Recovery Studies for Microbial Sampling – Revisited

In the recent (2020) ISPE Guide: Cleaning Validation Lifecycle – Applications, Methods, and Controls there is one section and a related appendix on bioburden sampling recovery methods (including methods for swab, contact plate and rinse) that state the following:

“The recovery study results should be assessed by the QC laboratory when reviewing cleaning validation and routine microbiological data. If the recovery results are low, then an RCF should be applied to the final test results to compensate for sampling method limitations and to determine if the acceptance criteria were met.” [RCF is “recovery correction factor”.]

This appears to be stating that a recovery from surfaces should be done for microbial sampling methods much as is done for sampling methods for “chemical’ residues such as the API and the cleaning agent. This recommendation flies in the face of what we know from science about microbial enumeration and what we know about sampling of microorganisms from surfaces, as well as how microbial limits are typically set for cleaning validation protocols. I have written on this extensively in the past, but the basic issues are as follows:

1. Microbial enumeration using traditional growth techniques is highly variable. If a spiked level is 50 CFU and the amount recovered is 35 CFU, that is not a 70% recovery from the surface, but those values are essentially the same. The variability is illustrated by USP <1111>, where a specification of 102 CFU means that the result has to be no more than 200 CFU. With this high level of variably, how can any meaningful recovery percentage be determined?
   
2. In recovery studies, as soon as the microorganisms are applied and allowed to dry (or partially dry), there will some level of death or loss of viability for the microorganisms such that the measured CFU value will be less than what the theoretical spiked level is supposed to be. If microbial sampling in protocols is done on dry equipment surfaces, then drying the spiked coupon in a recovery study will result in a lower measured recovery.
   
3. In traditional growth techniques one measures colony forming units, not individual cells. The number of colony forming units may be increased by the swabbing procedure, by the swab extraction procedure, or by the measurement procedure because of the separation of one colony into two or more colonies. This may possibly lead to higher CFU values for the results after sampling.
   
4. In cleaning validation protocols, limits for bioburden are not set the same way as the carryover calculations for APIs and cleaning agents. Microbial limits are generally set on an industry standard practice that is much lower than what would be established by a carryover calculation. So even with recoveries of 25%, the quality of the next product is not compromised.

In a sense, the appendix (“Appendix 4 – Example: Bioburden Swab and Rinse Recovery Methods”) referred to in the ISPE document actually supports the reasons for not doing a recovery study for microorganisms. The “method” does go through all the steps that would be required for doing a sampling recovery for microorganisms. But, in section 15.1.1 is one bullet point stating “Use a recovery solution (inoculation) such as PBST or other effective solution to spread, stabilize, and prevent desiccation (drying) of inoculated (spiked) microorganisms on coupons” and a second bullet point stating “Spiked (inoculation) microorganisms on test coupons are not allowed to dry too long (validated drying time) or consider using a wet method.” [Emphasis added for both quotes]

For these reasons, this appendix provides methods for both “wet swab” sampling (on dried or partially dried inoculated coupons) and for “dry swab” sampling (on inoculated coupons before any drying). This appendix also recommends a variety of organisms for the challenging the sampling methods, including “a Gram negative rod, a Gram positive cocci, yeast, mold, a spore former, as well as the most common and virulent (resistant) environmental isolate”. Furthermore, this appendix recommends an “acceptance criteria” of not less than 50%.

In my opinion (and for the reasons given), doing recovery studies for microorganisms from surfaces is a waste of time and is “busy work” (a term I have recently adopted that has been used by a pharmaceutical microbiology consultant whose opinions I particularly respect).

Let me also clarify that recoveries from surfaces is different from the recovery covered by USP <1227>. In that USP document, the recovery is done to insure that sampling conditions (including possible interferences from swabs, from product residues, and from cleaning/sanitizing agents) do not inhibit the growth of microorganisms in the measurement technique. I should also clarify that many of my objections to microbial recovery studies from surfaces may be overcome if newer methods that count cells (which may be viable and non-viable) rather than “colony forming units” were used. However, other than in big pharma those newer techniques are not widely used.