USP 797 Environmental Monitoring Program Design and Application
By Joseph P. Manfrida, Ph.D., EMLab P&K Analyst
The proper design and execution of an environmental sampling plan is a central component of USP <797>. A good environmental sampling program will not only allow a pharmaceutical compounding laboratory to know whether or not it is within the recommended action levels of USP <797>, but will also provide valuable information for determining sources of potential contamination and counteracting them.
Testing Requirements and Methodology
Successful implementation of a USP <797> environmental sampling program starts with an understanding of the different tests required by USP <797>. The most basic division between the required tests is nonviable particle sampling and viable particle sampling. Nonviable airborne particle testing seeks to measure the density of airborne particles based strictly on their size (0.5 µm in diameter or larger) without regard for the nature of the particles themselves. Viable particle sampling only measures those particles that are living organisms (typically bacterial and fungal spores). USP <797> breaks viable particle testing into several different categories, each of which is designed to test a separate aspect of pharmaceutical compounding for potential contamination. These tests are viable airborne particle testing, viable surface particle testing, gloved fingertip sampling and media-fill testing (also called aseptic manipulation testing).
Nonviable airborne particle sampling must be performed by a qualified operator using an electronic particle counter. Testing needs to occur once every six months at a minimum. Additional testing is required any time that the primary engineering controls (laminar flow hoods, isolators, etc.) are moved or altered, or in the event of major changes to the facilities surrounding the primary engineering controls. At a minimum, each separate ISO class 5, 7 or 8 area needs to be tested (1). A thorough testing plan will also investigate areas and items that could be potential sources of nonviable particulates. Potential sources of nonviable particles include, but are not limited to, potential leaks in a clean room's containment (possibly at windows, doors and pass-through cabinets), mechanical and electrical equipment (refrigerators, computer printers, etc.) and areas with high personnel traffic during compounding operations. A map of the laboratory being sampled can be very useful for pinpointing critical areas for sampling. If a map or blueprint of the compounding laboratory cannot be obtained from the laboratory manager, a hand-sketched map can prove to be useful. All data collected must be thoroughly documented. Documentation should include the specific locations sampled, the time of day sampling took place, copies of the calibration certificate for the particle counter used to collect the data, and training certificates for the individual(s) who performed the sampling.
Airborne bacteria and fungi can pose a significant threat of contamination during the manufacture of compounded sterile preparations. Viable airborne particle testing is performed to monitor this threat and to ascertain that physical and procedural controls in place are keeping the airborne microbial load of a facility's air to an acceptable level. The frequency of testing for airborne viable particulates is identical to that for airborne nonviable particulates (2). It is recommended that sampling take place during ongoing compounding operations. If it is not possible to take viable air samples while compounding is taking place, then sampling should take place immediately after compounding has ended for the day (3). Volumetric sampling devices must be used and 400 to 1000 liters of air must be tested for each sample (4). Gravimetric sampling may be used to supplement volumetric sampling, but gravimetric sampling is not sufficient for meeting the requirements of USP <797>. Air samples taken for the purpose of measuring bacterial load utilize TSA (Tryptic Soy Agar) or soybean casein digest as growth media for collected organisms, while air samples taken to examine airborne fungal load are collected using MEA (Maltose Extract Agar) or Sabouraud Dextrose Agar. It is important to note that testing for fungi is only required for compounding categorized as high risk (5).
Viable surface sampling is required by USP <797> in order to assess the success of the laboratory's cleaning program in keeping surfaces free of microbial contamination. While USP <797> mandates periodic surface sampling, it does not specify a definitive period or time frame for sampling. A feasible option is for surface sampling to take place at the same time as air sampling. Sampling surfaces in parallel with air sampling allows for a more thorough investigation of any detected contamination and maximizes convenience for personnel performing the sampling. At least one surface sample must be taken from each ISO 5, 7 and 8 area after compounding has concluded (6). Additional samples can be taken from any surface that personnel, materials or produced compounds are exposed to regularly. Work surfaces, storage surfaces, door handles and equipment are all good targets for surface sampling. Surface samples may be collected using TSA contact plates with added lecithin and Polysorbate 80 (TWEEN 80) or swabs. Contact plates are generally the preferred method for surface testing because of their ease of use, but sample collection with swabs is perfectly acceptable under USP <797>. Swabs are also the only means of sampling from curved and oddly shaped surfaces, such as door handles or sink faucets.
Gloved fingertip sampling is performed to evaluate the efficacy of compounding personnel's hand washing and garbing techniques. Workers wash their hands and don all of their protective equipment while being observed by their supervisor, in order to make certain that their technique is in accordance with the laboratory's standard operating procedures. Once they are completely garbed, the worker presses four fingers and a thumb to a TSA plate (one for each hand) with lecithin and Polysorbate 80 (7). It should be noted that only TSA plates with lecithin and Polysorbate 80 are utilized for this test. There are no requirements for a separate gloved fingertip test specifically to check for fungal contamination. Gloved fingertip tests must be performed prior to any new personnel beginning work at a compounding facility, annually for established personnel performing low and medium risk compounding, and once every six months for personnel performing high risk compounding.
Media-fill testing (also called aseptic manipulation testing) is performed to measure the ability of procedures, personnel and equipment to successfully produce a sterile end product. This test is a simulation of the actual compounding taking place in a facility. During media-fill testing, personnel perform all of the steps for a given compounding procedure using the same equipment and facilities as they would when compounding, except the typical medications and diluents normally used in the procedure, are replaced with soybean casein digest media. It is important for the laboratory manager or head pharmacist of a facility to participate extensively in the development and execution of this particular test. This is because extensive knowledge of the laboratory's compounding procedures is necessary to properly set up a media-fill test that closely resembles the compounding taking place in their lab or facility. A media-fill test is considered successful if no growth is seen in the final containers prepared by the test after incubation. Media-fill tests must be performed by all personnel prior to being allowed to begin compounding at a given facility, annually for personnel working in low and medium risk compounding facilities, and once every six months for personnel engaging in high risk compounding (8).
While performing viable or nonviable sampling it is important to be aware of quality control. Copies of calibration certificates should be provided to the laboratory manager for all equipment used in sampling. Likewise, a copy of the sampling personnel's training qualifications should also be made available. All media used for viable sampling should have an accompanying certificate of analysis that also should be provided to the laboratory manager. It is generally considered good practice to utilize positive and negative controls for viable testing, however USP <797> does not require these controls. Positive controls require that a viable sample plate be exposed to a known quantity of microorganisms and incubated alongside samples taken in a compounding facility. In practice, it is very difficult for personnel working in the field to obtain and maintain stock cultures of organisms with known concentrations. In the field, personnel seeking to make a positive control, sometimes sample from an area that is not ISO rated. Sampling air outside of an ISO classified area is likely to detect microorganisms of an unknown concentration. This type of "positive control" will demonstrate the media's ability to support microbial growth if microorganisms are in the area sampled, but without any specific data available for microbial concentrations in a specific area, it is impossible to be certain how well the media performs. As a result, this type of sample represents a compromise between the precision of a true positive control and the ambiguity of having no positive control at all. Negative controls are much easier for field personnel to obtain. A negative control is provided by sealing an unopened media plate taken from the same batch as the sample plates, then sending it to a laboratory for incubation and analysis without ever exposing the plate to an air sample. For media plates that are received sterile, the negative controls should show no growth after incubation.
Data interpretation begins by comparing the results of nonviable and viable air particle testing and viable surface particle testing to the recommended action levels in USP <797>. These recommended action levels have been reproduced in Table 1 (9). If the number of particles detected by nonviable air particle testing, viable air particle testing or viable surface particle testing, exceeds the levels given in table 1, then it is necessary to begin a full root cause investigation of the source of contamination, followed by whatever steps are necessary to bring the particulate levels detected to within acceptable limits.
|Table 1: Recommended Action Levels. (9)|
|ISO Class||≥ 0.5 µm Nonviable
|3||3,520||> 1||> 3|
|7||352,000||> 10||> 5|
|8||3,520,000||> 100||> 100|
For example, if a viable airborne particle sample from an ISO 7 clean room yielded a total of 11 cfu/m3, it would be higher than the recommended action level of greater than 10 cfu/m3 and a full investigation into the source of contamination would be necessary. Alternatively, if the same sample had only 9 cfu/m3, the recommended action level would not have been exceeded and a full investigation would not need to be initiated. It is also necessary to implement an investigation and remediation in the event that there are particulates identified above historically detected levels for a given facility.
There is also a special requirement for viable air particle samples. If any cfu's are detected on a viable airborne particle test plate from an ISO 5, 7 or 8 area, then USP <797> requires that the colonies growing on that plate be identified to at least the genus level, even if the number of colonies is below the recommended action level (10). The reasoning behind this requirement for genus identification is that there are some organisms that cannot be tolerated in a clinical environment at any concentration.
For example, Methicillin-resistant Staphylococcus aureas (MRSA) is a very dangerous organism in hospitals, and has been discussed extensively in the popular media. If even one cfu of MRSA is detected in the air of a pharmaceutical compounding laboratory, then extensive investigation and remediation would be warranted. Full speciation of viable colonies is highly recommended in order to avoid expending extensive resources for remediation of non-dangerous organisms in the same genus as highly dangerous ones.
To return to our previous example, Staphylococcus epidermidis would not warrant the same response as Staphylococcus aureas. However, a genus identification for these two organisms would yield the same result, Staphyloccoccus. A laboratory manager would not be able to distinguish which of the two organisms were present in the laboratory and would have to respond as if the more dangerous organism was present. Species level identification allows for a more precise response by laboratory personnel to the actual threat posed by any given organism.
Interpretation of data from gloved fingertip and media-fill tests is less complex than interpretation of air and surface plates. For gloved fingertip plates, a new worker must have three successful tests with 0 cfu's, prior to beginning work in a compounding laboratory or facility. For experienced workers, the recommended action level is >3 cfu's per test total, whereby the counts from both the left and right hand plates are added together. Media-fill tests are either positive or negative for growth. Any positive media-fill tests require a root cause investigation and the implementation of remediation to fix the problem (11).
Investigation and Remediation
Investigation of viable particle concentrations above action levels, or away from historical baseline levels of contamination, must take into account both physical and personnel factors. Physical factors include items such as making certain that all equipment is working properly. The maintenance records of all equipment should be reviewed to be certain that everything in the lab has been properly maintained. Regularly scheduled cleaning of the lab itself is also critical. Cleaning logs should be reviewed and cleaning equipment inspected to be certain that it is still capable of cleaning properly. Cleaning equipment should be non-shedding and dedicated for use strictly in the controlled environment. Cleaning agents need to be rotated on a regular basis. Not all cleaning agents are equally effective against all microorganisms. For example, 70% ethanol is a cleaning agent that is highly effective against bacteria, but has very little effect on bacterial endospores, some fungal spores and certain viruses (12, 13). Rotating through a series of different cleaning agents over time increases the likelihood that all organisms in a laboratory are eventually exposed to a susceptible substance. Cleaning agents must also be stored and used according to their labels. Changes in the weather or in the building containing the compounding facility also need to be investigated. A rapid climate change can cause previously quiescent organisms to begin growing. Changes to the physical structure of the building could affect the ability of the compounding laboratory to prevent environmental incursions. Personnel factors are the ability of compounding personnel to successfully implement the laboratory's standard operating procedures to prevent contamination. Direct observation of personnel during ongoing compounding operations should be conducted to verify proper implementation of all laboratory standard operating procedures. The procedures themselves should be reviewed to make certain they are adequate to the task of preventing contamination.
At all stages of USP <797> implementation it is very important to remember to document every action taken and every test result. The historical record is the only means that a laboratory manager has for recognizing trends and spotting potential problems before they become severe. Ideally, observing how the contamination levels change over time in a laboratory will allow potential contamination issues to be isolated and solved early, before they have a chance to cause injury or illness in pharmacy patients or laboratory personnel. In the event that a patient illness leads to an investigation by legal authorities, these records are the only proof that a laboratory has been following the regulations established by USP <797>. Without thorough documentation that the laboratory has been meeting the requirements of USP <797>, they will likely be subject to legal consequences.
Design and execution of a USP <797> environmental sampling plan is a task that requires attention to detail, extensive knowledge of the tests required and precision in reporting data. At EMLab P&K we are dedicated to providing the technical expertise and USP <797> testing laboratory capabilities necessary to meet the challenge of implementing a rigorous USP <797> program. We are prepared to provide the commitment to quality that you have come to expect from EMLab P&K. Contact us for USP <797> services.
1. The United States Pharmacopeial Convention. <797> Pharmaceutical Compounding - Sterile Preparations. Revision Bulletin. 2008, p. 1-61.
2. Ibid., Revision Bulletin, p. 25.
3. The United States Pharmacopeial Convention. <1116> Microbiological Evaluation of Clean Rooms and Other Controlled Environments. National Formulary. 2000, p. 2099-2106.
4. Ibid., Revision Bulletin, p. 25.
5. Ibid., Revision Bulletin, p. 25.
6. Ibid., Revision Bulletin, p. 33.
7. Ibid., Revision Bulletin, p. 31-32.
8. Ibid., Revision Bulletin, p. 30.
9. Ibid., Revision Bulletin, p. 2, 26 and 34.
10. Ibid., Revision Bulletin, p. 26.
11. Ibid., Revision Bulletin, p. 31-32.
12. Allen, L.V., Jr. and Okeke, C.C. 2007. Basics of Compounding: Considerations for Implementing United States Pharmacopeia Chapter <797> Pharmaceutical Compounding - Sterile Preparations, Part 4: Considerations in Selection and Use of Disinfectants and Antiseptics. International Journal of Pharmaceutical Compounding. 11(6): 492-496.
13. Utama, I.M.S., Wills, R.B.H., Ben-yehoshua, S. and Kuek, C. 2002. In Vitro Efficacy of Plant Volatiles for Inhibiting the Growth of Fruit and Vegetable Decay Microorganisms. Journal of Agricultural Food Chemistry. 50(22): 6371-6377.
This article was originally published on February 2010.