Impact of physical and chemical parameters on spinoculation for chimeric antigen receptor T cell manufacturing using a quality-by-design approach

Chimeric antigen receptor (CAR) T cell therapies represent a significant advancement for treating hematological malignancies, particularly in relapsed/refractory cases. Despite their clinical success, the high cost of CAR T cell therapies remains a major barrier to broader implementation. A significant proportion of these costs stems from the dependency on viral vectors and the limited understanding of transduction mechanisms. This work evaluates the impact of physical and chemical parameters during transduction using a spinoculation process. Physical parameters, such as spinoculation duration and speed, were identified as key drivers of transduction efficiency, contributing to a 20%-30% increase in transduction. Similarly, the addition of LentiBOOST and polybrene enhanced transduction efficiency by approximately 1-2-fold compared with control conditions without these supplements. Given that both physical and chemical parameters influence transduction efficiency, a quality-by-design approach was used to systematically investigate their potential synergistic or antagonistic interactions. This systematic approach highlighted the cytotoxic impact of polybrene and demonstrated that LentiBOOST is critical to drive transduction, particularly in CD4 subsets. The optimized process led to a 2-3-fold improvement in transduction without compromising CAR T cell growth or functionality and was shown to be compatible with serum- and xeno-free medium, supporting its translational potential.