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 provides an introduction to cleanroom microbiology, discussion of guidance documents and FDA observations, and a summary of common sources of microbial contamination in cleanrooms. Subsequent parts will address some cleanroom design basics, discussion of proper material transfer, aspects of cleanroom gowning, concepts of environmental monitoring, and the importance of disinfectant efficacy and proper cleaning.
An Introduction To Cleanroom Microbiology
Cleanroom microbiology encompasses a wide variety of subjects, including microbial identification, cleanroom design, material transfer, personnel and material flow, cleanroom gowning, personnel behavior, aseptic technique, environmental monitoring, data trending, cleaning, sanitization, and disinfection. Understanding the concepts of cleanroom microbiology is important to help control the cleanroom environment and ultimately ensure the consumer is safe.
Tainted environments or materials can potentially contaminate the product, lead to product recalls, regulatory observations, or fines, harm the product efficacy, or even kill the patient. A well-known example of this is the New England Compounding Pharmacy meningitis outbreak of 2012. More than 800 people were sickened and 64 people died due to product contaminated with fungus.7
Microorganisms are present in most habitats. This includes soil, water, plants, animals, hot springs, the deep sea, food, beverages, skin, and hair. To be able to survive in harsh habitats, certain species of microorganisms must be resilient. In fact, 25-million-year-old bacterial spores have been revived from the belly of a honey bee that was preserved in amber.11
Not only are microorganisms resilient, they are also easy to spread. They can be transferred directly from one surface to another by touching the surface with an object that is contaminated with microorganisms.4 Or, microorganisms can be transferred indirectly, such as when microorganisms are distributed through the air.4 In cleanroom environments in particular, humans are the primary source of contamination. Typically, 80 to 90 percent of normal microbial flora identified in a cleanroom environment is generated from humans.5
Particular practices and procedures must be followed in order to minimize and control microbial contamination in the cleanroom environment. One of these practices is widely known as aseptic technique, a practice used to maintain sterility and prevent the spread of contamination.3 Some aspects of aseptic technique include:
- Never breaking first air or reaching over exposed product, components, or fill lines.
- Not touching sterile items with nonsterile items.
- Not exposing sterile items to nonsterile environments.
- Using sterile components and gloves.
In order to control microbial contamination, all activities that happen in the cleanroom need to be controlled.
- Processes should be demonstrated and validated to be successful.2
- Procedures should be controlled and precisely followed.
- Personnel should be properly trained, have good hygiene, attitudes, and behaviors, gown appropriately, follow procedures, practice good aseptic technique, move slowly and deliberately in the cleanroom, and do the right thing even when no one is watching.2
- Raw materials and components should pass quality control testing prior to use in manufacturing. They should not add microbial contamination to the process.2
- The facility and equipment should be properly maintained, cleaned, and disinfected.2
- The facility should be constructed with quality by design concepts in mind and be fit for the manufacturing purpose.
- Cleanrooms should have proper positive pressure air cascades and high-efficiency particulate air (HEPA) filtration.2
- The environment and utilities must be properly maintained, monitored, and controlled to prevent the ingress of microorganisms, correct any microbial excursions, and prevent cross contamination.2
Guidance Documents And Regulatory Observations
There are multiple guidance documents and regulations that speak to cleanroom microbiology, including those on the following list. This is not intended to be all-inclusive.
- European Commission EudraLex “The Rules Governing Medicinal Products in the European Union” Volume 4, EU Guidelines for Good Manufacturing Practice, Medicinal Products for Human and Veterinary Use, Part II: Basic Requirements for Active Substances used as Starting Materials. Brussels, 03Feb2010
- European Commission EudraLex “The Rules Governing Medicinal Products in the European Union” Volume 4, EU Guidelines for Good Manufacturing Practice, Medicinal Products for Human and Veterinary Use, Annex 1 – Manufacture of Sterile Medicinal Products, 25Nov08.
- FDA Guidance for Industry – Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice. September 2004.
- Parenteral Drug Association (PDA) Technical Report (TR) No. 13 – Fundamentals of an Environmental Monitoring Program
- PDA Technical Report No. 69 – Bioburden and Biofilm Management in Pharmaceutical Manufacturing Operations
- PDA Technical Report No. 70 – Fundamentals of Cleaning and Disinfection Programs for Aseptic Manufacturing Facilities
- Pharmaceutical Microbiology Manual (PMM) 2014, Version 1.1, ORA. 007, 25Apr14.
- Pharmaceutical Inspection Convention (PIC/S) PI 007-6 “Recommendation on the Validation of Aseptic Processes” January 2011
- United States Pharmacopeia (USP) <1116> Microbiological Control and Monitoring of Aseptic Processing Environments
- USP <1072> Disinfectants and Antiseptics
- Code of Federal Regulations (CFR) Title 21, Volume 8. Cite 21 CFR 820.70 – Production and Process Controls.
- 21 CFR 211.42
- 21 CFR 211.25
- 21 CFR 211.28
- 21 CFR 211.113
- 21 CFR 211.25
- 21 CFR 211.28
When regulatory expectations are not met, warning letters may follow. Warning letters can be researched on the FDA’s website (https://www.fda.gov/iceci/enforcementactions/WarningLetters/default.htm). They can be a useful audit tool to help companies find, correct, and prevent similar findings. The following three summaries of FDA warning letters pertain to cleanroom microbiology. There are many more complete warning letters for review on the FDA website.
- The first summary is from an FDA warning letter that was written in July of 2014. During this inspection, the FDA noted insanitary conditions. The company violated cleanroom microbiology principles as follows:
- The employees were not properly sanitizing their hands after touching nonsterile equipment.
- The employees were wearing nonsterile masks.
- The employees had exposed skin in the cleanroom.
- The employees were reusing cleanroom gowning.
- The company also did not perform personnel monitoring of all operators as required.
- The second FDA warning letter was also written in July 2014. The FDA observed some cGMP violations during an inspection. Specifically, the FDA observed personnel not wearing clothing appropriate to protect drug product from contamination. This was a 21 CFR 211.28 violation.
- The third FDA warning letter was written in February 2012. According to the FDA, the firm that received the letter violated cleanroom microbiology principles in the following ways:
- The employees were using poor aseptic gowning technique.
- The employees touched the outsides of the gowns while gowning.
- The employees exposed equipment to a dirtier classification of air before introduction to the cleaner classification of air.
- The employees broke first air during manufacturing.
- The employees kept touching the parts of the filling machine.
Reading these warning letters, one can infer the following principles are key to maintaining a cleanroom environment:
- The outside of the gowns must not be touched.
- First air should never be broken.
- Equipment should not be exposed to a dirtier classification of air prior to introduction to the cleaner classification of air.
- Hands should be sanitized properly.
- Sterile components should be worn.
- Sterile gowning supplies should not be reused.
- Skin should not be exposed.
Sources Of Microbial Contamination In Cleanrooms
Understanding the sources of contamination can aid in developing corrective and preventive actions when excursions occur. Microorganisms are easily spread and are practically everywhere.
Microorganisms can be classified based on their mode of mobility, their shape, their atmospheric needs, their Gram stain reaction, their optimal growth temperatures, and whether they produce spores. With regard to shape, microorganisms can be round (cocci), rod shaped (bacilli), spiral shaped, or even vibrio shaped, which looks similar to a comma.
The atmospheric needs of microorganisms will vary depending on the microorganism. Microorganisms can be aerobic, meaning they require oxygen to grow. They can be anaerobic, meaning they do not require oxygen to grow. Some microorganisms may require various other combinations of atmospheric conditions for cell growth.
Microorganisms also vary in their optimal growth temperatures. For example, psychrophiles enjoy colder temperatures, usually below 15 C.14 Mesophiles enjoy moderate temperatures, typically 20 to 45 C.14Thermophiles like hotter temperatures, usually 45° to 80° C.14 If the cleanroom is kept at a moderate room temperature, the recovered microbial flora is usually mesophilic bacteria.
A Gram stain helps to determine the structure of the outer cell wall of bacteria. Most bacteria will fall into one of two groups as a result of the reaction to the Gram stain. Gram negative cells are pink and have a thin peptidoglycan cell wall, which is surrounded by an outer membrane containing lipopolysaccharide.14When Gram negative rods are recovered in cleanrooms, sources of stagnant water or moisture are usually present. Gram negative bacteria can be found in water systems, on surfaces due to improper disinfection practices, in standing water, in piping or hoses with moisture, and in sinks or drains. Depending on the species, Gram negative rods may also originate from human, animal, plant, or food sources. Poor aseptic technique can contribute to spreading the contamination.
Gram negative bacteria are also of the main source of endotoxin. Endotoxins are a complex lipopolysaccharide molecule located in the outer membrane of Gram negative bacteria.14 They are released when the bacterial cell is destroyed. This is a concern because endotoxins can trigger fevers, shock, or even death in the patient.
Gram positive cells are purple. They are surrounded by thick layers of peptidoglycan, but lack the outer cell membrane.14 Gram positive cells are the most recovered microorganisms in cleanrooms. They are usually associated with humans and are found on skin, hair, and clothing. Poor aseptic technique, poor material transfer practices, poor disinfection practices, and improper gowning can lead to contamination with Gram positive cocci microorganisms.
In cleanrooms, the phrase “Gram positive rod” typically sparks thoughts of contamination from dirt or soil. Gram positive rods are often isolated from dirt, soil, dust, air, cardboard, paper, water sources, mops, humans, food, and clothing. Gram positive rod microorganisms can either be spore formers or non-spore formers. Spores make bacteria harder to kill by allowing the bacteria to survive extreme habitat fluctuations. For example, the bacterium Geobacillus stearothermophilus is commonly used in autoclaves and isolator qualifications because of its resistance to heat and vaporized hydrogen peroxide. Poor aseptic technique, poor material transfer practices, poor disinfection practices, and improper gowning can lead to contamination with Gram positive rod microorganisms.
Yeasts usually appear to be very large oval cells under the microscope. When isolated from clean rooms, common sources of contamination are humans or food items. Poor aseptic technique, poor material transfer practices, poor disinfection practices, improper cleaning, and improper gowning can lead to contamination with yeast.
Molds can grow almost anywhere and are typically found in damp, dark, and humid areas. When molds are isolated in cleanrooms, paper, cardboard, wet drywall, heating, ventilation, and air conditioning (HVAC) systems, or wooden pallets are the typical sources of contamination.1 They are also associated with the building, walls, ceilings, dirt, and even dust. Improper disinfection practices, improper cleaning, poor material transfer practices, poor gowning practices, or improper aseptic technique can lead to contamination with mold.