James Polarine Jr. - Senior Technical Service Manager, STERIS Corporation
Thomas Walker - Senior Quality Assurance Manager, Novartis Pharmaceuticals
Introduction
A primary tenant of aseptic manufacturing is controlling the environment microbiologically before, during, and after a manufacturing batch. The static and operational controls include the combined effects of engineering and other controls such as cleaning and disinfection, personnel garbing, etc. Together, they provide the assurance required for sterile product manufacturing.
A Contamination Control Strategy (CCS) is a formal risk assessment document for identifying, controlling, and preventing the transfer of product/process risk(s) to the patient. The defined process controls ensure the final product is sterile, within endotoxin specifications, and practically free of particles as required for the product. The formal assessment must include all process inputs to adequately use the Quality Risk Management (QRM) principles to identify the risk(s).
An effective cleaning and disinfection program is a fundamental component of a CCS. Selecting a disinfecting solution and the required regimen requires multiple evaluation elements. Chemical capability and efficacy are primary concerns for a site but may be influenced by solution availability, site logistics/design, and application requirements. The overall sanitization regimen must include all elements and the effectiveness demonstrated during site start-up, following site changes or shutdowns, and through routine monitoring.
Background
Controlling microbiological contamination includes cleanroom design, defined process flows (e.g., personnel, materials, product components), engineering controls (e.g., HVAC), utility supply controls, and operational procedures. A critical operational procedure includes a well-established cleaning and disinfection program to manage the indirect and ancillary cleanroom surfaces. The process must complement and be commensurate with the operational control limits (e.g., Grade A, Grade B, Grade C, Grade D, etc.).
A quick overview of the primary risk areas includes:
Facility/Cleanroom Design
Production facilities, cleanrooms, and equipment are located, designed, constructed, qualified, and maintained to suit operations carried out. Cleanroom areas include non-porous surfaces easily cleaned (e.g., minimize irregular surfaces with unnecessary surface area, avoid horizontal solid surfaces, constructed from compatible materials, etc.) and maintained with adequate process flows. Access areas are divided into cleanliness zones (e.g., UC, CNC, Grade D, Grade C, etc.) to minimize zone-to-zone cross-contamination from known bioburden areas to areas with little to no bioburden. The design and operational controls protect the manufacturing process from extrinsic contamination when considered together.
Material and Personnel Flows
The required material and personnel flows must avoid and minimize unnecessary movement. All movements must be intentional and assessed for contamination risk. Each process flow for material and personnel must be separate and accessed through separate airlocks.
Personnel zone-to-zone transitions include retentive garbing procedures commensurate with operations and expected cleanliness levels. The transition points within the required flow must focus on the primary source of contamination via any contact point.
Material flows through transition zones must incorporate disinfection processes to minimize microbiological cross-contamination from lower cleanliness zones to higher cleanliness zones. Transfer flows may also include the removal of coverings intended to protect the article from contamination while moving through lower to higher cleanliness zones.
Engineering Controls
The primary engineering control for cleanroom areas is the air supply. All supplied air is conditioned to control temperature, humidity, and delivered quantities (e.g., differential pressure cascade, etc.) via High-Efficiency Particulate Air (HEPA) filters. The constant flow of air must meet quality and velocity limits for the identified cleanliness zone. Airflow within the zones is generally described as displacement airflow (i.e., high-velocity unidirectional Grade A air) or dilution airflow (i.e., lower cleanliness zones measured by air movement and change rates).
Utility Supplies
Processing utility supplies (e.g., water, steam, compressed gas, etc.) are divided into two main categories, critical utilities and less-critical utilities, based on risk. The critical utilities serve the sterile product pathways and present the greatest risk to the product or process. These include WFI supplies for formulations and clean steam used to steam sterilize materials and equipment. Utilities with indirect product impact pose a risk but can be measured in product or process support status, such as instrument air or facility steam. Ultimately, the design of the utility must not allow for contamination but also allow for monitoring to mitigate the identified risk.
Operational Procedures
The ability to effectively work within a contamination control strategy requires well-defined procedures. When engineering controls cannot or do not adequately mitigate risk, clear and direct operational procedures must provide the direction to control the cross-contamination risk. The high-risk direction for aseptic operations includes garbing, aseptic behavior, material transfer, and cleaning and disinfection. Each process must be repeatable and have objective means to evaluate effectiveness. Suppose the cleaning and disinfecting processes are not effective before production. The contamination risk is transferred to the product/process with limited detectability and no chance to remediate with subsequent disinfection.
Case Study
Selecting a disinfection solution requires multiple evaluation elements. Initially, the type of manufacturing process, associated risk, and the expected control level may narrow the list of possible cleaning/disinfecting agents. Additionally, chemical capability and efficacy are primary concerns for a site but may be influenced by solution availability, site logistics/design, and application requirements. The overall sanitization regimen must include all elements and effectiveness demonstrated during site start-up or following site changes.
A clinical supply manufacturer added a new integrated filling operation to their aseptic filling capabilities. The new area provided 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 reviewed the existing CCS to leverage current mitigation efforts and to identify new risk(s) associated with the added filling operation, the need for an alternative regimen was recognized to meet the local needs and synergize global initiatives.
The site reviewed several available formulas in the United States (US) and Europe. Not all formulations are available globally; 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 using multiple application methods
- With minimal residue after application
- Not containing 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 including the coupon testing comprised of multiple cleanroom surfaces consistent with the site construction. The review of the Disinfectant Efficacy Testing (DET) studies passed all acceptance criteria and was 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 executing the Environmental Monitoring Performance Qualification (EMPQ), also referred to as in situ testing. Furthermore, as this was a newly constructed area, there is no microbiological history to evaluate for environmental isolate testing. Functional testing verified that the manufacturer’s DET results and the newly implemented sanitization process provided 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. 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 and magnitude) compared to the zero limit in Grade A areas. This limit allows recovered microbial counts to 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 Ready-to-Use (RTU) focused on efficacy determined by direct application following site construction. The 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, including Gram staining, was characterized 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, including (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 hydrogen peroxide/ peracetic acid RTU application using the same techniques used for the quaternary ammonium RTU.
Completing the baseline sampling, T=0, T=1, and T=2 lead to the (1X) static and (3X) dynamic sampling described in ISO-14644.
The site map below details the facility layout and includes the environmental classification 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 was 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 window
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.
The tables below summarize the air and surface data from the T=0, T=1, and T=2 sampling results, as expressed in 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.
Baseline Sample Regimen Summary:
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 the total number of CFUs per plate are summarized in Table 1-5.
The data expressed in total change (percent change) in growth from T=0, T=1, and T=2 is summarized in Table 1-6.
The percent change for air sampling is the result of increased room activity. Additional personnel performed sampling-related activities in minimal garments commensurate with Grade C/D classification. The data does not reflect the solution or regimen effectivity but rather the result of activity within the Grade C/D areas.
Conclusion
The sanitization process reduced the total bioburden from T=0 to T=2 (reference Table 1-8). 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 Gram-negative rods.
The final baseline environmental flora represented an expected makeup, from highest to lowest, Gram-positive cocci, Gram-negative rods, and mold (reference Table 1-2). The bioburden reduction at each baseline sampling (including expected morphology reduction and overall percentage makeup) and the solution manufacturer quaternary ammonium RTU disinfectant efficacy testing results concluded that the new sanitization process is effective. It will provide a sufficient bioburden reduction to maintain Grade C/D environmental monitoring limits.
The final sanitization process leveraged the data recovered from the baseline sampling and the solution manufacturer’s 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, continuously evaluate the environmental results. Changes in typical and seasonal flora, growth isolation/location, and shifts in the bioburden gradient will prompt 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 that growth levels are reduced consistently with the expected bioburden of the magnitude within Grade C/D areas using the in-situ efficacy test data. Following the routine operation’s site cleaning and disinfecting procedures and the triple cleaning steps related to production shutdown conditions, the controlled conditions are expected to be established and maintained to viable particulate limits using quaternary ammonium RTU. As a result of the study, the defined cleaning and disinfecting regimen supports the Contamination Control Strategy in mitigating the microbiological contamination risk to the product/process.
Author Biographies
Thomas Walker is a Senior Quality Assurance Manager for Novartis Pharmaceuticals. He has been with Novartis for 30 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 Biology. He enjoys family time, flying, beekeeping, and traveling when not working.
Jim Polarine is a senior technical service manager at STERIS Corporation, where he has been for 21 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, the Middle East, Asia, and Latin America on cleaning and disinfection, microbial control in cleanrooms, and validation of disinfectants. Mr. Polarine is a frequent international industry speaker and published several PDA book chapters and articles related to cleaning, disinfection, and contamination control. He is active on the PDA’s COVID-19 Task Force. He was a co-author of PDA’s Technical Report #70 on Cleaning and Disinfection and Technical Report #88 on Microbial Deviations. Mr. Polarine teaches industry regulators and 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, IEST, and the University of Tennessee Parenteral Medications Course. Mr. Polarine is the current President of the PDA Missouri Valley Chapter and Technical Coordinator for the IEST. He is also a leader on the PDA’s Chapter Council Steering Committee. Mr. Polarine graduated from the University of Illinois with a Master of Arts in Biology. He previously worked as a clinical research manager 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. He is very active in tornado research and tornado safety.
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