Lamin Jallow - Microbiology Technology Specialist, MilliporeSigma; Burlington ; MA; USA, An affiliate of Merck, KGaA Darmstadt, Germany; Dr. Anne-Grit Klees Lead Expert, Product & Portfolio Manager, BioMonitoring Environmental Monitoring, Merck KGaA, Darmstadt, Germany.
Every day counts for manufacturers of short-shelf-life biologics/treatments who need to release their products and have them administered to patients as fast as possible. This is particularly the case for biologics as well as cell and gene therapies that are based on hybridoma or cultures of the patient’s cells, for example, T cells or stem cells. Classical sterility testing takes 14 days to complete following 21 CFR 610.12 and USP <71> due to lengthy incubation because microorganisms vary considerably with respect to their proliferation rate on or in the culture media used for sterility testing, so unfortunately it is the slowest growers that ultimately determine the time-to-result. In addition to the microorganisms that grow slowly by nature, there may be others that are in a dormant stage or have been injured or stressed, for example by microbicidal compounds or depletion of certain nutrients during cell cultivation. These organisms need to recover and can take longer to grow than under optimal conditions. Manufacturers of advanced therapy medicinal products are therefore increasingly turning to alternative rapid microbiological methods to perform their sterility tests and reduce the time to result.
Rapid testing methods suitable for biologics
USP <1071> , titled “Rapid Microbial Tests for Release of Sterile Short-Life Products: A Risk-Based Approach”, gives an overview of recommended rapid technologies. These are based on either adenosine triphosphate (ATP) bioluminescence, flow cytometry, isothermal microcalorimetry, nucleic acid amplification, respiration or solid phase cytometry. Scientists at the FDA’s Division of Biological Standards and Quality Control examined three rapid systems for their suitability for sterility testing of biological products: the Milliflex® Rapid system for membrane filtration and microcolony detection as CFUs via ATP bioluminescence and two systems that detect the CO2 released in the course of microbial growth after direct inoculation (contact us for study details).
The Milliflex® Rapid system achieved results the fastest, detecting all the 11 bacterial, yeast and mold strains in the panel within five days, crucially including slow growers such as Cutibacterium acnes, a bacterium that took the other two rapid methods almost as long as the compendial methods to detect. The Milliflex® Rapid method was found to be significantly more sensitive at detection than both other rapid methods as well as the compendial membrane filtration and direct inoculation methods. The Milliflex® Rapid filtration procedure rinses away inhibiting substances such as thimerosal so the method was unaffected by the addition of this preservative to the matrix, whereas the two other rapid methods did not consistently recover all strains under this condition. The Milliflex® Rapid method also proved compatible with inactivated influenza vaccine and aluminum-containing adjuvants, with no interfering background bioluminescence.
Removing the ATP emanating from mammalian cells
ATP-based sterility testing of mammalian cell cultures has to take into account that ATP is contained in all living cells, not just microorganisms. When the density of cells in a sample is high (e.g. 108 cells/mL), the Milliflex® Rapid method may require an extra step to remove background luminescence. In a study of ours aimed at establishing a rapid sterility testing procedure for cell culture samples that does not harm microorganisms and that complies with USP and EP guidelines, we treated two different immune cell preparations with a proprietary mammalian cell lysis buffer (MCLB) and the enzyme apyrase. This selectively lyses mammalian cells and subsequent filtration removes the ATP they have produced, paving the way for selective detection of the remaining microbial ATP. The Milliflex® Rapid method captures the widest possible range of microbes using Schaedler Blood Agar/RSTM (Rapid Sterility Test Media) for aerobic and anaerobic incubation at 30 to 35 °C as well as aerobic incubation at 25 to 30 °C. The fast and slow growing bacteria, yeast and mold strains in our study all showed recovery rates above the 70% threshold criterion after one to three days of incubation (contact us for study details).
Although these results are an indication that the Milliflex® Rapid method will work with mammalian cell cultures in general, each manufacturer or contract lab must qualify it in their own laboratory using their own products, cell cultures and in-house microbial strains. Getting the method implemented as an alternative to compendial sterility testing usually reduces the time-to-result from 14 to 5 days or, depending on the individual risk assessment, to as little as four days. We off er expert services and consultation to support a successful validation.
Find out more about the 21 CFR Part 11 compliant, second-generation Milliflex® Rapid System 2.0 for rapid sterility and bioburden testing on a small footprint platform & get in touch with our sterility testing experts.
www.SigmaAldrich.com/milliflex-rapid
Publication Detail
This article appeared in American Pharmaceutical Review:
Vol. 26, No. 8
Nov/Dec 2023
Pages: 70-77
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