Despite ambiguities inherent in the regulatory inspection process, a few absolute expectations should be met
By Elaine Kopis Sartain, Steris Corporation
I recently attended a meeting with several members of the pharmaceutical, biotechnology and medical device industries. During our discussions of cleanroom decontamination, it quickly became apparent that the subject of disinfectant validation was a considerable source of both regulatory pressure and internal frustration. One of the key concerns was that regulatory authorities have provided very little guidance as to how they expect a disinfectant validation to look. Some also stated that what may be acceptable to one investigator might not be acceptable to another.
While I agree that there may be some variability inherent in the regulatory inspection process and consequently, some variability in what is considered an acceptable validation study, there are a few absolute expectations that anyone involved in disinfectant validation should meet.
A: Acquire thorough knowledge of your process before designing your validation protocol
One cannot validate a process without first understanding it. Knowledge in the following areas is essential for developing a disinfectant validation protocol:
Antimicrobial products (i.e., disinfectants, sporicides, and decontamination agents )
Environmental conditions (e.g., airflow, temperature, humidity)
Microorganisms: Include environmental isolates
When discussing microorganisms, regulatory agencies have been clear and consistent: isolates from the processing environment must be included in disinfectant studies. Because these are the microorganisms most likely to present a contamination risk to your product, it is critical tosubstantiate that your microbial control program is capable of controlling them. It is no accident that firms receive FDA-483 observations for failure to identify microorganisms: “Organisms are not identified…when counts exceed the firm’s established specification of…organisms recovered from gowning validations and routine monitoring are not identified.”1
Knowledge of antimicrobial products is important for developing protocol.
The fact that a microorganism was isolated may be an indication that your microbial control program is not capable of handling that particular organism. The only way to rule it out as the potential cause of the excursion (and thus prevent future excursions) is to identify the organism and to confirm that your disinfectant validation has substantiated performance against it or a very similar strain. Although all isolates need not be included in the disinfectant validation, there should be a valid rationale for the organisms that are included in the study.
What is sufficient microbial reduction?
Another question that must be addressed during protocol development is: What level of microbial reduction is sufficient to demonstrate satisfactory antimicrobial efficacy? The answer to this will not be found in the new Aseptic Processing Guideline or in any other regulatory document. However, the USP informational chapter 1072, Disinfectants and Antiseptics, In-process Revision, states the following: “In practice, sufficient organisms need to be inoculated on a 2-inch x 2-inch square of the surface being decontaminated, i.e., a coupon, to demonstrate at least a 2-(for bacterial spores) to 3-(for vegetative bacteria) log reduction…”2
It is important to consider that the microbial load on cleanroom surfaces, especially those that are kept dry, is extremely low to begin with; therefore, a 2- to 3-log reduction should be sufficient. Additionally, during testing the organisms are dried onto the surface and then recovered using additional steps, all of which may lead to reductions in recoverable and viable organisms. This makes it very difficult to prove higher log reductions. Suspension tests may allow for better recoveries and overall higher log reductions, but they do not provide substantive proof of performance under application conditions.
There are fairly significant differences in antimicrobial capabilities among different types of antimicrobial agents. More than one type is required to achieve the proper balance of effective microbial control, minimal substrate damage, and personnel comfort and safety.
Disinfectants typically contain a surfactant (synthetic detergent) and an antimicrobial agent (e.g., phenol). The surfactant allows the disinfectant to handle a fairly substantial soil load, making it an ideal product for heavy traffic areas such as floors. These agents are also designed for frequent use and are relatively safe for both personnel and surfaces.
Sporicides are typically formulated with highly reactive chemicals (e.g., sodium hypochlorite and peroxygen compounds) and may cause damage to surfaces, even stainless steel, if used too frequently. They are a “necessary evil” for cleanrooms, since routine disinfectants are not capable of controlling bacterial endospores or resistant molds (i.e., A. niger). However, they should be used judiciously to avoid damage to substrates and to reduce the potential for irritation to the personnel involved in cleaning operations.
More than one type of antimicrobial agent is required to achieve a proper balance.
Isopropyl alcohol (IPA) is capable of good broad-spectrum efficacy, but the manner in which it is routinely used (i.e., minimal contact time) and its flammability effectively limit its broad applicability as a primary mic robial control product. It is more appropriately classified as a decontamination or residue control agent.
All three product types are required to properly maintain a cleanroom environment and control microorganisms. Therefore all should be included in the disinfectant validation study, although they may not necessarily be tested against all of the same challenge organisms.
To some degree, antimicrobial products are only as effective as the manner in which they are applied. Cleanroom housekeeping guidelines generally emphasize the importance of “clean to dirty” application techniques.3 The purpose here is to ensure that a previously clean surface, such as a wall, is not inadvertently contaminated with residue from another surface (i.e., the floor) via the cleaning device and procedure. This technique is generally included in cleaning SOPs, but there are other application-related issues, just as important to the success of the microbial control program, that are not always well understood, documented, or executed. These include maintaining sufficient wet contact time and balancing the surface area-to-antimicrobial solution ratio.
Cleaning SOPs often indicate that a surface should be allowed to remain wet for a defined period (for example, ten minutes) after disinfectant application. This timing is often derived from product label instructions or from a validation study. However, seldom have I seen an operator actually time the application to determine if the “ten minutes” is being achieved. It is also rare to see a validation protocol in which a predefined wet contact time was qualified during in situ evaluations. What I have seen, however, is FDA-483 observations that make a point of this oversight: “The qualification of the various disinfectants used in sanitizing surfaces in the aseptic processing area (sterile core)…failed to assess the disinfectants in the manner that they are used, including …disinfectant exposure time.”4
The surface area-to-antimicrobial agent ratio is somewhat related to contact time in that if sufficient solution is applied to a surface, and environmental conditions (temperature, humidity, airflow) are accounted for, then sufficient wetting will be achieved to attain the desired contact time and thus, the expected microbial control.
Surface wetting effectiveness may also be related to the application device (e.g., wiper, mop, or sprayer) and to the technique. Therefore, application procedures and methods should be included in the disinfectant validation work, in order to avoid a citation: “…Disinfectant agents used to sanitize surfaces in the aseptic process areas (APA) have not been adequately qualified to assure that they provide the intended microbial decontamination when used in the manner as specified in standard operating procedures … The qualification study immersed the test surface in the disinfectant …instead of wiping the surface as specified in cleaning SOPs.”5
The actual cleanroom surfaces being cleaned also affect the antimicrobial efficacy of agents. At one time, surface studies were conducted using either stainless steel or glass almost exclusively. In fact, most industrial protocols (e.g., AOAC, EN) involve the use of suspension testing or stainless-steel panels. Over the last few years I have reviewed the results of disinfectant validation studies that document the differences in performance between suspension and surface testing methods and show that surface condition and type have an impact on cleanability (i.e., particulate removal efficiency). Consider the difference in surface texture between a cast iron skillet and a Teflon -coated pan; or more to the point, between a heavily-textured, epoxy-coated floor and a smooth, seamless, polymeric floor.
The regulatory authorities have made the same observations. They have made it clear that surface evaluations should be included in the disinfectant validation and that in vitro studies should include surface materials that represent the types of surfaces being cleaned: “The qualification of the various disinfectants used in sanitizing surfaces in the aseptic processing area …failed to assess the disinfectants in the manner that they are used, including types of surfaces disinfected…”6
Another consideration is whether or not the actual environmental conditions of the cleanroom can be captured during an in vitro study. Airflow, humidity and temperature are all important parameters that have an impact on wet contact time and consequently on disinfectant performance. Temperature is an especially important consideration where cold-room applications are involved. An attempt should be made to replicate these conditions during in vitro testing. For example, a laminar flow hood can be used for drying inoculated coupons.
B: Build a consensus on the validation protocol and prevalidation steps based on a solid scientific rationale and the best available guidance tools.
The steps leading to development of a protocol, and ultimately the protocol itself, should at least address the following questions:
Do SOPs need to be revised?
Which antimicrobial products are going to be included in the validation study?
Which microorganisms will be included in testing and what criteria will be used for selection of these microorganisms?
Which substrates will be included in surface studies and what criteria will be used to select the substrates?
What application techniques will be utilized for the surface studies?
Will suspension testing be conducted?
How will the in situ evaluation be conducted?
What recovery techniques will be used?
Will testing be done internally or at a contract-testing facility?
How will the testing facility be selected and qualified?
How will failing data be addressed?
How often and under what circumstances will revalidation be conducted?
The results from in vitro studies give an indication of how the disinfectants will perform under actual use conditions, but no matter how sound the in vitro protocol is, FDA-483 observations have shown that there is no substitute for evaluation under actual use conditions: “The Disinfection Qualification Testing Report was incomplete in that the study failed to simulate actual use (i.e., contact on production surfaces).”7 Hence most facilities will agree to conduct an in situ qualification as a part of the protocol.
The in situ study can be conducted under worst-case conditions, such as during a PM shut-down. This type of qualification generally involves sampling several locations, including a preponderance of worst-case locations, prior to and after disinfectant application. Data is subsequently reviewed to determine that the disinfectant effectively reduced the bioburden in those areas.
C: Correlate data gathered during the in vitro and in situ studies with results obtained during routine EM monitoring
This approach is also supported by the USP 1072 In-process Revision: “To demonstrate the efficacy of a disinfectant within the pharmaceutical manufacturing environment, it may be deemed necessary to conduct the following tests: … a statistical comparison of the frequency of isolation and numbers of microorganisms isolated prior to and after the implementation of a new disinfectant.”8
There is an “X factor” involved in disinfectant performance. This factor may be defined as the manner in which the disinfectant performs under use conditions when considering the following elements:
The condition of working cleanroom surfaces
The room environment (e.g., temperature, humidity), including seasonal fluctuations
The condition of application devices (e.g., integrity, bioload)
The compliance of cleanroom personnel (i.e., disinfectant preparation, application, cleanroom-appropriate behavior)
The cleaning frequency
The use of other, potentially interfering, substances in the room
The X factor may have tremendous impact on the success of a microbial control program. The only way to evaluate the impact of this X factor is by reviewing EM data and correlating the results to those obtained during qualification studies. Careful evaluation of this data should provide an indication of the origin of cleanroom microbial control problems and may help in the design of a roadmap toward greater regulatory compliance.
ABCs can yield results
Disinfectant validation is a precise and demanding function that must account for many potential variables in order to secure a validatable aseptic manufacturing environment. These ABCs address key issues raised in FDA-483 observations, and provide basic guidelines that can help to establish an effective validation protocol.
Elaine Kopis Sartain is the director of technical service for STERIS Corporation. She can be reached at [email protected].
1. GMP Trends, Issue No. 622, December 15, 2002.
2. USP Chapter 1072, Disinfectants and Antiseptics, In-Process Revision, Vol. 30 No. 6, Nov.-Dec. 2004.
3. IEST-RP-CC018.3 Cleanroom Housekeeping: Operating and Monitoring Procedures, December 2002.
4. GMP Trends, Issue No. 666, October 15, 2004.
5. GMP Trends, Issue No. 631, May1, 2003.
6. See reference 4.
7. GMP Trends, Issue No. 665, October 1, 2004.
8. See reference 2.