Issue



Getting the bugs out


04/01/2008







By Fran McAteer, Microbiology Research Associates, Inc., and Ryan Burke, Acusphere, Inc.

Environmental monitoring (E/M) is a surveillance system for microbiological control of cleanrooms and other controlled environments. The process provides monitoring, testing, and feedback regarding the microbiological quality levels in aseptic environments. Environmental surveillance monitors the effectiveness of various controls for microbiological contamination, sources of which can come from air, personnel, equipment, cleaning agents, containers, water and compressed gases, among other things.

Sound monitoring requires understanding the stringent regulatory specifications put into effect by organizations such as the U.S. Food and Drug Administration (FDA), International Standards Organization (ISO), Parenteral Drug Associates (PDA), European Union (EU), and United States Pharmacopeia (USP). Tables 1???3 provide a summary and comparison of various parameters. The tables are separated by cleanroom classification and are found in Parenteral Drug Associates (PDA) Technical Report No. 13, Fundamentals of Environmental Monitoring.1

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Note that Federal Standard 209E has been retired and superseded by ISO 14644-1.

Understanding cleanliness and microbiological risk classifications

As one can see, there are many differences in both terminology and measurement parameters that can confuse personnel developing the E/M process. Acceptance criteria for microbiological contamination are different for air, surfaces, and personnel, forcing companies to look at setting appropriate surveillance based on where they may be selling their products. Cleanroom classifications differ between the United States, Europe, and ISO classifications. ISO classes 5???8 are the applicable classifications for aseptic processing companies.

USP <797> links sterile compounding in hospital pharmacies to the same standards used in the pharmaceutical industry and further illustrates regulatory attempts to harmonize specifications in order to reduce potential contamination in the drug-to-patient cycle. These aseptic specifications in ISO 14644-1, USP <1116>, and USP <797> represent uniformity in stringent asepsis from the pharmaceutical drug manufacturing operations through hospital pharmacy drug compounding for immediate patient care. Such coordinated parameters may provide more consistency from fill/finish manufacturing to IV compounding and play an important role in lowering hospital-acquired infections (HAIs).

The European Union Guidance documentation, The Rules Governing Medicinal Products in the European Union, takes cleanrooms and further breaks them down to their conditional state specifying either 1) at rest, static, or 2) operational, dynamic. Each state is further segregated based on air quality from Grade A through Grade D, with Grade A being the cleanest. Associated with each grade are the maximum allowable viable and non-viable particulates. Grade A areas are associated with high risk applications such as fill/finish operations. Grade B usually designates preparation areas for fill/finish operations where open containers may be present. Grade C indicates clean zones for less critical applications such as media and buffer preparations. Grade D areas are the least clean and involve cleaning, wash, or other preparatory requirements.

In the United States, aseptic manufactur-ing is classified as Level I, Level II, or Level III and refers to the microbiological risk of contamination; with Level I being associated with areas that have open product exposure and Level III associated with ancillary functions such as cleaning or other prepara-tion activities.

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Most large biotechnology, pharmaceutical, and medical device companies today market their products in both the United State and Europe. In order to compete in the appropriate region, they must ensure that the manufacturing environment can meet all of the regulations set by the USP, ISO 14644-1, and European Commission Annex One. There are slight inconsistencies among these different documents, which may add unnecessary confusion and difficulty to the already extensive process of validating a pharmaceutical, biotechnology, or medical device manufacturing facility. Current efforts to harmonize these specifications have not been finalized.

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One of the most critical aspects within an aseptic manufacturing facility is the amount of viable microorganisms and non-viable particulates within a controlled area. However, this is where the different guidance documents fail to harmonize and integrate into unified environmental monitoring specifications.

In order to control the levels or amount of microorganisms and non-viable particulates within the different areas used for manufacturing purposes, most aseptic processing operations identify the zones within the facility by different grades based on the levels of cleanliness. The grades of the rooms are specifically set up to keep microorganisms and particulates away from any sensitive, exposed parts of the manufacturing process where the product may be susceptible to contamination. For newer companies—and maybe even for the established ones—trying to decipher the terminology describing the various grades in the guidance documents can be one of the first hurdles in setting appropriate specifications.

Whether manufacturing for the United States and/or European drug market, the specifications for non-viable particulates are mainly located within three separate guidance documents: USP <1116>, ISO 14644-1, and the European Commission Annex One. However, the three sets of guidance are not completely harmonized. Table 4 offers a more condensed summary and comparison of various parameters within the different classifications.

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As one can see, there are many discrepancies about the particulate limits for different classifications among the different guidance documents. The ISO 14644-1 covers the most ground in all the different classifications; however, the majority of the aseptic manufacturing companies within the U.S. and European market are mainly concerned with the 0.5- and 5.0-μm size particulates. The ISO 14644-1 and the European Commission both list specifications for the 0.5- and 5.0-μm size particulates, but the USP <1116> does not specify any limits for the larger 5.0-μm size particulate or for any larger size particle for that matter; it only deals with the 0.5-μm particulate size. Another inconsistency is that the European Commission has specifications for both operational (dynamic) and at rest (static) conditions whereas the USP <1116> and the ISO 14644-1 have just one general specification for each limit. Having separate specifications for static and dynamic conditions does make good sense in that adding people and activity to a room will normally result in increased particulates.

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A further inconsistency involves the particulate limits within each classification with the European Union (EU) being slightly more conservative. Last and arguably the largest issue concerning the particulate limits for these classified areas is the zero specification for the 5.0-μm size particulate per cubic meter of air in the EU Grade A and Grade B at rest. The zero specification is not practical because this number is too small to be statistically significant and is virtually impossible to obtain due to the fact that the distribution of particulate matter within a classified area is not homogeneous. Furthermore, false counts, associated with electronic and other background noise within the room, can cause the particle counter device to measure trace amounts of larger particulates. This issue has precipitated discussions within the European Commission to consider a limit of twenty 5.0-μm size particulates per cubic meter of air for these classifications.

Viable counts—colony forming units (CFU) specifications—concerning aseptic manufacturing companies within the U.S. and European market are listed in the USP <1116> and the European Commission Annex One. The ISO 14644-1 is for non-viable particulates only. Again, there are discrepancies and inconsistencies regarding the acceptable limits with the different classifications of rooms.

Table 5 displays the inconsistencies between the two guidance documents throughout all the different classifications. First off, some allowable limits are detailed in one guidance document and not specified in the other. Second, there are notable differences in acceptable limits for each classification specified by each of the guidance documents. For example, surface viables are both very low in Class 100 space with USP <1116> specifying =3 CFU while EU limits it to <1 CFU. While the EU is certainly more conservative and strives for sterility, the question is whether this level can be maintained in practical applications. This very stringent specification can lead to increased deviations and product rejections.

In some cases, the regulatory specifications flip-flop in regard to a conservative approach as shown in Grade A (USP Class 100) with the European Commission being more conservative than the USP <1116>; however, this is not the case in Grades C (USP Class 10,000) and Grade D (USP Class 100,000) where USP <1116> has set the more conservative limits. Either way, the discrepancies displayed in the table are significant. Confusion in translating appropriate specifications causes interpretational differences and therefore demonstrates the pressing need for these documents to be harmonized as soon as possible. Currently, our clients are advised to go with the most conservative specification that will ensure all acceptance criteria are met. However, this certainly increases overall quality and facility costs.

Monitoring and sampling techniques

Environmental monitoring evaluates existing cleanrooms, HVAC systems, personnel, cleaning, and sanitization activities. It monitors both viable and non-viable particles. Viables would include microorganisms such as bacteria, yeast, and molds. Non-viables would be air particulates of various sizes. For viable counts, E/M programs test air, surfaces, and personnel. Aseptic processors check their cleanroom environments routinely to ensure they meet required standards.

Air monitoring can be performed by active air sampling or by settling plates. Active air samplers such as rotary centrifugal air samplers (RCSs) measure an exact amount of air with a quantifiable number of viable microorganisms. Contamination can be measured per cubic foot or cubic meter of air. Generally, active air monitoring devices directly correlate microbial contamination with measured airflow. The most widely used methods involve media impaction instruments. They include:

  • Slit to agar (STA)
  • Sieve impactors
  • Centrifugal impactors
  • Liquid impingement
  • Filtration

Considerations for active air monitoring are a function of the final product, the aforementioned regulatory specifications, methods of production, facility design, and so on. They may include such simple considerations as ease of use, portability, and cost. Other considerations may be more substantive such as calibration of airflow, capability for steam sterilization, and capacity of air volume measurement. Whatever the reason, each type of active air sampler has unique advantages and disadvantages. Careful evaluation for compliance to the appropriate regulatory specification coupled with a comprehensive appraisal of operational parameters including manufacturing, quality, and facility layout should optimize the selection of the appropriate active air monitoring methodology.

Gravitational or settling plates are Petri dishes that contain sterile growth media and can also be used to test cleanroom air. They are passively exposed to the environment, usually for 30???60 minutes. Viable microorganisms that settle onto the media surface will grow when the plates are incubated. Settling plates are easy to use and cost effective. However, they do not directly correlate microbial contamination with measured air volume and do not provide a quantitative measurement of air contamination. The FDA desires active air sampling to be used and not passive monitoring, while the EU details settling plate specifications for air viables. Again, the enigma of which limit to use complicates matters. Thus, air monitoring may involve both types of testing to satisfy compliance. Settling plates do offer minimal environmental interruption and can be easily used in dynamic indications such as USP <71> sterility tests to provide feedback regarding the controlled environment during actual test conditions. Take care to ensure that media desiccation does not occur over dynamic conditions over extended time periods.

Surface monitoring involves contact or RODAC (replicate organism detection and counting) plates, which contain sterile growth medium in 50-mm plates or a sample area of 25 cm2. The agar protrudes above the sides of the plate. This convex contact plate is pressed against any flat regular surface that needs to be sampled. Any viable microorganisms on that surface will adhere to the agar and grow upon proper incubation. This technique replicates the number of viable microorganisms on a surface. Contact plates are easy to use and widely available, but they may not be appropriate for irregular surfaces. Neutralizers are usually in the media to minimize inhibitory effects of disinfectants used in the cleaning process. Contact plate residue must be removed immediately after testing the sample site. Otherwise, active microbiological growth media is left in the aseptic processing area, increasing contamination risk.

Swabs are used for sampling surfaces that are not flat, such as tubing or equipment. Swabbing is qualitative, and reliability depends on technique. A 2x2 in.2 sample site (approximately 25 cm2 surface area) is swabbed by using a back and forth technique and rotating 90?? and repeating. The swabs are then streaked onto microbiological agar plates for identification. Swabs can also be quantified by using transport media in pour plates or membrane filtration techniques. Swab technique can vary greatly with technicians; training should be documented. Swab microbial recovery tests should be considered for method validation.

Personnel working in a cleanroom need to be monitored for microbial contamination. Personnel monitoring is a useful indicator of cleanroom personnel gowning proficiency. Monitoring sites are located on gloves, forearms, shoulders, etc. Action levels would be low since these sites represent areas that may be exposed to product. It is recommended that gowning qualification programs be required for new employees and be designed to demonstrate compliance over time (e.g., three successive gownings). Annual or periodic requalification should be done for all cleanroom technicians. USP <797>, for example, is a bit more stringent in requiring aseptic technique proficiency to be demonstrated by all cleanroom or compounding personnel. Touch plates (contact plates) can be used to dynamically monitor technicians’ hands immediately after a critical process, such as product fill or USP <71> sterility testing. Along with monitoring, thorough personnel hygiene training should be introduced and conducted for all aseptic processing personnel. One example of such protocol is that cleanroom technicians should report upper respiratory infections to their supervisors because these conditions may restrict them from working in critical manufacturing environments.

Surface rinse methods are an E/M technique for fermentation vessels, hold tanks, and kettles. They are used for large surface areas and tested microbiologically by membrane filtration. These methods provide ease of use and test data for vessels that are difficult to access. Rinse methods are widely used in cleaning validations, especially in clean-in-place (CIP) systems.

For non-viable measurements, particle counts measure airborne particulates. These counts help qualify the cleanroom or controlled environment by demonstrating control of potential contamination. Some particle count systems offer continuous monitoring (24/7). The equipment uses light scattering technology based on the principle of passing an aerosol through a light source. Selection of the appropriate particle counter is based on size range, sensitivity, and flow rate. Most specifications for particle count are based on 0.5-μm size associated with the overall size of bacteria. As previously discussed, the EU is also concerned about larger particles, which are felt to possibly carry multiple microorganisms.

Sample sites for environmental monitoring depend on the manufacturing process. Evaluation of sample sites is based on the risk of microbiological contamination. Sites should be clustered at areas where the product is exposed to the environment such as a) filling lines for parenterals, especially at fill heads; b) inoculation vessels for fermentation activities; or c) loading of the freeze-dried or lyophilized product. Sample sites should include areas that may be inaccessible or difficult to clean, such as stopper bowls, chromatography columns, transfer lines, or fill nozzles. Other site selection factors may include areas that have a greater impact to add to bioburden levels. This may involve water point-of-use valves, compressed air lines, or general sites such as door handles. Sample site selection is customized to the manufactured product and unique to the individual manufacturing facility. Sample site selection can also be done uniformly by using grid profiling usually associated with particle counts.

Sample frequency is also dependent on multiple factors such as product type, equipment used, facility layout, type of processing, etc. The high quantity of hands-on processing by personnel will necessitate increased frequency of monitoring activities. Changes in sample frequency are dictated by changes in facility design, construction activities, processing changes, microbiological trends, and new equipment acquisition. Frequency changes should be accompanied by documentation summarizing potential upcoming activities and historical monitoring data and be reviewed by a qualified microbiologist.

Alerts, actions, and analysis

Action and alert levels are parameters designed to signal drifts in data from historical performance measurements. They are meant to point out changes in data before the quality of the product is affected. Alert levels are set below action levels and based on quality levels; they may signify a potential deviation from normal case scenarios. These alerts are usually associated with increased formal communication to quality and manufacturing management. Action levels are based on accepted regulatory specifications such as FDA, EU, ISO, PDA, or USP as previously discussed. Action level deviations require a corrective action and root cause analysis as detailed in a company’s corrective and preventative action (CAPA) standard operating procedures (SOPs).

A hot topic in today’s environmental monitoring is data management: data collection, analysis, and interpretation. Based on the larger number of E/M sites, sampling frequency, and conditional states, many companies are utilizing computer programs specifically for E/M. Image scanner and label systems help improve data collection. Trending analysis can also be obtained from both manual and computer-based programs. Trends can be depicted by histograms showing action levels. Data distribution will be different due to cleanroom classification, processing activities, the amount of human intervention, seasonal effects, and cleanroom conditional states (i.e., dynamic vs. static). Interpretation of trend analysis is based on statistical process control (SPC). SPC may show increases or decreases over time (e.g., seasonal effect), change in flora, possible patterns, or clusters, according to the process outlined. Risk analysis techniques can make determinations to assess potential contamination or other consequences with a process outline.

Characterizing E/M isolates to species levels helps categorize house organisms. The house organisms profile will be useful in future investigational analysis involving sterility test failures, disinfectant efficacy challenges, and positive media tables. Vegetative cells can be characterized by gram stain, colony morphology, sporulation, selective media, or automated identification systems. Sporulating organisms such as Bacillus species and/or molds are of particular concern due to their high resistance. Characterizations of isolates can be helpful in determining the source of contamination. For example, a gram-positive coccus like Micrococcus luteus may indicate human intervention (skin bacteria) while a gram-negative rod Sphigomonas maltophilia may indicate water contamination (water bacteria).

Validation and surveillance testing

Environmental monitoring methods need validation to demonstrate accuracy and robustness. Equipment such as air samplers and particle counters should have installation, operation, and performance qualifications (IQ, OQ, and PQ). These validation tests verify and document that the equipment performs consistently over time. For microbiological media utilized in air and surface monitoring, growth promotion should exhibit acceptable recovery of challenged microorganisms including bacteria, yeast, and molds. If media are being used with neutralizers such as polysorbate 80 or lecithin, then neutralizer efficacy testing should be done. Neutralizers are put into media to cancel out the effects of sanitizers or disinfectants that are present from tough cleaning programs. A neutralizer efficacy test checks the effectiveness of the neutralizer in counteracting the active microbial inhibitory effects of the sanitizer. Even swab methodology can be validated for consistency and recovery.

Other aspects of a strong surveillance program include:

  • Water testing
  • Compressed air testing
  • Media fill
  • Terminal sterilization
  • Utilities

Water is ubiquitous in an aseptic processing facility—as a raw material, buffer, rinse agent, etc. Microbiological quality of water is of particular importance. Feed water, pre-treatment, reverse osmosis (RO), deionization (DI), and water for injection (WFI) need to be tested and assessed for microbial counts. United States Regulatory Guidance is provided by USP <1231>—Water for Pharmaceutical Purposes, American Public Health Association (APHA) Standard Methods for Examination of Water and Wastewater, and Environmental Protection Agency (EPA)National Primary Drinking Water Regulations. Water systems should be monitored at points of use, storage tanks, and loop circulation. Water sampling personnel should be trained in aseptic technique. Microbiological media, such as R2A, should be selective for slow-growing, injured bacteria that may be present in the very harsh environment of a pharmaceutical water system. Water pH, conductivity, total oxidizable carbon (TOC), and chemistry are routinely tested. Water results should be trended to look for seasonal effects, especially in the feed water source. These seasonal effects may change salt concentration, pH, and microbiological flora, which may all affect product quality.

Compressed air systems are used in cleanroom operations to overlay products, pressurize tanks, and dry and supply energy to equipment. Compressed air including nitrogen, CO2, etc. should be filtered with hydrophobic vent filters. It is important to test for both viables and non-viables. Use pressure reduction orifices to provide a steady stream of air, and ensure validation of media, paying special attention to agar desiccation.

Media fills are product simulation studies using general bacterial growth media like trypticase soy broth (TSB). This broth is usually inserted into a production fill/finish process at the end of a normal manufacturing campaign. This end run demonstrates “worst case scenario” testing. The TSB is filled into the same product container/closure system in statistically significant quantities (e.g, =1,000 units) and incubated for 14 days. The units are then inspected for turbidity or microbial growth. Sterility of all units validates the entire fill/finish system including tanks, lines, conveyors, containers, fill lines, closures, personnel, and cleanroom facility. Media fills should be done frequently as specified by quality systems specifications and to validate any changes to the fill/finish operation.

Terminal sterilization may be used in aseptic processing industries to sterilize raw materials, equipment, buffers, and containers. Autoclaves or steam-in-place (SIP) systems should be monitored on a routine basis for temperature time and pressure. Biological indicators (BIs) demonstrate appropriate lethality for sterility assurance level (SAL). Terminal sterilization systems assist in controlling overall bioburden and endotoxin levels in final product quality and require monitoring for operational consistency. Periodic validations should be completed on empty and full load scenarios. Load maps should be developed to allow for consistent packing of the autoclave chamber to maximize microbial lethality.

Product bioburden is another aspect in some environmental monitoring programs. Bioburden testing provides microbial surveillance of various components in the manufacturing process including raw material, water, components, buffer prep, equipment, and upstream processing. The bioburden levels can be cumulative and overwhelm filtration in aseptic processing. Knowing these microbial loads helps in building appropriate process controls to improve final product quality. Bioburden methodology includes pour plates, spread plates, membrane filtration, most probable number (MPN), water activity (Aw), and some automated rapid microbiology systems. All bioburden methods should be validated for plate count recovery.

HVAC systems in controlled environments must also undergo validation under both static???at rest testing and dynamic???at operation testing. These two conditional states will demonstrate the consistency of HEPA filtration over time at both baseline and stressed conditions. Dynamic operational or stressed conditions should include the personnel typically working in the cleanroom and the equipment in operation. Typical tests include air and surface monitoring for viable counts, particle counts, temperature humidity control, HEPA leak tests, velocity, and smoke tests for laminar flow. Utilities such as electrical, gas, and steam should be validated upon initial startup and be consistently monitored.

Documentation for a robust E/M system includes the following:

  • SOPs
  • E/M test procedures
  • Equipment use, maintenance, and calibration procedures and logs
  • Cleaning and sanitization data sheets
  • Sample site maps
  • Alert and action levels
  • Test results, date of results, incubation temperature deviation, and quality review
  • Microbial identifications
  • Environmental trending spreadsheets, histograms, etc.
  • Deviation reporting and/or CAPA procedures

Documentation should be completed using Good Documentation Practices (GDPs).

Summary

Overall, a thorough E/M system will provide management with process feedback for compliance activities. Environmental monitoring primarily focuses on microbiological control. Surveillance of the aseptic processing environment confirms overall production quality and provides significant insight into potential contamination risk. Environmental specifications can be dictated by the region where aseptic products are being marketed. Regulatory guidelines offer various measurement parameters but are inconsistent with acceptable limits. This can cause confusion in the development and data interpretation of environmental monitoring programs. In some cases, limits are not specified or are completely different. It is important for quality management personnel to be aware of these discrepancies in attempting to meet compliance standards. U.S. and EU harmonization of both viable and non-viable counts in various cleanroom conditional states is needed to provide better consistency and quality levels.

Corresponding author Fran McAteer is vice president at Microbiology Research Associates, Inc. (Acton, MA; www.mra-bact.com) and has expertise and experience in E/M programs, aseptic manufacturing, and USP <797> compliance. He acts as a consultant for many pharmaceutical, biotechnology, medical device, and hospital compounding operations.

Ryan Burke is a microbiologist at Acusphere, Inc. (Tewksbury, MA; www.acusphere.com). He manages the Quality Systems Microbiology Laboratory and helped to develop the E/M programs for the facility.

References

  1. Parenteral Drug Association (PDA), Technical Report No. 13, Fundamentals of an Environmental Monitoring Program.
  2. United States Pharmacopeia and National Formulary, USP <1116>—Microbiological Evaluation of Cleanrooms and Controlled Environments.
  3. International Standards Organization, ISO 14644-1—Classification of Air Cleanliness.
  4. European Union (EU) Annex on the Manufacture of Sterile Medicinal Products.
  5. U.S. Food and Drug Administration (FDA), Guideline on Sterile Products Produced by Aseptic Processing.