Background
A clinical supply manufacturer was adding a new integrated filling operation to its aseptic filling capabilities. The new area provides a Grade C background environment with adjacent Grade D transition airlocks for material and personnel. The traditional sanitization/disinfection used by the facility included the routine use of hydrogen peroxide/peracetic acid RTU, 70% isopropyl alcohol (IPA), and hot Water for Injection (WFI). The site explored cleaning agents currently available and utilized at the adjacent affiliated commercial sites after realizing the limited cleaning effectiveness of the established regimen. As the site strategized to determine the revised regimen, the need for a global approach was considered to synergize all related activities.
The site reviewed several formulas currently available in the United States (US) as well as globally. Not all formulations are available globally and, thus, the choices were limited. In addition to global availability, the site needed a formulation that met the following criteria:
- Demonstrated efficacy
- Chemically compatible with hydrogen peroxide/peracetic acid RTU
- Suitable for hard, non-porous surfaces common in cleanrooms
- Ready-to-use formulations
- Capable of handling multiple application methods
- With minimal residue after application
- Did not contain a phenolic active ingredient
- Capable of controlling bioburden to room classification limits per ISO-14644.
Once the formula was selected, the site reviewed all efficacy data provided by the solution manufacturer to include the coupon testing comprised of multiple cleanroom surfaces consistent with the site construction. The Disinfectant Efficacy Testing (DET) studies reviewed passed all acceptance criteria and were accepted by the site to support the initial efficacy evaluation. Because of the design and results of the solution manufacturer DET studies, an in-house DET study was not performed before the execution of the Environmental Monitoring Performance Qualification (EMPQ), which is also referred to as in situ testing. Furthermore, as this was a newly constructed area, there is no microbiological history to evaluate for an environmental isolate testing. An objective of this functional testing was to verify that the solution manufacturer’s DET results, coupled with the newly implemented sanitization process, provide a robust system for microbiological organism reduction to comply with Grade C/D classifications. The site would leverage the efficacy data available through the solution manufacturer and rely on empirical data collected during routine environmental monitoring (EM).
Real-time EM data collection and data trending must support the routine use of sanitization solutions. Typical organisms isolated represent the actual flora recovered, and solution efficacy is directly linked to this worst-case performance in a Grade C environment. Site bioburden limits for Grade C areas allow for measurable growth (percent growth as well as magnitude) as compared to the zero limit in Grade A areas. This limit enables recovered microbial counts to directly measure/calculate the expected solution efficacy versus actual amounts delivered.
The validation study links empirical data generated to establish and verify solution efficacy (in situ) with real application in controlled areas.
Evaluation Protocol
The site evaluation of quaternary ammonium RTU (Ready-to-Use) focused on efficacy as determined through direct application following site construction. Phase I construction activities were completed, and the area was ready for the EMPQ.
Before beginning the EMPQ, the constructed area was cleaned of all debris and residual construction materials. Once the debris and materials were removed, the area was washed with hot WFI followed by baseline sampling of all air and surface sites in multiple phases. The baseline 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 (i.e., fungal, spore-forming, non-spore forming) and changes between each baseline phase.
The time zero (T=0) testing followed the post-construction debris removal and hot WFI wash included (105) Replicate Organism Detection and Counting (RODAC) surface samples and (100) active air samples using Tryptic Soy Agar (TSA) as the test media. Once sampled, the area was disinfected with quaternary ammonium RTU using overlapping, top to bottom, clean to dirty spray and wiping patterns. Surface mopping included overlapping fi gure-8 stroke patterns using the double bucket (dirty/rinse buckets) system on floors. After a 15-minute drying time, the area was sampled using the same surface and air sites. This sampling was identified as T=1. The final baseline surface and air sampling, T=2, followed the application of hydrogen peroxide/peracetic acid RTU using the same techniques used for the quaternary ammonium RTU.
The completion of the baseline sampling, T=0, T=1, and T=2, leads then to the (1X) static and (3X) dynamic sampling, as described in ISO-14644.
The site map below details the facility layout and includes the environmental classification as related to the cleanroom areas.
Results
Each surface and air EM sample plate using TSA media was incubated for a minimum of 2-4 days at 30-35°C, followed by a minimum of 3-5 days at 20-25°C. Once incubated, each sample was examined under controlled conditions to evaluate the presence of colony-forming units (CFU). The enumerated growth was evaluated against the acceptance criteria for the Grade C/D controlled area and any impact determined.
The EMPQ data collected from the areas cleaned and disinfected were used to establish the various regimen used for routine facility cleaning and disinfection. The procedural requirements for routine cleaning and disinfection are as follows:
- Weekly – Quaternary ammonium RTU disinfection, followed by WFI wash of the windows
- Monthly – Quaternary ammonium RTU disinfection, followed by hydrogen peroxide/peracetic acid RTU for sporicidal disinfection, followed by WFI wash of the windows
- Shutdown – Residue Removal Process - Hot WFI water wash, followed by 70% IPA wipe down
NOTE: The EMPQ test period was extended to a 7-day period to cover the weekly regimen. Surface and air samples were pulled following static and dynamic conditions. The controlled areas were not cleaned or disinfected following each condition simulation.
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The tables below summarize the air and surface data from the T=0, T=1, and T=2 sampling results as expressed in terms of total isolate characterization (Gram stain results), total organism recovery (bacteria and mold)/total samples performed and percent reduction from T=0, T=1, and T=2.
The organisms recovered from the surface and air samples were characterized, and Gram-stained as summarized in Table 1-2.
The data expressed in total growth and total number of CFUs per plate are summarized in Table 1-3.
The data expressed in total change (percent change) in growth from T=0, T=1, and T=2 is summarized in Table 1-4.
Conclusion
The sanitization process achieved a reduction in total bioburden from T=0 to T=2). Baseline sampling of environmental flora resulted in an expected organism type percent recovery throughout the sanitization process; T=0 reduced total bioburden, T=1 reduced total bioburden and Gram-positive cocci and T=2 reduced total bioburden and Gramnegative rods.
Final baseline environmental flora represented an expected make up, from highest to lowest, Gram-positive cocci, Gram-negative rods, and mold (reference Table 1-2). The reduction in bioburden at each baseline sampling (including expected morphology reduction and overall percentage make up) coupled with the solution manufacturer quaternary ammonium RTU (Ready-to-Use) disinfectant efficacy testing results concluded that the new sanitization process is effective, and provide a sufficient bioburden reduction to maintain Grade C/D environmental monitoring limits.
The final sanitization process implemented leverages the data recovered from the baseline sampling along with the solution manufacturer coupon testing data. The routine regimen includes:
- Weekly – Quaternary ammonium RTU disinfection, followed by WFI wash of the windows
- Monthly – Quaternary ammonium RTU disinfection, followed by hydrogen peroxide/peracetic acid RTU sporicidal application, followed by a WFI wash of the windows
- Shutdown – Residue Removal Process - Hot WFI water wash, followed by 70% IPA wipe down
Data trending, including percent growth, percent excursion, growth maps, and heat maps, are used to evaluate the environmental results continuously. Changes in typical and seasonal flora, growth isolation/location, and shifts in the bioburden gradient prompts a re-evaluation of the sanitization/disinfection program.
The study demonstrates the real-time efficacy of quaternary ammonium RTU and hydrogen peroxide/peracetic acid RTU used in Grade C/D clinical production environments. The study indicates growth levels are reduced consistently with the expected bioburden in terms of the magnitude within Grade C/D areas using the in situ efficacy test data. Following the site cleaning and disinfecting procedures for routine operations along with the triple cleaning steps related to production shutdown conditions, it is expected that the controlled conditions are established and maintained to viable particulate limits using quaternary ammonium RTU.
About the Authors
Jim Polarine is a senior technical service manager at STERIS Corporation. He has been with STERIS Corporation for twenty years. His current technical focus is microbial control in cleanrooms and other critical environments. Mr. Polarine is a 2019 PDA Michael S. Korczynski Award recipient. He has lectured in North America, Europe, Middle East, Asia, and Latin America on issues related to cleaning and disinfection, microbial control in cleanrooms and validation of disinfectants. Mr. Polarine is a frequent industry speaker and published several PDA book chapters and articles related to cleaning and disinfection and contamination control. He is active as co-chair on the PDA’s microbial investigations task force. He was a co-author on PDA’s Technical Report #70 on Cleaning and Disinfection. Mr. Polarine teaches industry regulators as well as the pharmaceutical, biotech, and medical device industries at the PDA and the University of Tennessee. Mr. Polarine currently teaches the cleaning and disinfection course as part of the PDA Aseptic Processing Course and at the University of Tennessee Parenteral Medications Course. Mr. Polarine is current President for the PDA Missouri Valley Chapter and Technical Coordinator for the IEST. Mr. Polarine graduated from the University of Illinois with a Master of Arts in Biology. He previously worked as a clinical research coordinator with the Department of Veterans Affairs in St. Louis, MO and as a biology and microbiology instructor at the University of Illinois. His main hobby is storm chasing and is very active in tornado research and tornado safety.
Thomas Walker is a Senior Quality Assurance Manager for Novartis Pharmaceuticals. He has been with Novartis for 27 years. In his current role, he serves as the quality representative for the new integrated isolator filling and lyophilization line. Mr. Walker has had multiple professional affiliations during his career including PDA and ISPE. He has presented webinars for Novartis on topics including cleanroom design and operation, cleanroom cleaning and disinfection and disinfectant efficacy testing. Mr. Walker graduated from the University of Texas at Arlington with a Bachelor of Science degree in in Biology. When not working, he enjoys family time, flying, beekeeping and traveling.