Quasi-perfusion studies for intensified lentiviral vector production using a continuous stable producer cell line

Abstract

Quasi-perfusion culture was employed to intensify lentiviral vector (LV) manufacturing using a continuous stable producer cell line in an 8-day process. Initial studies aimed to identify a scalable seeding density, with 3, 4, and 5 x 104 cells cm-2 providing similar specific productivities of infectious LV. Seeding at 3 x 104 cells cm-2 was selected, and the quasi-perfusion was modulated to minimise inhibitory metabolite accumulation and vector exposure at 37 °C. Similar specific productivities of infectious and physical LV were achieved at 1, 2, and 3 vessel volumes per day (VVD), with 1 VVD selected to minimise downstream processing volumes. The optimised process was scaled 50-fold to 1,264 cm2 flasks, achieving similar LV titres. However, scaling up beyond this to a 6,320 cm2 multilayer flask reduced titres, possibly from suboptimal gas exchange. Across three independent processes in 25 cm2 to 6,320 cm2 flasks, reproducibility was high with a coefficient of variation of (7.7 ± 2.9)% and (11.9 ± 3.0)% for infectious and physical LV titres, respectively. The optimised flask process was successfully transferred to the iCELLis™ Nano (Cytiva) fixed-bed bioreactor, with quasi-perfusion at 1 VVD yielding 1.62 x 108 TU.