Tony Cundell- Microbiological Consulting, LLC, Rye, NY
Introduction
This commentary highlights how the advances in technology, risk assessment, and regulatory guidelines have excluded human intervention in aseptic filling, improving both product quality and patient safety.
The risk of microbiological contamination during aseptic processing during the past four decades is largely a reflection of technological advances, the application of microbiological risk management tools, anthe d emphasis on contamination control by regulators. These changes have significantly reduced the exposure of packaging components and drug product to the manufacturing environment, especially to human operators making sterile injectable products. These advances and their supporting activities in terms of risk mitigation can be summarized as follows:
- Technological advances in aseptic processing.
- Widespread application of quality risk management tools to aseptic processing.
- Development of industry and regulatory standards applied to sterile product manufacturing.
- Transition from small molecule to large molecule products and, more recently, to cell and gene therapies requiring aseptic processing.
Note: After I wrote this article, I became aware of Jim Agaloco’s excellent article on the design and operation of aseptic filling areas in the July-August 2025 issue of American Pharmaceutical Review that contains graphic illustrations of these different areas.
Technological Advances in Aseptic Processing
Advancement of aseptic surgery by the Victorian physician Lord Lister (1827-1912), innovations like the carbolic acid spray, the introduction of surgical gloves, masks, and gowns, and unidirectional airflow enclosure, were slowly implemented for routine surgery. Technical innovations that have been more recently adopted for aseptic processing, listed in a rough chronological order, include the following:
- High-efficiency particulate absorbing (HEPA) filters
- Laminar flow cleanrooms developed by Willis Whitfield at the Sandia National Laboratories
- Advances in sterilization processes for package components
- Non-viable particulate monitoring systems
- Isolator systems were initially derived from the nuclear industry
- Advance aseptic connections
- Vapor phase hydrogen peroxide (VPHP) decontamination
- Restrictive Access Barrier Systems (RABS)
- Blow/Fill/Seal technology
- Bio-fluorescence particle monitoring systems
- Robotic systems compatible with aseptic processing in closed isolators
In terms of cleanroom design for aseptic filling operations, the pharmaceutical industry has progressed from traditional cleanrooms with vinyl curtains surrounding an ISO 5 filling line to restricted access barrier systems (RABS) to open-gloved and half-suit isolator systems to closed gloveless isolators containing robotic systems. This progression limited and then finally excluded human interventions during aseptic processing. Other changes include reduction of air cleanliness grades for the surrounding area as the manufacturing become more separated from human operators, manual sanitization being replaced with vapor-phase sporicidal decontamination of the interior surfaces within an isolator, remote air cleanliness sampling, and adding robotic systems replacing human manipulations using gloves and half suites that are non-particle sheading and amenable to cleaning and decontamination with a closed isolator. Isolator systems are most applicable to small batch sizes associated with cell and gene therapies than large-scale batches of small molecule products. In addition, the employment of blow-form-seal systems for lower-cost sterile products, such as sterile saline eyedrops, is possible and delivers the product with a high sterility assurance. Other processing equipment, including centrifuges, chromatographic columns, transfer devices, remote sample collection, and incubators that may be built into an isolator, reduces microbial contamination risk throughout the manufacturing process.
The long-awaited 2022 revision to the EU Good Manufacturing Practices Annex 1 Manufacture of Sterile Medicinal Drugs wisely emphasized Quality Risk Management (QRM) and required manufacturers to develop site Contamination Control Strategies (CCS) to minimize microbial contamination of their sterile drug products. This document, with minor revision, has been adopted by the PIC/S organization, which includes the FDA and other major regulatory agencies are members, creating a de facto global standard for aseptic processing, largely superseding the 2004 FDA aseptic processing guideline.
The pharmaceutical literature contains a series of publications applying risk management to aseptic processing, beginning with the 1996 publication by Whyte, which was extended by a 2004 publication co-authored by W. Whyte from the University of Glasgow and T. Eaton from AstraZeneca entitled Microbial Risk Assessment in Pharmaceutical Cleanrooms. Other significant contributors, in the opinion of the authors, include Ljungqvist and Reinmuller from the Royal Institute of Technology, Stockholm, Sweden, American industry consultants Akers and Agalloco, Sekiya from Tokyo University Center 16 | | September/October 2025 for Stem Cells and Regenerative Medicine, Boom and his co-workers in sterile compounding from the Zaans Medical Center, Zaandam, The Netherlands, Tidswell and his co-workers from Merck & Co, Baseman and Long from the consulting company ValSource, and McCall and the other members of an industry consortium from ADMA Biologics. Typically, these publications are not reviewed together in a single review article, so their relative merits are nosystematicallyly examined. It is the intention of the author to briefly summarize the positions taken and hope that someone more mathematically adept will establish a more unified approach, based on the probability of microbial contamination, to the state-of-the-art aseptic processing technology. Risk assessment may be qualitative, merely identifying and subjectively ranking process steps as high or low risk activities, or quantitatively assigning a probability of microbial contamination associated with the activity and time of potential exposure to risk. The author believes that only probabilistic methods have the necessary rigor to be objective and highly effective. What follows is a summary of the contributions of different pharmaceutical scientists and engineers to risk management.
- Whyte and Eaton (WE method). This pioneering approach can be viewed as semi-quantitative. The innovation in Whyte’s thinking was from the number of microorganisms within the air and their rate of deposition, the diameter of the neck of an open vial, and dwell time on an aseptic f illing line, the probability of contamination during a f illing operation could be calculated and was found to be extremely low (Whyte, 1986; Whyte, 2004; Whyte and Eaton, 2004; Whyte and Eaton, 2014; Whyte, Agricola and Derks, 2016;; Whyte and Eaton, 2017).
- Ljungqvist and Reinmuller (LR method). This approach can be viewed as quantitative with an assignment of genuine empirical data giving a true probability of contamination (Ljungqvist and Reinmuller, Ljungqvist and Reinmuller,1995; Ljungqvist, Reinmuller et al, 2024).
- Akers and Agalloco (AA method). This approach is numeric with the assignment of surrogate values, but it is not a true probability of contamination (Akers and Agalloco, 2006).
- Boom and his co-workers (Boom Method) risk assessment associated with sterile compounding in a pharmacy setting (Boom, Ris et al, 2021a&b).
- Tidswell (Quality Risk Management). This approach depends on the assignment of actual empirical data, which results in the derivation of a true probability of contamination during an aseptic manipulation (Tidswell and McGarvey, 2016; Tidswell, Rockwell, and Wright, 2019).
- Baseman and Long (Long Method). The approach is a risk assessment largely based on the number of human interventions, which can be reduced to mitigate risk (Baseman, Chakraborty, and Long, 2022).
- McCall and other ADMA Biologics consortium members. The argument that the microbial contamination risk in a closed, gloveless isolator is so low that routine environmental monitoring can be eliminated was accepted by the FDA Center for Biologics Evaluation and Research ( McCall, Barnard et al, 2022).
Critical Interventions
The criticality of operator inventions will depend on the extent of the barrier system, the intervention complexity, exposure of product or packaging component to contact surfaces or first air, duration of the intervention, and the air classification of the area. The next section will discuss risk mitigation more extensively.
Identified Risk Factors for Traditional Cleanrooms, Restrictive Access Barrier Systems, Open Isolators, and Closed Isolators
As the industry progressed from open cleanrooms to isolator systems by eliminating human interventions, the risk of microbial contamination of the drug product has been significantly reduced. This section lists the major sources of microbial contamination risk and their mitigation in aseptic processing environments that range from traditional cleanrooms to closed isolator systems with robotic manipulations.
Traditional Cleanrooms
Risk Factors
Limited barriers for operator entry to critical ISO 5/Grade A areas. The filling line is segregated from less-controlled areas by vinyl curtains and Perspex shields.
Frequent operator intervention in critical locations in the component and product exposure pathways.
Extended exposure time of package components in controlled areas on conveyor belts and rotary accumulators.
Manual cleaning and disinfection of the cleanroom.
Risk Mitigation:
Depyrogenation tunnels to sterilize glass vials for product filling.
Stopper cleaning and steam sterilization before manual loading of stopper bowls and feeds.
Steam sterilization and aseptic assembly of the product contact parts.
Continued use must be justified to regulatory agencies.
Extensive air, surface, and personnel monitoring required to confirm environmental control.
Relative Risk Level: Moderate to High
Restrictive Access Barrier Systems
Risk Factors:
Barriers can be open during manufacturing and cleaning, and disinfection.
Human interventions are ot fully eliminated.
Glove port leakage is highly possible due to pin-hole formation and tears.
Manual cleaning and disinfection of the cleanroom and inside the barrier.
Risk Mitigation:
Barriers limit the access of operators to critical areas during aseptic processing.
Barriers are surrounded by an ISO 6/Grade B environment.
Multiple glove ports are incorporated into the barrier system for human interventions.
Sterilization-in-Place of the product contact parts.
Reduced air, surface, and personnel monitoring is possible when justified. Relative
Risk Level: Moderate
Open Isolator Systems
Risk Factors:
Container entry and exit on conveyor belts through mouse holes.
Glove ports and half suits are incorporated into the hard-walled isolator systems.
Glove port leakage is possible.
Risk Mitigation:
Isolators limit the access of operators to critical areas.
Glove ports are incorporated into the hard-walled isolator systems.
Glove port leakage is possible, but is monitored by physical integrity testing.
Open isolators have a positive space pressurization to a surrounding ISO 6/Grade B environment.
Vapor phase hydrogen peroxide decontamination of transfer and aseptic processing isolators.
Reduced air, surface, and personnel monitoring when justified.
Relative Risk Level: Low to Moderate
Closed Isolator Systems
Risk Factors:
Largely eliminated.
Risk Mitigation:
Isolators totally exclude the access of operators from critical areas during operation.
Typically, they are hard-walled isolator systems not prone to damage and leakage.
No glove ports.
Closed isolators may be surrounded by an ISO 8/Grade D environment.
Vapor phase hydrogen peroxide decontamination has become a reliable process.
Container entry and exit through transfer hoods, i.e., decontamination chambers.
Set-up and manipulation by robotic systems, not human operators.
Greater reliance on single-use processing components.
Often use horizontal HEPA airflow to reduce particulate contamination.
Elimination of routine environmental monitoring can be justified.
Relative Risk Level: Low
It is often valuable to assign a numerical value to the relative risk level. This is my attempt to assign a number to the risk level: Traditional Cleanrooms – 10; Restrictive Access Barrier Systems – 5; Open Isolator Systems – 3; Closed Isolator Systems -1.
Other technologies have been utilized to mitigate the risk of microbial contamination. They include blow-fill-seal technology and aseptic filling of prefilled syringes, which are discussed briefly in the next two sections.
Blow-Fill-Seal Technology
Blow-Fill-Seal (BFS) technology uses plastic granules, typically low-density polyethylene, as the source material of the containers, which are fed into a rotating extruder producing molten polymer. This melt is extruded through an orifice, producing a continuous tube of molten plastic termed a parison. The mold encloses the parison with each container formed by either vacuum or blown air to shape the container. The formed containers within the mold are shuttled to the filling station, filled, and the upper part of the mold is closed to seal the container. The operation takes place within a constant stream of sterile, HEPA-filtered air.
Aseptic Processing Simulation data comparing traditional aseptic filling with BFS technology established that BFS technology had a higher sterility assurance level than traditional filling operations (Leo et al, 2024).
Pre-Filled Syringes
Single-use pre-filled syringes offer the advantages of convenience in delivery, cost savings in the elimination of product overage that are necessary with filled vials, accuracy of delivery, improved safety, and a well-established manufacturing process, which may include online radiation sterilization of plastic syringes with stainless steel needles and aseptic filling in an open isolator. The entry and exit of the syringes could be through a restricted barrier system.
Development of Industry and Regulatory Standards Applied to Sterile Product Manufacturing
Industry standards and good manufacturing practice regulations are major drivers of microbial contamination risk mitigation. The major standards introduced, listed chronologically, are as follows:
- U.S. Federal Standard 209 Airborne Particulate Cleanliness Classes in Cleanrooms and Clean Zones A-E (1963 to 1999)
- ISO 14644 Cleanrooms and Associated Controlled Environments Parts 1 -10 (1999)
- ISO 14698 Cleanrooms and Associated Controlled Environments Parts 1: General Principles and Methods and Part 2: Evaluation and Interpretation of Data (2003)
- FDA Guidance for Industry - Guideline on Sterile Drug Products Produced by Aseptic Processing 2004
- PDA Technical Report No. 44: Quality Risk Management for Aseptic Processes (2008)
- SPE Baseline Guide Volume 3 Sterile Product Manufacturing Facilities Second Edition, September 2011
- ASME BPE-2022 Standard for Bioprocessing Equipment 2022
- ICH Q9 (R1) Quality Risk Management June 2022
- EU Good Manufacturing Practices Annex 1 Manufacture of Sterile Medicinal Drugs 1997 and 2022 (Revised)
- PIC/S -EU GMP Annex 1 on Sterile Manufacturing 2022
- PDA Technical Report No. 90: Contamination Control Strategy Development in Pharmaceutical Manufacturing (2023)
The U.S Federal Standard 209 E was cancelled in 1999 and replaced d the International Standards Organization (ISO) 14698 Cleanrooms and Associated Controlled Environments Parts 1 and 2, which is the engineering standard for the design, construction, and operation of cleanrooms. For example, the air cleanliness classification for CRA critical aseptic processing step, where the product is exposed, is designated an ISO 5 area. The 2022 EU Good Manufacturing Practices Regulation Annex 1 Manufacture of Sterile Medicinal Drugs, due to its wide acceptance by national boards of health, including the F, D, and its establishment as a PIC/S GMP standard, is now the governing document for our industry. ISO 5 areas are designed as Grade A areas. These GMP requirements mandate that manufacturers establish a documented Contamination Control Strategy, which will more systematically eliminate risk in aseptic processing.
Transition from Small Molecule Products to Large Molecule Products and Cell and Gene Therapies Requiring Aseptic Processing
Chanlikee of pharmaceutical drug products has increased the importance of aseptic processing. In the period 1980–2022, the FDA approved 1355 new drugs, with an annual drug approval average ± standard deviation of 31.5 ± 12.0 drugs. The annual average number of approvals increased from 23.1 ± 6.1 (1980-1992) to 29.8 ± 15.6 (1992-2012) and 45.0 ± 11.9 (2012-2022) due to increased funding through user fees and better management. FDA approvals included 1103 (81.4%) new molecular entities (NME), 235 (17.3%) therapeutic biologics, and 17 (1.3%) gene and cell therapies. The annual average number of approved NME increased from 21.8 ± 6.4 (1980-1992) to 24.7 ± 15.3 (1992-2012) and 32.1 ± 9.7 in 2012-2022. Whereas the annual average number of approved therapeutic biologics increased from 1.2 ± 1.0 (1980-1992) to 5.0 ± 1.8 (1992-2012) and 11.5 ± 4.4 in 2012-2022. The FDA approved 17 gene and cell therapies in the period 2010 through 2022 (Seoane‐Vazquez et al, 2024).
Notable is the significant increase in therapeutic biologics and the emergence of gene and cell therapies amongst the FDA approvals, all of which are produced using aseptic processing. The FDA leads all other regulatory agencies in both the number and the shortest time for approval. By year-end 2022, the number of biologic approvals narrowly outpaced that of small-molecule NMEs, a landmark in biologics’ steady rise since the end of the twentieth century. It can be suggested that higher prices obtained for biologics and their smaller lot sizes will inevitably support the investment in isolator systems. In 2003, the FDA Center for Drug Evaluation and Research (CDER) approved an average of 46 novel new drugs annually, with 55 new drugs approved in 202,3, of which 20 were first-in-class drugs, while the FDA Center for Biologics Evaluation and Research (CBER) approved 25 Biological License Applications.
Conclusions
As the aseptic processing technology has advanced along with the evolution of global GMP requirements in response to these advances, the risk of microbial contamination has been significantly reduced. The most frequent routes of microbial ingress from human operators contaminating the air and processing equipment have largely been eliminated. Based on the reduction of the risk profile, the industry standards, GMP requirements, and the environment monitoring strategy should all be re-visited. Although a closed isolator with robotic manipulation has the lowest risk, justifying the elimination of routine air and surface monitoring, they are usually only suitable for smaller batch sizes associated with cellular therapies and clinical supplies. Yes, Bill Whyte was right in pioneering the emphasis on the probability of contamination on a filling line.
Acknowledgements
The author thanks Ed Tidswell for his comments on the classification of the risk assessment publications.
References
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