Rapid Microbial Monitoring
Adoption, Applications, and Regulatory Guidance
Rapid microbial monitoring (RMM) is the real-time or near-real-time determination of microbial presence in a sample without the need for incubation, laboratory services, or intervention. RMM is a deﬁnition, not a single methodology.
Regulatory guidelines accept many methods of bioburden detection and measurement. The detection method is less important than its ability to determine microbial presence, especially in continuous manufacturing processes. Evaluative guidelines are prescribed to show equivalency between different methods and devices but not the number duplication in the colony forming unit (CFU) format. This paper describes applications for RMM in pharmaceutical waters; it also delves into the regulatory guidelines that support its adoption and use, with a focus on biopharmaceutical production and critical utilities.
USP38-NF33, CHAPTER <1223>5
Validation of alternative microbial methods
This revised chapter stresses that analyses conducted with alternative methods may differ signiﬁcantly from traditional growth-based incubation, but that does not necessarily indicate a greater risk to the patient or a greater chance of pathogenic species.
The chapter also repudiates a common comparison, stating: “The statistical comparison of results expressed as CFU obtained by signals analysis made by biochemical, genetic, or physiological methods is of little value.” The monograph then identiﬁes possible protocols for validating any RMM instrument regardless of its method of detection:
… It is critical to consider that in microbiology, the ﬁnding of “no microorganisms present” does not mean in absolute term that zero cells are actually present. A result of “no growth” in a current compendial method is properly interpreted as “no growth was detected under the speciﬁed conditions” … Studies on the recovery of microorganisms from potable and environmental waters have demonstrated that traditional plate count methods reporting cell count estimates as colony forming units (CFUs) may recover 0.1%–1% of the actual microbial cells present in a sample.
Understanding the strengths and weaknesses of the CFU as a signal is vital in the validation of an alternative method that uses an alternative signal. The CFU cannot be considered the only unit of microbiological enumeration, because it is only an estimate of cells present rather than an absolute measure.
The enumerative values, given as CFU results in association with reference methods, typically cannot be used as acceptance criteria for the assessment of articles via candidate alternative methods. Instead, it is the users’ responsibility to propose values that they consider acceptable and unacceptable for the method that they have chosen; this will be done independently of existing standards expressed in terms of CFU.
Absolute CFU-to-CFU comparisons between RMM and traditional growth-based methods are impossible. Bacteria culturable in R2A media (agar) may represent < 1% of the total bacteria count in the sample. Fricker notes that “[t]he pharmaceutical industry routinely uses Tryptic Soy Agar (TSA) as a culture medium with incubation at 37°C, but this tends to detect fewer bacteria than using either the Reasoner’s 2A agar (R2A) medium and incubation at 22°C, or yeast extract agar at either temperature. Waterborne bacteria generally prefer lower temperatures and lower levels of nutrients. Choosing the right conditions for the culture are essential. An RMM that can measure all bacteria will have a large discrepancy in comparison to growth-based CFUs. Methods outlined in USP <1223> describe how to validate an RMM without growth-based-media CFUs.6
USP <1223> explains the formulation of equivalence, not exactitude. You do not have to read the same number for validation to ensure your method of detection is sound. Performance is demonstrated by the consistency of readings when tested against known bacteria concentrations, as shown in Table A.
- Acceptable procedures
- Does not require a direct comparison with a compendial method
- Does require a reference material as a standardized inoculum of a specific microorganism
- Performance equivalence
- Demonstrate that the alternative method is equivalent or better than the compendial
- Validation criteria include accuracy, precision, speciﬁcity, limit of detection, limit of quantiﬁcation, robustness, and reproducibility
- RMM may have worse results for one or more of the seven validation criteria but still be considered acceptable because of the advantages it offers, especially if it is relevant to assessing the quality of the material in question (scan time in the case of cytotherapy, regenerative medicine, or radiopharmaceuticals, for example)
- Results equivalence
- Demonstrate that the alternative and compendial methods give the same result
- Microbiological analysis must be established for a tolerance interval with the alternative method, which must be numerically superior or inferior. Using statistics and relative standard deviation in fairly high percentages can help with acceptance.
- Because alternative methods, not based on growth, provide signiﬁcantly higher cell-count values, the two methods can be compared using calibration curves and R2 calculations
- Decision equivalence
- Generates a pass/fail indication, not a numeric result. With this approach, the frequency of positive and negative results should not be pejorative to the compendial
- To qualify the RMM unit, laboratory tests that use spiking techniques with varying levels of microorganisms are preferred.
|Option||Demonstration||Comparison to Official Compendial Method||Based on Numerical Results or Conclusions||Number of Characteristics|
|1. Acceptable Procedures||Acceptable||No||Results||Multiple|
|2. Performance Equivalence||Equivalent or Better||Yes||Results||Multiple|
|3. Results Equivalence||Equivalent||Yes||Results||Single|
|2. Decision Equivalence||Equivalent||Yes||Conclusions||Single|
If the RMM instrument reads X and the culturable bacteria reads Y, it does not mean that either or both are right or wrong. It only means that the detection methods are different and consistent readings are needed to show equivalency of the RMM method to gain conﬁdence in the data.
EU PHARMACOPOEIA, CHAPTER 5.1.6 (50106)
Alternative methods for control of microbiological quality
This has aspects and mandates similar to USP <1223>, with two additional validation criteria: range and speciﬁcity of the response. The chapter includes an example of alternative microbial detection validation, titled, “Example validation of an alternative method: detailed protocol followed by a laboratory for the implementation of bioluminescence.” The European Pharmacopoeia example1 for a specific type of RMM instrument displays the validation protocol and results during validation, providing a needed touchstone for the uninitiated:
Primary validation in order to characterise a speciﬁc microbiological method, the principle of detection must be clearly described by the supplier… The method must be fully detailed with respect to the conditions required for application, the materials and equipment needed, and the expected signal. The application principle should be described in a peer-reviewed journal. The principle of detection must be characterised in a model system and/or with a panel of test microorganisms, by at least:
- Prerequisite treatment of sample or microorganisms
- Type of response
- Speciﬁcity of the response
- Limit of detection
- Linearity of the response
- Accuracy and precision of the response
- Robustness of the method in a model system
- Limits of suitability
Once the method has been characterised in this way by the supplier, the principle of detection need not be veriﬁed by each user.
EP 5.1.6 was written in 2008, with little or no revision since then. The monograph cites different types of RMM detection methods and lists three major areas of concern for each: “principles of measurement,” “critical aspects,” and “potential uses.” It also cites risk-beneﬁt analysis and validation methods to enhance acceptance of RMM technology.
Parenteral Drug Association Technical Report 332 is the most up-to-date document for those who need a guide on choosing and validating RMMs. The ﬁrst part of the document contains a series of considerations on alternative methods, more rapid than traditional ones, and covers the following points:
- Limits and weaknesses of classic methods:
- Long response times.
- Potential inability to highlight microorganisms (stressed or viable nonculturable).
- Inability to bind to principles of quality by design and quality risk management.
- The positions of leading authorities (US Food and Drug Administration3 , European Medicines Agency, Australia’s Therapeutic Goods Association, Japan’s Pharmaceutical and Medical Devices Agency), with particular attention to validation requirements and assessment of the need to submit a formal request. Considerations of regulatory agencies in other countries are also included.
- Economic considerations and return on investment.
- Potential quality risks arising from the use of quick or alternative methods and tools for identifying and assessing risks.
- Automation of classical methods and simpliﬁed validation requirements.
- Reviews of methodologies and alternatives (available or under development) and their scientiﬁc bases, citing over 60 methods for bacteria detection under six classiﬁcations: growth-based, viability-based, cellular-component-based, optical spectroscopy, nucleic acid ampliﬁcation, and micro-electro-mechanical systems. There is no advantage to any detection method when the guidelines are applied as long as the instrument or technology provides the option to follow the guidelines for application in a pharmaceutical environment.
ASSUMPTIONS AND REALITIES
All RMM methods, regardless of detection method, will most likely have false positives or false negatives. There will be virtually no equivalence between any two detection methods.
It’s important to establish conﬁdence in the RMM by having continuous data, and to evaluate trending levels. Both manual and online or rapid sampling can be used to compare different detection methods. The data can trend very similarly, even though there may be a signiﬁcant difference in actual values.
RMM will be the main online tool for monitoring microbials in the near future. Grab sampling with growth-based incubation will continue to be used for product release. RMM will monitor the microbials in real time, and if there should be a value or consistent values above the standard deviation of 1σ, then a grab sample is performed to conﬁrm the microbial values deemed trending.
Installing an RMM unit in the 24/7 puriﬁed water system to record the health and operation of the water system can complement the 24/7 online instrumentation, analysis, and readings for all mandated and performance measurements, while adhering to process analytical technology (PAT) guidance.4 This provides complete knowledge and interaction with the water system. Product liability, unplanned shutdowns due to bad microbial tests, loss of production, and investigations can be severely curtailed, increasing uptime, productivity, quality, throughput, and compliance.
“Cycle time” is the period between the beginning of a production cycle and the beginning of the next product or batch initiation using the same vessels. This includes the use of a clean-in-place (CIP) regimen for 3–5 hours, after which production vessels are idle until the CIP residual microbial data is confirmed by sampling and incubation, which can take from 2–7 days, depending on the protocol. Using RMM technology, the vessels could have been released for the next cycle immediately after the completion of the CIP regimen. This can dramatically decrease cycle time, allow for dozens more cycles per year, and increase revenue without additional investment in infrastructure, utilities, or vessels.
Online RMM with other online instrumentation can determine the health, operation, and status of a water system in real time, preventing unplanned specification excursions and downtime, while maintaining 24/7 compliance.
Upfront evaluation of RMM using the regulatory guidelines in USP <1223>, EP 5.1.6, and PDA Technical Report 33 will help increase acceptance of RMM with internal and external regulatory entities. Online RMM, after proper validation and installation of the instrument, can increase cycles, increase revenue, shorten idle time, minimize downtime, and provide compliance with PAT guidance.7
- 5US Pharmacopeia and National Formulary. “Validation of Alternative Microbiological Methods.” USP38/NF33, chapter <1223>.
- 6Stewart, Eric J. “Growing Unculturable Bacteria,” Journal of Bacteriology 194, no. 16 (August 2012): 4151–4160.
- 1European Pharmacopoeia 6.0., Chapter 5.1.6, “Alternative Methods for Control of Microbiological Quality.”
- 2Parenteral Drug Association. “Evaluation, Validation and Implementation of Alternative and Rapid Microbiological Methods.” Technical Report No. 33. Revised 2013.
- 3US Food and Drug Administration. Pharmaceutical CGMPS for the 21st Century—A Risk-Based Approach.” September 2004. https://www.fda.gov/downloads/drugs/developmentapprovalprocess/ manufacturing/questionsandanswersoncurrentgoodmanufacturingpracticescgmpfordrugs/ucm176374.pdf
- 4———. Guidance for Industry: “PAT – A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance.” September 2004. https://www.fda.gov/downloads/drugs/guidances/ucm070305.pdf
- 7Fricker, Colin. “Detection of Viable but Non-Culturable Organisms.” Clean Room Technology, March 30, 2016. https://www.cleanroomtechnology.com/technical/article_page/Detection_of_viable_but_nonculturable_organisms/116926