Corporate Partner Spotlight: EMD Millipore Corporation
Author: Anne Connors
Regional Marketing Manager-Environmental Monitoring/Media Fill Testing
EMD Millipore Corporation
290 Concord Road
Billerica, MA 01821 USA
Phone: +1 781.533.5438
Identifying Contamination Faster Using New Environmental Monitoring Technologies
Environmental monitoring is an essential component of any QC microbiology program and is used to determine the microbial and particulate content of air and surfaces and operators. Identifying and addressing contamination in compounding pharmacy production is critical since it can damage product, lead to product recall, slow product’s time to market and compromise consumer safety. A variety of approaches and new technologies for environmental monitoring exist to help identify conditions contributing to excessive microbial and particulate levels, as well as to alert personnel to conditions compromising the area classification.
Surface monitoring is conducted to determine the presence of viable microorganisms on a variety of critical surfaces, including workbenches, floors, laboratory personnel and difficult to reach areas such as the interior of tubing and filling needles.
Contact plates are used for sampling flat or convex surfaces. The 50mm diameter plates are filled with either Tryptic Soy Agar (TSA) or Sabouraud Dextrose Agar (SDA), which are ideally suited for the growth of yeasts and molds. The solid media protrudes from the plate and is pressed against the surface to be tested. The plate is then incubated to determine the number of colony forming units (cfu’s)present.
Contact plates can pick up residues of sanitizing agents used for cleaning or disinfecting cleanrooms when coming into contact with test surfaces. Sanitizing substances can inhibit the growth of contaminants on the contact plate. To counteract the growth-inhibiting properties of these sanitizers, neutralizers have been added to the culture media found in contact plates. Appropriate combinations of lecithin, polysorbate 80, histidine and sodium thiosulfate are believed to neutralize up to 80% of the sanitizers typically used. However, some of the remaining substances, such as polyhexamethylene biguanides, have proved to be a challenge. A new neutralizing mixture called Neutralizer A (EMD Millipore, Billerica, MA) was recently introduced to cover a larger proportion of the sanitizers. It has proven successful in neutralizing all sanitizers tested so far, with recovery rates all above 50% compared to control plate counts.1
Residues of antibiotics such as β-lactam derivatives can also inhibit microbial growth on contact plates. However, a combination of a new cephalosporinase and penicillinase provides inactivation of a broad spectrum of β-lactam antibiotics, including cephalosporins of the third and fourth generation.
Contact plates are impractical for irregular surfaces such as equipment recesses, nooks, crevices, tubing and filling needles. Instead, pre-moistened swabs can be used to validate cleaning and sanitation procedures and to verify that a required hygiene level has been attained.
The most critical component of the swab is the head. It must be lint-free and extremely low in non-volatile residues and particulates. Swab heads can be made of various materials, including polyester, polyurethane, nylon and cotton, which can be structured or knitted in different ways. The type of material determines the extent to which abrasion may occur, and some materials may be degraded by aggressive solvents and subsequently leave residues. The swab’s shaft is also important and needs to be flexible and long enough for difficult-to-reach locations, such as tubing interiors. For maximum recovery, the swab is carefully removed from its tube and rubbed across a surface area using a twisting motion. Conventional swabs are re-suspended in a specified amount of rinse solution and agitated to transfer any microorganisms present on the swab head into the solution. The collection medium is then tested, usually by direct plating or membrane filtration, followed by incubation of filters on culture media.
A disadvantage of using conventional swabs is that the recommended procedure involves a number of steps, each of which includes the potential for handling errors and cross-contamination, potentially leading to false positives. To avoid having to open the tube several times, an all-in-one swab system can be used. This system requires only one opening because it already carries a reservoir containing the culture broth (ICR swabs, EMD Millipore). After returning the swab to the tube, the reservoir at the top is snapped and squeezed until the broth fully saturates the swab head. The broth is monitored after incubation for turbidity as an indicator of contamination.
This ICR swab is gamma-sterilized in its final packaging at a relatively high dose of 25-35 kGy. Both the moistened swab tip and the medium were shown to be non-inhibitory against challenges of eight microbial species that are more commonly isolated from aseptic processing environments.2 This swab is also suited for use in isolators because it is triple-packaged, with the inner bag possessing a pre-punched hole at the top to hang it up, minimizing surface area for vaporous decontamination.
There are several standard ways of monitoring potential airborne contaminants. Passive air sampling uses solid media settle plates exposed to the air for a pre-determined period of time. Active air sampling utilizes an instrument that draws ambient air and then directs the air stream at an attached agar plate for collection. Particle counters are used to quantify all potential contaminants in the air.
Settle plates usually contain TSA or SDA, and they are placed throughout the test area with their lids removed. After exposure, the plates are closed and incubated. The number of colonies is then counted and the microorganisms are identified. For safe and efficient documentation, pre-barcoded culture media plates are used, enabling each plate to be fully traceable back to its date of use and the location of sampling.
A concern when using settle plates is that they lose water due to evaporation when exposed, leading to an increasingly dry skin on the agar surface. Dry agar can cause poor growth of certain microbes on the media and lead to an underestimation of the proportion of these organisms in the air. TSA plates have been found to lose up to 16% of their original weight over a typical four-hour exposure period in a unidirectional airflow cabinet. However, when such plates were inoculated with typical contaminants and subsequently incubated, all recovery rates were above 70%.3
To enable prolonged exposure and incubation periods while ensuring that the plates still deliver reliable results, settle plates can be poured to a particularly high filling level, as found with ICR Settle Plates (EMD Millipore). The maximum exposure time should be validated for each production line, taking into consideration air flow, temperature, relative humidity of the air and turbulences.
When using active air samplers in critical areas, there are several ways to optimize the process. First, it is important that the air flow is not severely disrupted by the instrument’s operation, placement or removal, as disturbance makes it easier for contaminants to infiltrate the product or process being monitored. To save space and minimize air flow disruption in isolators and other confined areas, manufacturers have developed variants of their instruments. For example, air ducting is directed from the sampling points outside the controlled area where all electronic and moving parts remain. In addition, the instruments’ internal pumps enable easy decontamination of the sample heads and aspiration tubes. To analyze the disruption propensity of an instrument in use, we conduct smoke studies that visualize the movement of air in decommissioned cleanrooms.
MAS-100 NT and RCS High Flow Touch air samplers (EMD Millipore) are specifically designed with rounded edges and other features to minimize air flow disruption. Autoclavable sampling heads make the instrument’s handling safer to reduce air flow disruption and the likelihood of human error. Any easy-handling features likely to minimize human activity in critical areas, such as touch screen operation or high battery capacities for longer cycles, can help to prevent contaminations. A built-in anemometer found in the MAS-100 NT automatically adjusts the flow speed if there are any variations in temperature or pressure.
Particle monitoring of ambient air is conducted to quantify non-viable contaminants in the air and to determine the quality of air in controlled environments. Quantitative particle data often also provides an indication of the status regarding viable contaminants because most airborne microbes adhere to particles.
One decision compounding pharmacies must make when selecting systems is whether to choose pre-installed or portable particle counters. In many facilities, the particle counters are pre-installed at specified locations. Measurement data are automatically collected by cleanroom monitoring systems for centralized analysis and storage, along with data on other parameters such as temperature, relative humidity and air pressure differentials. When the infrastructure of the facility allows for pre-installed particle counters, the systems have proved very convenient.
However, many facilities opt for the flexibility of portable particle counters. For example, the APC SmartTouch system (EMD Millipore) can be programmed from a touch screen and is able to display a visual representation of a cleanroom’s sampling locations, thus reducing the likelihood of human error. Because it is equipped with two high-capacity batteries, it can sample continuously without the need for downtime during recharging.
In addition to utilizing environmental monitoring technologies, companies can implement sterility testing program products. Sterility testing monitors samples from pharmaceutical products that are purported to be sterile. The final article of the series will describe the range of new technologies for sterility testing and media fill testing, and how they can be used to recover potential contamination and impact product release.
As the regulatory environment for compounding pharmacies continues to evolve, facilities can take immediate steps to strengthen their QC microbiology program with new environmental monitoring technologies. Surface and air monitoring can help prevent or address contamination events sooner to avoid contamination of compounding pharmacy products, reduce risk and ensure consumer safety. It is ultimately up to the company responsible for the final product to determine that their process and product is in a state of control.
1Hedderich R. and Klees A. (2012): Neutralization of Disinfectants by Culture Media used in Environmental Monitoring in Environmental Monitoring – A comprehensive Handbook, Volume 6: 159-180
2Sandle, T. (2011): A study of a new type of swab for the environmental monitoring of isolators and cleanrooms (the heipha ICR-Swab). European Journal of Parenteral and Pharmaceutical Sciences, Vol. 16, No.2, pp42-48.
3Sandle, T. (2011): Microbial recovery on settle plates in unidirectional airflow cabinets. Clean Air and Containment Review 6/2011.