Opinion: Revisit Regulatory Expectations for Micro ID in Grade A Environments for Non-growth-based Methods?
[Editor’s Note: The opinions presented here are those of the authors and do not represent the official opinion of PDA.]
Biofluorescent particle counters (BFPCs) allow for the detection and enumeration of microorganisms in the environment and rely on intrinsic fluorescence detection instead of growth, constituting a paradigm shift in microbial monitoring.
BFPC systems count total and biologic particles, commonly called auto-fluorescent units (AFU), by detecting Mie scatter for particle size and presence information and intrinsic fluorescence to categorize particles as biologic or inert. All microorganisms contain fluorophores, like nicotinamide adenine dinucleotide phosphate (NADPH) and riboflavin, that fluoresce when excited by 405 nanometer laser light. BFPC systems commonly use this wavelength of light. The AFU is not equivalent to the colony-forming unit (CFU) as these methods of detection are fundamentally different.
There are several BFPCs commercially available for use. Unlike with traditional growth-based methods, there is no need to change plates within the aseptic core during set-up and every four hours during processing. BFPCs are capable of remote sampling and generate results frequently and on a continuous basis with no interruptions or interventions needed. Since the method of detection for a BFPC is different from the traditional growth-based method, there is no associated agar plate that is incubated. This means there is also no CFU from which to perform microbial identification.
BFPC systems fall into the category of rapid methods, which are encouraged by the new European Union GMP Annex 1: Manufacture of Sterile Medicinal Products (1). This document explores the advantages of using a BFPC in a Grade A aseptic processing environment. It discusses the lack of a need for microbial identification in the case of a positive event. While this discussion is limited to the use of BFPC in Grade A environments, the salient points related to microbial identification, where BFPC monitoring is employed, may be applicable to other situations, such as water monitoring.
BFPCs Benefits and Challenges
BFPCs offer huge advantages in controlling aseptic processing operations. These instruments generate results continuously without intervention, allowing for real-time evaluation of the filling environment and the ability to react to action-level excursions and adverse trends during the filling operation, including the ability to segregate material with a lower sterility assurance from a batch. This is something that cannot be achieved with growth-based methods, which rely on incubation over a period of days, only a retrospective evaluation can be conducted, and no actions at the time of the event can be taken.
BFPCs also offer continuous data during setup and filling, a requirement in Annex 1 (1). There is no pause in monitoring to change out agar plates as with traditional growth-based methods. As such, an entire operation is continuously monitored, improving the aspect of demonstrating environmental control. There is also a significant reduction in microbial contamination risks as interventions associated with environmental monitoring are eliminated. At the same time, because the number of potential findings is linked to the frequency of monitoring, moving from active air sampling once per eight-hour shift to continuous sampling may uncover excursions not captured previously.
The implementation of BFPCs during routine filling operations is not without challenges (2). In general, BFPCs detect viable microorganisms and do not provide colonies on a plate suitable for microbial identification. In contrast, the traditional growth-based methods provide for both a CFU count and isolates for microbial identification. The inability to identify isolates is compensated by the advantages of real-time monitoring afforded by the implementation of BPFC technology, which allows for the detection of stressed and viable but nonculturable (VBNC) microorganisms and replaces delayed confirmation of environmental control with real-time in-process control.
In addition, applying BFPCs might occasionally lead to signals of nonviable origin, which are derived from interferent materials located in the vicinity of the respective sampling site. Evaluation of such potential interferents (e.g., by nearby equipment, disinfectants, or products), combined with knowledge about common interferences provided by the supplier of the BFPC system, will lead to a deeper understanding of the system and its potential background influencing factors.
Micro ID in Grade A Environment When Using BFPCs
We argue that in a Grade A environment, where mostly zero microorganisms are to be expected, you do not need microbial identification information unless there is an extensive contamination event. This could only occur by a breach in the containment of the isolator, which would also be indicated by the physical parameter alarms and would result in high AFU values with repeated hits. Due to the real-time aspect, in these cases, there would be immediate awareness and the possibility of additional sampling with agar plates in an attempt to recover and identify the organisms.
We further argue that there are no significant advantages to routinely attempting microbial identification of rare events detected in grade A air monitoring when BFPC monitoring is employed. The use of BFPC provides for immediate and continuous knowledge of the environment. As a result, immediate corrections such as line clearance are possible when an event is observed. This means that concerns related to product impact (normally alleviated by microorganism identification) are significantly reduced, if not eliminated. The same cannot be said for growth-based methods, as data is not available until days after the conclusion of the process.
In these cases, microbial identification is useful in determining the impact on the product as one may determine, such as which toxins are produced by the microorganism and assess the subsequent potential impact on the product. It is precisely this retrospective viewpoint that requires the microorganism’s identity since corrections to prevent product impact could not be taken immediately when the event occurred. When using BFPCs, this need is eliminated.
Trend Analysis Without BFPC ID in Grade A Areas
Annex 1 requires that trending includes changes in the microbial flora with particular attention paid to those microorganisms that are recovered from events where there was a loss of control, spore formers, and those that may indicate a “deterioration in cleanliness.” The 2004 U.S. FDA guidance document Sterile Drug Products Produced by Aseptic Processing echoes this sentiment, indicating that identification should be part of “monitoring critical and immediate surrounding clean areas as well as personnel” (3).
Replacing the traditional, growth-based method for air sampling in a Grade A environment with non-growth-based methods, like BFPCs—without identification—does not mean that you lose information about your environment in terms of the ability of trending and determining the microbial flora of the environment.
Traditional growth-based monitoring on surfaces and personnel is still in place, and recovery from these locations will result in microbial identification. Sampling plans for other aseptic areas (Grades B, C, or D) continue to be performed, providing identification and trending information.
Surface sampling locations for BPFCs in a Grade A isolator are part of the overall contamination control strategy of a facility, with sampling points being defined via a risk assessment. Special emphasis is laid on the surrounding background environment of the isolator and the potential entry points for contamination of the facility, airlocks, filter systems, and infeed processes, following the overall material and personnel flows. Consequently, traditional microbial data about the microbial flora will still be available to determine the in-house isolates and perform trending.
A Shift in Mindset is Needed
What will be lost with this transition? We argue very little. The authors have found that the utility of microbial identification has been overemphasized. The species most identified from sterility failures, testing, and manufacturing areas are typically the same 15-20 bacterial species, so their identification adds little or no value to the investigation (4).
Modern Isolator technology greatly reduces the contamination risk (very low recovery rates <0.1%). Rare findings in modern isolators are ascribed in most cases to secondary contaminations. Since the BFPC method eliminates the risk of inadvertent contamination during human sampling, the need for identification would be reduced to the rare event of a “real” finding in Grade A. Since BFPCs also detect nonculturable microorganisms, the expectation of an attempt to cultivate them for identification is quite unrealistic.
Moving away from long-accepted traditional monitoring techniques, which are based on classical microbiology and the growth of microorganisms on nutrient media, requires revisiting our expectations and a novel operational approach to applying these methods. We must embrace the advantages gathered when considering/employing new alternative approaches and reexamine long-standing paradigms in environmental monitoring.
Authors' Note
The authors are part of a collaboration of industry working groups that joined forces in 2021 to support the awareness and adoption of modern microbial methods. These groups include the BioPhorum Alternative and Rapid Micro Methods (ARMM) Biofluorescent Particle Counting team (part of the BioPhorum Operations Group), the Kilmer Community Rapid Microbiology Methods group, the Online Water Bioburden Analyzer (OWBA) Working Group, and the Process and Environmental Monitoring Methods (PEMM) working group. The group has already published an article in the PDA Letter called Initial Evaluation Roadmap for Modern Microbial Methods, an article in the PDA Journal on Challenges Encountered in the Implementation of Bio-Fluorescent Particle Counting Systems as a Routine Microbial Monitoring Tool, and another article in the PDA Journal on Understanding the Non-equivalency of Bio-fluorescent Particle Counts versus the Colony-forming Unit.
The following subject-matter experts contributed to the development of this opinion article.
Allison Scott is a Principal Scientist at MicronView LLC and a member of the PEMM Working Group.
Caroline Dreyer is an Aseptics Specialist at Novo Nordisk A/S, supporting production facilities globally and a member of the BioPhorum ARMM BFPC Team.
Chris Knutsen, PhD, is a scientific director with the microbiology center of Excellence for Bristol Myers Squibb and a member of the BioPhorum ARMM BFPC Team.
Hans-Joachim Anders, PhD, is the Quality Team Leader within Novartis Pharma Stein AG, Switzerland, an analytical science and technology organization. Anders is a member of the OWBA Working Group.
Jim Cannon is the Head of OEM and Markets in Mettler-Toledo Thornton Inc. Cannon is a member of the PEMM and OWBA Working Group.
Joanny Salvas is Senior Manager Manufacturing Intelligence in Pfizer, Inc. Salvas is a member of the ARMM BFPC Team.
Mike Dingle is a Senior Product Specialist at TSI. Dingle is a member of the PEMM Working Group.
Phil Villari is an Associate Principal Scientist at Merck & Co., Inc., in Rahway, New Jersey. Villari is a member of the BioPhorum ARMM BFPC Team and the PEMM Working Group.
Victoria Navarro is Engineering Validation and Quality Manager within Pfizer, Inc. at Spain Manufacturing site. Navarro is a member of the BioPhorum ARMM BFPC Team.
Conclusion
The advantages of continuous monitoring in Grade A using the BFPC technology are so overwhelming in improving sterility assurance that when moving from once-per-shift confirmatory testing to in-process monitoring, our industry can forgo the ability to isolate and identify microbial isolates in Grade A. As emphasized in the 2022 revision of Annex 1, BFPCs have a broader measurement than traditional growth-based microbial methods detecting viable microorganisms that may not grow on microbiological culture media. Our experience with microbial investigations indicates that the value of microbial identification in Grade A has been overemphasized by both regulators and the industry and should not be a GMP requirement in situations where more advanced methods are used and deliver better process knowledge and control.
References
- European Union; GUIDELINES: The Rules Governing Medicinal Products in the European Union, Annex I: Manufacture of Sterile Medical Products, 2022, https://health.ec.europa.eu/system/files/2022-08/20220825_gmp-an1_en_0.pdf
- Scott, A., et al.; “Challenges Encountered in the Implementation of Bio-Fluorescent Particle Counting Systems as a Routine Microbial Monitoring Tool,” PDA Journal of Pharmaceutical Science and Technology; January 2022, doi: 10.5731/pdajpst.2021.012726, https://journal.pda.org/content/early/2022/07/15/pdajpst.2021.012726.
- U.S. FDA; Guidance for Industry Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice; 2004, https://www.fda.gov/media/71026/download
- Guilfoyle, D. E. and A. M. Cundell; Do Plant Isolates have a Role in Method Suitability and Growth Promotion Testing in the Microbiology Laboratory? Is it a Matter of Science versus Compliance?; PDA Journal of Pharmaceutical Science and Technology; February 2022, pdajpst.2021.012675; DOI: https://doi.org/10.5731/pdajpst.2021.012675
About the Authors
Petra Merker, PhD, is a Biological Quality Control Expert at Bayer AG and a member of the BioPhorum ARMM BFPC Team.
Tony Cundell, PhD, is the Principal Consultant at Microbiological Consulting, LLC, Rye, New York and a member of the PEMM Working Group.
Cynthia Martindale is the Principal Consultant at Applied Rapid Microbiology Specialists, Ltd., and a member of the PEMM and OWBA Working Group.