Toward Translation of Cowpea Mosaic Virus Intratumoral Immunotherapy With a Scalable Production Process.

The plant virus cowpea mosaic virus (CPMV) is being developed as a drug candidate for intratumoral immunotherapy for solid tumours. Data from preclinical studies in mice and rats as well as clinical studies in canine cancer patients demonstrate potent efficacy and an excellent safety profile. Toward Investigational New Drug (IND)-enabling studies, a scalable and Good Manufacturing Practice (GMP)-compatible manufacturing process must be developed. Therefore, here, we focused on the downstream process and developed a scalable purification process. We systematically screened the following processes: acidic extraction conditions making use of the virion pH stability, ultrafiltration making use of the nanoparticle character and the virion size, and finally ion exchange chromatography based on the virion's surface charge. The three processes were then combined into a 7-step protocol, providing efficiency compared to the contemporary laboratory procedures involving various centrifugation steps (and a total of 15 steps to yield pure CPMV). Furthermore, we streamlined the removal of endotoxins by combining detergent-based endotoxin removal with ion exchange chromatography. The new ultrafiltration-based process reduced the number of unit operations by more than half and the processing time from ~20 to ~7 h compared to centrifugation-based purification of CPMV. Importantly, toxic organic solvents, ultracentrifugation and isopycnic ultracentrifugation which are difficult to scale were omitted. We used a battery of characterisation methods to validate CPMV's structural integrity and biological activity.