Primary T-cell transduction performance as the guiding principle behind LV process development and platform innovation 

Lentiviral vectors (LV) are the most common vehicle for the manufacture of autologous CAR-T cells for cancer immunotherapy. The upstream component of the complex LV manufacturing process involves transient transfection of production cells by four plasmids; Rev, Gag-Pol, Genome, and envelope (VSV-G) at an optimal ratio. Downstream processing is used to purify the produced LV through multiple steps including but not limited to anion exchange chromatography, ultra-diafiltration and filter-sterilization to produce the final drug product. Biological variability due to product differences, upstream and downstream processes play an important role in determining LV drug product potency.  

Transduction titres, usually measured in an in-house cell line, is currently considered as the industry benchmark of LV batch manufacture success. Typically all early process optimisation/platform development activities are geared towards obtaining maximum LV titres on generic cell lines. However, efficient transduction of primary human T-cells by LV is critical for successful CAR-T cell generation. We will present data to show that optimising titre of vector on cell lines does not always correlate with highest vector performance on target cells such as primary T-cells in the case of CAR-T therapy. Through the development of a miniaturized LV purification and T-cell transduction workflow, we have now integrated T-cell transduction efficiency as an important Design of Experiments (DoE) parameter in our early client process development and platform innovation activities. Case studies involving finding the optimal design space for high T-cell transduction efficiency coupled with improved overall process robustness and its impact on vector functionality will be discussed.