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These facts underscore the importance of good personal hygiene and a careful attention to detail in the aseptic gowning procedure used by personnel entering the controlled environment. Once these employees are properly gowned—including complete facial coverage—they must be careful to maintain the integrity of their gloves and suits at all times. Since the major threat of contamination of product being aseptically processed comes from the operating personnel, the control of microbial contamination associated with these personnel is one of the most important elements of the environmental control program.
The importance of thorough training of personnel working in controlled environments, including aseptic techniques, cannot be overemphasized. The environmental monitoring program, by itself, will not be able to detect all events in aseptic processing that could compromise the microbiological quality of the environment. Therefore, periodic media-fill or process simulation studies to revalidate the process are necessary to assure that the appropriate operating controls and training are effectively maintained.
Critical Factors Involved in the Design and Implementation of a Microbiological Environmental Control Program
An environmental control program should be capable of detecting an adverse drift in microbiological conditions in a timely manner that would allow for meaningful and effective corrective actions. It is the responsibility of the manufacturer to develop, initiate, implement, and document such a microbial environmental monitoring program.
Although general recommendations for an environmental control program will be discussed, it is imperative that such a program be tailored to specific facilities and conditions. A general microbiological growth medium such as Soybean Casein Digest Medium should be suitable in most cases. This medium may be supplemented with additives to overcome or to minimize the effects of sanitizing agents, or of antibiotics if used or processed in these environments. The detection and quantitation of yeasts and molds should be considered. General mycological media, such as Sabouraud's, Modified Sabouraud's, or Inhibitory Mold Agar are acceptable. Other media that have been validated for promoting the growth of fungi, such as Soybean–Casein Digest Agar, can be used. In general, testing for obligatory anaerobes is not performed routinely. However, should conditions or investigations warrant, such as the identification of these organisms in sterility testing facilities, more frequent testing is indicated. The ability of the selected media to detect and quantitate these anaerobes or microaerophilic microorganisms should be evaluated.
The selection of time and incubation temperatures is made once the appropriate media have been selected. Typically, incubation temperatures in the 22.5 ± 2.5 and 32.5 ± 2.5 ranges have been used with an incubation time of 72 and 48 hours, respectively. Sterilization processes used to prepare growth media for the environmental program should be validated and, in addition, media should be examined for sterility and for growth promotion as indicated under Sterility Tests 71. In addition, for the Growth Promotion test, representative microflora isolated from the controlled environment or ATCC strain preparations of these isolates may also be used to test media. Media must be able to support growth when inoculated with less than 100 colony-forming units (cfu) of the challenge organisms.
An appropriate environmental control program should include identification and evaluation of sampling sites and validation of methods for microbiological sampling of the environment.
The methods used for identification of isolates should be verified using indicator microorganisms (see Microbial Limit Tests 61).
Establishment of Sampling Plan and Sites
During initial start-up or commissioning of a clean room or other controlled environment, specific locations for air and surface sampling should be determined. Consideration should be given to the proximity to the product and whether air and surfaces might be in contact with a product or sensitive surfaces of container-closure systems. Such areas should be considered critical areas requiring more monitoring than non-product-contact areas. In a parenteral vial filling operation, areas of operation would typically include the container-closure supply, paths of opened containers, and other inanimate objects (e.g., fomites) that personnel routinely handle.
The frequency of sampling will depend on the criticality of specified sites and the subsequent treatment received by the product after it has been aseptically processed. Table 2 shows suggested frequencies of sampling in decreasing order of frequency of sampling and in relation to the criticality of the area of the controlled environment being sampled.
Table 2. Suggested Frequency of Sampling on the Basis of Criticality of Controlled Environment
Sampling Area Frequency of
Sampling
Class 100 or better room designations Each operating shift
Supporting areas immediately adjacent Each operating shift
to Class 100 (e.g., Class 10,000)
Other support areas (Class 100,000) Twice/week
Potential product/container contact areas Twice/week
Other support areas to aseptic Once/week
processing areas but non-product con-
tact (Class 100,000 or lower)
As manual interventions during operation increase, and as the potential for personnel contact with the product increases, the relative importance of an environmental monitoring program increases. Environmental monitoring is more critical for products that are aseptically processed than for products that are processed and then terminally sterilized. The determination and quantitation of microorganisms resistant to the subsequent sterilization treatment is more critical than the microbiological environmental monitoring of the surrounding manufacturing environments. If the terminal sterilization cycle is not based on the overkill cycle concept but on the bioburden prior to sterilization, the value of the bioburden program is critical.
The sampling plans should be dynamic with monitoring frequencies and sample plan locations adjusted based on trending performance. It is appropriate to increase or decrease sampling based on this performance.
Establishment of Microbiological Alert and Action Levels in Controlled Environments
The principles and concepts of statistical process control are useful in establishing Alert and Action levels and in reacting to trends.
An Alert level in microbiological environmental monitoring is that level of microorganisms that shows a potential drift from normal operating conditions. Exceeding the Alert level is not necessarily grounds for definitive corrective action, but it should at least prompt a documented follow-up investigation that could include sampling plan modifications.
An Action level in microbiological environmental monitoring is that level of microorganisms that when exceeded requires immediate follow-up and, if necessary, corrective action.
Alert levels are usually based upon historical information gained from the routine operation of the process in a specific controlled environment.
In a new facility, these levels are generally based on prior experience from similar facilities and processes; and at least several weeks of data on microbial environmental levels should be evaluated to establish a baseline.
These levels are usually re-examined for appropriateness at an established frequency. When the historical data demonstrate improved conditions, these levels can be re-examined and changed to reflect the conditions. Trends that show a deterioration of the environmental quality require attention in determining the assignable cause and in instituting a corrective action plan to bring the conditions back to the expected ranges. However, an investigation should be implemented and an evaluation of the potential impact this has on a product should be made.
Microbial Considerations and Action Levels for Controlled Environments
Classification of clean rooms and other controlled environments is based on Federal Standard 209E based on total particulate counts for these environments. The pharmaceutical and medical devices industries have generally adopted the classification of Class 100, Class 10,000, and Class 100,000, especially in terms of construction specifications for the facilities.
Although there is no direct relationship established between the 209E controlled environment classes and microbiological levels, the pharmaceutical industry has been using microbial levels corresponding to these classes for a number of years; and these levels have been those used for evaluation of current GMP compliance.2 These levels have been shown to be readily achievable with the current technology for controlled environments. There have been reports and concerns about differences in these values obtained using different sampling systems, media variability, and incubation temperatures. It should be recognized that, although no system is absolute, it can help in detecting changes, and thus trends, in environmental quality. The values shown in Tables 3, 4, and 5 represent individual test results and are suggested only as guides. Each manufacturer's data must be evaluated as part of an overall monitoring program.
Table 3. Air Cleanliness Guidelines in Colony-Forming Units (cfu) in Controlled Environments (Using a Slit-to-Agar Sampler or Equivalent)
Class* cfu per cubic meter of air** cfu per cubic feet of air
SI U.S. Customary
M3.5 100 Less than 3 Less than 0.1
M5.5 10,000 Less than 20 Less than 0.5
M6.5 100,000 Less than 100 Less than 2.5
* As defined in Federal Standard 209E, September 1992.
** A sufficient volume of air should be sampled to detect excursions above the limits specified.
Table 4. Surface Cleanliness Guidelines of Equipment and Facilities in cfu in Controlled Environments
Class cfu per Contact Plate*
SI U.S. Customary
M3.5 100 3 (including floor)
M5.5 10,000 5
10 (floor)
* Contact plate areas vary from 24 to 30 cm2. When swabbing is used in sampling, the area covered should be greater than or equal to 24 cm2 but no larger than 30 cm2.
Table 5. Surface Cleanliness Guidelines in Controlled Environments of Operating Personnel Gear in cfu
Class cfu per Contact Plate*
SI U.S. Gloves Personnel Clothing
Customary & Garb
M3.5 100 3 5
M5.5 10,000 10 20
* See in Table 4 under (*).
Methodology and Instrumentation for Quantitation of Viable Airborne Microorganisms
It is generally accepted by scientists that airborne microorganisms in controlled environments can influence the microbiological quality of the intermediate or final products manufactured in these areas. Also, it generally is accepted that estimation of the airborne microorganisms can be affected by instruments and procedures used to perform these assays. Therefore, where alternative methods or equipment is used, the general equivalence of the results obtained should be ascertained. Advances in technology in the future are expected to bring innovations that would offer greater precision and sensitivity than the current available methodology and may justify a change in the absolute numbers of organisms that are detected.
Today, the most commonly used samplers in the U.S. pharmaceutical and medical device industry are the impaction and centrifugal samplers. A number of commercially available samplers are listed for informational purposes. The selection, appropriateness, and adequacy of using any particular sampler is the responsibility of the user.
Slit-to-Agar Air Sampler (STA)— This sampler is the instrument upon which the microbial guidelines given in Table 3 for the various controlled environments are based. The unit is powered by an attached source of controllable vacuum. The air intake is obtained through a standardized slit below which is placed a slowly revolving Petri dish containing a nutrient agar. Particles in the air that have sufficient mass impact on the agar surface and viable organisms are allowed to grow out. A remote air intake is often used to minimize disturbance of the laminar flow field.
Sieve Impactor— The apparatus consists of a container designed to accommodate a Petri dish containing a nutrient agar. The cover of the unit is perforated, with the perforations of a predetermined size. A vacuum pump draws a known volume of air through the cover, and the particles in the air containing microorganisms impact on the agar medium in the Petri dish. Some samplers are available with a cascaded series of containers containing perforations of decreasing size. These units allow for the determination of the distribution of the size ranges of particulates containing viable microorganisms, based on which size perforations admit the particles onto the agar plates.
Centrifugal Sampler— The unit consists of a propeller or turbine that pulls a known volume of air into the unit and then propels the air outward to impact on a tangentially placed nutrient agar strip set on a flexible plastic base.
Sterilizable Microbiological Atrium— The unit is a variant of the single-stage sieve impactor. The unit's cover contains uniformly spaced orifices approximately 0.25 inch in size. The base of the unit accommodates one Petri dish containing a nutrient agar. A vacuum pump controls the movement of air through the unit, and a multiple-unit control center as well as a remote sampling probe are available. |
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