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Microbial control is (Cleanroom Microbiology) critical in cleanroom environments. Cleanroom Microbiology.

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.¹

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.²

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.³ Or, microorganisms can be transferred indirectly, such as when microorganisms are distributed through the air.³ 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.

Particular practices

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.⁵ Some aspects of aseptic technique include: Cleanroom Microbiology

• 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.⁶
• 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.⁶
• Raw materials and components should pass quality control testing prior to use in manufacturing. They should not add microbial contamination to the process.⁶
• The facility and equipment should be properly maintained, cleaned, and disinfected.⁶
• 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.⁶
• 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.⁶

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. Cleanroom Microbiology

FDA warning letter

• 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 Cleanroom Microbiology

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.⁷ Mesophiles enjoy moderate temperatures, typically 20 to 45 C.⁷ Thermophiles like hotter temperatures, usually 45° to 80° C.⁷ 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.⁷ When 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. Cleanroom Microbiology

Gram negative bacteria

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.⁷ 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.⁷ 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.⁸ 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.

The next article in this series reviews the basics of cleanroom design and best practices for proper material transfer.

References:

1. Eichenwald, K. “Killer Pharmacy: Inside a Medical Mass Murder Case.” Newsweek. April 16, 2015.
2. Fischman, J. “Have 25-million-year-old bacteria returned to life?” Science, v.268, May, 19, 1995, pp 1060-1064.
3. Brandes, R. Aseptic Processing: Qualification of Personnel, Mass & Peither AG-GMP Publishing, April 12, 2012.
4. Hayes, B. “Managing Aseptic Gowning with in Classified Environments.” Cleanroom Technology. April 8, 2015. Accessed on Apr. 5, 2017: http://www.cleanroomtechnology.com/technical/article_page/Managing_aseptic_gowning_within_classified_environments/107339
5. Aseptic technique (n.d.). Mosby’s Medical Dictionary, 8th Edition. Elsevier Health Sciences, 2008. Accessed on May 10, 2017 from http://medical-dictionary.thefreedictionary.com/aseptic+technique
6. European Commission 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, November 25, 2008.
7. Madigan, M. et al., Brock Biology of Microorganisms, Tenth Edition. Pearson Education, Inc. New Jersey, 2003.
8. Abraham, Z. “Understanding, Preventing, and Remediating Mold in Cleanrooms.” IVT. December 17, 2013.

A Clean Room, or cleanroom, is a laboratory environment that ensures that airborne particles are maintained at a very low concentration. This room is isolated, actively cleansed, and prevented from contamination from a range of particles and biological material including dust, airborne organisms, or vaporized particles. These rooms are necessary to protect human health and industrial product assembly, and their primary aim is to prevent the release of whatever particle is being handled inside it. They are highly specific environments as several parameters are controlled.

By Hidaya Aliouche, B.Sc.Reviewed by Sophia Coveney

The international organization for standardization (ISO) is a worldwide federation comprised of several national standard bodies that were created to set standards for similar technologies in different countries. In the context of cleanrooms, standards and grading have been developed to assess the air quality and cleanliness.

International standards for monitoring airborne particles in cleanrooms

In Federal Standard 209 (A to D) of the USA, the number of particles ≥0.5mm is measured in one cubic foot of air, and this count is used to classify the cleanroom. The Federal Standard 209Ewas the preliminary standard for the classification of clean zones and cleanrooms; however, a new global standard replaced this in 1999 (ISO 14644-1).

Satisfactory cleanroom air quality is essential to ensure the quality of the product being produced or the effectiveness of the process being performed in the cleanroom. To determine whether the cleanroom complies with the global standard, some form of airborne sampling is necessary.

The important thing

It is important to note that there is frequent variability in readings of airborne particles in cleanrooms due to the contributions of dynamic particulate levels, variability in sampling technique, and differences or inconsistencies between the calibration and maintenance of instruments. However, ISO classification offers a complete platform to control for the discrepancy between different instruments.

An ISO 14644-1 classified cleanroom is a room or contained environment where it is crucial to keep particle counts low. ISO 14644-1 cleanrooms are classified from ISO 1 to ISO 9. Each cleanroom is classified according to the maximum concentration of particles per cubic meter or cubic foot. ISO 8 is the second lowest cleanroom classification and is considered to be the least clean cleanroom classification.

When Is a Cleanroom Required?

A cleanroom can be used for several applications across several industries. They are necessary for applications for which sterile and clean environments are necessary. With regards to the life sciences setting in which production is occurring,  the safety and quality of products is the primary objective. If too many particles enter the cleanroom raw materials, manufacturing processes, and finished products can be adversely affected.

An ISO 1 cleanroom classification is rare and is only implemented in cases of the most sensitive research and development processes is, for instance, those involving the construction of semiconductors or bioscience. An ISO 5 cleanroom is considered to be a high-grade cleanroom and is necessary for pharmaceutical applications. This may include the sterile filling of pharmaceutical drugs into their respective packaging. An ISO 7 facility is necessary for sub-sterile applications.

the primary objective

An ISO class 8 cleanroom is the lowest acceptable level for sterility. It is used when sterility is not considered to be the primary objective, but it is necessary to ensure a basic level of hygiene. Examples of such applications include the storage of pharmaceuticals.

It is important to note that these examples are indicative of these standard applications in practice. The standards are often set by a combination of several factors, which depend on the application of the cleanroom. Some applications may therefore fall somewhere between the levels indicated in the standards.

Cleanrooms ensure:

• Maintenance of effective contamination control measures and their assessments
• Identification of specific threats to product purity.
• Informing on the performance of heating, ventilation, and air conditioning systems, cleanliness of personnel, gowning practices, equipment, and cleaning operations
• Protection of the end-user (patients and healthcare providers) by protecting against biological contamination.
• Protection of the workforce:  some raw materials in product manufacture can produce contaminants that could be hazardous to operators. Cleanrooms ensure the immediate safety of the environment for Personnel in the cleanroom through the use of drowning areas, protective equipment, and air filtration systems.
• Secure product or process certification:  successfully and globally marketed products ranging from medical devices to pharmaceuticals require the manufacture to occur in a cleanroom that meets all parameters of international standards where the device is to be sold. By implementing these international standards, manufacturers can market to a larger customer base and therefore increase their product profitability.

Requirements For Setting Up a Cleanroom

It is the responsibility of the manufacturers and research labs to meet minimum requirements and standards that apply in their geographical region.

Manufacturers and research laboratories must remain vigilant to changes to the international standards and be able to respond to them appropriately. There are a comprehensive set of requirements and regulations that have been described in documentation.  The most common/ ubiquitous of these include:

• Cleanroom air monitoring system: as air quality is a critical determinant of cleanliness and air quality, enabling particle thresholds and therefore regulations to be adhered to.

• Continuous testing for contaminant particles:  the average person in a cleanroom can add 100,000 0.3 microparticles per minute while in a standing position. Mitigating the risk generated as a result of particle production begins with testing such particles to determine strategies to prevent the build-up of excess particulates. The choice of filtration systems is dependent on the type of work, the personnel, and the quantities of particles generated in the cleanroom workspace.
• Written procedures: To prevent harm to the users of any products generated in a cleanroom, contamination prevention measures need to be documented and submitted to the appropriate regulatory agency. Regulators require documentation as proof of preventative contamination strategies applied to both the product itself and the process used to generate it.

new challenges

The development of more complex products, including device miniaturization under nanotechnologies will increasingly present new challenges for cleanroom manufacturers. It is believed that automation will be used to leverage manufacturing, with the early implementation of automation in the assembly process enabling a low-cost means of ensuring cleanliness. It is also believed that artificial intelligence and machine learning can increase quality standards and reliability.

References

• Mahnert A, Vaishampayan P, Probst AJ, et al. Cleanroom Maintenance Significantly Reduces Abundance but Not Diversity of Indoor Microbiomes. PLoS One. 2015;10(8):e0134848. doi:10.1371/journal.pone.0134848
• Cleanroom Technology.  What is ISO 8 cleanroom classification? Available at: https://www.cleanroomtechnology.com/news/article_page/What_is_ISO_8_cleanroom_classification/180543. Accessed January 2022.
• National Research Council (US) Committee on Prudent Practices in the Laboratory. Prudent Practices in the Laboratory: Handling and Management of Chemical Hazards: Updated Version. Washington (DC): National Academies Press (US); 2011. 9, Laboratory Facilities. Available from: https://www.ncbi.nlm.nih.gov/books/NBK55867/
• ISO 8 Cleanrooms. Available at: https://www.cleanroomsbyunited.com/modular-cleanrooms/class-100000-cleanrooms. Accessed January 2022.
• Xu Z. Characteristics of Cleanroom. Fundamentals of Air Cleaning Technology and Its Application in Cleanrooms. 2013;587-617. doi:10.1007/978-3-642-39374-7_12 .