Dan Klein- Senior Technical Service Manager, STERIS Corporation; Jim Polarine, Jr.- Senior Technical Service Manager, STERIS Corporation; Kiara Brooks- Lab Instructor, The University of Tennessee Health Science Center, UT Plough Center, Memphis, TN; Paul Jeffry Pulliam- Production Manager, The University of Tennessee Health Science Center, UT Plough Center, Memphis, TN; Harry Kochat, PhD- Director, Business Development & Operations, The University of Tennessee Health Science Center, Plough Center for Sterile Drug Delivery Solutions, Memphis, TN.
Abstract
There remains debate on both how to conduct a triple clean evaluation as well as what data generation is optimal during the process in order to demonstrate in situ effectiveness of a disinfection program. A triple clean can be performed at any time following a worst-case event such as a shutdown or equipment failure at which times the microbial challenge is expected to be much higher than typically encountered in a cleanroom environment.
In this study, we outlined a triple clean remediation as a quaternary ammonium compound disinfectant used twice followed by a peracetic acid/hydrogen peroxide sporicide. This regimen allows for excellent microbial remediation as a long-term disinfectant rotational program which can be demonstrated by observing reductions in microbial bioburden during the process of reinstituting the cleanroom for use. During a triple clean event, the worst-case conditions present in the cleanroom give an excellent indicator of future performance of a rotational program when significantly lower bioburden challenges are expected.
Introduction
There is increasing focus on an overall, well thought out contamination control strategy (CCS). As the CCS is developed and implemented across the cleanroom site it is critical to have an effective cleaning and disinfection program. Annex I conveys that the disinfection process should be validated.9 Validation studies should demonstrate the suitability and effectiveness of disinfectants in the specific manner in which they are used. Therefore, a well-planned CCS will include disinfectant field trials to support the validation of the disinfectants and sporicides being implemented onsite.
This disinfectant in-situ validation is one of the key activities for evaluating the effectiveness of the cleaning and disinfection program.1,5 The most common definition of the triple cleaning process is to use the disinfectant two times followed by the sporicide.2,3,4 There are some other processes utilized in the industry for bringing the facility online after a worst-case event such as fogging application, VHP application or a 9X cleaning.3
A triple clean consists of an in-situ evaluation following a worst case event, including a facility shut down, maintenance work or power failure. After these events, bioburden levels in a cleanroom environment are expected to be at their highest when compared to standard operation.
The qualification process typically begins with a disinfectant efficacy study with in-vitro studies where the efficacy of sanitizers/ disinfectants/sporicidal agents are evaluated on representative cleanroom substrates with in-house isolates, or reference strains (for a new facility). The in-vitro tests simulate as closely as possible the actual cleanroom conditions such as temperature, cleanroom substrates, and the type of microorganisms. After the sanitizers/disinfectants/ sporicidal agents successfully pass the efficacy tests, the qualification process moves into stage 2, in-situ testing, where the performance of the sanitizers/disinfectants/sporicidal agents will be evaluated under the actual worst-case conditions in the cleanroom. Stage 3 of the process is the continuous monitoring and trending of the environmental data over time to ensure the cleaning and disinfection program continues to be effective at controlling bioburden in the cleanroom environment.
In this case study from a pharmaceutical manufacturer, the ISO 5, ISO-7 and ISO-8 cleanrooms in this facility needed to be shut down for engineering and construction work. The engineering and construction work involved equipment installation, room pressure modification, and facility construction including shutting down HVAC systems that supply HEPA filtered air to the cleanrooms. The site conducted Environmental Monitoring Performance Qualification (EMPQ) in order to re-establish their cleanroom status after the maintenance shut down.
The goal of this testing was to evaluate the effectiveness of the triple cleaning process involving two applications of a quaternary ammonium compound disinfectant, followed by the application of a peracetic acid/hydrogen peroxide sporicide under these worst case conditions.
Materials and Methods
This evaluation consisted of two separate events performed with the same regimen of products. The two evaluations, Evaluation I and Evaluation II, were performed after separate shutdown events approximately four months apart with the redundant testing designed to incorporate different seasons, microbial load and personnel entry requirements. During the maintenance shutdown prior to Evaluation II, workers were not required to wear cleanroom PPE and gowning materials, which could affect the initial microbial load. Both evaluations were performed in the same room areas with the same disinfectant and sporicide regiment. The regimen consisted of first cleaning all debris and residual construction materials. Once the debris and materials were removed, baseline counts were determined as it is critical to understand the existing bioburden after the shutdown occurs. The area was then disinfected with a quaternary ammonium compound ready to use disinfectant followed by baseline sampling of all surface sites. A second application of a quaternary ammonium compound ready to use disinfectant then occurred with surface sampling after treatment. Finally, the area was disinfected with a peracetic acid/ hydrogen peroxide sporicide as a final step of the triple clean and all surfaces sampled.
All cleaning and disinfection were performed using a two-bucket system. The two-bucket system consists of a stainless-steel trolley system equipped with color-coded buckets, a DuoPress™ mop wringer, Duo™ Plus frame, extendable Universal Handle and individually sterile wrapped microfiber laminated mop.
The test phases (i.e., T=0, T=1, and T=2) were intended to assess the change in bioburden levels following each cleaning phase. The recovered growth was characterized, including Gram staining, to understand the nature of the microorganisms (i.e., fungal, spore-forming, non-spore forming) and changes between each baseline phase.
Environmental monitoring was performed on three consecutive days with cleaning and disinfection performed between T0 and T1 sampling and before sampling on T2 and T3 (Refer to Figure 1). All environmental monitoring locations remain the same for T0 (baseline monitoring), after the 1st sanitization (T1), after the 2nd sanitization (T2) and after the 3rd sanitization (T3), including floor surface, wall surface, counter/equipment surface and air viable.
Evaluation I and Evaluation II followed the same procedures and product utilization. The environmental sampling points were conducted at worst case and high-traffic areas in the cleanroom operation. The environmental monitoring consisted of RODAC® plating, settle plating, active air sampling, and swabbing at these worst-case locations in the cleanrooms. For each evaluation, the media plates used were Trypticase™ Soy Agar (TSA) Sterile Plates with Lecithin and Polysorbate 80, RODAC® plates, and Trypticase™ Soy Agar Settling plates. All lots of media plates were tested for Growth promotion and passed. The plates used for Environmental Monitoring of this study were stored in a 2-8° refrigerator.
Immediately after sampling, all media plates were incubated between 20 to 25 °C for no less than 72 hours. After initial incubation, all media plates were transferred to an incubator set between 30 to 35 °C for no less than 48 hours. After all of the sampled plates were pulled from the 30 to 35° incubators, they were read using the colony counter and examined visually for morphology and Gram stained.
Results
The tables below summarize the air and surface data from the T=0, T=1, and T=2 sampling results, as expressed in both bacteria and mold recoveries expressed as CFU.
For each of the thirteen sample areas, the number of microorganisms present decreased significantly from the initial baseline levels with only one site showing a single colony present of all 132 samples taken after the triple clean process. When all sites are pooled, the data suggest an initial reduction resulting from the use of the quaternary ammonium disinfectant with a reduction to nearly zero with the final application of the sporicide.
Similar to Evaluation I, Evaluation II showed a progressive reduction in total microbial CFU’s recovered over the application of the two disinfectants and the sporicidal treatment. This evaluation had a higher initial value of CFU recovered and demonstrated a more pronounced reduction between the first and second disinfectant step. Despite some larger initial values as predicted for this evaluation, the counts for 11 of the 12 sample sites after the sporicidal treatment still demonstrated between 0-4 CFU present. There was an outlier in the data set from the B125 Personnel Entrance sample which demonstrated an increase in microorganisms from a single CFU after the second disinfectant treatment to 27 CFU after the subsequent sporicide application. Since a sporicide could not increase the number of microorganism present, the outlier was omitted from the graph below. The investigator in the study theorized that the outlier could be a result of unauthorized foot traffic during the study.
Discussion
A triple clean event serves to return an area to operational conditions following a worst-case event such as a shutdown, room failure or maintenance intervention. Following these events, a cleanroom or controlled surface will have higher levels of bioburden present than typically expected during normal operation as a result of uncontrolled access and environmental conditions. Additionally, the triple clean activities can provide excellent documentation of the overall expected success of a disinfectant program. Three main elements often comprise a well-designed disinfectant qualification and implementation program. These include in vitro analyses (Disinfectant Efficacy Studies), in situ evaluation (triple clean studies) and active interpretation and surveillance through environmental monitoring.
These elements are key components to an overall Contamination Control Strategy to demonstrate the suitability and effectiveness strategy of the disinfectant program in place.
In this study, we followed the regimen of disinfectant/disinfectant/ sporicide to show the reduction of microorganisms per application after a shutdown event. This approach combines the surfactant-based one-step cleaning/disinfection of a quaternary ammonium compound disinfectant with the enhanced sporicidal and fungicidal efficacy of an effective PAA/hydrogen peroxide sporicide.
The similar results obtained from Evaluation I and Evaluation II, despite being conducted months apart with each demonstrating a unique bioburden, further suggest the robustness of the disinfection program. Although the design of the studies were identical, inherent variability exists during microbiological evaluations, so the observed results demonstrating the same trend from the use of the product rotation adds additional confidence that the products selected will function well under normal conditions.
Conclusion
A progressive reduction in microbial count was observed from baseline T0 to T3 for each sample site within the ISO-5, ISO-7 and ISO-8 cleanrooms. The total recovered CFU after the final disinfection using the peracetic acid/hydrogen peroxide sporicide gave very low counts with zero CFU’s recovered in 17 out of 25 samples.
This case study demonstrates the effectiveness of the triple cleaning process involving two applications of a Quaternary Ammonium Disinfectant, followed by the application of a peracetic acid/hydrogen peroxide sporicide under these worst-case conditions which serves as an excellent indicator of future performance of this regimen. The case study supports the sites disinfectant validation program and is an integral part of their ongoing CCS which can be assessed routinely for ongoing improvements. The disinfectant field trials are always an important part of a robust CCS which encompasses all facets of the cleanroom operation.
References
- Polarine, J. and Walker, T. Evaluation of a Quaternary Ammonium Ready-to-Use (RTU) Disinfectant and Hydrogen Peroxide/Peracetic Ready to Use (RTU) Combination Sanitization Regimen for Cleanroom Start-Up. American Pharmaceutical Review, May/June 2020.
- Polarine J., Kroeger, B. HOW ISSUES RELATED TO UTILITIES, SURFACES AND PRACTICES IMPACT CLEANROOM ENVIRONMENTS, Contamination Control in Healthcare Product Manufacturing, Volume 4 PDA/DHI Publishing, June 1, 2016
- Polarine J., Kroeger, B. Bringing Cleanrooms Online Initially and After a Worst-Case Event, PDA Poster at PDA Annual Meeting 2015, March 10, 2015.
- Polarine J., Kroeger B., Start-up of cleanrooms, initially and after a worst-case event., Clean Air and Containment Review, August 15, 2017.
- IEST-RP-CC-018.5 Cleanroom Cleaning and Sanitization: Operating and Monitoring Procedures., IEST May 2021.
- Ildikó no Ziegler, Judit no Borbély-Jakab, Lilla no Lilla Sugó and Réka J. Kovács. Revision of Viable Environmental Monitoring in a Developmental Pilot Plant Based on Quality Risk Assessment: A Case Study. PDA Journal of Pharmaceutical Science and Technology January 2017.
- Anne Marie Dixon, Ch. 11, Cleaning of Non-Product Contact Surfaces in Cleaning and Cleaning Validation for the Pharmaceutical and Medical Device Industries, Vol. 1 Basics, Expectations, and Principles. Paul L. Pluta, Ed., PDA, Bethesda, MD, and DHI Publishing, LLC, River Grove, IL. 2009.
- PDA Technical Report Number 70, “Fundamentals of Cleaning and Disinfection Programs for Aseptic Manufacturing Facilities”.
- Annex I Manufacture of Sterile Medicinal Products. August 2022.
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