USP Rapid Method Update Disappoints at ECA Conference

During the recent ECA Rapid Microbiological Methods conference, Dr. Tony Cundell (Vice Chair of the USP Microbiology Committee of Experts) provided an update on the revision to USP informational chapter <1223> and the development of a new compendial rapid sterility test chapter. The presentation left attendees with more questions than answers, and for some rapid method suppliers and end-users, a disappointment in the direction the USP is taking.

Dr. Cundell first described the objective of a proposed revision to chapter <1223>, Validation of Alternative Microbiological Methods.  The revised chapter is intended to provide general guidance in the implementation of alternative methods for compendial microbial tests. He stated that the chapter would address a number of factors that must be considered prior to the selection of equipment and the qualification of assays with actual product. The factors to consider include the identification of suitable alternate assay methodologies, the development of user-specified stations for the selected equipment, demonstration of the applicability of the method as a replacement for a standard compendial method, installation and operational qualification of equipment, and laboratory qualification of the test method.

Next, Dr. Cundell provided an outline of the individual chapter sections. These include user requirements, components of data quality, instrument qualification, installation, operational, and performance qualifications, demonstration of equivalency, traditional validation criteria for quantitative and qualitative tests, user and instruments supplier responsibilities, method suitability, equivalency, statistical tools, and key references. 

It is of interest to note, that a number of these proposed chapter sections have already been addressed in the recently published revision to PDA Technical Report No. 33 (click here for an overview). However, when Dr. Cundell was asked if the teachings from TR33 would be considered, his response was that there might be diverging guidance from the technical report (and even Ph. Eur. 5.1.6, which is also in revision) because as an informational chapter, the USP does not have to harmonize with other documents. This statement was a surprise to many of the conference attendees, given the fact that the guidance in TR33 represents industry best practices for the validation and implementation of rapid methods, and that the strategies utilized by multinational firms for the regulatory approval of such technologies have been incorporated into the new TR33.

When discussing method equivalency, Dr. Cundell referenced a stimuli article that was previously published in the Pharmacopeial Forum (Hauck et al., 2009, Acceptable, equivalent or better approaches for alternatives to official compendial procedures. Pharm. Forum. 35(3): 772-778). He presented Hauck’s “comparison to alternative method options” matrix that would be included in the revised chapter <1223> for the purpose of providing guidance on determining whether an alternative method was equivalent or better to the compendial method. Unfortunately, Dr. Cundell did not spend sufficient time on this topic for the attendees (and this author) to fully understand the impact of this matrix on the validation of alternative or rapid microbiological methods. We are hopeful that this will be thoroughly explained when the revised chapter <1223> is available for review.

Next, Dr. Cundell provided a brief history of the plate count technique and the colony forming unit (CFU), including limitations when using this microbial viability “signal.” This included the inability of microorganisms to grow in a defined medium. For example, species that would normally be culturable may fail to grow because their growth state in nature, such as dormancy, prevents adjustment to conditions found in the medium.  He then described microbial viability signals other than the CFU, including ATP levels, PCR amplified target sequences, autofluorescence via flow cytometry, and the detection of cells stained with viability markers. In any case, Dr. Cundell stated that to take advantage of emerging technologies, we might need to cut the ties to the CFU as a so-called gold standard. Interestingly, there are a number of alternative and rapid technologies currently available that still rely on the growth of microorganisms, especially when large numbers of organisms are required to generate an alternate viability signal. An example may be found with most ATP bioluminescence systems, in that a single bacterial cell does not produce a sufficient quantity of ATP to be directly detected by these technologies.

When asked about whether specific recommendations were to be provided with regard to validation criteria, Dr. Cundell noted that internal USP statisticians were working on appropriate models and that these would be communicated in a draft in-process revision that will be available for review and public comment sometime in mid-2014.  We are hopeful that the USP takes current industry best practices into consideration, such as those that are provided by PDA TR33 (which, by the way, have been accepted by worldwide regulators) and not to completely reinvent the wheel. We will just have to wait and see.

The second half of Dr. Cundell’s presentation concentrated on the development of a new rapid sterility test chapter; however, the focus was on a rapid sterility test for short-lived, cell-based biological and radioactive injectable products. For these products, the USP has designated that a rapid sterility test be completed in 48 hours, preferably in real time, is non-proprietary, can be conducted in any competent microbiology laboratory, and would meet the method validation requirements as outlined in chapter <1223>.  Technologies that the USP committee considered include ATP bioluminescence, carbon dioxide detection, advanced imaging, flow or solid phase cytometry, and PCR.

Surprisingly, the USP rejected ATP bioluminescence and carbon dioxide detection as not being suitable because they are growth-based with an “incubation time of the order of 7 days.” However, Dr. Cundell stated that these could still be validated as alternative methods using the validation criteria provided in USP <1223>.  It is of interest to note that a number of cell-based biologic manufacturers have already obtained regulatory approvals for using carbon dioxide detection technologies as alternatives to the compendial sterility test, and ATP bioluminescence has also been validated as an alternative sterility test for conventional pharmaceuticals with a less than 7-day incubation period. And the USP ignored solid phase cytometry, which has been validated and approved (FDA and EMA) as an alternative sterility test within our industry, with single cell detection and a time to result in about 4-6 hours.

Moving forward, Dr. Cundell stated that the USP chose two technologies that will be developed for a rapid sterility test: flow cytometry of vital stained microbial cells and RT-PCR using universal primers and probes.

I question this decision.  And so did many rapid method suppliers who attended Dr. Cundell’s presentation.

For instance, knowing that flow cytometry will not necessarily provide single cell detection, and that this method can only analyze small volumes during the test (e.g., 1 mL or less), there will need to be an appropriate enrichment or growth period (of a suitable amount of the test sample) to allow for single cells to proliferate to a level that can be detected by flow cytometry. It is unclear whether this can occur within the USP’s 48-hour rapid sterility test requirement.  Incidentally, ATP bioluminescence may also be used under this same scenario (enrichment followed by detection), but ATP systems were rejected by the USP as being inappropriate for a rapid sterility test methodology.  

The same may be said for RT-PCR.  If the starting material for this test is DNA, then it will be necessary to enrich the sample to generate a sufficient number of target sequences to be amplified by the PCR system. Otherwise, the potential for false positives due to the presence of residual DNA (from dead cells and/or cross contamination) may increase.  And it is also unclear how long the enrichment period will be required for in order to detect all types of microorganisms, including those are fastidious, slow growing or injured.  

Dr. Cundell concluded his presentation by stating that the next step will be the development of a draft chapter, Rapid Sterility Tests, numbered 71.1. [Author note: because the chapter number will be less than 1000, this would be a compendial procedure for which compliance is enforceable by the FDA.]

Dr. Cundell also stated that protocols would be developed for external collaborators to conduct proof of concept studies to demonstrate the robustness of the proposed methods. And based on these experiences, the chapter will be finalized and published as an in-process revision for public review and comment.