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.