New rapid mycoplasma testing service delivers speed, sensitivity and specificity to a broad scope of sample matrices

Jeri Ann Boose, Ph.D., director, Biopharmaceutical Services; Robert Donatelli, M.S., principal scientist, Cell and Molecular Biology; Weihong Wang, Ph.D., manager, Cell and Molecular Biology

Mycoplasmas are the smallest known self-replicating prokaryotes. They parasitize a wide range of host organisms, including humans, animals, plants and insects and are common contaminants of mammalian cell culture. Due to their small size, mycoplasmas can readily pass through 0.2 μm filters used to maintain sterility. Sources for the introduction of mycoplasmas into cell culture systems include: cell culture media and additives, the use of previously infected cells and laboratory personnel.

Although visual signs indicative of mycoplasma contamination are often lacking, mycoplasma contamination of cell lines used to produce biopharmaceutical products can disrupt cellular growth and metabolism and lead to changes in gene expression. These adverse cytopathological events can result in decreased product quantity and quality.

Importantly, mycoplasma infections have been associated with respiratory illness, urethritis and arthritis, and they act as a co-factor in numerous infectious diseases. It is for these reasons that world-wide regulatory agencies require that biotechnological products produced in cell substrates be tested to ensure the absence of mycoplasma contamination.

Compendial mycoplasma testing procedures for the detection of viable mycoplasma contaminants are culture based and have three components: direct cultivation on agar plates, enrichment in broth followed by detection on agar plates and detection in indicator cell culture.

The current procedures are time consuming as they are 28 days in length. This time requirement is not amenable for obtaining the rapid lot release testing results needed for biopharmaceutical products that have short half-lives or for which there is high market demand.

The lengthy assay period is also not conducive to the rapid screening of raw materials intended for use in future production, nor to the rapid in-process screening of intermediates for the purpose of detecting and containing contamination events. This summer, Eurofins Lancaster Laboratories will offer a rapid mycoplasma test comparable in sensitivity and specificity to the 28 day compendial method. After several months of working with the EMD Millipore MilliPROBE® Real Time Detection System for Mycoplasma, Eurofins Lancaster Laboratories’ scientists have unequivocally demonstrated the value of the method for the rapid testing of manufacturing samples without observing matrix interference effects.

An additional, important advantage of the MilliPROBE System is that it has the capability of processing a volume of material comparable to that tested in the compendial method, making it preferable to rapid mycoplasma detection methods that have volume limitations.

Finally, the system has two features that allow it to preferentially detect viable mycoplasmas: (1) a mycoplasma retention filter, which retains mycoplasma cells but allows free nucleic acids to be rinsed away into waste and (2) a detection system designed to target and amplify mycoplasma rRNA from mycoplasma cells captured and lysed within the sample prep device. RNA is not only a better viability marker than DNA, but it increases the sensitivity of the method as a single mycoplasma contains approximately 1000 rRNAs compared to only 1-2 DNAs. Validation of the MilliPROBE System is currently underway with plans to commercially offer both GMP and non-GMP versions of the assay this summer.