[Development of a rapid chimeric antigen receptor (CAR)-T production process and assessment of critical quality...

Chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated remarkable clinical efficacy in certain hematologic malignancies. However, its broader clinical application is constrained by the prolonged duration of conventional ex vivo manufacturing. The extended production cycle not only delays treatment and increases costs but may also exacerbate terminal differentiation and functional exhaustion of T cells, thereby reducing their in vivo persistence. To address these bottlenecks, shortening the ex vivo culture duration to preserve the early differentiation phenotype and antitumor activity of T cells has emerged as an important strategy for optimizing CAR-T cell therapy. In this study, a rapid CAR-T cell production process requiring only 24 h was established. Through systematical optimization of T-cell activation conditions and the viral transduction time window, efficient CAR gene delivery and cell preparation were achieved within 24 h. The rapidly manufactured CAR-T cells exhibited an early differentiation phenotype and enhanced proliferative potential. In vitro functional assays demonstrated potent tumor-killing activity of the CAR-T cells. In a xenograft mouse model, the cells showed antitumor efficacy and in vivo persistence comparable to those produced with the conventional manufacturing process. These results indicate that the 24 h rapid production strategy is feasible and provides a promising technical approach to shorten patient waiting time, reduce production costs, and generate functionally improved CAR-T cell products for clinical translation.