By Crystal M. Booth, PSC Biotech
Microbial control is critical in cleanroom environments. Contaminated environments can lead to product recalls, regulatory observations, fines, or even consumer deaths. To prevent, destroy, and monitor microbial contamination in cleanrooms, several aspects of cleanroom microbiology must be understood. This foundational introduction to cleanroom microbiology article series discusses some of those aspects. Parts 1 and 2 introduced cleanroom microbiology, discussed guidance documents and FDA observations, and summarized common sources of microbial contamination in cleanrooms. Part 3 provided an overview of cleanroom gowning procedures. This final article will address concepts of environmental monitoring and the importance of disinfectant efficacy and proper cleaning.
According to 21 CFR 211.113(b): “Firms must follow appropriate written procedures designed to prevent microbiological contamination of drug products claiming to be sterile.” In order to control microbial contamination, the sources must first be understood. Environmental monitoring, utility monitoring, and product testing are used to gain an understanding of these sources. For environmental monitoring, the air is sampled for viable and nonviable particulates. In addition, personnel and surfaces are sampled for microbial contamination. If microorganisms are recovered, their identification can be used to help identify the potential sources of the contamination and any corrective and preventive actions that may be required.
Monitoring trends in the environment and facilities assists in ensuring that procedures for preventing contamination are effective and that the facility is in a state of control. Other benefits include allowing the facility to be proactive by identifying areas of concern before an out-of-limits event occurs, helping to establish relevant alert levels, helping to establish a list of predominant microorganisms and resident flora of the facility, and providing historical data so that abnormalities in the data can be detected.
It is important to provide a clear definition of what a “trend” looks like. This definition should be included in procedures to provide consistent interpretation across the facility and over time. In general, a trend is understood to be a shift from historical data or a shift in a specified direction.1
Alert levels should be used as an early warning system for when processes are drifting from their established state of control. Utilizing 50 percent of the action level without proper justification is not scientifically sound and can be frowned upon during regulatory audits. Statistical analysis can be performed on trend data to establish alert limits based on the process capabilities of the facility. For example, one can utilize historical data and establish a 95 percent confidence interval, where 95 percent of samples taken will be expected to be within the proposed alert limits.
The majority of expected action levels have been established in multiple regulatory documents. In cases where action limits are not described by regulatory bodies, such as personnel gowning qualifications (other than gloved hands), risk assessments and statistical analysis may provide justification for establishing action limits.
Contamination recovery rates are described in USP <1116>. It is a useful tool for trending results that frequently contain zero colony forming units (CFU), like in aseptic processing environmental monitoring. USP <1116> states that “Because of the inherent variability of microbial sampling methods, contamination recovery rates are a more useful measure of trending results than is focusing on the number of colonies recovered from a given sample.”2 The contamination recovery rate is calculated by taking the total number of plates containing growth of any kind, divided by the total number of plates, times 100 to give a percentage. USP <1116> also provides a table for suggested initial contamination recovery rates in aseptic environments.
Disinfectant Efficacy And The Importance Of Proper Cleaning
Control of the introduction of contamination into the cleanroom is the most critical concern for the entire cleaning and disinfection process.3 The cleanliness of components, personnel, carts, tanks, tools, and instruments that are transferred into the clean area is of upmost importance. If these items are not properly disinfected or cleaned, or if personnel are not properly gowned, microorganisms can be easily transported into the cleanroom. Multiple guidance documents and articles are available that discuss disinfectant efficacy testing. Two very good sources include USP <1072> and the PDA technical report 70.
Items must be cleaned before they can be disinfected or sterilized. Cleaning is the process of removing foreign material from objects, and it is necessary for disinfectants to work as intended.3 Foreign material that is not removed may block the disinfectant from coming into contact with microorganisms on the surface. Foreign materials may also negatively react with disinfectants and inactivate them.3
Difficult to clean areas are governed by 21 CFR 211.42. The regulation requires smooth, hard surfaces that are easily cleanable. Porous surfaces can harbor microorganisms, making it difficult to recover them for environmental monitoring. Porous surfaces can also make it difficult to destroy microorganisms during cleaning and disinfection.3 Hard to reach surfaces pose the risk of not being cleaned properly.3
Rushing the cleaning process, being sloppy, or doing it incorrectly are dangerous practices. Even worse is skipping the cleaning process entirely. Dirt, oil, fingerprints, and disinfectant residues that are left behind create hospitable habitats for microorganisms or even protect underlying microorganisms from applied disinfectants.3 It is extremely important to follow SOPs and clean properly to remove dirt sources, even if they are not visible to the naked eye. This will help to ensure that the disinfectants can work properly.
Appropriate concentrations of disinfectants are established during the disinfectant efficacy study. If disinfectants are not properly prepared, handled, or used, their effectiveness to destroy microorganisms can be compromised.3 This can lead to the survival of microorganisms. The effectiveness of disinfectants can be compromised if they are not applied and utilized correctly. For example, shortcutting the established disinfectant contact time can lead to microbial survival.3 Even if disinfectant solutions are properly made and changed frequently, dirty equipment, such as mop heads or buckets, can impact the effectiveness of the disinfectant and lead to microbial survival.3 The contamination may be unknowingly spread throughout the facility. It is also important to ensure the cleaning equipment is clean and properly maintained.
Disinfectants must be qualified for use in the facility. When qualifying the disinfectants, the testing should include a variety of surfaces found in the facility that represent the worst-case porosity and the most difficult to clean surfaces.3 The microorganisms chosen for the study should include environmental isolates from the facility, when possible.3 During the study, the suggested contact times will also be established. The term “contact time” refers to the amount of time that disinfectants will remain wet and in contact with the surface being disinfected.3
The performance of the disinfectant should be evaluated throughout the in-use period to establish the in-use expiration dates. PDA technical report 70 recommends using disinfectants that are close to or slightly beyond their expiration date for the study. It is important to note that disinfectants should be sterile-filtered or sterilized prior to use in Grades A/B to prevent the disinfectant from being a source of contamination that can be spread throughout the cleanroom.3
Disinfection efficacy testing and disinfection programs are usually hot topics in regulatory audits. To make the process move along smoothly, be prepared and have all the data summarized and neatly organized in clean reports.
Cleanroom microbiology encompasses a variety of subjects. Multiple guidance documents and regulations are available referencing cleanroom microbiology concepts. To avoid potential regulatory findings, be proactive and research warning letters published online, and correct any noted procedural gaps as soon as possible.
To help regulate microbial contamination, all activities that happen in the cleanroom must be controlled, including processes, procedures, people, raw materials, excipients, components, APIs, the facility, equipment, machines, environment, surfaces, air supply, utilities, water sources, material transfer, adjacent areas, and disinfectant and cleaning regimens. Employees must follow procedures, use proper aseptic technique, pay attention to details, and use utmost care each time they deal with aseptic products. A consumer’s life could depend on the employee’s behavior, their integrity, properly controlled cleanrooms, and cleanroom microbiology principles.
- Trend. (n.d.) American Heritage® Dictionary of the English Language, Fifth Edition. (2011). Retrieved May 11, 2017 from http://www.thefreedictionary.com/trend
- USP <1116> Microbiological Control and Monitoring of Aseptic Processing Environments
- PDA Technical Report No. 70 – Fundamentals of Cleaning and Disinfection Programs for Aseptic Manufacturing Facilities, Parenteral Drug Association, Bethesda, MD (2015)
About The Author:
Crystal M. Booth is president of Azzur Labs, LLC. She has over 19 years of experience in pharmaceutical microbiology, working in quality assurance, CDMOs, R&D, and quality control laboratories, including startup companies. During her career, she has developed and validated methods for antibiotics, otic products, topical creams, topical ointments, oral solid dose products, oral liquid dose products, veterinary products, human parenterals, vaccines, biologics, aseptically filled products, and terminally sterilized products. Those methods include microbial limits testing, bacterial endotoxins testing, particulate testing, sterility testing, pharmaceutical water system validations, EM programs, surface recovery validations, disinfectant efficacy studies, minimum inhibitory concentration testing, antimicrobial effectiveness testing, hold time studies, and various equipment validations. Booth earned her bachelor’s degree in biology from Old Dominion University and her master’s in microbiology from North Carolina State University.