Comparison of Five Commercial Molecular Assays for Mycoplasma Testing of Cellular Therapy Products

• Published in: Journal of clinical microbiology 2023-02, Vol.61 (2), p.e0149822-e0149822
• Main Authors: Totten, Arthur H, Adams, Anna Julia, Halas, Hyunmi K, Gebo, James E T, East, Amanda D, Lau, Anna F
• Format: Article
• Language: English
• Subjects: Bacteriology; Cell Culture Techniques; Cell- and Tissue-Based Therapy; Humans; Limit of Detection; Mycoplasma; Reference Standards
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/jcm.01498-22

Testing of cellular therapy products for is a regulatory requirement by the United States Food and Drug Administration (FDA) to ensure the sterility and safety of the product prior to release for patient infusion. The risk of contamination in cell culture is high. Gold standard testing follows USP 63 which requires a 28-day agar and broth cultivation method that is impractical for short shelf-life biologics. Several commercial molecular platforms have been marketed for faster raw material and product release testing; however, little performance data are available in the literature. In this study, we performed a proof-of-principle analysis to evaluate the performance of five commercial molecular assays, including the MycoSEQ detection kit (Life Technologies), the MycoTOOL real-time detection kit (Roche), the VenorGEM qOneStep kit (Minerva Biolabs), the ATCC universal detection kit, and the Biofire assay (bioMérieux Industry) using 10 cultured spp., with each at four log-fold dilutions (1,000 CFU/mL to 1 CFU/mL) in biological duplicates with three replicates per condition (  = 6) to assess limit of detection (LOD) and repeatability. Additional testing was performed in the presence of tumor infiltrating lymphocytes (TILs). Based on LOD alone, the Biofire assay was most sensitive followed by the MycoSEQ and MycoTOOL which were comparable. We showed that not all assays were capable of meeting the ≤10 CFU/mL LOD to replace culture-based methods according to European and Japanese pharmacopeia standards. No assay interference was observed when testing in the presence of TILs.