E/M Associates, Inc. P.O. Box 2077 Salem, NH 03079 978.686.

0215

ENVIRONMENTAL MONITORING OVERVIEW

Environmental Monitoring (E/M) is a surveillance system for microbiological control of cleanrooms and other controlled environments. It is a process which provides monitoring, testing and feedback to the microbiological quality levels in aseptic environments. Environmental surveillance monitors the effectiveness of various controls for microbiological contamination of the environments. Sources of contamination in the cleanroom can come from air, personnel, equipment, cleaning agents, containers, water and compressed gases amongst other things. Sound monitoring required understanding the various stringent regulatory specifications by various organizations such as the Food and Drug Administration (FDA), International Standards Organization (ISO), Parenteral Drug Associates (PDA), European Union (EU) and United States Pharmacopeia (USP). The following table is a synopsis of the regulatory publications. It provides 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. 131.

Table 1. Class 100 Monitoring Table (max. values are given).

COUNTY DOCUMENT CLASSIFICATION FREQUENCY U.S. 209E M 3.5 (100) Not stated 3.500/m3 (>0.5m) 100/cu. ft. U.S. USP <1116> M 3.5 Each operational shift 100/cu. ft. (>0.5m) EU (at rest, static) A and B Not stated 3.500/m3 (equal to or above 0.5m) 0/m3 (>5m) Not stated EU (operational, dynamic) A Frequent, using a variety of methods 3.500/m3 (equal to or above 0.5m) 0/m3 (>5m) <1 CFU/m3 Settle plate 90 mm <1 CFU/4 hours <1 CFU per contact plate (not distinction for floors and walls) <1 CFU per contact plate (not distinction for floors and walls) Not stated Glove print 5 fingers <1 CFU per glove 0.45 m/s + 20% Continuous EU (operational, dynamic) B Frequent, using a variety of methods 350,000/m3 (equal to or above 0.5m) 2,0000/m3 (>5m) <10 CFU/m3 Settle plate 90 mm 5 CFU/4 hours 5 CFU per contact plate (not distinction for floors and walls) 5 CFU per contact plate (not distinction for floors and walls) Not stated Glove print 5 fingers 5 CFU per glove Not appropriate Continuous ISO 14644-1 5 Not stated 3,520/m3 (equal to or above 0.5m) 29/m3 (5.0 m) Not stated

TOTAL PARTICULATE COUNT

AIRBORNE VIABLES

Not stated

0.1 CFU per cu. ft.

SURFACE VIABLES (except floors)

Not stated

3 CFU per contact plate*

Not stated

Not stated

SURFACE VIABLES (floors)

Not stated

3 CFU per contact plate

Not stated

Not stated

PERSONNEL GOWN PERSONNEL GLOVES AIR VELOCITY UNIDIRECTIONAL FREQUENCY OF P MONITORING

P = Differential pressure

Not stated Not stated

5 CFU per contact plate 3 CFU per contact plate Not stated Each shift

Not stated Not stated

Not stated Not stated

Not stated Not stated

0.45 m/s + 20% Not stated

Not stated Not stated

*Contact plate areas vary from 24-20 cm2

Comment: Fed-Std-209E indicates that SI names and units are preferred for naming and describing the classes, but the English (U.S. customary) units may be used. With the publication of ISO 14644-1 and 14644-2, it is expected that Fed-Std-209E will be retired by the end of 2001. Grade A Terminally sterilized: Filling of terminally sterilized products, when usually at risk. Aseptically prepared: Aseptic preparation and filling. Handling of sterile starting material and components. Transfer of partially closed containers and open trays. Background for grade A. Transfer of partially closed containers in sealed trays.

Grade B

Table 2. Class 10,000 Monitoring Table (max. values are given).

COUNTY DOCUMENT CLASSIFICATION FREQUENCY TOTAL PARTICULATE COUNT U.S. FS 209E M 5.5 (10,000) Not stated 353,000/m3 (>0.5m) 10,000/cu. ft. AIRBORNE VIABLES Not stated 0.5 CFU per cu. ft. Not stated 100 CFU/m3 Settle plate 90 mm 50 CFU/4 hours 25 CFU per contact plate Not stated Not stated Not stated Not stated U.S. USP <1116> M 5.5 Each Operating Shift 10,000/cu. ft. (>0.5m) EU (at rest, static) C Not stated 350,000/m3 (equal to or above 0.5m) 2,0000/m3 (>5m) EU (operational, dynamic) C Not stated 3,500,000/m3 (equal to or above 0.5m) 20,0000/m3 (>5m) ISO 14644-1 7 Not stated 352,000/m3 (equal to or above 0.5m) 930/m3 (5.0 m) Not stated

SURFACE VIABLES (except floors) SURFACE VIABLES (floors) PERSONNEL GOWN PERSONNEL GLOVES FREQUENCY OF P MONITORING
P = Differential pressure

Not stated Not stated Not stated Not stated Not stated

5 CFU per contact plate* 10 CFU per contact plate 20 CFU per contact plate 10 CFU per contact plate Each shift1 2x/week2

Not stated Not stated Not stated Not stated Not stated

Not stated Not stated Not stated Not stated Not stated

*Contact plate areas vary from 24-20 cm2

Comment: Fed-Std-209E indicates that SI names and units are preferred for naming and describing the classes, but the English (U.S. customary) units may be used. With the publication of ISO 14644-1 and 14644-2, it is expected that Fed-Std-209E will be retired by the end of 2001. Grade C
1 2

Terminally sterilized: Preparation of solutions, when usually at risk. Filling of products Aseptically prepared: Preparation of solutions to be sterile filtered.

Adjacent to Class 100 Support Areas Product

Table 3. Class 100,000 Monitoring Table (max. values are given).

COUNTY DOCUMENT CLASSIFICATION FREQUENCY TOTAL PARTICULATE COUNT U.S. FS 209E M 6.5 (100,000) Not stated 3,530,000/m3 (>0.5m) 100,000/cu. ft. AIRBORNE VIABLES Not stated 2.5 CFU per cu. ft. U.S. USP <1116> M 6.5 Twice Weekly 10,000/cu. ft. (>0.5m) EU (at rest, static) D Not stated 3,500,000/m3 (equal to or above 5m) 20,0000/m3 (>5m) Not stated EU (operational, dynamic) D Not stated Not defined ISO 14644-1 8 Not stated 3,520,000/m3 (equal to or above 0.5m) 29,300/m3 (>5 m) Not stated

SURFACE VIABLES (except floors) SURFACE VIABLES (floors) FREQUENCY OF P MONITORING

P = Differential pressure

Not stated Not stated Not stated

Not stated Not stated Weekly

Not stated Not stated Not stated

200 CFU/m3 Settle plate 90 mm 100 CFU/4 hours 50 CFU per contact plate Not stated Not stated

Not stated Not stated Not stated

*Contact plate areas vary from 24-20 cm2

Comment: Fed-Std-209E indicates that SI names and units are preferred for naming and describing the classes, but the English (U.S. customary) units may be used. With the publication of ISO 14644-1 and 14644-2, it is expected that Fed-Std-209E will be retired by the end of 2001. Grade D
1 2

Terminally sterilized: Preparation of solutions and components for subsequent filling. Aseptically prepared: Handling of components after washing.

Adjacent to Class 100 Support Areas Product

Please note Federal Standard 209E has been retired and superseded by ISO 14644-1.

As one can plainly view, there are many differences in both terminology and measurement parameters. This can cause confusion by personnel developing environmental monitoring progress. Acceptance criteria for microbiological contamination are different for air surfaces, and personnel. This difference forces companies to look at setting appropriate surveillance based on where they may be selling their products. Cleanroom Classifications differ between the United States and Europe ISO classifications. Rank cleanliness of cleanrooms from Class 1 thru 9 based on particle size of interest. The table below illustrates ISO Standard in comparison to the cancelled Federal Standard 209E. From USP30 <797> International Organization of Standardization (SIO) Classification of Particulate Matter in Room Air [Limits are in Particles 0.5m and Larger per cubic meter (Current ISO) and cubic feet (former Federal Standard No. 209E, FS209E.] Class Name ISO Class 3 4 5 6 7 8 U.S. FS209E Class 1 Class 10 Class 100 Class 1000 Class 10,000 Class 100,000 ISO, m 35.2 352 3520 35,200 352,000 3,520,000
3

Particle Size FS 209E, ft3 1 10 100 1000 10,000 100,000

Adapted from the Federal Standard No. 209E General Services Administration, Washington, DC 20407 (September 11, 1992) and ISO [4644-1: 1999 Cleanrooms and Associated Controlled Environments Part 1: Classification of Air Cleanliness. For example, 3520 particles of 0.5m per m3 or larger (ISO Classes) is equivalent to 100 particles per ft3 (Class 100) (1m3 = 34.314ft3). 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 thru 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 is usually preparation areas for fill/finish operations where open containers may be present. Grade C are 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, the classification of aseptic manufacturing is classified as either Level I, Level II or Level III and the classification 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 preparation activities. Most major biotechnology, pharmaceutical, and medical device companies today market their products in both the United State and in Europe. In order to compete in the appropriate country, 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. Current efforts to harmonize these specifications have not been

finalized. However, there are slight inconsistencies among these different documents which may add unnecessary confusion and difficulty to the already extensive process of validating a pharmaceutical, biotechnological, medical device manufacturing facility. 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 harmonize and integrate into a 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 identify the zones within the facility into different grades based on the levels of cleanliness. The different grades of the rooms are specifically designed and set up in an order 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 possibly even for some established ones, trying to decipher the different terminology of the various grades described 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 nonviable 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.
Specifications: Limits for Particulates per cubic meter of air Particle Size .1m .2 m .3 m .5 m 1 m 5 m ISO 146441 Class 5 100,000 237,00 10,200 3,520 832 29 USP <1116> Class 100 NS NS NS 3,530 NS NS EU Grade A NS NS NS 3,500* 3,500** NS 0* 0** ISO 14644-1 Class 6 1,000,000 237,000 102,000 35,200 8,320 293 USP <1116> Class 1,000 NS NS NS 35,300 NS NS EU Grade B NS NS NS 3,500* 350,000** NS 0* 2,000** ISO 146441 Class 7 NS NS NS 352,000 83,200 2,930 USP <1116> Class 10,000 NS NS NS 353,000 NS NS EU Grade C NS NS NS 350,000* 3,500,000** NS 2,000* 20,000** ISO 14644-1 Class 8 NS NS NS 3,520,000 832,000 29,300 USP <1116> Class 100,000 NS NS NS 3,530,000 NS NS EU Grade D NS NS NS 3,500,000* NS** NS 20,000* NS**

* At rest ** Dynamic NS Not stated 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 micron and 5.0 micron size particulates. The ISO 14644-1 and the European Commission both list specifications for the 0.5 micron and 5.0 micron size particulates but the USP <1116> does not specify any limits for the larger 5.0 micron size particulate. It

only deals with the 0.5 micron particulate size. Another inconsistency is that the European Commission has specifications for both dynamic and at rest (static) conditions where 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 sense due to the fact that adding people and activity to a room will normally result in adding more particulates to the environment as well. Another discrepancy involves the particulate limits within each classification. The European Commission (EC) is generally the most conservative. Lastly, and arguably the largest issue, concerning the particulate limits for these classified areas is the zero specification for the 5.0 micron 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. Due to this issue there have been discussions amongst the European Commission to consider a limit of 20 5.0 micron size particulates per cubic meter of air within 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.

Specifications: For Viable Counts - Colony Forming Units (CFU) Sample Type USP <1116> Class 100 <3 CFU NS 3 CFU 3 CFU 3 CFU 5 CFU EU Grade A NS* <1 CFU** NS* <1 CFU** NS* <1 CFU** NS* <1 CFU** NS* <1 CFU** NS USP <1116> Class 1,000 NS NS NS NS NS NS EU Grade B NS* 10 CFU** NS* 5 CFU** NS* 5 CFU** NS* 5 CFU** NS* 5 CFU** NS USP <1116> Class 10,000 <20 CFU NS 5 CFU 10 CFU 10 CFU 20 CFU EU Grade C NS* 100 CFU** NS* 50 CFU** 25 CFU NS NS NS USP <1116> Class 100,000 <100 NS NS NS NS NS EU Grade D NS* 200 CFU** NS* 100 CFU** NS* 50 CFU** NS NS NS

Air sample / M3 Air Settling Plates / 4 hr. exposure Surface Viables (except floors) / plate Surface Viables (floors) / plate Gloves / plate Personal Gown / plate

* At rest ** Dynamic NS Not stated

The table above displays the inconsistencies between the two guidance documents throughout all the different classifications. First off, one can see that some items are specified in one guidance and not in the other. Also, the table exhibits the different acceptable limits for each classification specified by each of the guidance documents. In the Grade A (USP Class 100) area the European Commission is 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 the more conservative limits. Either way the discrepancies displayed in the above table are very significant in each of the classified areas. One can see how this guidance can be very confusing and difficult to interpret, therefore the pressing need for these guidance documents to be harmonized as soon as possible. Currently, our clients are advised to go with the most conservative specification which will ensure that all acceptance criteria are met. However, this certainly increases overall quality and facility costs. 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, environmental monitoring programs test air, surfaces and personnel. Viable particles are living microscopic organisms present in the aseptic environment. Is especially quantifies bacteria, yeast, and mold in the air and on surfaces. Testing usually includes surface monitoring, personnel monitoring and air monitoring. Companies check their clean room environments are testing routinely to insure they meet required standards. Monitoring of air can be by rotary centrifuged sampling on settling plates. Rotary Centrifugal Air Samplers (RCS) are also used. These instruments measure an exact amount of air with a quantifiable number of viable microorganisms. The most widely used organisms are media impaction instruments. Contamination can be measured per cubic foot or cubic meter of air. Rotary Centrifugal Air samplers are active monitoring devices that do correlate microbial contamination with measured air flow. They include: Slit to Agar (STA) Seive Impactors Cetrifugal Impactors Liquid Impingement Filtration

Gravitational or settling plates are Petri dishes that contain sterile growth media. 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 offer ease of use and remain cost effective. However, they do not directly correlate microbial contamination with measured air volume and do not provide a quantitative measurement of air contamination. Surface monitoring involves contact or rodac plates which contain sterile growth medium in 50mm plates or a sample area of 25cm2. 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 plates residue must be removed immediately after testing the sample site.

Swabs are used for sampling surfaces that are not flat, such as tubing or equipment. Swabbing is qualitative and is technique dependent. A 2x2 sq. in. sample site approximately 25cm2 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 from technician and training should be enforced. Personnel working in a clean room need to be monitored for microbial contamination. Personnel monitoring is an indication of gowning proficiency. Sampling sites are located on both gloves and gowns. Touch plates (contact plates) can be used to dynamically monitor technicians hands immediately after a critical process, such as sterility. Personnel hygiene training should be conducted for all aseptic processing personnel. Personnel monitoring is a good tool to assess cleanroom gowning techniques. Surface rinse methods are another monitoring technique for fermentation vessels, hold tanks and kettles. It is used for large surface areas and tested by membrane filtration. For non-viable measurements, particle counts measure airborne particulates. These counts help qualify the cleanroom or controlled environments 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 anaerobia 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 micron size associated with the overall size of bacteria. As previously discussed, the European Union (EU) is also concerned about larger particles which are felt to possibly carry multiple microorganisms. Samples sites for environmental monitoring are dependent 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 for lyophilized product. Sample sites should include areas that may be inaccessible or difficult to clean. This may include 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 simply sites such as door handles. Sample site selection is customized to the product produced by the company and unique to the individual manufacturing facility. Sampling sites 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, a summary of historical monitoring data and be reviewed by a qualified microbiologist.

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 may signify a potential deviation from normal case scenarios. Alert levels 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 companies Corrective Action and Preventative Action (CAPA) Standard Operating Procedures (SOP). A hot topic in todays environmental monitoring is E/M Data Management. This includes data collection, analysis and interpretation. Based on the larger number of E/M sites, sampling frequency and conditional states, many companies are utilizing computer data informational 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 (i.e. seasonal effect), change in floras, possible patterns or clusters and 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 medias 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 possible source of contamination. For example, a gram positive coccus may indicate human intervention (skin bacteria). Environmental monitoring methods need validation to demonstrate accuracy and robustness. Equipment such as air samples and particle counters should have installation, operation and performance qualifications (i.e. IQ, OQ, PQ). These validation tests should verify and document that the equipment performs consistently over time. For microbiological medias utilized in air and surface monitoring, growth promotion should exhibit acceptable recovery of challenged microorganisms including bacteria, yeast and molds. If medias 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 cleanroom cleaning programs. A neutralizer efficacy test tests 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: 1. 2. 3. 4. Water Testing Compressed Air Testing Media Fill Terminal Sterilization

5. Utilities Water is ubiquitous in an aseptic processing facility. It includes uses 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 is critical and sampling personnel should be trained in aseptic technique. Microbiological medias, such as R2A, should be selective for slow growing, injured bacteria which may be present in the very harsh environment of a pharmaceutical water system. pH, conductivity, total oxidizable carbon (TOC) and water 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 contamination, pH and in microbiological flora which can have an effect on product quality. Compressed air systems are used in cleanroom operations to overlay products, pressurize tanks, dry equipment and energize equipment. Compressed air including air nitrogen, CO2, etc. should be filtered with hydrophobic vent filters. Testing for both viables and non-viables should take place. Use pressure reduction orifices to provide a steady stream of air. Insure validation of media especially paying attention to 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 (i.e. >1000 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 be done 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. The used of biological indicators (BIs) will demonstrate appropriate lethality for sterility assurance level (SAL). Terminal sterilization system assists in controlling overall bioburden and endotoxin levels in final product quality and should be monitored for consistency of operation. 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 can overwhelm filtration in aseptic processing. Knowing these microbial loads can build 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 should also undergo validation under both static, at rest, testing and dynamic, at operation, testing. These two conditional states will demonstrate the consistency of the 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 start-up and consistently monitored. Documentation for a strong environmental monitoring system should include the following: A. B. C. D. E. F. G. H. I. J. Standard Operating Procedures (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 (GDP). Overall, a good environmental monitoring system will provide management with process feedback to 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 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 totally different. It is important for quality management to be aware of these discrepancies in meeting or attempting to meet compliance standards. US and EU harmonization of both viable and non-viable counts in various cleanroom conditional states is needed to provide better consistency and quality levels.

References Parenteral Drug Association PDA Technical Report No. 13 Fundamentals of an Environmental Monitoring Program United States Pharmacopeia and National Formulary USP30 <1116> Microbiological Evaluation of Cleanrooms and Controlled Environments International Standards Organization (ISO) 14644-1 Classification of Air Cleanliness European Union (EU) Annex on the Manufacture of Sterile Medicinal Products Food and Drug Administration (FDA) Guideline on Sterile Products Produced by Aseptic Processing