Equivalence Testing of BFPCs in Grade A Environments Challenges and Alternative Strategy Industry Position Statement

Environmental monitoring (EM) in Grade A cleanrooms is an essential control measure aimed at sustaining the strict requirements necessary for sterile manufacturing to ensure product integrity and safety.

Bio-fluorescent particle counting (BFPC) is an advanced technology designed to detect, count and classify airborne particles based on their biofluorescent properties. These instruments use an internal laser to distinguish biological particles (viable) from inert particles by analyzing their fluorescence signals, offering rapid detection of microbial contamination in critical environments.

BFPCs provide real-time detection and quantification of airborne microorganisms in controlled environments, significantly increasing their detection and allowing for immediate response and corrective action. At the same time, potential contamination risks are significantly reduced by eliminating the human interventions required for manual sampling steps connected with traditional methods. They detect adverse trends as they occur, allowing for immediate reaction and intervention (2). The benefits and limitations associated with the transition to nongrowth-based air-monitoring methods, like BFPCs, are also discussed in several PDA publications (3–5).

The validation of BFPCs presents several unique challenges, especially when it comes to comparing them to the traditional colony-forming unit (CFU)-based methods. One of the primary challenges is the fundamental difference in their detection principles. While traditional methods rely on the growth of microorganisms on nutrient agar plates, BFPCs detect particles based on their biofluorescent properties, measuring the presence of viable particles in terms of autofluorescent units (AFU), independent of growth. Consequently, BFPCs detect culturable as well as “viable but not culturable” (VBNC) microorganisms. This fundamental difference means that no direct correlation between AFUs and CFUs is possible (5).

The industry lacks clear guidance on validation and qualification approaches of nongrowth-based methods like BFPCs, and expectations from regulatory agencies are not fully harmonized. The core principles of validation involve comparing new alternative methods with established standards, thereby posing the critical challenge of directly comparing fundamentally disparate technologies and defining appropriate specifications, such as alert and action limits. The lack of standardized guidance can impede the implementation of BFPCs, as end users may face difficulties in making decisions on investment proposals and may require significant resources to implement the technology.

Scope and Terminology

The scope of this position statement is framed within the overall validation strategy for using BFPCs as a routine environmental monitoring tool in Grade A environments. It is set in the context of secondary user-specific validation (rather than primary supplier-based validation) where it is expected that the alternative method is tested in parallel to the traditional method to demonstrate that the alternative method produces results that will allow for the same or higher level of environmental control.

In this article, the authors discuss the drawbacks of parallel testing in Grade A environments and a potential alternative strategy of validating BFPC systems in lower-grade environments but with intended use in a Grade A environment. This approach would enable parallel testing of low levels of microorganisms in controlled environments.

When discussing the validation of BFPCs compared to traditional methods, several terms are often used interchangeably, including "equivalence," "comparability" and "non-inferiority." Each of these terms has specific implications and nuances that are important to understand in the context of EM.

Equivalence generally refers to the idea that two methods produce results that are statistically indistinguishable from each other. In the case of BFPCs, this approach would require rigorous statistical analysis to demonstrate that the BFPC method can consistently produce results that match those obtained by traditional methods. This would be possible only if the amount of data collected is large enough to reach statistical significance and if the signals and detection of the two methods are comparable.

Comparability is a broader term that implies that two methods produce results that are sufficiently similar, to be used interchangeably for the same purpose. While equivalence focuses on statistical indistinguishability, comparability allows for some degree of variation as long as the overall performance and outcomes are aligned. In the context of BFPCs, comparability testing would involve demonstrating that BFPCs can reliably detect and quantify microorganisms in a manner that the outcome is comparable to traditional methods, even if there are differences in the actual numbers.

Non-inferiority is a term often used in regulatory contexts to indicate that a new method is not worse than an existing method by a specified margin. Non-inferiority testing involves statistical comparisons to show that the new method performs at least as well as the traditional method. For BFPCs, non-inferiority testing would require demonstrating that the BFPC method is capable of detecting microorganisms equally or better than traditional CFU-based methods. This approach is particularly relevant when the new method offers additional benefits, such as real-time detection and reduced contamination risks.

While "equivalence," "comparability" and "non-inferiority" are often used interchangeably, they each have specific implications for validation testing. According to the European Pharmacopoeia (Ph. Eur.), Chapter 5.1.6, the goal is “to demonstrate that the results of the alternative method enable an unequivocal decision as to whether compliance with the standards of the monographs would be achieved if the official method was used” (6).

For the purpose of this article, the term "equivalence" will be used in the context of "decision equivalence" as defined by the United States Pharmacopeia (USP) General Chapter <1223> (7).

Current State of Guidance and Regulations on BFPC Validation

The validation of alternative rapid microbiological methods (ARMM) is governed by various regulatory documents and industry standards. As the term “alternative methods” comprises a variety of different technologies and detection principles, there is currently no clear guidance for the validation of BFPCs, and the applicability of validation parameters given in the regulatory documents for this technology is limited.

The U.S. Food and Drug Administration (FDA) confirmed that companies should use the USP chapter <1223> “Validation of Alternative Microbiological Methods” as guidance for implementing BFPC systems. If the BFPC system is used alongside a traditional method for process monitoring or control, there is flexibility in how equivalence of the BFPC technology to the traditional method is shown. However, if the BFPC system is replacing a traditional method, the end users must demonstrate that the BFPC technology is equivalent to or superior to the methods currently in use.

Ph. Eur. Chapter 5.1.6 (3.3.2.9) provides specific guidance for equivalence testing and discusses the principles of equivalence testing between Alternative or Rapid Microbiological Methods (ARMMs) and traditional methods. It emphasizes the need for an adequate comparison experiment at low levels of inoculation (e.g., less than 5 CFU) with a sufficient number of replicates of relevant strains of test microorganisms (6). The expectation is that manufacturers of BFPC equipment would perform volume-to-volume testing as part of their primary validation package. Nonetheless, Ph. Eur. 5.1.6 (3.3.2.9) does not preclude the necessity of conducting these tests during the user-specific validation stage as well. This approach is impractical for BFPCs as the implementation in a filling line necessitates specialized equipment and conditions, such as an aerosol generator or a Grade A test chamber. Introducing microorganisms into Grade A environments is counterintuitivef and conflicts with all the measures in place to avoid contamination. Consequently, pharmaceutical end users are left with the necessity of waiting for natural contamination events.

The Annex 1 revision encourages explicitly the implementation of alternative and rapid technologies, such as BFPCs, for EM in aseptic filling. It also emphasizes the importance of continuous monitoring using a quality risk management approach as part of the contamination control strategy (1). The specific language in the EU GMP guideline does not explicitly mention comparative testing between ARMMs and traditional microbiological methods. However, this concept stems from the general GMP principles and best practices in validation and qualification of alternative methods when introduced into sterile manufacturing environments, such as Grade A areas.

On the other hand, the Swiss Medic Inspectorate has published a technical interpretation reflecting the general opinion of the Swiss inspectors on the Annex 1 revisions. Commenting on EM and process monitoring (Ph. Eur. chapters 9.22, 9.23, 9.28, 9.29, and 9.31), the Inspectorate asks for actual process data of the real-time viable particle counting to be collected and compared to standard EM data. It is further specified that data must be available for at least 12 months. We propose calling into question this expectation, as there is no explicit guidance or requirement for such a prolonged data collection period in Annex 1, and the stipulation of a one-year duration appears impractical and unwarranted.

Overall, there is a notable absence of standardized approaches and expectations across different health agencies, leading to varied interpretations and implementation challenges. This lack of uniformity complicates the validation process and poses significant hurdles for end users seeking to adopt BFPC technology.

Equivalence testing is an integral part of the secondary user-specific validation testing to be performed in the target environment. This means that the validation efforts are tailored to the specific needs and conditions of the end user's environment, ensuring that BFPCs perform effectively in the actual operational setting. The publication Understanding the Non-Equivalency of Bio-Fluorescent Particle Counts versus the Colony-Forming Unit explains that the noncorrelation between AFU and CFU should not be a barrier to leveraging BFPC technology (5). USP <1223> leaves the option for an alternative method and the traditional method not to produce a match in terms of results. What is important is that the candidate method can allow a microbiologist to make an equivalent decision regarding product quality consistently.

Stochastic Nature of Particle Detection in Aseptic Environments

When employing two methods to detect a single particle, where each method inherently consumes the particle upon detection, several stochastic factors come into play that influence the process. First, the movement of one particle in an isolator is to some extent random along the unidirectional flow. Thus, if only one particle is present, one needs to carefully consider the probability distribution associated with its detection. Unlike bulk measurements, where statistical averages smooth over individual variability, single-particle detection is highly sensitive to stochastic variability. Once collected, the particle is consumed by the detection process and thus, each method essentially has a single opportunity to register the particle's existence. This one-shot nature increases dependence on initial conditions and enhances variability since there is no possibility of repeated measurements from the same sample. Second, each detection method has an intrinsic efficiency rate, defined as the probability of successfully detecting a particle. For agar-based methods, factors such as the impaction efficiency and the concept of VBNC microorganisms are considered. Likewise, for BFPCs, factors such as the physical efficiency of the particle detector, the efficiency or accuracy of the fluorescence detector, and the viability algorithm. Thus, both methods have a risk that, even upon successful collection, the particle might not be detected.

Statistical Challenges of Analyzing Mostly Zero Values

When evaluating if a new method is non-inferior to a traditional method, the presence of predominantly zero values in the dataset presents significant statistical limitations. Non-inferiority tests aim to demonstrate that the effectiveness of the new method is not worse than the traditional method by a small prespecified margin (the non-inferiority margin). However, when the data is mostly zeros, several challenges arise. Most statistical analyses rely on variations in data to identify differences, and a dataset primarily composed of zeros lacks this essential feature, making it difficult to detect subtle differences. With limited data variability due to numerous zeros, the power of statistical tests is also significantly reduced, increasing the chances of a Type II error (also known as a false negative). In the context of a non-inferiority study, a Type II error occurs when the study fails to reject the null hypothesis even though the alternative hypothesis is true. This means that the conclusion of the study incorrectly suggests that the new treatment is inferior to the traditional method. In other words, the study will not detect non-inferiority when it actually exists. To this end, any single detection event and/or recording error (including both false negatives and false positives) can have an outsized effect on the outcome of the analysis, which can potentially lead to the wrong conclusion about the non-inferiority of the method. To address these limitations, significantly larger sample sizes are necessary to achieve enough statistical power. However, maintaining cleanroom conditions is costly and the extended sample-size requirement to achieve enough statistical power is thus impractical and resource-intensive.

In combining these factors, the detection of a single, consumable particle is a highly stochastic process, one that must be analyzed with an eye toward probability and statistical inference. Comparing two methods under such conditions necessitates careful consideration of both intrinsic uncertainties and practical constraints. For datasets that are dominated by zero-values, statistical methods such as zero-inflated models or other specialized approaches to appropriately handle such data structures do exist. However, in Grade A environments, the action limit is 1 CFU/m³, and thus no positive events are expected in controlled conditions rendering statistical comparisons unattainable.

Potential Conclusions of Equivalence Testing in Grade A Environments

To meet the USP <1223> equivalence demonstration requirements in a BFPC validation for use in a Grade A environments, the expectation would be a comparison study with a traditional growth-based method during routine aseptic processing. However, whether such a study design adds valuable input to the validation package remains debatable. The potential outcome of such a study can be summarized within four scenarios, provided all other sterility assurance factors are in a state of control:

1. Occurrences of AFUs, but no recoveries of CFUs using the standard growth-based method. This environment would be classified as an uncompromised sterile environment using traditional methods, with particles present only detectable by the BFPC technology, for example, VBNC, damaged cells or false-positive results. The scenario of potential false-positive results should be investigated and covered through a dedicated and controlled interference study as part of a risk assessment for the use of the BFPC technology in the specific surrounding. Common potential interferences already need to be assessed by the equipment supplier and be part of the primary validation package.
2. Occurrences of CFUs together with AFU alerts. This points to a breach of the aseptic environment and would basically be an ideal situation for the parallel evaluation of this new technology as opposed to traditional methods. This scenario is unlikely to happen, however, since breaches of sterility in modern isolators are extremely rare. An active contamination of the Grade A environment by protocol design, that is, on purpose, is not justifiable and is usually prohibited through the facility contamination control strategy. The assessment of whether the BFPC equipment is capable of discovering microbiological contamination should be covered within the supplier’s primary validation package.
3. Occurrences of CFUs without AFU alerts. This could be the result of a low-level contamination of the grade A environment or, alternatively, a secondary contamination of the growth plate, which is an inherent risk of the traditional method. Because single microbes can either land on the growth plate or in the BFPC probe head, but not both, this falsely leads to an overestimation of the performance capability of the traditional plate method, which is a statistical probability. The risk of a secondary contamination event can be avoided using the BFPC technology.
4. No occurrences of CFUs or AFUs. This is the most likely outcome, after subtracting justified false-positive interferent signals from the BFPC. These will be the final results to be analyzed for the study conclusion. In other words, the analysis report to determine non-inferiority will include the comparison of zeros from CFUs and zeros from AFUs and will require theoretical assumptions of small numbers to replace zeros to be able to calculate the results on statistical terms. Obviously, this provides no additional value.

Alternative Proposal for Equivalence Study Design

An equivalence study in an aseptic environment to demonstrate non-inferiority of the BFPC technology will not yield useful results. For the demonstration of equivalent performance capabilities, a low level of microbiological contamination is a prerequisite. Appropriate conditions for a non-inferiority test study can therefore be found in lower cleanroom Grade C or Grade D areas, where the presence of microorganisms representing the “in-house flora” is to be expected. The results would have a direct relevance on the intended implementation of BFPCs in Grade A settings. This approach would offer the potential for a statistical analysis and a direct comparison between the methods, delivering the data package that health authorities are seeking with the required confidence in the approval process.

Operational and Regulatory Implications

Shifting to non-inferiority testing in lower-grade environments means gaining statistically meaningful datasets, thereby reducing the need to go through the burdens of prolonged parallel testing approaches in Grade A areas. At the same time, end users may experience reduced complexity in validation studies, leading to fewer disruptions of aseptic operations and more streamlined performance qualification approaches.

Conducting parallel testing in Grade A environments requires introducing validation protocols during routine manufacturing and, potentially, new process flows to accommodate testing. This can disrupt normal operations, requiring careful scheduling to avoid affecting negative batch production. By moving non-inferiority testing to lower-grade areas, such disruptions can be minimized, allowing routine aseptic processing to continue without added complexity.

Similarly, the operational burden associated with investigations triggered by potential BFPC signals during parallel testing can be reduced. The European regulatory landscape does not explicitly allow for a “research exemption” (i.e., “Safe Harbor” principle in FDA regulations) for the validation of new alternative methods, like BFPCs. The Annex 1 mandates that all excursions in critical areas be investigated to ensure product sterility and patient safety. For this reason, Quality Assurance teams often express reluctance towards parallel testing during routine production, as every viable particle count detected using BFPCs in Grade A areas must be thoroughly investigated. This could negatively impact batch release, up to batch rejection if a contamination cannot be ruled out. Such a scenario is particularly concerning where decisions regarding product loss are based on individual signals from a system that has not yet been fully validated.

Shifting from equivalence testing in Grade A areas to testing in lower-grade environments does not eliminate the general need for conducting some Grade A-specific assessments before full implementation. Verification testing in Grade A settings is recommended with the instrument installed at its intended position to confirm that the BFPC performs as expected under aseptic conditions. Moreover, before full implementation, operators must be trained in the proper handling, operation and interpretation of BFPC data. Training should include procedures for recognizing and responding to real-time alarms (AFUs), ensuring alignment with existing EM practices, for example, from nonviable particle counting. Evidence of operator qualification will have to be documented to demonstrate that the new technology is used correctly and consistently.

We propose these activities be performed during an initial feasibility study, not requiring a full GMP response to signals that might arise from the system. This could be achieved either by collecting data during technical runs that do not require full GMP conditions, during aseptic process simulations, or in routine production under the Safe Harbor principle. The data thus raised can be used to streamline the operational approach and to assign the rules for dealing with excursions after full implementation into routine production. This includes also the possibility of deciding on the need to set new baselines.

By maintaining steps above, end users can ensure a smooth transition to the BFPC technology while maintaining compliance with regulatory expectations for monitoring in Grade A environments.

Our suggested approach has the potential to lead to a more standardized and globally harmonized validation framework for BFPCs, reducing variability in validation approaches across different end users. This could streamline regulatory reviews and inspections by setting clearer expectations across the industry and would create a more consistent and predictable regulatory landscape.

While the shift could be beneficial, inspectors may place greater emphasis on the scientific rationale behind non-inferiority studies, including statistical evaluation. End-users adopting this approach may need to engage proactively with their country-specific inspectorates and regulators through scientific advice meetings or pilot programs to ensure alignment with expectations. This may include approaching them in a harmonized way through industry interest groups.

Successfully implemented, this approach could set a precedent for validating other real-time monitoring technologies using non-inferiority testing in lower-grade environments. Overall, it can standardize BFPC validation strategies and improve efficiency across the industry, while maintaining regulatory compliance — provided that strong scientific justification and post-implementation controls support the approach.

Conclusion

Demonstrating the equivalence of BFPCs to the traditional methods in Grade A environments presents unique challenges.

Our proposed approach not only reduces the operational strain associated with parallel testing in Grade A aseptic environments but also addresses the inherent stochastic nature of particle detection and the statistical challenges posed by datasets dominated by zero values. By conducting validation studies in lower-grade environments, end users can generate meaningful datasets with direct implications for Grade A settings, ensuring robust validation without compromising production integrity.

This strategy aims to align with regulatory expectations for continuous real-time monitoring and supports a more standardized and globally harmonized validation framework. Engaging proactively with regulators and providing strong scientific justification will be crucial to gaining acceptance for this approach. Ultimately, this shift has the potential to set a precedent for validating other real-time monitoring technologies, fostering innovation while maintaining stringent quality and safety standards.

The industry can thus enhance the efficiency and reliability of environmental monitoring in sterile manufacturing, ensuring product integrity and patient safety while navigating the evolving regulatory landscape.

References

1. European Commission. EudraLex Volume 4 – Guidelines for Good Manufacturing Practices for Medicinal Products for Human and Veterinary Use, Annex 1: Manufacture of Sterile Medicinal Products; August 2022.
2. Barensteiner R, Calisi L, Denk R, et al. Bio-Fluorescent Particle Counter (BFPC) Continuous Environmental Viable Particle Monitoring Strategy for Aseptic Filling; ISPE White Paper; February 2023.
3. Merker P, Cundell T, Martindale C. Opinion: Revisit Regulatory Expectations for Micro ID in Grade A Environments for Non-Growth-Based Methods? PDA Letter; November 2023.
4. Scott A, Vanbroekhoven A, Joosen C, et al. Challenges Encountered in the Implementation of Bio-Fluorescent Particle Counting Systems as a Routine Microbial Monitoring Tool. PDA J Pharm Sci Technol, Vol. 77(1); January 2023.
5. Salvas J, Merker P, Dingle M, et al. Understanding the Non-Equivalency of Bio-Fluorescent Particle Counts versus the Colony-Forming Unit. PDA J Pharm Sci Technol, Vol. 77(6); November 2023.
6. Council of Europe. Chapter 5.1.6, Alternative methods for Control of microbiological Quality. In European Pharmacopoeia (Ph. Eur.) 11^th Edition.
7. United States Pharmacopeial Convention. General Chapter <1223>, Validation of Alternative Microbiological Methods. In USP–NF; 2023. DOI: https://doi.org/10.31003/USPNF_M99943_04_01.

About the Authors

Mads Lichtenberg, Ph.D., is a Senior Process Scientist at Fujifilm Biotechnologies, Denmark, and a member of the BioPhorum ARMM BFPC workstream.

Petra Merker, Ph.D., is a Quality Control Expert, member of the Modern Microbial Methods Collaboration and formerly part of the BioPhorum ARMM BFPC workstream.

 

Roger Slavik, Ph.D., is an Associate Director Compliance R&D Manufacturing at Johnson & Johnson and a member of the BioPhorum ARMM BFPC workstream.

 

Alice Le Gatt is a Global Change Facilitator at BioPhorum, part of the Fill Finish Phorum and leading the ARMM BFPC workstream.