Assessment of Q-PCR based vector copy numbers and viral titration in CAR T cell products, Probe vs SYBR Green.
Assessment of vector copy numbers (VCNs) in comprised of the chimeric antigen receptor (CAR (T cells as a safety parameter and viral titration in the vector bank (VB) as an efficacy parameter are essential in the CAR T cell production procedure. Here, we developed a quantitative-PCR (Q-PCR) based VCN and viral titration analysis using both Probe and SYBR Green methods. Cellular viral titration was also applied for comparison of viral titration results. The VCN in CAR T cells with SYBR Green was 1.72, 2.31, and 2.46 respectively, while in the Probe based analysis, it was 1.33, 1.83, and 2.20. The molecular viral titration in SYBR Green resulted in 2.90*109, 5.24*1010, and 9.76*1010, while in the Probe it was 1.54*109, 1.13*109, and 7.73*108. The cellular titration resulted in 1.73*106, 2.16*106, and 2.49*106. In summary, both SYBR Green and Probe revealing that Probe strategy had a lower dependency on sample contamination and could be a better strategy for the Q-PCR method. The SYBR Green based viral titration method is significantly different from the Probe based method, with the Probe based method yielding better results compared to the cellular titration method. However, no significant difference was reported in both methods for VCNs.