Intensified lentiviral vector perfusion bioprocessing with a spiral inertial microfluidic cell retention device.

Abstract

Scalable production of cell therapy doses relies on inexpensive, efficient production of gene delivery vectors, such as lentiviral vectors, in HEK293 cell culture. Intensified perfusion processes improve the volumetric productivity of cell culture by continuously supplying nutrients, oxygen, and media to cells while removing harmful metabolites, thereby enabling higher producer cell densities. Membrane filter-based cell retention devices commonly used in perfusion bioprocessing can experience significant clogging and fouling over long-term processes, which leads to the undesired retention of lentiviral vectors in the filter matrix. In this work, we used spiral microfluidic technology as a cell retention device to continuously harvest lentiviral vectors and remove metabolic waste from HEK293 cells in a bioreactor running high cell density perfusion cultures. With the spiral microfluidic device, we performed four perfusion culture runs with maximum cell densities between 15 × 10⁶ and 25 × 10⁶ cells per mL, achieving up to seven days of continuous lentiviral vector production and lossless harvesting with maximum, unconcentrated, functional titers on the order of 10⁸ transducing units (TU) per mL. These production titers are competitive with other bioprocessing approaches in industry and academia. The highest cell-specific productivity (over 50 TU per cell per day) and cell-specific yields of our study (over 80 TU per cell) were achieved when using spiral device-mediated perfusion bioprocessing to cultivate cells and then transferring the cells to a shake flask environment with daily media replacement to generate lentiviral vectors.