moistened swab tip and the medium were shown to be non-inhibitory against challenges of eight microbial species that are more commonly isolated from aseptic processing environments2. This swab is also suited for use in isolators because it is triple-packaged, with the inner bag possessing a pre-punched hole at the top to hang it up, leaving no uncleanable surface underneath.
There are several standard ways of monitoring airborne potential contaminants. Passive air sampling uses solid media settle plates exposed to the air for a predetermined period of time. Active air sampling utilizes an instrument that draws ambient air and then directs the air stream at an attached agar plate or strip for collection. Particle counters are used to quantify potential contaminants in the air.
Settle plates usually contain TSA or SDA, and they are placed throughout the test area with their lids removed. After exposure, the plates are closed and incubated. The number of colonies is then counted and the microorganisms are identified. For safe and efficient documentation, pre-barcoded culture media plates are used, enabling each plate to be fully traceable back to its date of use and the location of sampling.
A key issue with settle plates is that they lose water due to evaporation when exposed, leading to an increasingly dry skin on the agar surface. This can cause poor growth of certain microbes on the media and thus 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 have been found to lose as much as 16% of their original weight. However, when such plates were inoculated with typical contaminants and subsequently incubated, all recovery rates were above 70%3. To enable prolonged exposure and incubation periods while ensuring that the plates still deliver reliable results, settle plates can be poured to a particularly high filling level. However, the maximum exposure time should be validated for each production line, taking into consideration air flow, temperature, relative humidity of the air and turbulences.
Active Air Samplers
When using active air samplers in critical areas, there are several ways to help avoid contamination. First, it is important that the airflow is not severely disrupted by the instrument’s operation, placement or removal, since disturbance makes it easier for contaminants to attach to instrument housing. To minimize air flow disruption in isolators and other confined areas, as well as to save space, manufacturers have developed variants of their instruments. For example, air ducting is directed from the sampling points outside the controlled area where all electronic and moving parts remain. In addition, the instruments’ internal pumps enable easy decontamination of the sample heads and aspiration tubes. To analyze the disruption propensity of an instrument in use, EMD Millipore conducts smoke studies that visualize the movement of air in decommissioned cleanrooms.
Autoclavable sampling heads also make the instrument’s handling safer and, as a consequence, reduce airflow disruption and the likelihood of human error. In fact, any easy-handling features likely to minimize human activity in critical areas, such as touchscreen operation or high battery capacities for longer cycles, can help to prevent contaminations. Finally, fine polished stainless steel makes it more difficult for particle-attached microbes to adhere to instrument housing. The material is also relatively easy to clean and disinfect.
Particle monitoring of ambient air is conducted to quantify non-viable contaminants in the air and to determine the quality of air in controlled environments. Quantitative particle data often also provides an indication of the status regarding viable contaminants because most airborne microbes adhere to particles.
One decision pharma companies must make when selecting their systems is whether to choose pre-installed or portable particle counters. In many production facilities, the particle counters are pre-installed at specified locations. Their measurement data is automatically collected by cleanroom monitoring systems for centralized analysis and storage, along with data on other parameters such as temperature, relative humidity and air pressure differentials. When the infrastructure of the facility allows for pre-installed particle counters, the systems have proved very convenient.
Once installed, however, modifying the particle counting system can be very disruptive. For instance, new sample locations will need to be added to many cleanrooms when the planned revision of ISO 14644-1 takes effect (pending, following a 2010 Draft International Standard). With the new standards, the minimum number of systems will no longer be determined on the basis of the square root of the cleanroom’s total area, but on the values in a table; a cleanroom of 36 square meters will require nine sample locations, three more than now4. For a pre-installed system, retrofitting may entail constructional changes as well as modifications to a complex data processing system.
For this reason, many pharma companies opt for the flexibility of portable particle counters. Some can be programmed from a touchscreen and display a visual representation of a cleanroom’s sampling locations, thus reducing the likelihood of human error.
Media Fill Trials
Media fill trials simulate the actual filling process as closely as possible, using media in place of the liquid pharmaceutical ingredients. They are performed to help validate or revalidate the robustness of aseptic manufacturing processes, demonstrating whether a product is likely to become contaminated during the filling process.
In these trials, the media comes into contact with the same production line components that the pharmaceutical product will eventually touch. Using material guaranteed to be free of BSE/TSE is therefore pivotal, and the use of non-animal origin media fill is increasing. Like animal-based peptone media, these vegetable media allow growth of a similarly broad range of microorganisms. In quantitative tests of 20 Gram-positive and eight Gram-negative bacterial strains as well as eight fungal strains, the growth-promoting properties of vegetable peptone broth were found to be similar to those of Tryptic Soy Broth (TSB)5.
Supply of Monitoring Equipment
The equipment outlined above, intended for use in critical environments, should be produced under equally controlled environmental conditions. Disposables used in cleanrooms and isolators, such as settle plates, contact plates and swabs, should therefore be filled in cleanrooms that meet high microbiological standards. As a result, even if there were a contamination at a supplier production facility, it would be much less likely to be passed on to the user without being noticed.
Disposables 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 after entering the next highest class of cleanroom. For class A (ISO 5) cleanrooms and isolators, transparent triple bagging is very well suited.
For use in isolators, the material of the innermost bag has to meet a further requirement: it must be impermeable to H2O2 vapor to ensure this universally used bio-decontaminant will not be transferred on the culture media and inhibit microbial growth after sampling. To neutralize a certain amount of H2O2 that may, nevertheless, accumulate on the culture media (e.g. as a consequence of active air sampling), one can supplement culture media designed for use in isolators. Another important feature for use in these highly sensitive environments is that the inner bag possesses a mechanism for it to be hung up within the isolator, leaving no uncleanable surface underneath.
Because the final packaged product cannot be sterilized outright, it is gamma-irradiated. The dose has to be finely tuned to ensure a contaminant-free product while also making sure the media does not degenerate to become impacted in its capacity to grow microorganisms. In addition, the plastic packaging material must be qualified to withstand the irradiation dose without becoming easily damaged when handled or shedding particles that would compromise cleanroom safety. In our experience, doses between 9 and 20 kGy provide the right balance.
It should be noted that because of the aseptic filling process without a terminal sterilization process, a Sterility Assurance Level (SAL) cannot be defined. However, the presence of supplier-introduced colonies on gamma-irradiated media is an extremely rare event. When contaminants are indeed encountered, these are usually not viable. In 2011, our companies irradiated a total of 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, gamma-irradiated culture media should be visually checked for contamination prior to usage.
It is important that plates are stored upright and at roughly the temperature they will be used for sampling. Their shelf life is based on a storage temperature of approximately 20°C. Refrigeration is not advised; when stored in the cold, drops of condensation will form that may result in contaminations when these moist plates are handled. To be entirely sure that even during transportation the plates are kept at the right temperature, it is necessary to use temperature-controlled shipment.
A key concern of decision-makers in pharmaceutical companies is that the instruments and disposables they purchase 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 worldwide, knowing that this gives them a competitive edge by saving their customers considerable time and costs. Providers of culture media disposables perform studies to support the in-house validation that end-users need to perform.
Environmental monitoring of ambient air, surfaces and personnel in critical areas is a significant risk and cost issue in pharmaceutical production. Bearing in mind that many pharma companies involve a significant number of staff in their environmental monitoring activities, the routines need to be as straightforward and fail-safe as possible in order to yield reliable results every time. To improve results, pharmaceutical suppliers are working to develop improved environmental monitoring products. This trend is likely to continue, boosting growth in the supplier market and making the production process, and therefore pharmaceutical products, ever safer.
- Hedderich 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
- Sandle, T. (2011): A study of a new type of swab for the environmental monitoring of isolators and cleanrooms (the heipha ICR-Swab). European Journal of Parenteral and Pharmaceutical Sciences, Vol. 16, No.2, pp42-48.
- Sandle, T. (2011): Microbial recovery on settle plates in unidirectional airflow cabinets. Clean Air and Containment Review 6/2011.
- Væver Hartvig, N., Farquharson, G. J., Mielke, R., Varney, M. and Foster, M. (2011): Sampling Plan for Cleanroom Classification with Respect to Airborne Particles. Journal of the IEST, V. 54, Number 1, Special ISO Issue.
- Hedderich, R., Klees, A., Eiermann, K., Greulich, Y. and Müller, R. (2009): Growth promoting properties of a vegetable peptone broth (VPB) in comparison to tryptic soy broth. Poster at PDA Annual Global Conference on Pharmaceutical Microbiology.
Anne-Grit Klees is product manager at heipha Dr. Müller GmbH, a subsidiary of Merck KGaA, She can be reached at firstname.lastname@example.org.
Tony Ancrum is technical marketing manager Microbiology & Hygiene at Merck KGaA.