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MYCOPLASMA
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Lessons Learned from Validation of a Real-Time PCR Mycoplasma Test for Autologous Cell Therapy Products
John Duguid
Cell Therapy Manufacturing Technical Services Genzyme, a Sanofi company
Introduction
In June 2013, the European Medicines Agency (EMA) approved a real- time polymerase chain reaction (Real-Time PCR) Mycoplasma test for lot release of matrix-applied characterized autologous cultured chondrocytes (MACI®). MACI® is an autologous cell therapy product indicated for the repair of articular cartilage defects in the knee in the European Union (EU). Implementing rapid microbiological methods (RMM) for lot release of cell therapy products with short shelf lives improves the safety of these products and ultimately leads to increased protection for patients. Mycoplasma species are the smallest known free-living organisms, ranging in size from 0.15 to 0.3 μm in diameter. They are self-replicating bacteria that lack cell walls and are relatively common contaminants of mammalian cell culture [1] in a research setting. Mycoplasma present particular challenges as they are difficult to culture and detect using traditional microbiological detection techniques. Conventional test methods for Mycoplasma detection [2-4] take at least 28 days to complete. Developing a rapid method to detect Mycoplasma was necessary for lot release of MACI®, which has a 6-day shelf life. Selecting a method based on Real-Time PCR facilitated successful development, validation, and technology transfer. The test takes less than six hours to complete; automation of sample preparation steps further reduces that time to five hours. Implementing the newly approved method in a production environment provided an excellent opportunity to reflect on the validation and technology transfer process.
Methodology Overview
The sample configuration for the Real-Time PCR Mycoplasma test is the same as for the conventional growth-based test. It contains drug product in conditioned cell culture medium. Centrifugation pellets any Mycoplasma organisms present in the sample and concentrates them to improve detection. After lysing the organisms and enzymatically digesting proteins that could interfere with nucleic acid recovery, an extraction with proprietary magnetic particles and a magnet collects and purifies any DNA in the sample. Final preparation of the DNA extract requires elution of the DNA and removal of the magnetic particles. Using a Real-Time PCR Instrument, the assay analyzes the DNA extract using optimized multiplexed primer design for specific and comprehensive Mycoplasma species detection. Positive samples exhibit a threshold cycle (Ct) inversely proportional to the amount of
96 | | September/October 2013 - 15TH ANNIVERSARY ISSUE
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