Supplementary file 1_A novel cultivation strategy to recover NK cell cytotoxicity.pdf

Natural killer cells are emerging as promising “off-the-shelf” effectors for cancer immunotherapy, yet expansion of the NK-92 cell line in batch cultivation leads to rapid loss of cytotoxicity concomitant with lactate accumulation. In this study we developed and validated a two-phase manufacturing strategy that decouples cell proliferation from functional recovery in order to obtain an improved final product potency. Our 8-day kinetic survey determined declines in viability, metabolite profiles and cytotoxicity during static batch expansion. Guided by these data, in a 32-run full-factorial design-of-experiments approach we varied fresh cultivation medium proportion, temperature, dissolved oxygen, and recovery duration; partial least squares modeling identified fresh-medium ratio and recovery time as the primary drivers of cytotoxicity restoration. Identified optimal conditions (90% fresh medium, 37.2 °C, 3.7 days recovery) recovered cytotoxicity and maximized cytotoxic capacity. These setpoints were then translated to a 2 L stirred-tank bioreactor, where a fed-batch expansion under controlled pH and lactate levels produced 2.0 × 109 cells, followed by recovery that achieved 43% ± 8% cytotoxicity. This scalable, two-phase paradigm minimizes medium usage and obviates continuous perfusion, offering a potential workflow to increase NK-92 potency and a base for manufacturing high-quality advanced therapy medicinal products.