By Art Vellutato, Jr., Veltek Associates, Inc.
Developing a compliant and effective cleaning and disinfection system for controlled environments in pharmaceutical and biotechnology operations requires firms to consider the most important element of what is to be accomplished: the development of a system that attains repeatable results.
Too many times in this industry, firms haphazardly focus their efforts too narrowly on just one aspect of the requirements relating to cleaning and disinfection. By doing so, the system is not complete and its lopsided nature proves a failure. The experienced cleaning and disinfection professional knows this story all too well. At times, quality assurance departments worry too much about validation data and forget how personnel will carry out cleaning procedures. Likewise, production personnel sometimes forget what the quality assurance and validation departments have proven through their testing efforts to be critical during the cleaning process. And thus, the lack of awareness of the necessity for a complete system by all involved undermines the success of the task at hand.
What is a complete system?
A complete system revolves around six basic principles. They are:
1. Controlling what contamination enters the facility
2. Cleaning the controlled areas
3. Conducting and carefully reviewing environmental monitoring data
4. Using environmental monitoring data to determine the validation protocol for sanitizers, disinfectants and sporicides
5. Validating the mechanical cleaning and disinfectant application methodologies
6. Training and supervising personnel
Carefully reviewing each principle and creating a well-defined action list to address each element will prove the basis for success.
Controlling what enters the environment
A complete system starts with controlling what enters the facility. Most GMP operations struggle with this task each and every day. Simple reasoning tells us that if one reduces contamination entering the controlled environment, then its presence doesn’t have to be addressed. If contamination is controlled, the cleaning and disinfection process becomes much simpler since, as existent particulates and microbes are reduced, cleaning and disinfection efforts can also be reduced. Although it’s a simple concept, it’s more difficult to implement than it sounds. However, on a scale of importance, the control of particulates and microbes entering a controlled environment is probably the most critical area of concern in the entire cleaning and disinfection process. Controlling contamination from introduced components, personnel, carts, tanks, tools and other sources is also important. Lack of control over the cleanliness and sterility of items entering the clean space can compromise the environmental conditions during manufacturing, particularly as relates to the introduction of particulates and microbes, and the remains of dead organisms, or pyrogens. In the industry, pyrogens have proven to be an equal threat to live cells. Fevers and even death have been attributed to their presence. There are two ways to eliminate a pyrogen: disintegrate it at high temperatures using a dry-heat process called depyrogenation, or clean it. Depyrogenation of cleanroom surfaces is not possible, so pyrogens must be cleaned from the surface like a particulate.
There are two high-risk time periods when contamination enters the cleanroom. The most critical time is after disinfection and prior to the beginning of manufacturing. Production personnel enter the just-cleaned facility and riddle it with particulates and microbes that may shed from their entry and whatever components they bring into the room. Such particulates and bioburden will now exist in the area during manufacturing. Although it is well understood in the industry that, because it is an aseptic process and not a sterile process, the area will always have some level of particulates and bioburden in it, the goal in the aseptic cleanroom is to reduce bioburden to its lowest level.
The other high-risk time is after manufacturing and before cleaning. In general, we are less conscious of cleanliness after the manufacturing is complete. And so, breaches tend to occur during this time.
To reduce particulates and microbes, we must first define the cleanliness and sterility (or disinfection when sterilization is not an option) of what we bring into the environment. Those responsible for developing disinfection systems need to first make a list of every item and/or person that enters the controlled area, then evaluate each to determine whether it is clean and appropriately sterilized (or disinfected) prior to entry. Organizations that institute a very strict entry system for all items and personnel have fewer excursions.
Cleaning the controlled areas
Too often, cleaning is confused with disinfection. They are not the same. Cleaning removes contamination from the surface while disinfection attempts to destroy what viable cells exist on the surface. Consider a toothbrush and mouthwash, for example. If we were to discuss with our dentist the option of not using a toothbrush anymore and only utilizing a daily mouthwash rinse, he/she would inform us that we would soon have no teeth. Residues, particulates and microbials would build up on the surface of our teeth, which would eventually deteriorate. This scenario illustrates what occurs too often in the pharmaceutical and biotechnology industry: We forget to “brush” and instead try to kill anything that exists on the surface. Every day we coat the walls with disinfectants and sporicides, but do we ever clean them? The answer is rarely. Eventually surfaces become residue-laden and more difficult to disinfect, and ultimately will begin to deteriorate. And if they do, a major and costly shutdown will be required.
The removal of particulates, microbes and possibly existent residues from surfaces is characterized as cleaning, and it requires that a non-destructive mechanical action be applied to loosen and remove contaminants from the area. Procedurally, contaminants and residues are loosened and rinsed to the floor. Subsequently, the dirtied solution on the floor is collected and removed from the area. By lessening the level of particulates, microbes and residues on the surface, disinfection efforts become simpler. First, there are fewer organisms to destroy because most have already been removed from the area. And secondly, as bioburden and residue levels are decreased, the possible obstructions blocking the chemical agent from contacting the organism are minimized. In short, cleaning prepares a surface for disinfection.
Disinfection relates to the saturation and penetration of the cell wall of an organism by a chemical agent. It further requires that an organism remain wetted for a specified contact time with a chemical agent capable of killing the organism in question. Disinfection depends upon temperature, saturation and penetration of the cell wall, contact time, surface and bioburden of the surface, existent soil load, concentration of the chemical agent, and pH. Provided the appropriate chemical agent is utilized, the key to disinfection in the cleanroom is keeping the surface wetted for 5 to 10 minutes. This can sometimes be difficult, however, because the movement of air via laminar flow tends to dry surfaces more quickly.
The effect of the build-up of residues, particulates and possibly microbials is also exacerbated by the surface itself. Cleanroom surfaces are irregular in nature as (see Fig. 1) and trap residues and other contaminants, making the surface more difficult to disinfect.
Environmental monitoring and data review
Through an environmental monitoring program, isolates will be recovered. The list of isolates is normally recorded and trends identified. The predominant recurring cells will be used to validate our sanitizers, disinfectants and sporicides. Normality is to test 6 to 8 organisms of varying types. The selection is based on organism type and frequency of occurrence. Careful review of trending data now and over time is critical to gauge how effectively we are addressing bioburden in our controlled environments. As time progresses, we may notice a recurring organism that has not been tested. If this occurs, the organism is subsequently tested and a determination is made regarding the effectiveness of the cleaning agents.
At the same time, a review of the isolates will help determine which sanitizers, disinfectants and sporicides we will require in our system. Normality is to qualify one sanitizer (either a 70 percent isopropyl alcohol or 70 percent denatured ethanol solution), one disinfectant (a phenol, a quarternary ammonium or a hydrogen peroxide) and one sporicide (a sodium hypochlorite, a sodium hypochlorite-based product, a peracetic acid and hydrogen peroxide, or a gluteralde-hyde product) that will be rotated on a given frequency.
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In very simple terms, of the three products, a sanitizer has the lowest ability to destroy vegetative cells. A disinfectant destroys a broader spectrum of vegetative cells than a sanitizer does, and incorporates some log reduction of spores. A sporicide, however, destroys all vegetative cells and spores. Specifically, the United States Environmental Protection Agency (EPA) under FIFRA, CFR Title 40, governs and registers by U.S. law antimicrobial effectiveness claims for hard-surface disinfection. Table 1 gives a basic overview of what is required of a disinfectant manufacturing company by the U.S. EPA in order to make certain claims on marketed products.
When choosing a sanitizer, 70 percent isopropyl alcohol (IPA) has been proven to be more effective against cleanroom isolates than 70 percent ethanol (EtOH) solutions, which have a greater impact on viral cells. Alcohol solutions are used during cleaning operations and as a sanitizer during manufacturing.
When selecting a disinfectant, one needs to choose one broad-spectrum agent that incorporates a surfactant (detergent) in its formulation to help clean surfaces. In the past, it was traditional in the industry to rotate two disinfecting agents to counteract the theoretical build-up of a cell’s resistance to a particular disinfectant. This became predominant in the early 90s. However, since 1999, most firms have discounted this theory since no conclusive data has ever been published to prove that such resistance should be a concern. Now the industry standard is to use one disinfecting agent on a routine basis and supplement its lack of sporicidal kill with a sporicide that is also used on a routine basis. In reality, the disinfectant/sporicide rotation is a stronger prescription for bioburden than the past practice of rotating two disinfecting agents.
 Figure 2. Because vapors and residues that are characteristic of some disinfectants and sporicides pose a risk of contamination, they cannot be used during manufacturing. |
In today’s world, most firms choose either a low pH phenol, a quarternary ammonium or a hydrogen peroxide solution as their routine disinfectant. Normal frequency for application is dependent upon the environmental monitoring data. Normality in the industry is to clean surfaces daily to every few days to weekly (dependent upon the classification of the area). The disinfectant is used during cleaning operation but not during manufacturing due to possible product contamination from vapors or residues that are characteristic of these products (see Fig. 2).
For a sporicide, the industry normally chooses either a sodium hypochlorite at 0.52 percent or a mixture of peracetic acid and hydrogen peroxide. Of the two agents, sodium hypochlorite at 0.52 percent is more predominantly used on structural cleanroom surfaces. The sporicide is used on a routine basis (maybe weekly or monthly) and its use is determined by environmental monitoring data. Prior to using the sporicide, most firms use a cleaning agent that incorporates high surfactant (detergent) characteristics. As discussed earlier, this cleans and prepares the surface for disinfection. Again, the sporicide is used during cleaning operations but is not used during manufacturing due to residue issues.
Using environmental monitoring data to determine validation protocol
To test antimicrobial effectiveness, we will culture the chosen isolates to an enumeration nearing 1.0 x 104, though some may argue a higher enumeration is needed. The justification for the stated enumeration is that when RODAC samples are taken in a Class 100 (Grade A, ISO 5) area, most firms set their alert levels at 1 to 2 colony forming units (CFUs). In Class 10,000 (Grade C, ISO 7) areas, the alert levels are normally 5 to 10 CFUs and in Class 100,000 (Grade D, ISO 8) areas, most firms have set the alert levels at 25 to 50 CFUs. If we use the worst-case scenario of 100 CFUs, we realize that testing an enumeration nearing 1.0 x 104 equates to testing a bioburden level near 100 times higher than we will ever encounter.
At this juncture, a decision needs to be made whether to use the actual environmental isolate from internal stock cultures or an ATCC (American Type Culture Collection) culture purchased from an external source that matches the isolate we have identified. In this instance, the best choice is to use the actual environmental isolate that we have captured in our testing. The use of the in-house isolate meets the expectations of the FDA, EMEA and other agencies. Per the FDA’s Sterile Drug Products Produced by Aseptic Processing guideline: “Routinely used disinfectants should be effective against the normal microbial vegetative flora recovered from the facility.” And so, the isolate may be preferred and a more calculated and scientific choice than an ATCC culture.
The next step is to determine what test protocol to use for antimicrobial effectiveness testing. There are three basic protocols that are used as a template. The first protocol is defined as a suspension test. In this test, we culture the organism and pipet the prescribed volume directly to a test tube containing the sanitizer, disinfectant or sporicide. We then allow the organism to remain in suspension in the test tube for the required contact time and then either perform a serial dilution from 100 to106 in a neutralizing agent or filter the disinfectant/organism test tube onto a filter and rinse with the neutralizing agent. Subsequent plating of the positive and negative controls together with the neutralization confirmation and the test sample are completed. In this test, the organism will be surrounded on 360° by the disinfecting agent. In real-life situations in the cleanroom, however, this will not be the case. Rather, organisms will reside on surfaces.
Our second choice in protocols, the “time-contact-kill study” on carrier surfaces, may more precisely identify how to destroy viable cells in the cleanroom setting. The time-contact-kill study tests dried organisms on surface carriers made of varying material substrates from the facility. Some of these may be stainless steel, aluminum, epoxy, vinyl, plastic, terrazzo, mipolam, kidex, and others. Normally 6 to 7 surfaces are chosen. Once dried, the entire carrier is submersed in the test tube containing the disinfectant and subsequently (after the desired contact time) the solution is filtered and rinsed with the neutralizing agent, or a serial dilution is performed in the same manner as the suspension test. Subsequent plating of the positive and negative controls together with the neutralization confirmation and the test sample are completed. The time-contact-kill study represents real-life scenarios and is the best methodology to employ. Because we will clean surfaces that may be laden with microorganisms, this test emulates an actual in-operation scenario.
The last choice would be an Association of Analytical Chemists (AOAC) protocol test, which has existed for years and is used primarily for registration of an antimicrobial product with the U.S. EPA. In Europe, the new E.U. Biocide Directive looks for registering firms to conduct both the suspension test and time-contact-kill study. Registering a product as an antimicrobial agent is another venue, and its requirements are much more stringent than would be required to validate a sanitizer, disinfectant or sporicide in a GMP controlled environment. And thus, this testing should be avoided in GMP operations.
Validating mechanical cleaning and disinfectant application methodologies
We have proven in a controlled laboratory test that a validated sanitizer, disinfectant or sporicide can destroy a known enumeration of microorganisms on a carrier surface, but what happens when we combine the validated sanitizer, disinfectant or sporicide with our cleaning SOPs and our personnel? Will they all work together to attain success? Having validation data for the antimicrobial effectiveness of the agents does not guarantee the area will be cleaned and disinfected; therefore, most firms conduct an in situ or field study. The field study first tests a dirtied room for bioburden (via RODAC testing and microbial air testing). Then one of the approved disinfectants or sporicides, in conjunction with cleaning SOPs and personnel, are used to clean the area. Sanitizers such as IPA are not tested in this fashion but are rather a complement-such as an IPA wipe-down after the disinfection or sporicidal application. Upon completion, the area is monitored again. This is done separately for each disinfectant and sporicide we have approved in our scope, starting with a dirtied room each time.
At the same time, we must also consider what our disinfection regime will be if we encounter an excursion or after returning from a shutdown. If an excursion occurs, we know we have a problem and our scope may need to be grander than just applying a disinfectant or a sporicide. For example, maybe it requires cleaning the surface and then applying a sporicide. After a shutdown, we know we have encountered a major breach to the area: Construction, filter replacement, improperly gowned personnel traffic and other sources of contamination have all corrupted the environment. Normality among GMP firms is to use a multistep cleaning and disinfecting regime to bring the environment to acceptable levels. This testing may, as an example, first use a cleaner, followed by a disinfectant, followed by a sporicide. Again, a dirtied room is used and monitored first, then cleaned with the first cleaner and monitored again. After using the disinfectant and the sporicide, the room is monitored again. In the end, we hope to attain an acceptable environment. If not, we need to alter the scope so that we can meet such requirements.
Training and supervising personnel
One of the most disheartening endeavors any quality assurance professional can encounter is the audit review of the actual cleaning and disinfection procedures being done in the facility. Many times, cleaning and disinfection are done by the third shift. Those who develop the system and conduct the validation testing are not normally present for the actual cleaning. When one watches what is being done, many times the thought pattern shifts to, “What the heck are they doing?” Appropriate and repeated training, coupled with continued supervision, is critical to success. Most firms train personnel yearly, but this is not frequent enough-it should be conducted routinely at shorter intervals. Reinforcement is critical in this venue, and personnel and their supervisors naturally become lax over time. Without careful attention to training and supervision of personnel, the best-designed system will fail.
Conclusion
Developing, validating and assuring the implementation of an overall system for cleaning and disinfection is critical. Without a system-based approach, the efforts will be thwarted by problematic situations. The control of contamination, the review of environmental monitoring data, the conducting and review of validation data, the appropriate application, and continued supervision and training all combine to ensure success.
Art Vellutato, Jr. is vice president of technical support operations and one of the two founders of Veltek Associates, Inc. (VAI; Malvern, Pa.), an EPA- and FDA-registered facility founded in 1981. He is also the President of Aseptic Processing, Inc., one of the leading consulting groups in the industry. Mr. Vellutato is a frequent industry speaker with twenty-six industry publications and is one of the leading consultants specializing in cleaning and disinfection in the pharmaceutical and biotechnology industry. He has over nineteen years of experience, including his tenure as the director of quality assurance at VAI for nine years, and as the director of manufacturing for six years. He can be reached at (610) 644-8335 x110, or via e-mail at [email protected].
Resources
1. Vellutato, Arthur, Jr. “Implementing a Cleaning and Disinfection Program in Pharmaceutical and Biotechnology Clean Room Environments,” Laboratory Validation, A Practioner’s Guide, Davis-Horwood Publishing, pp. 170-230.
2. Center for Drugs and Biologics and Office of Regulatory Affairs, Food and Drug Administration. “Guidance for Industry, Sterile Drug Products Produced by Aseptic Processing,” Current Good Manufacturing Practice, September 2004.