Rapid two-step purification of Podoviridae bacteriophages using CIMmultus monolithic chromatography

Bacteriophages are promising biocontrol agents, but rigorous downstream studies require highly purified virions. We adapted a two-step monolithic ion-exchange workflow to purify the Podoviridae bacteriophage, BF25/12, which infects the plant pathogenic bacterium Dickeya fangzhongdai. Capturing the phage on a CIMmultus™ OH column, followed by polishing on a CIMmultus™ QA column, yielded a concentrated product (2.0 × 10¹⁰ PFU/mL) with >99% reduction in protein and 95-99% reduction in endotoxin levels, and an overall recovery of approximately 24%. Transmission electron microscopy observations were consistent with an increased proportion of intact virions during the polishing step and their separation from damaged particles. This workflow can be completed within hours using commonly available chromatography systems equipped for low-pressure operation. These results demonstrate the feasibility of applying a minimally adapted CIM-based workflow to Podoviridae phages and identify key parameters requiring further optimisation.