Qualification of an Environmental Monitoring Program

The microbiology department plays a critical role in the qualification, or requalification, of a facility. There are several areas where this is especially true:

• Cleaning Studies
• Contamination control planning (see PMF News v13 n6)
• Equipment Hold Time Studies (establishment of both clean and dirty hold times)
• Selection of sample sites for environmental monitoring.
• Establishment of alert and action limits

This article will focus on the selection of the sample sites as this issue causes a great deal of confusion in the field. This is not meant to describe the only possible approach to this selection but rather one that the author has used in the past. Due to the limitations of space this discussion will not include sampling of the water system, gasses or personnel (all important topics to be dealt with in later articles).
The number of sites to evaluate during a qualification study breaks down by test type and surface:

• Non-viable air
• Active sampling of air-borne viables
• Passive sampling of air-borne viable (settle plates)
• Surface sampling—Walls & Floors
• Surface Sampling—Equipment

Number of Sites for Qualification Studies

N_L = √A
Where N_L is the minimum number of sampling locations (rounded up to a whole number); and

A is the area of the clean room or zone in meters²

This might work well enough for non-viable particulate measures (which, after all is the intent and scope of 14644-1) but we also wish to consider viable air sampling (both passive and active), and viable surface monitoring. Let’s make this a bit easier and argue that both viable and non-viable active air sampling sites should be done at the same location (or as close as practical to avoid compromising the other measure, or the integrity of the product). Therefore the number of active air sampling sites is driven by the non-viable particulate calculation.
Passive air sampling (settle plates), are a frequently-used measure of clean room (or zone) control. They have the several advantages in this regard, chief among them the ability to remain in continuous exposure for up to 4 hours (exposure time must be demonstrated) and they are not disruptive to the immediate environment and so may possibly sample sites very near product exposure points (see Whyte, 1996 for a discussion of these, and other , advantages). In addition, they are not as prone to variation among different vendors as are active samplers (Yao and Mainelis 2006). However, it is not clear how to interpret the data in all cases from areas of laminar air flow at the rates used for modern clean rooms. However you view their usefulness, the current international regulatory expectation for air monitoring includes their use and the justification of sampling sites. A prudent measure is to use the same number of sampling sites for settle plates as used for the active viable and non-viable sampling programs. These will not be the same sites, but similar in number.

Remember, we are not talking about the sample sites for the on-going environmental monitoring program, but rather the qualification of the sample sites to be used in that routine program.
This leaves us with determination of the number of surface sampling sites for the qualification study. There is no regulatory guidance directed to this point for the international pharmaceutical industry (even PIC/S, which generally can be counted on to provide details on almost everything microbiological, is silent on this point – PIC/S 2004). Also silent on this point is the PDA (Parenteral Drug Association) Technical Report #13. We are left to our own devices. One approach to determination of the number of sites would be to address it in an manner similar to that of ISO 14644-1 for the walls and floors (as relevant). Each surface would then be treated as a separate item and the minimum number of sites determined for each as the square root of its surface area.
This leaves us with only the question of how to determine the number of surface sampling sites for equipment. This cannot be answered by this approach convincingly (in my opinion). This determination, quite frankly, may well be something that must be left to each facility as the numbers could be driven by the nature of the equipment and as well as the associated manufacturing process.
Having determined the number of sites for each room, we now need to determine their location.

Selection of Sample Sites

Here the PDA Technical Report provides some useful guidance:
“Factors to consider in selecting sites for routine surveillance are:

• At which sites would microbial contamination most likely have an adverse effect on product quality?
• What sites would most likely demonstrate heaviest microbial proliferation during actual production?
• Should site selection involve a statistical design (e.g.,following the calculations in Federal Standard 209E) or should site selection be made on the basis of grid profiling? Should some sites for routine monitoring be rotated? [Note from author: As 209e has been withdrawn in favor of ISO 14644, the answer is No]
• What sites would represent the most inaccessible or difficult areas to clean, sanitize, or disinfect?
• What activities in the area contribute to the spread of contamination?
• Would the act of sampling at a given site disturb the environment sufficiently to cause erroneous data to be collected or contaminate product?”

The FDA Aseptic Processing Guidance document (FDA 2004) also provides some guidance in section IVA:
“Air in the immediate proximity of exposed sterilized containers/closures and filling/ closing operations would be of appropriate particle quality when it has a per-cubic-meter particle count of no more than 3520 in a size range of 0.5 μm and larger when counted at representative locations normally not more than 1 foot away from the work site, within the airflow, and during filling/closing operations. This level of air cleanliness is also known as Class 100 (ISO 5).
We recommend that measurements to confirm air cleanliness in critical areas be taken at sites where there is most potential risk to the exposed sterilized product, containers, and closures. The particle counting probe should be placed in an orientation demonstrated to obtain a meaningful sample. Regular monitoring (Continued from page 3) should be performed during each production shift. We recommend conducting nonviable particle monitoring with a remote counting system. These systems are capable of collecting more comprehensive data and are generally less invasive than portable particle counters. See Section X.E. for additional guidance on particle monitoring.
Some operations can generate high levels of product (e.g., powder) particles that, by their nature, do not pose a risk of product contamination. It may not, in these cases, be feasible to measure air quality within the one-foot distance and still differentiate background levels of particles from air contaminants. In these instances, air can be sampled in a manner that, to the extent possible, characterizes the true level of extrinsic particle contamination to which the product is exposed. Initial qualification of the area under dynamic conditions without the actual filling function provides some baseline information on the non-product particle generation of the operation.”
Further on in Section XA we read:
“Sample timing, frequency, and location should be carefully selected based upon their relationship to the operation performed…
It is important that locations posing the most microbiological risk to the product be a key part of the program. It is especially important to monitor the microbiological quality of the critical area to determine whether or not aseptic conditions are maintained during filling and closing activities. Air and surface samples should be taken at the locations where significant activity or product exposure occurs during production. Critical surfaces that come in contact with the sterile product should remain sterile throughout an operation. When identifying critical sites to be sampled, consideration should be given to the points of contamination risk in a process, including factors such as difficulty of setup, length of processing time, and impact of interventions.”
The EU guidance document “Manufacture of Sterile Medicinal Products” (EU 2008) provides some site selection guidance:
“18. Where aseptic operations are performed monitoring should be frequent using methods such as settle plates, volumetric air and surface sampling (e.g. swabs and contact plates). Sampling methods used in operation should not interfere with zone protection.”
Similarly, guidance in the most recently proposed revision to USP chapter <1116> (USP 2007) is of general interest:
“Microbiological sampling sites are best selected when human activity during manufacturing operations are considered. Careful observation and mapping of a clean room during the qualification phase can provide information concerning the movement and positioning of personnel within these rooms. Such observation can also yield important information about the most frequently conducted manipulations and interventions.
Other areas of concern relative to introduction of contamination into clean rooms are at entry points where equipment and materials move from areas of lower classification to those of higher classification. Therefore, areas within and around doors and airlocks should be included in the monitoring scheme.”
Let’s summarize some specific considerations of sample site selection for the qualification study.
After we determine the minimal number of sites in a room, we have to determine their most useful location. This determination should be documented in a written justification and should consider:

• Contamination vectors (handles, control panels, doors, etc).
• High traffic areas
• Personnel flow
• Material flow
• Waste Flow
• Surfaces that are difficult to disinfect
• HVAC (primarily returns in this regard)
• Product risk
• Extent of product exposure
• The type of activity performed near that site
• The Potential for Contaminations from Interventions and manipulations
• Contamination vectors

Selection of Routine Sites After the Qualification Study

The qualification study should include sufficient replicates under conditions both “at rest” and “dynamic” to allow identification of sites that provide useful information. It should be clarified that the term “useful information” is not meant to describe “those sites that give the most desireable counts” but rather those sites which either give the highest counts (ie serve as the most sensitive measure of the state of control of the room) or were shown to be appropriately placed to herald a problem in the room. The number of sites in a room or zone should similarly be driven by data generated during this study. Both the number and location of sites or each clean room or zone should be justified in the report from this qualification study.
The following section (X.1.A) from the FDA guidance is relevant for consideration here:
“All environmental monitoring locations should be described in SOPs with sufficient detail to allow for reproducible sampling of a given location surveyed. Written SOPs should also address elements such as (1) frequency of sampling, (2) when the samples are taken (i.e., during or at the conclusion of operations), (3) duration of sampling, (4) sample size (e.g., surface area, air volume), (5) specific sampling equipment and techniques, (6) alert and action levels, and (7) appropriate response to deviations from alert or action levels.

A Note on the “Microorganism Catalog”

The FDA has clearly recommended establishment of a listing of common microorganisms found in the aseptic manufacturing environment. This expectation is laid out in section X.B. (FDA 2004).
“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. In some cases, environmental trending data have revealed migration of microorganisms into the aseptic processing room from either uncontrolled or lesser controlled areas. Establishing an adequate program for differentiating microorganisms in the lesser-controlled environments, such as Class 100,000 (ISO 8), can often be instrumental in detecting such trends. At minimum, the program should require species (or, where appropriate, genus) identification of microorganisms in these ancillary environments at frequent intervals to establish a valid, current database of contaminants present in the facility during processing (and to demonstrate that cleaning and sanitization procedures continue to be effective).”
The EM qualification study is an excellent opportunity to start this catalog, and to generate information on the effectiveness of the cleaning and sanitization program from a microbiological perspective.

References

1. EU. 2008. 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.
2. PIC/S. 2004. PI-006-2 Recommendations on Validation Master Plan, Installation and Operational Qualification, Non-sterile Process Validation, Cleaning Validation
3. PDA. 2001. PDA Tech Report #13 (Revised): Fundamentals of an Environmental Monitoring Program
4. PMF News v13 n6 http://microbiologyforum.org/PMFNews/PMFNews.13.06.0706.pdf
5. USP. 2007. <1116> Microbiological Control and Monitoring Environments Used for the Manufacture of Healthcare Products Pharm Forum. 33(3).
6. Whyte, W. 1996. In Support of Settle Plates. PDA J Pharm Sci Tech. 50(4):201-204.
7. Yao, M. and Mainelis, G. 2006. Investigation of Cut-Off Sizes and Collection Efficiencies of Portable Microbial Samplers. Aerosol Sci Technol. 40:595—606.