An MD&DI May 1998 Column
A case study examining the levels of airborne and surface contaminants through the use of three different incubation processes illustrates that manufacturers should ensure the effectiveness of their existing systems, especially if the safety of their devices could be compromised by increased levels of particulates.
Environmental monitoring is a key element in the maintenance and control of defined manufacturing conditions. Section 820.70 of the quality system regulation (QSR) requires that device manufacturers establish, conduct, maintain, document, and periodically inspect environmental control systems to "verify that the system, including necessary equipment, is adequate and functioning properly." An effective environmental control system provides information on many elements of the manufacturing operation, including the following:
- Process deviations.
- The ability to maintain validated conditions within the facility, equipment, and procedures.
- The effectiveness of controls on the manufacturing environment as well as on product contamination levels.
- The sterility assurance program for aseptically produced and terminally sterilized products.
- The effectiveness of facility and equipment cleaning procedures.
Environmental monitoring programs usually measure both viable and total particulate contamination. Programs measuring viable contamination levels should effectively capture and count the indigenous flora in the manufacturing environment. It is not necessarily important to identify isolated organisms by means of environmental sampling. Whether it is important to do so depends on many factors, including the nature of the product, potential consequences to product safety or performance, and whether the product is terminally sterilized.1 Alert and action limits need to be established based on the total number of organisms observed. There are a number of methods to choose from to calculate such limits.2 If the optimal medium and incubation conditions are calculated and then used, the quantitation method should recover a majority of the environmental organisms. A comprehensive facility validation program should include documentation of the testing used to determine the appropriate quantitation procedure.
A device manufacturer evaluated the effectiveness of its current quantitative procedure for viable contaminants by comparing the data generated from its standard method with data derived from the use of different media and incubation conditions. Such an evaluation could lead to a procedural change, depending on the study results. The manufacturing procedure requires the handling and distribution of large amounts of fibrous and corrugated materials. Based on experience, a number of fungi were expected in the indigenous flora of the manufacturing environment. The manufacturer had already documented that the presence of fungal species in the final product has no adverse affects. However, because of their potentially high numbers, the manufacturer wanted to account for fungal species in the reports of results of total and viable organism monitoring.
Under the standard procedure, viable contaminants were recovered for air sampling tests using Biotest RCS sampler with soybean casein digest agar (TSA) and for surface tests using equipment surface swabs wetted with Stewards transfer medium. After sampling, the swabs were extracted with soybean casein digest broth (TSB) and extractant aliquots plated with TSA. The swabs were incubated for 48 hours at 35° ± 2°C.
In addition to the company's standard culturing process described above, additional samples were cultured in different media and incubated under different schemes to collect data to study the effectiveness of the various methods. The other cultures and incubation periods are described as follows:
- TSA agar incubated for 48 hours at 25° ± 2°C, followed by incubation for 72 hours at 35° ± 2°C (cold/hot incubation).
- TSA agar incubated for 48 hours at 35° ± 2 °C, followed by incubation for 72 hours at 25° ± 2 °C (hot/cold incubation).
- Rose Bengal agar, a selective agar formulated for the recovery of yeast and mold species, incubated for 120 hours at 25° ± 2°C. Chloramphenicol, a component of this agar medium, inhibits bacterial growth.
Twenty sites were sampled for airborne contamination using the manufacturer's standard procedure, which involved sampling of 40 L of air with the Biotest RCS sampler during a 1-minute period. The sites represented a cross-sectional sampling of the entire manufacturing area. A total of three samples were taken at each sample location, two using TSA agar strips and one using Rose Bengal agar strips. Incubation was as described above. All airborne counts were reported as colony forming units (CFUs) per cubic foot of air sampled.
A total of 25 equipment surface sites were sampled by a Culturette II system with Stewards transfer medium. A 4-sq-in. area was sampled at each designated site. Within 4 hours of sampling, each swab was placed in TSB and extracted for viable organisms with 4 ml of TSB and 1 ml aliquots plated in either TSA or Rose Bengal agar. Incubation was as indicated above. The data from the surface samples were reported as CFUs per square inch.
Each plate was examined daily and all CFUs were measured after approximately 48 hours, when the incubation conditions were changed, and again after approximately 120 hours when the incubation period was concluded.
Airborne Samples. The optimal recovery of viable microorganisms from air and equipment surfaces occurred when using a TSA recovery medium and the cold/hot incubation scheme of 48 hours at 25° ± 2°C followed by 72 hours at 35° ± 2°C.
A total of 285 CFUs were observed from the TSA cold/hot recovery method of the 20 air sampling sites, as opposed to 187 CFUs recovered by the TSA hot/cold recovery method from the same sites. The hot/cold method therefore recovered only 66% as many CFUs as the cold/hot incubation scheme had. With Rose Bengal agar incubated for 5 days at 25° ± 2°C 141 CFUs were recovered, or 49% of the highest total from those sites.
Using the manufacturer's normal recovery method of incubating TSA for 48 hours at 35° ± 2°C, a total of 121 CFUs were observed from the 20 air sampling sites. This count is approximately 2.4 times lower than that observed with the TSA cold/hot incubation scheme and in fact this procedure yielded the worst results of all the investigated processes.
When the air counts from the manufacturer's current recovery method were added to the total observed with the Rose Bengal agar (for an estimate of total bacterial and fungal count), the total of 262 CFUs was approximately 92% of the air count results observed using the TSA cold/hot recovery method.
Treating the number of CFUs recovered from the TSA cold/hot procedure (285) as the total number of recovered microorganisms and comparing it to the total recovered by the Rose Bengal agar (141), it seems that approximately 50% of the recovered airborne microorganisms were fungi (Table I provides full results).
|TSA/25°C/35°C||TSA/35°C/25°C||Rose Bengal Agar
|Sample Number||After First
Table I. Results on airborne samples, indicating measurements of the number of CFUs per cubic foot taken after a 48-hour incubation period, followed by a 72-hour period at a second temperature setting. The Rose Bengal agar testing was performed at one setting. The column designated by an asterisk represents the findings obtained from what had been the manufacturer's standard procedure.
Surface Samples. A total of 23 CFUs were observed using the TSA cold/hot recovery method from the 25 equipment surface sites compared to 16 organisms from the TSA hot/cold method from these same sites. This number is approximately 70% of the CFUs recovered using the TSA cold/hot incubation scheme. Two CFUs were recovered with the Rose Bengal agar from the same 25 sites.
The manufacturer's recovery method used TSA with a 48-hour incubation period at 35° ± 2°C. Using this recovery procedure, one CFU was observed from the 25 equipment surface samples. This count is approximately 22 times lower than that observed using the TSA cold/hot incubation scheme.
When the equipment surface counts using the manufacturer's TSA method (48 hours at 35° ± 2°C) were added to the Rose Bengal counts, the total of three CFUs was approximately 13% of the result observed with the TSA cold/hot recovery method (Table II).
|TSA/25°C/35°C||TSA/35°C/25°C||Rose Bengal Agar
|Sample Number||After First
Table II. Results of the three environmental particulate tests performed on samples from equipment surfaces, indicating measurements of the number of CFUs per square inch taken after a 48-hour incubation period at the first temperature shown, followed by a 72-hour period at the second temperature setting. The Rose Bengal agar testing was all performed at one setting. The results in the column designated by an asterisk represent the findings obtained from what had been the manufacturer's standard procedure.
The data showed that the recovery method traditionally used by the manufacturer to recover viable environmental contaminants was not optimal, because it did not maximize the recovery of fungal species. Based on a preliminary review of the manufacturing process, it had been hypothesized that there would be a significant number of fungal species. The data supported this initial hypothesis; in fact, approximately half of the recovered airborne CFUs were estimated to be fungi.
The data from this study convinced the manufacturer to revise its current recovery practice to use a TSA medium and the two-tier cold/hot incubation scheme of 48 hours at 25° ± 2°C followed by 72 hours at 35° ± 2°C. This method optimizes the recovery of viable organisms, including fungi, from the manufacturer's environment. Fungal species were observed on the TSA strips after the 120-hour incubation period. The manufacturer concluded that including a specific differential medium for recovering fungi into its routine monitoring practice would not increase the total number of recoverable CFUs. Since fungi did not adversely affect product safety or performance, there was no reason to routinely differentiate fungal and bacterial species. The manufacturer believes that total counts are sufficient to monitor and assess the capabilities and performance of its environmental control practices.
This study was not intended to serve as a validation of the proposed method. Additional studies are necessary to confirm the initial findings and demonstrate the reproducibility of the method. The data suggested that the manufacturer's historical environmental microbial database was not indicative of actual contamination levels, which were probably several orders of magnitude greater than those reported. Thus, the manufacturer decided not to use this historical database to establish alert and action limits for environmental surveys. Rather, data generated by the cold/hot incubation method will be gathered monthly for 1 year, at which time revised alert and action limits will be calculated. Preliminary alert and action limits will be established based on the data from this study.
This study clearly demonstrates that a cold/hot incubation scheme with TSA is more effective in recovering organisms from a manufacturing environment than a hot/cold incubation scheme with TSA or a Rose Bengal agar with a cold incubation procedure. These results are intuitively reasonable because it can be assumed that the indigenous environmental organisms have adapted to ambient conditions. The colder temperature used in the tests is more representative of those conditions than the warmer test temperature. The lower initial incubation temperature may have allowed the slower-growing fungi to proliferate and develop into visible colonies before being overgrown by the faster-growing bacteria at the higher incubation temperature.
Manufacturers should not universally extrapolate the conclusions of this study to other environmental-monitoring situations. Many factors influence the presence of microbial flora, including the product being manufactured, the facility, its geographic location, and time of year. Additionally, the significance of what a particular microorganism means to a product's safety or performance may require customization of the recovery program to identify these specific organisms. Therefore, the optimum recovery program for one facility will not necessarily satisfy the requirements for another company. Individual recovery programs should be designed for each facility, based on documented data.
1. Reich RR, "Microbial Taxonomy: When Is It Appropriate in Environmental Monitoring?," Med Dev Diag Indust, 17(1):220221, 1995.
2. Wilson JD, "Setting Alert/Action Limits for Environmental Monitoring Programs," J Pharm Sci Tech, 51(4):161162, 1997.
Robert R. Reich is president, Pharmaceutical Systems, Inc. (Mundelein, IL).