Chapter 50: Environmental Monitoring

Environmental monitoring is considered by regulators (United States Food and Drug Administration [FDA]) necessary to ensure that the material/products produced are in accordance with 21 CFR 211.113, “Control of Microbiological Contamination.” This section of the Code of Federal Regulations (CFR) specifically states the “appropriate written procedures . . . shall be established and followed.”

A robust environmental monitoring program provides valuable information regarding the point-in-time conditions of the manufacturing environment as well as the ancillary clean areas. It allows for manufacturers to be able to potentially identify areas of contamination and be able to implement corrective and preventive action (CAPA) accordingly, such as additional cleaning regimens, different cleaning solutions, evaluation of personnel, and optimized material flow. It is important to note that a low level of contamination may not always be detected, and a trending program may alert manufacturers to future issues.

The specific cleanroom environment that a material/product would be manufactured in would dictate the frequency of monitoring and the specific techniques associated with the environmental monitoring program.

Nonviable Particle Monitoring

As part of any controlled environment, nonviable particle monitoring is important both for the identification and control of nonviable (nonliving particles, such as dust) and viable (living microorganisms) contamination. This monitoring is most critical in an ISO Class 5 (Class 100) environment, typically used for aseptic processing of injectable preparations. Meeting the specifications of the established cleanroom standards is a necessity to perform manufacturing according to the certification specific (and predefined) to the cleanroom.

Typically, the size of most concern is 0.5 microns. This size is representative of the approximate size of a single microorganism. It has been shown that microorganisms can attach themselves to particles and therefore become a biologic contaminant.

Testing may be performed in two different conditions, either dynamic or static. Dynamic conditions are those in which activities in the cleanrooms are occurring during normal operations. Testing in these types of conditions is more desirable when in production to ensure that the cleanrooms are meeting specifications during potentially worst-case operations. Static conditions are those in which no operations are occurring and minimal or no personnel are in the cleanroom (with the exception of the analyst), and do not give a representative picture of cleanliness in the room with personnel flow, components, and active machine operations.

With today’s technology, multiple ways exist to perform nonviable monitoring. One method is by a portable particle counting instrument, whereby an analyst performs a system verification of the instrument and, with the instrument’s internal vacuum, draws a programmed volume of sample air through the system for analysis (typically through use of a laser). Particle counter sensors may be installed in critical locations in cleanrooms, which are connected to a centralized unit and computer system. They enable alarms to be sent to multiple e-mail addresses or cell phones. These units are typically continuously run and can provide real-time results as well as significant amounts of data for trending purposes.

Contact Plates

Contact, surface, or RODAC (replicate organism detection and counting) testing is necessary in an environmental monitoring program. RODAC is a brand name commonly used in the pharmaceutical industry and is a registered trademark of Becton Dickinson, a manufacturer of these agar plates, although several other organizations sell such plates. Contact plates use a slightly convex surface of microbiological media to make direct contact with a flat surface. Predetermined work sites are tested by gently pressing the media surface onto the site and immediately recapping the plate. After testing, the site is wiped down with isopropyl alcohol and a lint-free wipe to ensure that no media residue is left behind. If media residue is left behind, it may become an inadvertent viable area for the growth of microorganisms. Critical areas should be tested at the end of processing to avoid contamination of a sterile processing area.

The contact plates are approximately 65 mm × 15 mm and provide a testing surface area of 25 cm². For routine monitoring, trypticase soy agar (TSA) with lecithin and polysorbate 80 is the media of choice. The trypticase soy agar is the nutrient media for the microorganisms (specifically bacteria) to grow in. Lecithin and polysorbate 80 are added to deactivate disinfectant residue. TSA contact plates are incubated at 30 ºC to 35 ºC for 48 to 72 hours.

Another formulation of media, Sabouraud dextrose agar (SDA), is used for monitoring of mold and yeast. The main difference between the TSA and SDA agar is that the lower pH of SDA provides for a more favorable environment of growth for fungi. SDA plates are incubated at 20 ºC to 25 ºC for five to seven days.

Contact plates may be used for personnel monitoring. Four fingers are closed together and the contact plate is lightly applied to the fingertips, and the thumb is lightly pressed on the surface as well. After testing, either the gloves are changed out (in the gowning area for an aseptic area) or disinfected with isopropyl alcohol and wiped with a lint-free wipe to prevent media transfer to work surfaces from gloves. In situations where media fills are occurring, sleeve monitoring may be performed as well. Contact plates should not be used on irregular or textured surfaces, only smooth surfaces. For irregular surfaces, a swab method may be used.

Swab Monitoring

In areas that are not able to be accessed and tested with contact plates (for example, machine parts, tubing, or rough or uneven areas), swab monitoring may be performed. A lint-free swab is moistened with a buffer and placed in a sterile, capped tube. When ready for use, which would be at the end of a sterile processing operation, the swab is carefully removed from the tube, using impeccable aseptic technique, and wiped across the surface, rotating the swab to ensure the entire swab is used. The swab is re-submerged in the buffer solution and vortexed (or similar mixing) to ensure that any microbiological contamination is transferred from the swab to the solution. The solution is aseptically poured onto agar plates (TSA or SDA) and incubated. Another swab method may be used: a slightly moistened swab housed in a sterile, capped tube is wiped across the surface and, using the same rotation motion, swabbed directly on the agar plate and incubated.

Regardless of swab method used, a large potential margin for error exists due to the sheer number of steps that may lead to unintentional personnel contamination. The swab monitoring method must undergo method validation to ensure that proper recovery is achieved.

Settle Plates

Settle plates are used as part of an environmental monitoring passive air-sampling program. This type of air testing is considered to be qualitative in that no predetermined quantity of air is being sampled, but rather the quality of the air (types of organisms present) is being tested by plates being placed in critical locations and the agar exposed for a specific amount of time, usually an hour.

Settle plates are composed of TSA or SDA. The plates are exposed and recapped, then incubated. One drawback of settle plates is that they desiccate with extensive exposure time and/or high airflows. In the pharmaceutical industry, this phenomenon displays a “potato chip” appearance when incubated and therefore would inhibit the potential growth of microorganisms. A maximum exposure time should be validated.

Active Viable Air Monitoring

A more representative method of monitoring air quality is active air monitoring. Varieties of samplers include impaction, centrifugal, and membrane. These devices allow testing of the number of organisms per volume of air sampled. This method of air testing is considered to be quantitative, that is, the number of organisms can be calculated by the volume of air sampled. Active air samplers draw in a predetermined volume of surrounding air and aim the air stream at the culture medium (agar plate or agar strip) for incubation. These active air samplers are placed in critical areas of an operation, although they should not disrupt the unidirectional airflow.