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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. [ABSTRACT FROM AUTHOR]

Oxygen limitation can be used as a simple environmental inducer for the expression of target genes. However, there is scarce information on the characteristics of microaerobic promoters potentially useful for cell engineering and synthetic biology applications. Here, we characterized the Vitreoscilla hemoglobin promoter (P vgb ) and a set of microaerobic endogenous promoters in Escherichia coli. Oxygen-limited cultures at different maximum oxygen transfer rates were carried out. The FMN-binding fluorescent protein (FbFP), which is a nonoxygen dependent marker protein, was used as a reporter. Fluorescence and fluorescence emission rates under oxygen-limited conditions were the highest when FbFP was under transcriptional control of P adhE , P pfl and P vgb . The lengths of the E. coli endogenous promoters were shortened by 60%, maintaining their key regulatory elements. This resulted in improved promoter activity in most cases, particularly for P adhE , P pfl and P narK . Selected promoters were also evaluated using an engineered E. coli strain expressing Vitreoscilla hemoglobin (VHb). The presence of the VHb resulted in a better repression using these promoters under aerobic conditions, and increased the specific growth and fluorescence emission rates under oxygen-limited conditions. These results are useful for the selection of promoters for specific applications and for the design of modified artificial promoters.