Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration

Abstract

Cell culture-based production of vector-based vaccines and virotherapeutics is of increasing interest. The vectors used not only retain their ability to infect cells but also induce robust immune responses. Using two recombinant vesicular stomatitis virus (rVSV)-based constructs, we performed a proof-of-concept study regarding an integrated closed single-use perfusion system that allows continuous virus harvesting and clarification. Using suspension BHK-21 cells and a fusogenic oncolytic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV), a modified alternating tangential flow device (mATF) or tangential flow depth filtration (TFDF) systems were used for cell retention. As the hollow fibers of the former are characterized by a large internal lumen (0.75 mm; pore size 0.65 μm), membrane blocking by the multi-nucleated syncytia formed during infection could be prevented. However, virus particles were completely retained. In contrast, the TFDF filter unit (lumen 3.15 mm, pore size 2–5 μm) allowed not only to achieve high viable cell concentrations (VCC, 16.4–20.6×106 cells/mL) but also continuous vector harvesting and clarification. Compared to an optimized batch process, 11-fold higher infectious virus titers were obtained in the clarified permeate (maximum 7.5×109 TCID50/mL). Using HEK293-SF cells and a rVSV vector expressing a green fluorescent protein, perfusion cultivations resulted in a maximum VCC of 11.3×106 cells/mL and infectious virus titers up to 7.1×1010 TCID50/mL in the permeate. Not only continuous harvesting but also clarification was possible. Although the cell-specific virus yield decreased relative to a batch process established as a control, an increased space-time yield was obtained. Key points • Viral vector production using a TFDF perfusion system resulted in a 460% increase in space-time yield • Use of a TFDF system allowed continuous virus harvesting and clarification • TFDF perfusion system has great potential towards the establishment of an intensified vector production

Digital Twin for Continuous Production of Virus-like Particles toward Autonomous Operation

“…For cell retention in the production of virus particles, the acoustic separator and an innovative combination of depth filter and hollow fiber module have proven successful. ,,,,− To accelerate cell settling in density-based separation, the g-force can be increased by an acoustic resonance field. , The acoustic separator not only enables separation of the product but also removal of dead cells, host cell proteins, and double-stranded RNA, leading to higher cell densities. ,, However, for scale-up and higher throughputs, increased input power is required to maintain separation efficiency. , This can result in higher temperatures in the separator, necessitating efficient temperature control. In addition, the decreasing concentration of dissolved oxygen in the acoustic separator can affect cell viability. ,, …”

“…A macroporous tubular filter is preferred for size-dependent separation, as it ensures efficient cell retention and good product permeability. ,,,,,− In contrast to the acoustic separator, a complete recirculation of the cells into the bioreactor is also possible since they do not pass the filter, and thus no cell loss occurs. In addition, the recirculation time is shorter, since no settling of the cells is required for cell separation. ,, …”