Determining Efficacy and Proper Methodology in Cleanroom Cleaning

Preventing contamination within classified areas of an operational cleanroom is a constant battle. Cleaning and disinfection programs help mitigate contamination by removing the microorganisms and particulates introduced into the classified areas during routine operations. In general, people are considered the number one source of contamination within a cleanroom, followed by materials and equipment that are transferred in and out of the classified areas. Following proper gowning and aseptic technique procedures help to mitigate contamination introduced by people. A properly developed and implemented cleaning and disinfection program is an integral component in ensuring a cleanroom is in a constant state of control.

Several components must be considered when developing and implementing a cleaning and disinfection program. To determine which cleaning and disinfectant agents will be used, firms must identify the natural flora within the facility. Performing baseline monitoring in the facility—otherwise known as in-situ monitoring—before cleaning any of the target areas ensures microorganisms can be recovered. Understanding what organisms are naturally present plays a significant role in choosing the appropriate disinfectants for a facility. The resistance of microorganisms to disinfectant agents can vary significantly. For example, vegetative bacteria are generally some of the easiest microorganisms to eradicate while mold and bacterial spores are some of the most difficult.

The effectiveness of a disinfectant depends on its intrinsic biocidal activity, the concentration of the disinfectant, the contact time, the nature of the surface disinfected, the hardness of the water used to dilute the disinfectant, the amount of organic materials present on the surface, and the type and amount of microorganisms present (1). Other key aspects to consider when choosing a disinfectant are the application method, ready to use vs. concentrate, how corrosive it is, and what the rotation of disinfectants is going to be. United States Pharmacopeia(USP) <1072>, Disinfectants and Antiseptics, recommends rotating a disinfectant with a sporicidal agent, typically on a monthly or weekly frequency, but sporicidal agents may cause corrosion of certain types of material if used too often (1). After selecting disinfectants, they must be evaluated to determine their effectiveness via a disinfectant efficacy study.

Determining efficacy

Disinfectant efficacy studies can become complex depending on the following criteria:

• Number of disinfectants
• Number of surface materials
• Duration of contact times for each disinfectant
• Number of challenge organisms that need to be included.

However, such studies demonstrate the bactericidal, fungicidal, and sporicidal efficacy of the disinfectant agents and are usually required by industry regulatory agencies.

The concept of the disinfectant efficacy test is to challenge the disinfectant agents by using a specific methodology that can produce measurable results. It is important to retain extra pieces of materials of construction from when the cleanroom is being built to make two-inch-by-two-inch coupons for the disinfectant efficacy study. The coupons are inoculated with enough microorganisms to demonstrate at least a two-log reduction for mold and bacterial spores and a three-log reduction for vegetative bacteria (1). A different coupon will be used for each organism and a predetermined contact time will be used for each disinfectant agent.

When choosing the challenge organisms, it is typical to begin by choosing from the ones listed in USP <1072>, such as Staphylococcus aureus, Bacillus subtilis, Aspergillus brasiliensis, Candida albicans, Pseudomonas aeruginosa, and Salmonella spp. These organisms represent a range of diverse organisms with varying resistivity to the disinfectant agents. However, most studies will tend to expand that list by selecting some of the organisms recovered during the baseline environmental monitoring (EM) and routine testing.

Once the criteria have been determined, the disinfectant efficacy study may begin. To briefly describe the process: coupons should be sterilized before the study to avoid contamination from other microorganisms outside of those selected for challenge organisms. Autoclaving is a typical sterilization method, but if materials are unfit for the autoclave, an alternative sterilization method must be used. Once ready, the coupons are inoculated on the upper surface with the predetermined amount of organism and then allowed to dry for a specified amount of time in an ISO 5 environment. The appropriate disinfectant is then applied to each inoculated coupon for a determined contact time. Each coupon is then swabbed using a neutralizing agent to stop the disinfectant activity and recover any surviving microorganisms. These swabs are placed into a test tube containing more of the neutralizing agent and agitated by vortexing them to remove the organisms from the swab. Log dilutions of this coupon wash are performed and plated either by pour plate method or membrane filtration onto appropriate medias. The plates are incubated at specified temperatures and durations of time, then the colony-forming units that develop are counted. A percent reduction is calculated by comparing the inoculum coupon control count plates to the plate counts from the coupon solutions. These results are analyzed to determine whether the minimum log reduction was achieved for each testing scenario.

Once the disinfectant study is completed successfully, the firms should keep the report document on file for future reference. The process of cleaning and disinfecting the classified areas should be specified in a standard operating procedure and documented every time the process is completed. Once the classified areas are in routine use, the effectiveness of the cleaning and disinfection program should be monitored on a routine frequency through an EM program.

Environmental monitoring

Establishing an EM program using a risk-based approach to determine the number of samples, where samples should be taken within the classified areas, and the frequency of sampling will help to maintain control of classified spaces. A failure mode and effects analysis (FMEA) risk assessment identifies actual and potential failure modes for a product or process. FMEA is both a qualitative and quantitative approach that anticipates potential process failures and attempts to provide mitigation factors to reduce the risk of these failures. The FMEA rating for risk is derived from three areas:

• Occurrence: the likelihood of the failure occurring
• Severity: references the consequences of the failure (effect on patient health, loss of time, loss of product)
• Detection: determines how likely the failure will be detected (a high detection rating can mitigate high occurrence).

Occurrence can be broken down further into two subcategories:

• P1: the probability of microbial contamination related to the environment. Hard-to-clean areas, areas near return vents, areas with high personnel and material traffic, and areas adjacent to uncontrolled spaces.
• P2: the probability of microbial contamination related to the product. Factors such as length of process time, process criticality, and areas where significant product activity will be considered during the scoring of P2.

This is due to the differentiation of contamination risk. Risk to product and risk from the environment are often mutually exclusive and should be assessed separately. Each risk category is given a numerical range from 1–4.

Risk is determined by multiplying each risk category to create a risk priority number (RPN). Therefore, probability occurrence (P1) x probability occurrence (P2) x detection x severity = RPN.

A risk priority can be determined without environmental monitoring (pre-EM) as a source of detection, and then later scored after the implementation of environmental monitoring (post-EM) at each sample location to show the risk reduction with the implementation of EM to increase detection of microbial contamination. Each sample is scored using this system and that score is used to justify its location within the classified area.

Once the risk assessment is completed, generate a sample location map for the performance qualification (PQ) and routine EM. A performance qualification protocol should outline the necessary qualification activities for areas that are usually classified as ISO 5, 7, or 8. The PQ testing determines whether the areas being tested are performing as intended and meeting the criteria defined in the protocol under both static and dynamic conditions. Various guidance documents may be referenced to determine the criteria for viable air and surface samples along with the non-viable particulates, such as USP <1116>, European Annex 1, and ISO 14644-1.

ISO-14644-1:2015 focuses on the classification of air cleanliness defined in table one of ISO 14644-1 (2) While ISO 14644-1 is strictly for non-viable particulates and room classification, the other two guidance documents, USP <1116> and European Annex 1, provide criteria for viable surface and air samples as well (1). If the criteria in these documents are used, those values will typically be considered the action levels for the PQ and the routine testing that immediately follows. However, once enough historical data is accumulated (typically, in one year) alert levels can be established, and the action levels can be revised based on these data as well.

A PQ normally will follow the room certification and consist of a specified number of days of at-rest (static) conditions followed by a specified number of days of operational (dynamic) conditions. The number of static and dynamic condition testing days can vary depending on a company’s needs. The EM usually encompasses non-viable particulate air sampling, viable air sampling, and viable surface sampling. Locations of each sampling type are determined by the risk assessment and are typically plotted on a diagram of the room to create a sample map. Viable air and surface sampling is performed using trypticase soy agar (TSA) media and sabouraud dextrose agar (SDA) media. TSA is a broad-spectrum general growth media formulated for bacterial growth, while SDA is formulated to better detect fungal growth. The two types of media are incubated at specified temperatures and durations to promote the growth of any microorganisms that may have been transferred to the media when sampling. After the PQ, implementing a routine EM program will aid in continually monitoring for any adverse microbial trends. Routine testing will help continually monitor the effectiveness of the cleaning and disinfection program. Implementing these critical components of the cleanroom program will help to ensure the cleanroom will operate properly and contamination levels will be minimized.

There will be some cost and time commitments associated with conducting these measures but, by taking the proper steps and implementing these critical components at the appropriate times, they will help mitigate contamination within the cleanroom areas. These steps are necessary to maintain a compliant cleanroom facility and to help ensure the integrity of the products being made within them.

References

1. USP, USP General Chapter <1072>, “Disinfectants and Antiseptics,” USP 43-NF 38 (Rockville, MD, 2022).

2. ISO, ISO 14644-1, Cleanrooms and associated controlled environments — Part 1: Classification of air cleanliness by particle concentration (2021).

About the authors

Ryan Burke is the environmental monitoring manager, and Madison Prifti is the senior environmental monitoring analyst, both for Element Materials Technology.