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

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

Cell and gene therapies (CGT) are one of the fastest growing areas of therapeutics and are at the very core of healthcare innovation. Production of cell and gene therapies, often use mammalian cells (cell banks), including human donor/patient cells. Mammalian cells are subject to contamination by viruses, microbes, fungus, or other agents and by mycobacterium. Mycobacterium causes tuberculosis in humans. According to the World Health Organization (WHO) (www.who.int/news-room/fact-sheets/ detail/tuberculosis), it is estimated that 10 million people globally developed tuberculosis in the year 2020.

Mycobacterium has been a concern in the vaccine industry for many years and now is also becoming a concern among those in the gene therapy industry. Testing for the absence of these organisms is outlined in various regulatory documents, including the Food and Drug Administration (FDA) 2010 Vaccine Guidance as well as WHO’s Annex 3 for the Evaluation of Animal Cell Cultures. As per FDA’s guidance, if the species from which cells are derived is susceptible to infection with Mycobacterium species, an appropriate test should be performed.

Eurofins BioPharma Product Testing has optimised 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 the 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 affecting human and animals). Validation was based on FDA ICH Harmonised 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. Matrix interference evaluation is included in standard sample testing with a control included in each assay performed. This rapid, standard assay has very quick turnaround time (one week or less).

Historical mycobacterium test methods utilised an in vitro approach. With the speed the CGT industry is moving, innovative approaches will be needed to address TAT required as well as increased testing volumes. The Eurofins BioPharma Product Testing 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 required to ensure a cell line is safe to be used for viral vector and vaccine production. Eurofins BioPharma Product Testing can provide the full GMP biosafety testing package for global clients working to meet regulatory requirements and ensure product safety for their clinical trials and/or commercial molecules. Contact us to learn more.