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. In order to properly prevent, destroy, and monitor microbial contamination in cleanrooms, several aspects of cleanroom microbiology must be understood. This foundational introduction to cleanroom microbiology discusses some of those aspects.
Part 1 of this article provided an introduction to cleanroom microbiology, discussion of guidance documents and FDA observations, and a summary of common sources of microbial contamination in cleanrooms. This part will address some fundamental concepts in cleanroom design, as well as some considerations for proper material transfer.
Basics Of Cleanroom Design
Properly designed facilities help control microbial contamination. There are multiple regulatory guidance documents on cleanroom design, including the EU Annex 1 and FDA 21 CFR 211.42. Annex 1 talks about minimizing particle and microbial contamination by having an appropriate environmental cleanliness level in the state of operation.
FDA 21 CFR 211.42 addresses design and construction features. According to the regulation:
- The facility must be of suitable size, construction, and location so that cleaning, maintenance, and operations can be properly performed.
- The facility must have adequate space and flow design to prevent mix-ups and cross contamination.
- Surfaces must be easy to clean.
- Temperature and humidity must be controlled as appropriate.
- Air must be positive pressure and HEPA filtered.
- Environmental conditions must be monitored.
- Room and equipment must be cleaned and disinfected properly.
- Equipment must remain in aseptic conditions.
The design of a cleanroom is typically established moving from the dirtiest areas (uncontrolled environments) to the cleanest areas (Grade A environments). As the room classifications increase, the requirements for gowning, cleaning, monitoring, and material transfer also increase. The classification of the room will depend on the process designed to occur in the area. For example, a Grade A room is usually used for aseptic and high-risk operations.1 Grade B rooms are usually the background rooms to Grade A rooms.1 People present in Grade A/B areas must be in full aseptic gowns. Grades C and D rooms are used for less critical operations. Grade C rooms are typically used for compounding aseptic products or for filling terminally sterilized products.1 Grade D rooms are typically used for compounding terminally sterilized products or washing equipment for manufacturing.1 The specifications for the room classifications are listed in the relevant guidance documents. Annex 1 from the EU provides the specifications for Grades A-D, and ISO 14464-1 provides the ISO specifications for cleanrooms and associated controlled environments.
By using a stepwise grade design, contaminates can be left behind or destroyed in the lower classified areas, which helps to maintain the cleanliness of the higher classified areas and minimize microbial contamination. This is often accomplished through material transfer procedures, gowning rooms, pass-through areas, support rooms, controlled access areas, and positive air pressure cascades. Access control of the areas helps to prevent employees who haven’t been properly trained from entering the controlled environments. In addition, by using the stepwise grade design, logical and sequential flow of personnel, materials, waste, and product can also be established.
Grade A areas are designed to be the cleanest. They have more HEPA-filtered air pumped into them than the surrounding areas and are used for high-risk operations, such as aseptic filling.1 The higher exchange rates of the HEPA-filtered air help to decrease the potential for microbial contamination during aseptic operations. Typically, HEPA filters are in the ceiling and the air is cascaded downward, providing the blanket of first air or clean air. The air then enters return air ducts and leaves the cleanroom.
People and objects present in the cleanroom areas create turbulence in the air flow patterns. The floor is considered the dirtiest part of the room. People walking can potentially stir up microorganisms that have settled on the floor. When working in clean areas, it is extremely important to move slowly and deliberately, avoid shouting to colleagues, and never break first air by reaching over objects required for aseptic manufacturing.1
One of the most successful means of controlling microbial contamination is the engineering to remove people from the process, such as through isolators or restricted access barrier systems (RABS). While the insides of both a RABS and an isolator provide a compliant Grade A environment for aseptic manufacturing, isolators provide the most protection for the product. Isolators are fully enclosed, and the inside can be decontaminated prior to manufacturing. The background environments for isolators can be downgraded, while a RABS still needs to operate within a Grade B environment with fully gowned operators.1
Not only are environmental controls required per the regulations, they also serve an important purpose for product safety. In terms of cleanroom microbiology, temperature control is required to provide stable conditions for materials and important instruments as well as for the comfort of personnel.1 Humidity control is necessary to prevent microbial proliferation, eliminate static electricity, and, again, provide a comfortable working environment.1 If an operator is uncomfortable, e.g., too hot or exposed to too much humidity, they may sweat. Personnel sweating through gowns can compromise the integrity of the gowns and introduce microorganisms into the manufacturing environment.
Proper Material Transfer
The primary goal of a material and personnel transfer program is to prevent microbial contamination from entering the cleanroom. Personnel are required to follow established gowning procedures and hygiene requirements to enter the cleanroom.1 Improper hygiene can lead to increased skin cell shedding and microbial contamination of the cleanroom. Open and honest communication with management is needed, and any sicknesses, open wounds, or compromised skin layers, such as sunburn or fresh tattoos, should be reported.1
The flow of personnel and materials into the cleanroom must follow established procedures and dedicated pathways. Typically, separate dedicated pathways are established and utilized to prevent cross contamination. Personnel can stage materials in a pass-through area and move to a separate gowning entry point. While the personnel gown, the material surfaces are disinfected in accordance with a validated disinfectant (or ultraviolet light, if used) contact time.
It is critical to adequately control materials that are transferred into classified clean areas to prevent the influx of contaminants.1 There are many considerations for proper material transfer, including the following:
- Generation of particles and microorganisms should be minimized when possible.
- For example, cardboard and wood pallets can shed and introduce mold into the facility. Those types of items should not be allowed into the cleanroom.
- Materials being transferred must be compatible with the disinfectant or transfer method being employed.
- For example, purchased media plates should not be transferred by using a double-sided autoclave, as the plates will be destroyed.
- Storing items on the floor should be avoided, as the floor is considered the dirtiest area.
- Instead, if items need to be stored, they should have dedicated locations on shelving.
- Items that are stored must contain proper cGMP labels, including the contents and expiration dates.
- Do not allow nonsterile materials, including active pharmaceutical ingredients (API) or raw materials, to be introduced into the cleanroom.
- Carts are usually essential, especially when transferring environmental monitoring equipment.
- When using carts, it is essential to ensure that they are properly disinfected, including the wheels of the carts. The wheels of the cart are exposed to dirty environments and if not cleaned appropriately, can transfer microorganisms into the cleanroom.
- The best practice is to have dedicated carts. Materials would be transitioned from the general use cart to the dedicated cleanroom cart inside the pass-through.
- Waste should be removed as soon as possible and should not come in contact with clean items.
- A separate dedicated waste pathway is a good practice to avoid cross contamination.
Procedures for material and personnel transfer will vary depending on the company, but the overall goal of contamination prevention remains the same. There are multiple ways in which materials can be properly transferred into a clean area. Some of these methods include utilizing disinfectants to spray or wipe materials, using multiple bags, or sterilizing items directly into the cleanroom. The method chosen will vary depending on the equipment, the compatibility of the materials with the disinfectant, and the materials being transferred.
The first example of chemical disinfection involves using a validated disinfectant to prepare the items for transport into the cleanroom. This may include spraying or wiping materials with the disinfectant, ensuring that all areas are exposed to the disinfectant. The minimum wet contact time with the disinfectant must be met. Some companies may implement procedures requiring overlapping strokes from the cleanest area on the item to the dirtiest area on the item.
The next method is the multiple bag method. In this method, materials are packaged in the number of bags (or wrappings) that are needed to get to the inner core of the cleanroom with one bag remaining intact for the Grade A area. Then, the material is sterilized. At each room classification transition, a single outer bag (or wrap) is removed as the item is placed into a transitional pass-through. At the final transition, the item should be in at least one bag. The purpose of this method is to remove the potentially contaminated outer bag and leave it, along with any microbial contaminants, in the lower classified area. This helps to prevent contaminants from migrating into the cleanroom.
The third method of discussion is the direct transfer through sterilization method. This is probably the most efficient method, but it requires the use of a double-sided sterilization apparatus, such as a double-sided autoclave. Items are loaded into a validated cycle pattern in the lower classified area. After the sterilization cycle is complete, the items are unloaded on the opposite side of the chamber in the higher classified area. This method can consist of autoclaves, vaporized hydrogen peroxide, ozone, dry heat ovens, and even washers.
The next article in this series covers various aspects of cleanroom gowning.
- EudraLex – The Rules Governing Medicinal Products in the European Union, Volume 4 – EU Guidelines to Good Manufacturing Practice, Medicinal Products for Human and Veterinary Use, Annex 1 – Manufacture of Sterile Medicinal Products, European Commission (Nov. 25, 2008).
- FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing-Current Good Manufacturing Practice, Food and Drug Administration, Rockville, MD (2004).
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.