Tim Cser- Senior Technology Specialist, MilliporeSigma, CA- An affiliate of Merck KGaA, Darmstadt, Germany; Yoann Gatineau- R&D Technical Leader, Millipore SAS, Molsheim, France- An affiliate of Merck KGaA, Darmstadt, Germany.
Mycoplasma contamination is a widespread and reoccurring problem in many cell and tissue culture systems that are designed to produce biologics such as recombinant proteins, monoclonal antibodies, and cell therapeutics. These ubiquitous bacteria can grow to high titers in culture media without exhibiting typical bacterial contamination signs such as turbidity or pH change. Their effects on cultured cells include altered metabolism, slowed proliferation, and chromosomal aberrations, potentially compromising the quality of the therapeutic and thus patient safety. This is why it is pivotal to verify that cell cultures for the manufacture of pharmaceuticals are, and remain, contamination-free. Unfortunately, compendial sterility testing is unsuitable to detect mycoplasmas as they are too small to be filtered and will not grow in the tryptic soy broth (TSB) or fluid thioglycollate medium (FTM) used for this test. For these reasons, there is dedicated regulation for mycoplasma testing, most notably USP <63> and Ph. Eur. 2.6.7, with guidance also given by CBER/FDA.
Based on a risk assessment, the manufacturer determines at which stages of the production process to test for mycoplasmas. Biopharmaceutical facilities that use eukaryotic cells for the production of vaccines have to test their cell banks and virus seed lots as well as perform in-process testing, particularly on the bulk vaccine during harvest. End-product tests usually do not yield as meaningful results because the bacteria would typically be less concentrated by this stage.
The complex compendial culture method
For many stages of the production process, compendial mycoplasma testing involves both a culture media test to detect cultivable strains and an indicator cell test for fastidious, non-cultivable strains, which is less sensitive and requires highly skilled staff to perform. The compendial culture media method is a painfully complex and long-drawn-out process. It requires up to three different media, Hayflick, Frey and Friis, that are specially formulated to jointly cover the detection of a broad spectrum of Mycoplasma species as well as related Mollicutes bacteria such as Acholeplasma laidlawii. These media are complicated to make in a lab due to some sensitive components. The shelf lives are also short, although we at MilliporeSigma, the only major supplier worldwide, have recently validated a longer four months for our agar and six months for our broths. The culture method also requires a relatively high volume of test sample and, most significantly, takes 28 days to deliver results—much too long for many modern-day biologics such as stem cell and regenerative medicine products, which have to be administered to patients within a few days. Although it is possible to treat patients with such products before the result of the culture-based test becomes available, this is not the ideal situation as freedom from mycoplasmas is part of a product’s safety profile.
Alternative nucleic acid methods save time
For the above reasons many facilities have turned to using rapid enzymatic activity-based methods or nucleic acid amplification techniques, in particular PCR, preferring to put up with the validation effort that implementing such alternative methods requires to avoid the culture method wherever possible. Equivalency of the alternative method to the compendial culture-based method is demonstrated using a performance comparison that includes sample suitability, robustness, sensitivity, specificity, and the ability to detect a broad spectrum of species. There are, as yet, no specific documents on the design of such comparative studies for an FDA review but some guidance can be taken from Ph. Eur. 2.6.7. It should be noted that validation of a direct PCR method without culturing can be challenging as there tends to be a relatively high risk of false negative and false positive results.
However, alternative methods are not making cultivation obsolete. Cultivation is regularly required for validation purposes. In addition, to achieve a suitable limit of detection of below 10 CFUs/mL or if the sample volume is below 1 mL, rapid methods such as PCR may need a prior enrichment step. Furthermore, inhibitory substances can impact rapid methods, with dilution by enrichment a possible remedy. In cases where the sample matrix renders direct PCR testing unreliable, centrifugation to clear the matrix has been a popular alternative to enrichment but will probably cease to be an option when the next revision of Ph. Eur. 2.6.7 comes into force. The reason is that mycoplasmas have the tendency to adhere to mammalian cells, so pelleting away the eukaryotic cells and debris will also remove bacteria from the sample, which can lead to false-negative test results. Nevertheless, rapid methods to test for mycoplasmas have resulted in valuable time savings for biologics’ manufacturers who have succeeded with implementation.
Find out about our full range of ready-to-use solid and liquid media for compendial mycoplasma testing.
sigmaaldrich.com/mycoplasma
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