Streamlining the purification of a clinical-grade oncolytic virus for therapeutic applications

•A robust process for complex oncolytic viruses such as chimeric recombinant VSV-NDV.•Enhanced DSP operations, including DNA digestion, clarification, chromatography, TFF, and sterilizing filtration.•Anion-exchange chromatography with external additives, achieving 86 % recovery of infectious particles.•Global recovery yield of 64 % with 99 % protein and 97 % DNA clearance across four DSP operations. Oncolytic viruses (OV) have emerged as a promising approach to mitigate the challenges of treating solid cancers. However, the lack of established manufacturing and downstream processing (DSP) platforms combined with the high treatment doses needed for clinical use (108–1011 TCID50/dose), hamper the widespread success of this therapeutic concept. Here, we present an efficient and scalable GMP-compliant process for the purification of a fusogenic oncolytic virus (rVSV-NDV). Non-GMO CCX.E10 cells grown in suspension in chemically defined medium were used for high titer rVSV-NDV batch production (3.2 × 108TCID50/mL) in stirred tank bioreactors. All DSP unit operations (DNA digestion, clarification, chromatography, TFF, and sterilizing filtration) were optimized to identify the best purification approach. Among several strategies evaluated, two filters enabled high throughput and turbidity reduction, while preventing any loss of infectious particles during the clarification step. For the intermediate purification, anion-exchange chromatography (AEX) was used in combination with the addition of polyprotic salts, which resulted in a maximum recovery yield of 86 % of infectious particles. The addition of citrate to the chromatography setup increased the separation resolution of rVSV-NDV particles from less negatively charged impurities. Global recovery yield after four operation units was 64 % with 99 % and 97 % of protein and DNA clearance, respectively. Together, with our previously optimized upstream process, we open up an avenue for large-scale manufacturing of oncolytic VSV-NDV for future clinical use.