Environmental sampling programs for the pharmacy cleanroom should be designed to provide information demonstrating that the engineering controls, sanitization processes, and work practices collectively provide an environment consistently capable of maintaining acceptable microbial levels. The program also should generate data capable of detecting adverse trends in microbial populations in a timely manner and should facilitate the identification of the source of such trends such as equipment failure, ineffective sanitization practices, poor personnel habits, or training deficiencies. Identification must be emphasized so those trends can be corrected before the environment, or ultimately the product, is adversely affected.
With these goals in mind, developing, instilling, and maintaining an environmental monitoring program for USP <797> compliance can be a daunting task and all too often, one of the most important aspects of such a program is the one that is most overlooked—the written program. This comprehensive document should describe the entire monitoring program from the sample plan to the response to exceeded concern levels. Once the written program is implemented, it should be reviewed annually for compliance to regulatory agencies’ as well as in-house requirements. For a list of the key program sections and what each should entail, see Figure 1.
Core Elements of Environmental Monitoring
When developing a program it is important to remember that compliance with more than one regulatory agency may apply and it is critical that the program meets all mandated regulatory requirements. After establishing this as the governing framework, the logical starting point for an environmental sampling program is the selection of appropriate equipment and materials. There are a multitude of materials and equipment available for sampling the cleanroom environment, so it is imperative to choose equipment supplied by a prominent manufacturer that adheres to federal requirements for good manufacturing practices. The time, money, and effort invested into developing and maintaining a program may be fruitless if that program is relying on improperly manufactured products. With this in mind, there are three necessary elements that must be evaluated—viable air samplers, surface samplers, and the type of media.
Viable Air Sampling
The most expensive piece of equipment is generally the viable air sampler. There are many types of air samplers available based on different technologies, but the following factors should be considered when choosing an air sampler:
- Disruption of Airflow—It is vital that the air sampler does not disrupt the flow of air in critical environments. This is most often encountered when purchasing used or older equipment.
- Flow Rate—The sampler should have a flow rate high enough to sample the required volume of air without drying out the media.
- Volume Selector—The sampler should be equipped with an adjustable air volume selector so that the required level of detection can be met and to obtain a countable plate.
- Supplies—The type of media used in the sampler should be confirmed ahead of time as well as its availability. Determine whether the media can be used in other sampling processes or if it is specific to that sampler only.
- Physical Aspects—The sampler should be portable, easily disinfected or autoclaved, sturdy, and manageable. Requiring tricky manipulations to place media in the sampler increases the risk of contamination by the operator.
- Calibration and Maintenance—Calibration services should be easily obtainable and the proper frequency of calibration should be determined.A simple way to assure that an air sampler is appropriate for use is to ask the manufacturer if it is compatible with ISO 14698 and if so, to provide the applicable study.
The use of contact plates and swabs are two effective ways to sample surfaces and consideration must be given to availability, ease of use, expiry dating, and storage requirements. Furthermore, there are specific uses for each type. Contact plates and slides should be used on relatively flat surfaces, while swabs should be used to sample irregular surfaces such as switches, where the agar surface in the contact plates may become disrupted. It is key to note that swabs need additional processing by the laboratory and are usually more expensive to analyze.
Selection of Media
Media is available in a variety of types and usually falls into three categories: general growth media, selective media, and differential media. For most pharmacy cleanroom applications, a general growth medium is sufficient, as it will support the growth of a large variety of organisms, including bacteria, yeast, and mold.
Media used for surface sampling will need to have additional ingredients incorporated into the agar to neutralize any residue left behind from the cleaning agents used in the cleanroom. Chemical residues left on equipment can be absorbed into the media during sampling and inhibit the growth of organisms resulting in false negatives. A review of the cleaning agents used in the cleanroom should be conducted and compared to the neutralizing capabilities of the media. Anytime the cleaning procedure is changed, likewise it is essential to review the neutralizing capabilities of the media.
As with the viable air sampler, it is crucial to ensure the selected media is manufactured correctly and is not only sterile but capable of supporting the growth of organisms. Media also can be affected by shipping and storage conditions, so it is wise to obtain a certificate of analysis from the manufacturer for each lot and file it along with the rest of the documentation for the program. This certificate of analysis should document studies completed by the manufacturer for growth support, pH, and sterility testing. Purchasing media that is double or triple bagged and irradiated, although generally much more expensive, will ensure sterility. Lastly, the laboratory should test a minimum of two unused, unopened media along with each sampling session for growth support and sterility. This will verify that the media was not exposed to conditions during shipping or storage that may have contaminated it or affected its ability to support the growth of organisms.
Proper Communication with the Lab
Once sampling is completed, another commonly overlooked aspect of monitoring programs is properly relaying to the laboratory what analysis you want completed with your samples. Incubation parameters, along with the type of analysis to be completed, should be fully described in your program and given to the laboratory. Verify with the laboratory that the incubators are validated, calibrated, and consistently monitored with some type of recording device. In addition, request that the laboratory perform a total microbial count, which will provide the total count of everything recovered on your samples including yeast and mold. It is critical to incubate and analyze the samples for yeast and mold since recovery of either requires immediate action. In high-risk areas, the addition of a mold agar may be incorporated into your sampling program if there is a chance that you have a high bacterial count that will interfere with the growth of molds. In most cases though, a general growth media incubated at 26 to 35°C will recover both bacterial and mold growth just as well.
In addition to requesting the total microbial count, all organisms recovered must be identified at least to genus and any Staphylococcus colonies should be tested for coagulase response. Recovery of any mold, gram negative, yeast, or coagulase positive Staphylococcus must result in immediate re-cleaning, resampling, and investigation of the facility. Have the laboratory notify you immediately if any of these organisms are recovered. Remember to request that the laboratory transform the total microbial count for air samples into CFU per cubic meter. This will aid in verifying that your results meet the acceptance criteria in USP General Chapter <797>.
Managing Exceeded Acceptance Levels
If you receive laboratory results that indicate levels higher than the acceptance criteria or the presence of objectionable organisms, you must initiate an investigation into the issue and remediate the area back into compliance. As with the written program description, this process should follow a basic structure:
- Analyze the lab data
- Compare to established levels
- Review types recovered
- Investigate the results
- Compare to established levels
- Establish best corrective actions
- Clean and resample
- Return to a state of facility control
- Correct the issue and install procedures to prevent it from reoccurring
Reign in Contamination Control
The three major sources of cleanroom contamination are people, physical conditions, and materials. Thus, it is critical that compounding personnel have good personal hygiene. There also are conditional situations that should affect staffing. For example, employees that have respiratory infections or sunburn, skin irritations, or open sores, should be reassigned to other duties for the duration of the affliction.
Temperature and humidity levels in the cleanroom also are critical. The temperature should be set such that personnel are comfortable when completely gowned. If working temperatures are too high, personnel will start to sweat and this can bring out the deeper skin flora. If personnel do start sweating, they are more likely to touch their face, increasing the risk of contamination with human flora. Conversely, if the temperature is too low, personnel will make unnecessary movements to keep warm; shivering also increases the release of microbes into the air.
Surrounding conditions can have a large impact on the contamination within a controlled environment. The presence of construction, heavy rain, or high winds will increase the amount of microbes in the outside air and personnel will be more likely to bring them into the cleanroom on their clothing and in their hair. Recognizing these conditions and acting accordingly goes hand in hand with proper gowning and PPE protocols.
How materials are stored, handled, and transferred into the cleanroom can play a significant role in contamination control. Any necessary cleanroom consumables should be stored in cleanroom-approved packaging until use. Garments and other gowning materials may be stored inside the cleanroom gowning area or anti-area, as well as the many cleanroom consumable supplies necessary to support the cleanroom operators and the processes inside the room. Not only is the storage of materials important, but the types of materials found within the cleanroom also can contribute to contamination. The following materials are not considered cleanroom compatible: wood pulp-based paper products, which includes regular paper, tissues, cardboard, books, and magazines; Styrofoam products; powders; erasers, pencils or felt-tipped pens; or any products that can easily shred or aerosolize. Packaging materials can carry a large number of microbes; therefore, material flow is another critical element in controlling cleanroom contamination. There should be a specified and documented pathway for the flow of materials and all materials brought into the cleanroom should be wiped with 70% IPA. Any unnecessary or inappropriate packaging materials, like cardboard, should be removed before the product is brought into the cleanroom.
Selection of cleaning materials, equipment, and cleaning agents should be appropriate to the type of cleaning required and all cleaning agents and subsequent cleaning protocols should be validated to assure efficacy. All chemicals and supplies used to clean the cleanroom also should be stored in original packaging until use, and used disposable mops and mop heads should be properly disposed of after use.
Personnel Habits Lead to Contamination
The behavior and habits of compounding personnel is another factor that can bring on contamination. For example, it is imperative to limit talking and other social activities in the cleanroom to what is required for compounding. Slow, purposeful movements are critical and personnel should not touch or move material unnecessarily, as this greatly increases viable particle counts. Particular care should be taken with cardboard packaging, which is usually filled with mold and bacillus spores. Personnel need to avoid scratching their heads or rubbing their hands and it is necessary to be discrete when coughing, sneezing, or blowing your nose. If possible these actions should be done outside the cleanroom and, as in the case of sunburn or other external health conditions, staff with established illnesses or afflictions should be reassigned for their duration.
The greater the number of people working in the cleanroom and/or the busier the workload, the greater the burden will be on the cleanroom making it more difficult to flush out the particles which are generated. Likewise, under these conditions, personnel are less likely to adhere to slow movements and aseptic techniques. Therefore, it is critical that the number of air changes is appropriate for the work being performed and for the number of people in the room.
When it comes to PPE, employees need to gown in the proper order before entering the cleanroom and all gowning materials need to fit properly. In the same respect, employees need to de-gown in reverse order if leaving the cleanroom for any reason. This is especially important if gowning materials, such as bunny suits or lab coats, are reused. The use of reusable gowning materials should be carefully analyzed in order to determine whether the cost savings are worth the increased risk of contamination. Employees also should be aware of the condition of their PPE, once gowned. They should inspect their gowning materials looking for holes or tears and all personal items such as keys, jewelry, cosmetics, and cell phones need to stay out of the cleanroom, as all of these harbor large numbers of microorganisms. Cell phones have become especially problematic, as employees will unzip bunny suits to access their phones exposing themselves and the environment. It is crucial that all personnel, including housekeeping and contractors such as certifiers, are trained and comply with institutional gowning requirements.
Specifics of Personnel Sampling
Compounding personnel are required to demonstrate proficiency in proper hand hygiene and gowning. This is also true for any support personnel, such as housekeeping, who may enter the cleanroom. All compounding personnel should be required to complete an initial sampling of gloved fingertips and thumb for both hands; the result must indicate zero CFU and this must be performed no less than three times prior to the pharmacist being allowed to compound. This sampling is to be done immediately after hand hygiene and gowning is performed, and be sure to remind those being sampled that they are not to disinfect their gloves prior to sampling. Once the initial gowning evaluation is completed, re-evaluation is required at least annually for those who compound low to medium risk and semi-annually for those who compound high risk. Fingertip sampling should also be completed after routine compounding on a regular basis.
To facilitate proper sampling, the employee is to lightly press four fingers and the thumb from one hand onto a contact plate filled with nutrient agar that contains neutralizers. If the agar is disrupted or cracked, the sample should be taken again. The plates should then be incubated at 30 to 35°C for 48 to 72 hours then at 26 to 28°C for five to seven days. The incubation at 26 to 28°C is not noted in the USP, but this is the responsible method of incubation. Each hand is to be reported separately, ie, CFU per employee per hand (left hand, right hand). The action level for gloved hands is based on the total number of CFU found on both hands.
Once data begins to accumulate it is important to evaluate for trends and establish alert and action levels specific to your facility. Sampling must be consistent in analysis, time, volume, media, and sites taken in order to recover meaningful data. Likewise, evaluation of environmental monitoring data needs to provide enough information to facilitate an investigation and show trends. If samples are taken on a monthly basis, data should be accumulated for at least one full year in order to incorporate seasonal, production, and facility changes into the baseline data. Samples taken just twice a year or less will require a much longer time period to gather enough data to evaluate for trends. It is worth noting that because of the inherent variability of environmental data, statistical analysis will vary depending on the type of data retrieved for your facility. Some facilities are unable to trend numbers because the analysis results in a high number of zeros. Therefore, there needs to be a change to the analysis to compensate for this, usually zero/non-zero trending. In addition, environmental monitoring data may need to be normalized before statistical analysis is started.
It is the responsibility of pharmacy to produce effective and safe drugs for patients and this can only be consistently accomplished by controlling the variables that can lead to contamination of the product. Exercising control begins by understanding and recognizing those factors that can contribute to contamination and extends through the development of approaches for controlling these factors in the cleanroom or aseptic area. As with any system, control is only as strong as the system’s weakest link, so while proper program development is essential, adhering to and improving the tenets of the program over time is key. A successful environmental monitoring and infection control program for the cleanroom trends data over time to recognize out-of-norm excursions, takes a proactive approach to correct potential and actual problems as soon as they are discovered, and instills procedures which will prevent them from re-occurring.
Kym Faylor is the director of Azzur Laboratories in Schnecksville, Pennsylvania. She has fifteen years’ experience managing a microbiology laboratory and assuring compliance with governing agencies, client specifications, and developing quality assurance aspects to conform to current advances in the pharmaceutical, medical device, and biotechnology industries. Kym has certification from the University of California, San Diego in regulatory affairs and is a certified quality auditor from the American Society of Quality. She has authored several publications and has served as a lecturer and trainer for USP <797> compliance.
Abby Roth is a microbiologist at Azzur Laboratories. Having worked in the pharmaceutical microbiology field since 2004, she is responsible for analyzing environmental monitoring samples and related client consultations, as well as microbiological examination of non-sterile product testing. Abby holds a BS in biology from York College of Pennsylvania, is a member of ASQ, and is a certified quality improvement associate. She recently spoke at the 2011 PDA Global Pharmaceutical Microbiology Conference on contamination control.
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