A Rapid and Sensitive Nucleic Acid Amplification Technique for Mycoplasma Screening of Cell Therapy Products

Abstract

Mycoplasma species (spp.) bacteria can infect cell cultures, posing a potential threat to recipients of cell therapy products. Conventional Mycoplasma testing methods are highly sensitive but typically require a minimum of 28 days to produce results. This delay is problematic if rapid results are needed to inform treatment decisions. Nucleic acid amplification technique (NAT) methods have been gaining favor for Mycoplasma testing due to their speed and specificity; however, they must first be qualified as meeting or exceeding the sensitivity of the compendial method. We present herein a NAT method for the detection of Mycoplasma that circumvents the need for live Mycoplasma spp. in the test procedure by instead being qualified using Mycoplasma spp. genomic DNA. We have demonstrated a lower limit of detection that exceeds the regulatory requirements set by Health Canada. This assay is now being used to screen clinical cell therapy products manufactured at our center.

The mechanism of MICU-dependent gating of the mitochondrial Ca2+uniporter

“…The MEF cell lines were authenticated by short tandem repeat profiling conducted by Labcorp. The cell lines were free of mycoplasma as determined by PCR based detection ( Dreolini et al, 2020 ).…”

In-house abbreviated qualification of a real-time polymerase chain reaction method and strategies to amplify mycoplasma detection in human mesenchymal stromal cells

“…Another limitation of this study is the use of mycoplasma gDNA for spiking instead of live or inactivated mycoplasma. NAT method qualifications using samples spiked with mycoplasma gDNA have been previously reviewed and accepted by Health Canada [8,27]. Additionally, as the authors wanted to leverage and compare our qualification of the realtime PCR method with our previous qualification of an endpoint PCR kit for the same MSC(M) investigational cell product [8], the authors wanted to stay with the same approach of using gDNA.…”

“…This qualification study was done specifically for MSCs(M), the same investigational cell product that was used in the authors' previous Health Canada-approved endpoint PCR kit qualification [8] to leverage the qualification data and strategy for regulatory approval. Recent studies have also reported NAT method qualification for mycoplasma detection in other therapeutic cell products, such as chimeric antigen receptor T cells [27] and EpsteinÀBarr virus-specific cytotoxic T lymphocytes [29].…”

Synthesis of 124I-labeled epichaperome probes and assessment in visualizing pathologic protein-protein interaction networks in tumor bearing mice

“…The use of another cell line is possible but needs adjustments and further troubleshooting for culture conditions and tumor induction in mice. Note: Cells should be authenticated using short tandem repeat profiling and tested for mycoplasma before experiments ( Dreolini et al., 2020 ). …”

“…Note: Cells should be authenticated using short tandem repeat profiling and tested for mycoplasma before experiments ( Dreolini et al., 2020 ).…”