Process intensification strategies toward cell culture‐based high‐yield production of a fusogenic oncolytic virus

Abstract

We present a proof‐of‐concept study for production of a recombinant vesicular stomatitis virus (rVSV)‐based fusogenic oncolytic virus (OV), rVSV‐Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi‐perfusion in small‐scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process‐related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK‐21, HEK293SF)infected at densities ranging from 15 to 30 × 106 cells/mL. The acoustic settler allowed continuous harvesting of rVSV‐NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 109 TCID50/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell‐specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large‐scale manufacturing of fusogenic rVSV‐NDV at HCD for all three candidate cell lines.

Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus

“…After infection, a steep decline in cell viability was noticeable starting from 24 hpi (Figure 5a). While a reduction of temperature to 34°C had no positive effect on virus production for rVSV-NDV, drastic declines in cell viability were prevented in STRs for both CCX.E10 and BHK-21 cells (Göbel, 2023; Figure 4a).…”

“…How to cite this article: Göbel, S., Jaén, K. E., Fernandes, R. P., Reiter, M., Altomonte, J., Reichl, U., & Genzel, Y. (2023).…”

“…These include mainly Vero cells (herpesvirus, measles virus, and vaccinia virus), A549 cells (adenovirus), HeLa cells (vaccinia virus), HEK293 cells (adenovirus, reovirus), and some proprietary cell lines (e.g., EB66, PER.C6, and CAP) (Ungerechts et al, 2016). Other continuous suspension cell lines, such as BHK-21, AGE1.CR, MDCK, and HEK293SF cells, have been evaluated for efficient production of fusogenic rVSV-NDV (Göbel, 2022(Göbel, , 2023. The highest reported rVSV-NDV yields were obtained in BHK-21 suspension cells with titers up to 5 × 10 8 TCID 50 /mL in optimized batch processes in stirred tanks and 2 × 10 9 TCID 50 /mL in perfusion cultures infected at high cell density (Göbel, 2023).…”

“…Other continuous suspension cell lines, such as BHK-21, AGE1.CR, MDCK, and HEK293SF cells, have been evaluated for efficient production of fusogenic rVSV-NDV (Göbel, 2022(Göbel, , 2023. The highest reported rVSV-NDV yields were obtained in BHK-21 suspension cells with titers up to 5 × 10 8 TCID 50 /mL in optimized batch processes in stirred tanks and 2 × 10 9 TCID 50 /mL in perfusion cultures infected at high cell density (Göbel, 2023). First studies performed with the avian cell line AGE1.CR, which is fully characterized and GMP-compliant, resulted in relatively low virus yields.…”

Advanced cell technologies: Making protein, cell, and gene therapies a reality

“…Oncolytic viruses, another emerging modality, shares the same bioprocess limitations as rAAV and LV. Göbel et al (2023) developed an optimized perfusion process for large-scale production of recombinant vesicular stomatitis virus-based fusogenic oncolytic virus (rVSV-OV). Three cell lines (AGE1.CR, BHK-21, and HEK293SF) were evaluated in HCD cultures, with a 15-30-fold increase in volumetric productivities being observed compared to the batch process, establishing perfusion as a viable process for large-scale production of rVSV-OV.…”