Detection of mycobacterium species in qPCR as a consideration for cell banking testing to support cell and gene therapy manufacturing

Klaudia Shick, M.Sc., Ph.D., Manager, Cell and Molecular Biology; Stan Prince, Sr Scientific Advisor; Eurofins BioPharma Product Testing

Cell and gene therapies (CGT) are one of the fastest-growing areas of therapeutics and are at the very focus of healthcare innovation. Production of cell and gene therapies, often use mammalian cells (cell banks), including human donors/patients cells. Mammalian cells are subject to contamination by viruses, microbes, fungus, or other agents. Another known contaminant is Mycobacterium. This organism causes tuberculosis in humans. According to WHO (www.who.int/newsroom/fact-sheets/detail/tuberculosis), it is estimated that 10 million people became ill with tuberculosis in the year 2020.

Mycobacterium has been a concern in the vaccine industry for many years and now for the gene therapy industry. Testing for the absence of these organisms is outlined in various regulatory documents including FDA 2010 Vaccine Guidance as well as World Health Organization’s Annex 3 for the Evaluation of Animal Cell Cultures. As per FDA recommendation, if the species from which your cells were derived is susceptible to infection with Mycobacterium species, an appropriate test should be performed (FDA 2010 Vaccine Guidance).

Eurofins has optimized and validated a platform real-time PCR method to test for the presence of mycobacterium species in cell lines used for vaccine and cell and gene therapy production. The assay has ability to detect Mycobacterium tuberculosis complex (MTBC) DNA, including M. bovis, M. tuberculosis, M. microti, M. caprae, M. pinnipedi, M. africanum, and M.canetti (species pathogen for human and animals). Validation was based on FDA ICH Harmonized Tripartite Guideline, Q2 (R1), and the standard testing is conducted under current Good Manufacturing Practices (cGMP). The PCR method has a detection limit (LOD) of 16 copies of nucleic acid per PCR reaction and does not require extensive suitability/interference testing. The matrix interference evaluation is included in the standard sample testing with a control included in each assay performed. This rapid, standard assay has very quick turnaround time (one week or less).

The historical mycobacterium test method was an in vitro approach. At the speed the CGT industry is moving, faster and alternative approaches are needed to address speed of results as well as quantity of material needed. The Eurofins Mycobacterium complex PCR detection method only requires 1-2mL of sample and the certificate of analysis can be issued in 10 days or less.

In addition to mycobacterium, microbial testing (sterility), mycoplasma testing, and viral adventitious agent testing are all needed to ensure a cell line is safe to be used for viral vector and vaccine production. Eurofins can provide the full GMP biosafety testing package for global clients needing to meet regulatory requirements and ensure product safety for their clinical trials and/or commercial molecules.