Scalable purification of cowpea chlorotic mottle virus (CCMV) and its bioconjugates from plant extracts using ultrafiltration

Plant virus-derived materials are finding applications in science, engineering, and technology – with some candidates advancing toward commercialization, scalable manufacturing methods are needed. We recently established ultrafiltration/diafiltration (UF/DF) for purification of cowpea mosaic virus (CPMV) and tested here broader applicability of this process by purifying cowpea chlorotic mottle virus (CCMV) and its bioconjugates. We optimized extraction, ultrafiltration and ion exchange chromatography for CCMV: acidic buffers (pH 4.0) were ideal for extraction, removing ~80% of plant host cell proteins while recovering nearly 100% of CCMV. Membranes with a 1000 kDa and 300 kDa molecular mass cut-off were best suited for ultrafiltration, allowing to remove impurities, both larger and smaller than CCMV, alongside bulk water. When combining these steps, the same level of purity (>99%) as the contemporary centrifugation process was achieved with only 7 instead of 13 process steps, thus shortening the processing time from ~2-3 days to ~7 hours. The ultrafiltration process was also compatible with virus purification by PEG-precipitation, allowing to harness the advantages of both methods. Importantly, UF/DF enabled purification of CCMV bioconjugates, not only shortening the processing time and improving the recovery up to ~95%, but also removing free dye more efficiently compared to centrifugation.