Qualification of a qPCR-based Mycoplasma assay under cGMP for release of effector T cell products

Mycoplasma contamination in cell therapy products poses a critical safety risk. Regulators require Mycoplasma testing for lot release of ATMP (Advanced Therapy Medicinal Products). We qualified the MycoSEQ™ Plus Mycoplasma Detection Assay (ThermoFisher Scientific) for release of effector T cell products manufactured in our GMP facility.

This qPCR-based method was validated by the kit manufacturer as equivalent to compendial methods described in EP 2.6.7 and USP <63>. The assay was performed precisely per the manufacturer`s protocol. We qualified a matrix of cryopreserved effector T cells mixed with supernatant from day 7 of T cell culture. These samples were spiked with purified genomic DNA isolated from the most common Mycoplasma contaminants found in cell cultures: M. arginini and M. fermentans. For each species, six independent extractions of DNA were performed, and each extraction was analyzed in quadruplicate qPCR reactions (total n=24 per species). The assay kit discriminatory positive control (DPC) was also included.

The spiking concentration of Mycoplasma DNA was 100 genome copies per sample, representing the assay`s stated limit of detection. Each species yielded at least 23 positive results out of 24 wells, meeting the pre-defined acceptance criteria. All negative controls, positive extraction controls, and internal assay controls met performance specifications.

The data from this qualification confirms that the MycoSEQ™ Plus assay is sensitive, specific, robust, and fit for purpose in detecting Mycoplasma contamination in effector T cell products. It offers a reliable, rapid alternative to traditional culture methods and expensive send-out testing. Sheba offers this test to outside customers.