Limitations of Microbial Environmental Monitoring Methods in Cleanrooms

• Janssen Research & Development

Introduction

Environmental Monitoring (EM)  program requirements are currently described in the 21 Code of Federal Regulation (CFR) 211.42, 21 CFR 211.46, 21 CFR 211.22,¹ USP <1116> Microbiological Evaluation of Clean Rooms and Other Controlled Environments,² European Medicine Agency (EMEA) Annex I³ , International Standard Organization (ISO) 14644-1,⁴ ISO 13408,⁵ ISO14698-1⁶ and Parenteral Drug Association (PDA) Technical Report #13, Fundamental of an Environmental Monitoring Program (Revised, 2014).⁷

Regulatory agencies like the Food and Drug Administration (FDA) and European Medicines Agency (EMA) requires pharmaceutical manufacturing companies to have an EM program and standard operating procedures (SOPs) in place as an important part of the drug manufacturing control process to ensure product safety attributes. Regulatory bodies have established the requirement for trending and identifying contamination sources. Major observations have specifically been issued to companies that lack adequate systems for monitoring environmental conditions in aseptic processing areas and for not having written procedures for EM including sampling frequency, sampling locations and procedures for alert and action levels.

The ISO 14644-1 regulation specifies the total particulate counts allowed for a clean environment to meet the defined air quality classifications. Unlike microbial contamination where humans are the main significant source, nonviable particulates can arise both from the environment, humans and from processing equipment. Non-viable (total) air particulate (i.e. total particles count) at rest and during normal operations is conducted to confirm that the environmental quality in ISO-classified areas is maintained. However, EM of total particulate does not correlate directly on the bioburden of the environment.

ISO 14644-1 and ISO 14698-1 do not set limits or assign classification values for viable airborne particles. The sampling of airborne viable particles is dealt with in ISO 14698, however action levels are to be determined by the user. Per ISO 14698-1, section 5.2 “Microbiological Alert, action and target levels are set by the user as appropriate to the field of the application”. These levels should be based upon target levels related to what product is manufactured as well as the product requirements. These levels may be adjusted based upon data collected during initial start-up and at intervals established by the contamination control policy or monitoring plan. Various regulatory bodies have guidance related to the manufacturing of sterile medicinal products. These guidances, link cleanroom class based upon non-viable particle levels to viable particle levels. These levels are provided for active air sampling, settle plates, surfaces and on operators.

Every microbial EM program should have a combination of the following methods: settle plates, contact plates and/or surface swabs, active air samples, and rinse samples (i.e. equipment). Air sampling is meant to assess the engineering/design controls performance as intended to clear aerial contamination and meeting classification requirements for total particulate per volume of air and personnel aseptic techniques practices and hygiene. Surface sampling are mainly intended to assess surface cleaning and sanitation effectiveness and personnel aseptic techniques practices and hygiene. All these methods are based on the ability of captured organisms to be visibly counted due to replication in nutrient media under aerobic conditions and mesophilic incubation temperatures.

At present, nearly all these methods rely on the growth and recovery of microorganisms, many of which are in environmental stress and therefore may be difficult to recover. The lack of accuracy and precision of the traditional enumeration methods and the restricted sample volumes that can be effectively analyzed suggest that microbial EM is incapable of providing direct quantitative information about sterility assurance. All known methods only sample at a single point in time, therefore would require repeat sampling to assess range of counts. On the other hand, microbial EM recover efficiency is undoubtedly influenced by many factors. Only viable microorganisms would be detected by EM methods. The type of surface, presence of product or chemical residues, organisms stress status, type of microorganism, technique bias by technician during sampling are among the facts that influence the recovery efficiency. The absence of recovery may give a false impression that the air or the surface sampled was “clean” if most of the microorganisms are non-cultivable or non-viable (incapable to reproduce), have special nutritional requirements, or are slow grower among other facts.^2,7 As a fact, the absence of growth does not mean the sample location is free of microorganisms and likewise a single sample point excursion does not indicate that the area is not in a state of microbial control. There is not a microbiological sampling plan that can 100% prove the absence of microbial contamination, even when no viable contamination is recovered.⁸

Microorganisms Recovery from Air

The distribution of microorganisms under the conditions of an air medium is strongly tied in with the colloidal properties of microorganisms. The length of occurrence of microorganisms in the air, diffusion, electrical charge, and their mixing by air flow are based completely on the laws of colloidal chemistry. A colloid, is a mixture in which one substance of microscopically dispersed insoluble particles is suspended throughout another substance. Unlike a solution, whose solute and solvent constitute only one phase, a colloid has a dispersed phase (i.e. the suspended particles) and a continuous phase (i.e. the medium of suspension).⁹ It is assumed that bacteria in the dust phase are not bound to dust particles, as occur with water, but freely suspended in air. Vlodavets (1964) reported that bacterial dust particles settled down even faster than bacterial drops. Initially a high concentration of microorganisms is created after the creation of the bacterial drop aerosol (i.e. bacteria with a water-based solution). Then, the concentration of bacterial in air gradually decrease. Decrease in concentration is dependent mainly in the settling of bacterial drops. It has been reported that a decrease in relatively humidity promote an increase of the time of occurrence of Staphylococci in the drop and dust phases of an aerosol. High humidity assists the settle down of bacterial drops as well as of particles of the bacterial dust and the lowering of the concentration of the bacteria on air. Vlodavets concluded that the concentration of viable microorganisms on air is influenced by two facts: 1) the settling of organisms (i.e. physical loss) and (2) the death rate (i.e. biological loss) of the microorganisms.¹⁰ Within cleanroom environments 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.^11-13 Engineering controls within cleanroom should maintain a low relative humidity hence the settlement of air organisms in cleanroom is decreased, facilitates the clearance of particulates.

Passive air sample by settle plate is a useful method for assessing air contamination by microorganisms. Passive sampling consists of letting particles settle by gravity on a flat surface that required long exposure time (i.e. approximately 1 hour). Only particles in larger size range >10 μm are likely to land on plates. Results are expressed in CFU/plate/ time or in CFU/m² /hour.¹⁴ Settle plates are not likely to be validated for recovery method because there is no accurate measurement of the volume of air sampled.

Volumetric air monitoring meant for a microbiological air sampler physically drawing a known volume of air through or over a particle collection device which can be a liquid or a solid culture media or a nitrocellulose membrane and the quantity of microorganism present is measured in colony forming units (CFU) per m³ of air. Active air sampling is ideal when monitoring a low bioburden environment. The most known methods are impaction, centrifugal and membrane (or gelatin) samplers. Instruments should be calibrated and the method is able to be validated.¹⁵

Several studies have attempted to compare the values of microbial loads on air obtained through both active and passive sampling methods, but with inconsistent results: in some cases, there was significant correlation^16-19 while in others there was none.^20-24 However, current active air can be more advantageous and effective in assessing airborne viable contamination in cleanrooms than settle plate monitoring. There may be no advantage in performing these two parallel methods for the detection of airborne contamination specifically because they may increase the number of interventions into critical areas, which may in turn increase the risk of contamination without providing any added benefit in terms of data collection and/ or process control.²⁴

Microorganisms Recovery from Surfaces

Microorganisms are easy to spread within the cleanroom. They can be transferred directly from one surface to another by touching the surface with an object that is contaminated with microorganisms.^12,25 When microorganisms are released into the manufacturing area air, they will be deposited onto these surfaces as either aerosol particles or as liquid droplets. There are many types of surfaces in the pharmaceutical production areas and cGMPs equipment, all with distinct physicalchemical properties. The type of surface greatly influences their ability to survive and their possibility to contaminate other materials.²⁶ The surface methods that are used most is the contact-plate method is suitable for flat, firm surfaces, (considering both recovery and repeatability), whereas swabbing is better for flexible and uneven surfaces and for heavily contaminated surfaces.^26-28 Recovery levels of surface-monitoring methods are typically low, due to variability in sampling procedure, analytical methods being employed (i.e., dilution), and the use of growth-based techniques.²⁵

Different media are employed, and in the case of swabs, different results have been reported for wet and dry swab methods. It is widely known the poor correlation between the amount of microbial contamination on surfaces and the recovery obtained. For example, a collaborative study comparing surface monitoring methods showed that artificially contaminated stainless steel at a theoretical level of 1.4 CFU per cm² (about 35 CFU per sample) gave recoveries of 25 to 30% when bacterial spores were employed.²⁹ As reported by Kang and colleagues (2007), recovery of Listeria monocytogenes from stainless steel inoculated test areas, or coupons, using a sonicating brush head (contact or noncontact) yielded a recovery level of about 60% compared with swab and direct agar contact methods (about 20%).³⁰ Many factors may contribute to this poor correlation, including differences in materials used (e.g., cotton, polyester, rayon, calcium alginate), the microorganisms targeted for culture, variations in surface, and differences in the personnel collecting and processing samples.³⁰ Additional sources of error are the potential for non-homogenous surface resulting in unequal or incomplete removal of microorganisms from surfaces.³¹ Two additional variables of swab method could be that optimum elution may vary with each organism and some media and diluents may be inhibitory to certain microorganisms.²⁵

The Replicate Organism Detection and Counting (RODAC) method was described first by Hall and Hartnett (1964) as a means of direct sampling of surfaces.³² The method is used not only for sampling of flat surfaces but also for personnel environmental (i.e., gowning) sampling.¹² A variant of this method is the touch plate where personnel will place their gloved finger-pads (i.e. fingertips) on the surface of the RODAC plate for getting an estimate of the microorganisms on the tips.^12,33 There are several limitations to RODAC method. The most known is the requirement of a flat uniform surface. A second limitation is that this method is very sensitive to residual disinfectant that may be on the sampled surface and could be transferred to the agar. This limitation can be overcome somehow by the addition of neutralizing agents into the nutrient agar. The third limitation is that the smaller size of the RODAC 50 mm limits the countable number of colonies on the plate. Therefore, the maximum range of 250 CFU for standard 100 mm standard Petri dishes³⁴ would not apply to RODAC (i.e. maximum range is 50 CFU). Surface sampling has been found to recover <50% even with high inoculum on standardized coupons. Recovery is also highly influenced by microorganism species and number of microorganisms present at the time of sampling. The fourth limitation is the residual agar transferred onto the surface being sampled; media residue on the surface must be promptly removed as media residue could serve as a nutrient source for microbial proliferation. Prior to their implementation the type of media and incubation conditions must be qualified. Such qualification may include laboratory studies using the compendial growth promotion microorganisms and/or representative microorganisms recovered from the facility environment.⁷

Swab and contact plate methods are not interchangeable because results may vary. If you decide to use both methods in the same area make sure the data is analyzed independently. This is because it has been reported that RODAC plates are superior to the swab technique for the detection of Gram-positive cocci, whereas Gramnegative rods can be detected more often by the swab technique.³⁵

These two techniques were not designed to obtain full recovery but to be suitable enough to establish trends for the assessment for environmental control.

Microbial Identification

Many factors influence the capacity of microorganism’s recovery by current EM methods. Microorganisms are likely to be found in air and surface forming clusters of one or distinct strains, associated to dead skin scalp, soils or inorganic material. In addition, metabolic active microorganisms normally are found in different stages of cell cycle. When conditions become unfavorable for growth bacteria stop replicating and viability starts to decrease. A high initial bacterial load increases the likelihood to be recovered and identified.³¹

USP <1116> suggests that microbial recoveries should be identified at a rate sufficient to support the EM program. Once isolation of the microorganism is achieved, microbial characterization and identification is performed.^36-37 Typically, microbial identification systems (either genotypic or phenotypic based) are employed after primary screening and characterization are performed through Gram staining. Identification platforms may vary between pharmaceuticals. Genotypic and Phenotypic automated systems are commercially available. The system chosen must be validated.^36-37

Characterization will represent the establishment of microbial profiles, including the evaluation of sources, routes of ingress and susceptibility to elimination or reduction. Characterizations can reveal useful clues as to the possible source of isolates. Routine characterization of isolates should continue to determine whether isolates are part of the normal microflora or represent something atypical.³⁸

A well-established microbial EM program must identify, at least at the genus level, microorganisms isolated from EM.³⁸ Class ISO 5 and ISO 6 microbial recoveries must be identified at the species level. Meanwhile, the level of identification of microorganisms from ISO 7 and ISO 8 class could be determined based on a risk assessment analysis. For example, for non-sterile class ISO 8 manufacturing or support areas it may be sufficient to identify isolates to morphology level by gram staining on a routine basis. Higher level of identification is recommended for microbes isolated from aseptic processing areas and may require identification to species level. The key concern is to determine the state of control for the facility with some relative level of confidence. Indeed, there is regulatory guidance that suggests the requirement to identify isolates to strain level when investigating microbial excursions or sterility failure. FDA 2004 Aseptic Guidance document, page 35 section B. Microbiological media and Identification establish that “Characterization of recovered microorganisms provides vital information for the environmental monitoring program. Environmental isolates often correlate with the contaminants found in a media fill or product sterility testing failure, and the overall environmental picture provides valuable information for an investigation. Monitoring critical and immediately surrounding clean areas as well as personnel should include routine identification of microorganisms to the species (or, where appropriate, genus) level”.³⁹

Characteristics such as colony morphology, cellular morphology, Gram stain and the presence, or absence, of endospores remain important in identification to genus level, while additional biochemical and physiological tests may often be able to differentiate to species level. The Gram stain method is prone to a significant level of operator error, which has encouraged the development of alternate methods for showing the difference in cell structures. Nowadays, automated gram staining systems have provided some level of reproducibility.

Most pharmaceutical companies identify isolates recovered from samples that have exceeded their alert and actions levels. Other pharmaceutical companies have designed a grid for randomly identified isolates. Less often others may identify the morphology of representative colonies captured. Most aseptic pharmaceutical laboratories identify microorganisms at the species level. Most Pharmaceutical companies identify filamentous fungi isolates to at least the genus level if the colony counts reach action level, but I would recommend that it be done sooner (at the alert level) to remediate a potential fungal plum within critical control manufacturing work spaces. Fast growing microorganisms should be identified at the species level, if possible. Fast growing bacteria grow in such a way that they appear to be “spread” across the plate. Such fast growing phenotype is a potential threat to the manufacturing environment and the product. Some species of Bacillus tend to form spreaders on moist semisolid media. Early read of test samples before the end of incubation or test plates transfer to the following incubation temperature is recommended to get an accurate count if spreaders are present. A final word of advice is to be consistent with the microbial identification platform used to identify isolates from different sources like EM samplings or finished product because the same strain tested using two different microbial identification platforms may give the laboratory results two different species names. This discrepancy may make it difficult to correlate matching microbes found in the finished product test with microbes recovered from the manufacturing area or specific raw materials used for product compounding.

Microorganism Prevalence in Cleanrooms

The low density of aerosolized particulates within cleanrooms should reduce the amount of both inorganic and biological contamination on and within the assembled products. The nutrient-deprived (i.e. oligotrophic), ultraclean, and desiccated conditions designed to be maintained within cleanrooms and most manufacturing areas strives to limit the proliferation and or survival of microbial life in these environments.^40-48 Rigorous maintenance procedures, such as regular cleaning,⁴¹ HEPA air filtration,⁴⁴ and constant low humidity and temperature control, make these facilities inhospitable to microbial life and become unlikely microbial persistence.^43-44

Microorganisms within the clean room are likely to be transient; accidental occurrences of microorganisms possibly introduced into the cleanroom by an assortment of external sources.⁴⁸ However, their path of passage may be the same each time. With a careful study of the EM data one may confidently predict this origin of microbial influx and ultimately design a mitigation step to prevent it from becoming a continued source of contamination for the controlled facilities. The identification of these entry sources is essential to improve microbial contamination prevention. There is no such thing as endogenous microflora for a constructed manufacturing facility. The establishment of any microorganism within the cleanroom shall be considered as a control breach and must be investigated and eradicated.

Bacteria can adapt to distinct environmental conditions. These include adaptations to changes in temperature, pH, concentrations of ions such as sodium, and the nature of the surrounding available nutrients. Bacteria react to a sudden change in their environment by expressing or repressing of various sets of genetic operons that control the expression of inducible proteins and other critical cellular components. These responses change the properties of both the interior of the microorganism and its surface chemistry. A well-known example of this adaptation is the so-called heat shock response (also known as stress shock response). The name derives from the fact that the response was first observed in bacteria suddenly shifted to a higher growth temperature.⁵⁰

It is widely recognized that the isolation of numerous bacterial species (including novel microorganisms) within cleanrooms capable of surviving diverse, unfavorable environmental conditions is most probably due to previous inherent resistant traits (i.e., spore formation, cytoplasmic condensation, stress shock proteins, etc.) rather than post adaption. The engineering controls as well as frequent cleaning and disinfection of clean rooms is the major challenge for the minimizing the establishment of any microbial entity.⁵¹ By not rotating nonsporicidal with periodic sporicidal disinfectants in the clean room one may encourage the establishment of one type of microbe not susceptible to the antimicrobial solution. For rapid growth in different environments, bacteria need to adjust their enzyme levels to rapidly benefit from the nutrient mix that is currently available in the surrounding. If the living environment undergoes rapid changes, the bacterium’s own production of proteins may need to be altered to adapt to these changes in an effective way. The opportunity for growth of bacteria is determined not only by the organic composition of their surroundings but also by sudden changes in the living environment. High rates of bacteria growth in a stable environment requires a certain kind of physiology, but environmental changes also require rapid adjustments of the bacteria’s inducible protein production. Therefore, this microbial phenotype type(s) must be available prior to the environmental change.^51-53

Most Gram positive rod-shaped bacteria are capable of existing in two forms, dormant spores and active vegetative cells. Vegetative cells form spores under adverse conditions as a means of survival. Spore formation protects the bacteria from starvation, drying, freezing, harsh chemicals, and extreme heat. When conditions become favorable, the spores germinate, allowing each spore to once again become a vegetative cell with the ability to reproduce. Among the bacteria, sporulation is not a means of reproduction since each cell forms a single spore which later germinates into a single cell again. Most sporulating bacteria that grow in the presence of air belong to the Genus Bacillus, and those microbes that grow only in the absence of air belong to the Genus Clostridium. The endospores of several Bacillus species isolated from spacecraft assembly facilities have previously exhibited various levels of resistance to H₂ O₂ treatment.^45,52 Gram positive bacteria are more likely to survive indoor low humidity conditions than Gram negative bacterium.^46-48

Microorganisms also vary in their optimal growth temperatures. For example, psychrophilic bacterium prefers colder temperatures, usually below 15°C. Mesophiles thrive best at moderate temperatures, typically 20 to 45°C. Thermophiles have adapted well to hotter temperatures, usually 45° to 80°C. Most human source microflora are mesophilic.⁵⁵ Mesophilic bacteria grow best at or near human body temperature, but are also capable of growth at room temperature.⁴⁸

Spore-forming Bacillus species are not the only ones that shows physiologically flexible phenotype able to persist in the inhospitable conditions of clean room environments. La Duc et al (2007) reported that the significant differences observed in the cultivable bacterial populations among the certified clean rooms are more likely attributable to the amounts of human activity and/or routine maintenance of each facility, rather than geographic location.^56-59 The isolation of numerous bacterial species (including novel microorganisms) capable of surviving diverse, unfavorable environmental conditions is a testament to the remarkable distribution of physiologically diverse unicellular life. The occurrence of thermophiles (Geobacillus spp.) in mesophilic condition, obligate anaerobes (Paenibacillus sp.) in oxygenrich environments, and halotolerant alkaliphiles (Oceanobacillus sp. and Exiguobacterium sp.) in neutral pH environments supports the adaptability and resistance of these microorganisms to environments that are not their first choice of growth.⁴⁶

Conclusion

The range and types of microorganisms able to be recovered from EM samples within cleanrooms is very limited. The parameters that contribute to the ability to recover these microorganisms include the microorganism’s physiology, environmental conditions (stress factors), and nutrient composition of the culture medium and the selected incubation conditions used to recover them. Due to the described limitations of the EM methods currently available for pharmaceutical manufacturing monitoring, it is unlikely to obtain all the microorganisms that may occur in the clean rooms that employ the need for human intervention. When manufacturing is performed in the presence of people, most likely the microorganisms recovered will be human source Gram positive, mesophilic aerobic or facultative aerobic bacteria. The current commercial growth media allows for the recovery and enumeration of these human and terrestrial microflora within a 7-day incubation period. Although, as stated in the introduction the need for microbial environmental monitoring is a global regulatory and compendia expectation and should not be taken lightly. The frustration and caveats with the available commercial methods to perform these tasks is that their recovery capability of natural microorganisms is not optimal. As industrial microbiologists, it is our responsibility to understand their limitations (with evidence available from the cited references) and how to apply these insights on their practical usefulness to monitor and indicate when our facilities are in or out of control and meeting our company’s compliance requirements as defined and documented in our validation studies and risk assessment reports.

Acknowledgments

I would like to thank Dennis E. Guilfoyle, Ph.D., Sr. Director, Microbiology & Analytical Regulatory Compliance, Johnson & Johnson for critical review and input of the manuscript.

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Author Biographies