COMPOUNDED STERILE PREPARATIONS (CSPs) CAN BE CONTAMINATED through poor aseptic technique (touch contamination) and poorly controlled environmental conditions. According to ISPE’s Sterile Manufacturing Facilities Guide, proper sterile compounding requires “a strict design regime, not only on the process area, but [also] on the interactions with surrounding areas and the movement of people, materials, and equipment, so as not to compromise the aseptic conditions.” It is crucial that the environment in which sterile preparations are compounded is clean and that factors—both human and otherwise—that can affect CSP microbial sterility or chemical stability are controlled. Several interconnected and dynamic variables form the foundation of a solid environmental monitoring program, and it is important to understand and detect environmental bioburden (air microbial contamination).
Viable and Nonviable Particles
Two types of particles can be found in the pharmacy compounding area: nonviable particles and viable particles. A viable particle contains one or more living microorganisms and can affect the sterility of the CSP. Viable particles generally range from ~0.2?m (micron) to ~30?m in size. Bacteria need a means to travel and inoculate the CSPs; their vectors can include bare or gloved hands and particles. A nonviable particle does not contain a living microorganism, but acts as transportation for viable particles. Examples of nonviable particles can include metal, rust, dust, dirt, pollen, fibers, cloth, and chemical compounds found in makeup, including silicone, magnesium, sodium, potassium, and chloride. These particles can be introduced through employee movement, cleanroom garb or employee clothing, and the movement of products. The amounts of these particles are measured twice a year during the certification of cleanroom and engineering controls, such as laminar airflow workbenches, biological safety cabinets, and barrier isolators. Nonviable particle quantities are reported as the number of particles 0.5 micron or larger per cubic meter of sampled air. The results of this test do not differentiate between viable and nonviable particles.
Bacteria can attach to particles of ~1.0?m or larger. Nonviable particles >5.0?m offer the same opportunity for viable particles to attach themselves, and they can also adversely affect a patient’s health and well-being; particles can block veins or capillaries if they get into the blood stream. The typical diameter of veins range from 7 to 50?m and capillaries from 4 to 7?m. Non-viable particles in poorly controlled environments can range from 0.01?m (viruses) to 120?m (dust), as reported in the January 2005 issue of A2C2 magazine, in an article entitled “Pharmaceutical Industry Cleanroom Monitoring: Viable and NonViable Particle Detection.”
Air Sampling Requirements
USP Chapter <797> currently requires air sampling of only controlled air environments, such as laminar airflow workbenches, barrier isolators, buffer rooms, cleanrooms, and anterooms. According to Chapter <797>, lowand medium-risk level compounding areas need to be tested on a monthly basis, and high-risk level compounding areas need to be tested on a weekly basis. Proposed changes to <797> would expand environmental testing to include surface testing in controlled air environments and the fingertips of gloves. These proposed changes also dictate the frequency of environmental sampling based on the volume of compounding activity in the compounding area. (See www.usp.org/standards/proposed797Revisions.html)
As mentioned earlier, the total amount of airborne particles (viable and nonviable) under static conditions are determined twice a year during routine hood and cleanroom certification. Air and surface sampling involve collecting environmental “snapshots” on settling, or tryptic soy agar (TSA), plates that support the growth of aerobic and facultative anaerobes. TSA plates are between 90 and 140 mm in diameter and should be exposed to cleanroom air for a period of one hour (for 140-mm plates) or four hours (for 90-mm plates). The sampling plates are incubated for 48 hours at 86 ?F to 95 ?F. According to the Encyclopedia of Pharmaceutical Technology, other types of microorganisms, such as yeasts and molds (which have been identified as sources of microbial contaminants in CSPs) require the use of Sabouraud dextrose agar or Rose Bengal agar. Two methods can be used to perform air sampling in the controlled air environments: passive sampling, via gravity settling plates placed in various locations throughout the controlled environment, or active sampling with a volumetric air sampler.
Passive sampling recovers microorganisms via the gravimetric properties of microbe-carrying particles (MCPs). As published in the International Journal of Pharmaceutical Compounding (2001; 5: 246-253), when monitoring horizontal laminar airflow environments, the use of settling plates for air sampling may not be the best method, because the settling velocity of MCPs is negligible. It is believed that settling plates do, however, have an advantage of predicting microbiological contamination that would result from disruptions of air caused by personnel and component movement and equipment. The graphic on page 4 illustrates some of the locations where settling plates can be used, along with some recommended CFU (colony-forming unit) counts for the plates. A CFU is any discrete microbial colony that grows on the plates and is counted at the completion of the incubation period. Air sampling is a quantitative test and, unless it is speciated by a qualified microbiologist, does not provide any information on the genus or species of microorganisms.
Active air sampling uses a device to pull a large volume of air over growth medium, where any MCPs are impinged or impacted onto the surface of the agar strip. Three different active air sampling devices can be used.
- Reuter centrifugal sampler. This device draws air through rotary blades, impacting MCPs against a nutrient agar strip by centrifugal force. The user determines the amount of air drawn through the blades. It is the most convenient and least expensive of the three active sampling devices.
- Slit-to-agar impaction sampler. With this device, air is drawn through a fixed slot and is then directed by a vacuum over a revolving plate containing the growth medium. This device can demonstrate changes in microbiological concentrations over time.
- Liquid impingement. Using a vacuum, air is drawn into the unit through fluid. Particles and microorganisms are “impinged” onto the liquid, which is then filtered. The filters are placed on nutrient media, incubated, and monitored for microbial growth. Liquid impingement has been recommended as the standard reference method for monitoring aerobic contamination.
Sampling sites, identified in Figure 1, should be chosen by the criticality of compounding that occurs at those locations. Ideally, environmental monitoring should occur under dynamic conditions—during regular work activity—and is best conducted toward the end of the compounding day, when the environment has been most stressed. Under these “worst case conditions” the environmental bioburden and particulate matter should be at their highest levels. Environmental monitoring should be done routinely to generate operating data that can be used to identify shifts in controls and trends. A baseline sample should also be taken under at-rest or static conditions. At-rest environmental bioburden monitoring can evaluate the operating status of engineering controls, operating equipment, and the effectiveness of cleaning and sanitizing procedures.
While the microbial air sample is being obtained, operating conditions can be rotated between production (dynamic) and non-production (static) times. Testing under dynamic conditions is useful in monitoring the effectiveness of hand washing, garbing, and gloving by personnel, and also records the microbial condition of the controlled work area when staff is present. Testing under static conditions provides information about the performance of high-efficiency particulate air (HEPA) filters and controls for pressure differentials, air exchanges, temperature, and humidity, and about the effectiveness of cleaning and sanitizing procedures.
Microbial air sampling data serves to observe the trends in microbial bioburden over time. Any sudden increase in established action limits or trended increases in bioburden over time signals that one or more systems or processes are failing. These shifts should prompt an investigation and the implementation of a corrective action plan to remediate the issues. Microbial air sampling is a simple and cost-effective quality-system metric that provides critical data on the functionality of engineering controls and employee compliance with policy and procedures.
Through his New Jersey-based consulting company, Clinical IQ, LLC, Eric Kastango, RPh, MBA, FASHP, provides expertise in aseptic processing, medical-device manufacturing, and the implementation of extemporaneous compounding-quality systems. He is also a pharmacy surveyor for the Accreditation Commission for Health Care, Inc.
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