Regulatory Perspectives on Changing Microbiological Acceptance Levels and Specifications

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Regulatory Perspectives on Changing Acceptance Levels and Specifications
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Regulatory agencies will generally accept a change in a manufacturing or testing process if the change has been proven to be equivalent to or better than the system currently in place. However, some non-growth-based technologies, especially those that do not rely on the growth of microorganisms (e.g., viability or spectroscopic systems), may provide a higher count or microbial recovery as compared with conventional methods. From a regulatory perspective, this is one of the most challenging aspects of working with RMMs, because Regulatory Affairs and Quality organizations have a very difficult time grasping the possibility that:

  • We may be "seeing" things we haven't in the past,
  • the RMM measurements may be different than what we have been used to historically, such as fluorescent units or relative light units, as compared with colony forming units (CFUs), and
  • we won't be able to meet the existing specifications or acceptance levels and/or we may need to change our levels to reflect the data generated by the new RMM.

A number of regulatory representatives have commented on these concerns both in print and during speaking engagements at international forums on rapid methods and pharmaceutical microbiology. For example, Dr. Brenda Uratani (CDER, FDA) provided guidance on modifying acceptance levels during her presentation at the 2007 PDA Global Pharmaceutical Microbiology Meeting. She indicated that the FDA expects higher counts will be recovered when using RMM technologies, especially if the methods are more sensitive than conventional methods, and that a GMP evaluation of the new method and the results must be driven by good science. The example she provided was for active air monitoring in an ISO 5 area, where the current 1 CFU per cubic meter specification may be changed, because this specification was originally based on a less sensitive, agar-based method.

In the 2006 Hussong and Mello paper, "Alternative Microbiology Methods and Pharmaceutical Quality Control," these FDA microbiologists addressed the use of new microbiological methods, environmental monitoring, and changing acceptance levels. They provided the following position:

"If data are to be compared over time, then test methods must remain the same, which is fundamental to trend analysis. However, to accelerate data collection, methods must change. Some changes will be insignificant (and test method validation may show no difference), and some will change data greatly. Often, new methods rely on a completely different body of information; some may be direct measurements, some indirect. In either event, previous acceptance criteria may not be applicable. Therefore, implementation of newly developed, or more rapid, microbiology methods may also require establishment of new acceptance criteria. Ultimately, trending of data may be lost in order to bridge the gap between "old" and "new" data analysis."

Finally, Paul Hargreaves (MHRA) presented an overview of revisions to EU Annex 1 during the 2008 PDA/EMEA conference. During his discussion on continuous monitoring, Hargreaves stated that more excursions are found as all events are now being monitored, and that alert limits and out-of trend limits may need to be revised when implementing continuous monitoring. Although these discussions did not focus on viable particle monitoring, Hargreaves did infer that the majority of the pharmaceutical industry has not introduced 21st Century technologies for the counting of microorganisms, and that a future revision of EU Annex 1 may consider specific requirements in this area.

More recently, Hargreaves made a statement at a 2009 PDA RMM regulatory meeting following a question from the audience on this subject:

Comment: "If a RMM does not provide CFU counts but provides a new measurement (a fluorescent unit), and you have demonstrated equivalence between the two methods, and the difference in counts are relatively small (historically zeros on an active air sampling plate vs. 1 or 2 counts using the RMM), we can't use the CFU spec of zero any more because the RMM does not provide CFU data."

Hargreaves Response: "In this case it is appropriate to change the limits as long as you have documentation and a rationale for making the change."

At this same meeting, Hargreaves explained that since most organisms won’t grow on agar plates, it is expected that newer, more sensitive methods will obtain higher counts than traditional methods. Therefore, limits can be changed as long as an appropriate rationale is provided, and the rationale is explained to the inspector.

The CFU vs. New RMM Signals

RMMs that do not rely on the growth of microorganisms may recover a higher number of organisms when compared to conventional growth-based methods. As such, there has been some debate on whether non-colony-forming-unit (CFU) signals, such as a fluorescent unit, can be compared with the traditional CFU.

USP <1223> addressed this potential challenge. The chapter states that attempts to use statistics to compare the CFU results to signals arising from biochemical, physiological, or genetic methods of analysis may have limited value because the different methods used cannot be expected to yield signals that could be compared statistically in terms of mean values and variability. As such, the chapter concludes the CFU cannot be used as acceptance criteria and it is the user’s responsibility to propose values (supported when necessary by scientific literature) that they can demonstrate are appropriate for the alternative method.

However, the chapter contradicts this position in its Equivalence section. Specifically, the section on Option 3 (Results Equivalence) states, "[r]eports on the use of alternative non-growth-based methods have shown that they may produce significantly higher cell count estimates than a growth method that reports outcomes in CFU. In this case, the analyst could use a calibration curve showing a correlation between the two methods in the product specification range."

If you experience a situation in which a non-growth-based RMM provides a greater recovery than a conventional growth-based method, but the results are still within the acceptable specification or acceptance level, then no changes to the specs may be necessary. However, if the RMM provides a significantly greater recovery in which the CFU spec would not be met, it may be possible to correlate the RMM signal to the control CFU counts and statistically justify a change in the specification to match the new signal and new recovery levels. Note that a sufficient amount of data may be needed to justify this change and discussions with regulatory authorities may be warranted, especially if the new specs would impact a previously-approved product submission.

New Guidance from the 2022 revision to Annex 1

After many years, the final revision to Annex 1 "Manufacture of Sterile Medicinal Products," under the The Rules Governing Medicinal Products in the European Union Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use, was published on August 25, 2022, and will go into effect August 25, 2023. The revision may be downloaded here:

Some of the most encouraging changes are associated with the use of rapid methods instead of the standard methods for environmental monitoring, and that non-colony-forming-unit (CFU) signals can be used instead of the historical limits based on the CFU.

For example, one of the notes in Table 2, which discusses the maximum permitted microbial contamination level during qualification of a cleanroom, states, "Limits are applied using CFU throughout the document. If different or new technologies are used that present results in a manner different from CFU, the manufacturer should scientifically justify the limits applied and where possible correlate them to CFU." Therefore, the EU allows us to revise the numerical values for the CFU-based maximum permitted microbial contamination level to new numerical values based on a rapid method's alternative microbial detection signal. One example of a new rapid signal is an intrinsic fluorescent count which can be derived in real-time and does not require growth on conventional microbiological media.

Similar guidance on the use of alternative limits is provided in the footnotes for Table 6, which discusses the maximum action limits for viable particle contamination during routine manufacturing. Additionally, although the table provides CFU-based limits for active air samples, settle plates, contact plates and glove prints, one footnote allows for the use of methods other than those specified in the table. Specifically, "[I]t should be noted that the types of monitoring methods listed in the table above are examples and other methods can be used provided they meet the intent of providing information across the whole of the critical process where product may be contaminated (e.g. aseptic line set-up, aseptic processing, filling and lyophilizer loading)."