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Surface Air System Sampler— This integrated unit consists of an entry section that accommodates an agar contact plate. Immediately behind the contact plate is a motor and turbine that pulls air through the unit's perforated cover over the agar contact plate and beyond the motor, where it is exhausted. Multiple mounted assemblies are also available.
Gelatin Filter Sampler— The unit consists of a vacuum pump with an extension hose terminating in a filter holder that can be located remotely in the critical space. The filter consists of random fibers of gelatin capable of retaining airborne microorganisms. After a specified exposure time, the filter is aseptically removed and dissolved in an appropriate diluent and then plated on an appropriate agar medium to estimate its microbial content.
Settling Plates— This method is still widely used as a simple and inexpensive way to qualitatively assess the environments over prolonged exposure times. The exposure of open agar-filled Petri dishes, or settling plates, is not to be used for quantitative estimations of the microbial contamination levels of critical enviroments.
One of the major limitations of mechanical air samplers is the limitation in sample size of air being sampled. Where the microbial level in the air of a controlled environment is expected to contain not more than three cfu per cubic meter, several cubic meters of air should be tested if results are to be assigned a reasonable level of precision and accuracy. Often this is not practical. To show that microbial counts present in the environment are not increasing over time, it might be necessary to extend the time of sampling to determine if the time of sampling is a limiting factor or not. Typically, slit-to-agar samplers have an 80-liter-per-minute sampling capacity (the capacity of the surface air system is somewhat higher). If one cubic meter of air is tested, then it would require an exposure time of 15 minutes. It may be necessary to use sampling times in excess of 15 minutes to obtain a representative environmental sample. Although there are samplers reported to be capable of very high sampling volume rates, consideration in these situations should be given to the potential for disruption of the airflow patterns in any critical area or to the creation of a turbulence that could increase the probability of contamination.
For centrifugal air samplers, a number of earlier studies showed that the samples demonstrated a selectivity for larger particles. The use of this type of sampler may have resulted in higher airborne counts than the other types of air samplers because of that inherent selectivity.
When selecting a centrifugal sampler, the effect of the sampler on the linearity of the airflow in the controlled zone where it is placed for sampling should be taken into consideration. Regardless of the type of sampler used, the use of a remote probe requires determining that the extra tubing does not have an adverse effect on the viable airborne count. This effect should either be eliminated or, if this is not possible, a correction factor should be introduced in the reporting of results.
Methodology and Equipment for Sampling of Surfaces for Quantitation of Viable Microbial Contaminants in Controlled Environments
Another component of the microbial environmental control program in controlled environments is surface sampling of equipment, facilities, and personnel gear used in these environments. The standardization of surface sampling methods and procedures has not been as widely addressed in the pharmaceutical industry as the standardization of air sampling procedures.3 To minimize disruptions to critical operations, surface sampling is performed at the conclusion of operations. Surface sampling may be accomplished by the use of contact plates or by the swabbing method. Surface monitoring is generally performed on areas that come in contact with the product and on areas adjacent to those contact areas. Contact plates filled with nutrient agar are used when sampling regular or flat surfaces and are directly incubated at the appropriate time for a given incubation temperature for quantitation of viable counts. Specialized agar can be used for specific quantitation of fungi, spores, etc.
The swabbing method may be used for sampling of irregular surfaces, especially for equipment. Swabbing is used to supplement contact plates for regular surfaces. The swab is then placed in an appropriate diluent and the estimate of microbial count is done by plating of an appropriate aliquot on or in specified nutrient agar. The area to be swabbed is defined using a sterile template of appropriate size. In general, it is in the range of 24 to 30 cm2. The microbial estimates are reported per contact plate or per swab.
Culture Media and Diluents Used for Sampling or Quantitation of Microorganisms
The type of medium, liquid or solid, that is used for sampling or quantitation of microorganisms in controlled environments will depend on the procedure and equipment used. A commonly used all-purpose medium is Soybean–Casein Digest Agar when a solid medium is needed. Other media, liquid or solid, are listed below.
Liquid Media* Solid Media*
Tryptone saline Soybean-casein digest agar
Peptone water Nutrient agar
Buffered saline Tryptone glucose extract agar
Buffered gelatin Lecithin agar
Enriched buffered gelatin Brain heart infusion agar
Brain heart infusion Contact plate agar
Soybean-casein medium
* Liquid and solid media are sterilized using a validated process.
These media are commercially available in dehydrated form. They are also available in ready-to-use form. When disinfectants or antibiotics are used in the controlled area, consideration should be given to using media with appropriate inactivating agents.
Alternative media to those listed can be used provided that they are validated for the purpose intended.
Identification of Microbial Isolates from the Environmental Control Program
The environmental control program includes an appropriate level of identification of the flora obtained from sampling. A knowledge of the normal flora in controlled environments aids in determining the usual microbial flora anticipated for the facility being monitored; evaluating the effectiveness of the cleaning and sanitization procedures, methods, and agents; and recovery methods. The information gathered by an identification program can also be useful in the investigation of the source of contamination, especially when the Action levels are exceeded.
Identification of isolates from critical areas and areas immediate to these critical areas should take precedence over identification of microorganisms from noncritical areas. Identification methods should be verified, and ready-to-use kits should be qualified for their intended purpose (see Critical Factors Involved in the Design and Implementation of Environmental Control Program).
Operational Evaluation of the Microbiological Status of Aseptically Filled Products in Clean Rooms and Other Controlled Environments
The controlled environment is monitored through an appropriate environmental monitoring program. To assure that minimal bioburden is achieved, additional information on the evaluation of the microbiological status of the controlled environment can be obtained by the use of media fills. An acceptable media fill shows that a successful simulated product run can be conducted on the manufacturing line at that point in time. However, other factors are important, such as appropriate construction of facilities, environmental monitoring and training of personnel.
When an aseptic process is developed and installed, it is generally necessary to qualify the microbiological status of the process by running at least three successful consecutive media fills. A media fill utilizes growth medium in lieu of products to detect the growth of microorganisms. Issues in the development of a media fill program that should be considered are the following: media-fill procedures, media selection, fill volume, incubation, time and temperature, inspection of filled units, documentation, interpretation of results, and possible corrective actions required.
Since a media fill is designed to simulate aseptic processing of a specified product, it is important that conditions during a normal product run are in effect during the media fill. This includes the full complement of personnel and all the processing steps and materials that constitute a normal production run. During the conduct of media fill, various predocumented interventions that are known to occur during actual product runs should be planned (e.g., changing filling needles, fixing component jams).
Alternatively, in order to add a safety margin, a combination of possible conditions can be used. Examples may include frequent start and stop sequences, unexpected repair of processing system, replacement of filters, etc. The qualification of an aseptic process need not be done for every product, but should be done for each processing line. Since the geometry of the container (size as well as opening of the container) and the speed of the line are factors that are variable in the use of an aseptic processing line, appropriate combination of these factors, preferably at the extremes, should be used in the qualification of the line. A rationale for products used should be documented.
The 1987 FDA Guideline on Sterile Drug Products Produced by Aseptic Processing indicates that media-fill runs be done to cover all production shifts for line/product/container combinations. This guideline should be considered not only for qualification media-fill runs, but also for periodic reevaluation or revalidation. Media fill programs should also simulate production practices over extended runs. This can be accomplished by doing media-fill runs at the end of production runs.
In general, an all-purpose, rich medium such as Soybean Casein Broth that has been checked for growth promotion with a battery of indicator organisms (see Sterility Tests 71) at a level of below 100 cfu/unit, can be used. Isolates from the controlled environment where aseptic processing is to be conducted may also be used. Following the aseptic processing of the medium, the filled containers are incubated at 22.5 ± 2.5 or at 32.5 ± 2.5. All media filled containers should be incubated for a minimum of 14 days. If two temperatures are used for incubation of media filled samples, then these filled containers should be incubated for at least 7 days at each temperature. Following incubation, the medium-filled containers should be inspected for growth. Media filled isolates are identified by genus and, when possible, by species in order to investigate the sources of contamination. |
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