Baculovirus purification with functionalized non-woven fibers: Impact of residence time, binding strength, and ligand chemistry

• Non-woven fibers bind up to 4 × 10 15 total particles/m 2 . • Mass transfer limitations for particles in convective media with grafted ligands. • Binding to quaternary amine ligand led to 13% recovery of infectious baculovirus • The highest recovery of infectious baculovirus was achieved on a cation exchanger. The purification of enveloped viruses, such as baculovirus, remains a significant challenge in bioprocessing. This study explores various factors affecting recombinant baculovirus purification using functionalized non-woven fibers in bind-elute mode, including contact time during adsorption, buffer pH and conductivity and ligand density and chemistry. Non-woven fibers with grafted quaternary ammonium groups demonstrated flowrate-dependent binding behavior, with mass transfer limitation observed for larger particles. Reducing the residence time decreased the binding capacity particles due to the mass transfer limitations. Adjusting the pH and conductivity of the equilibration, wash and elution buffers changed the elution profile; however infectious virus recovery could not be improved. Alternatively, reduction of the binding strength was attempted by reducing the ligand density. Surprisingly, fibers with reduced quaternary ammonium ligand density showed lower overall recovery of dsDNA, protein, total and infectious particles, suggesting unspecific binding interactions with the fiber backbone. Finally, a cation exchange fiber with carboxylic acid ligands was used and improved the total infectious virus recovery to 30%, with a yield of 26% in the main product fraction, doubling the results achieved with anion exchange fibers. This suggests that weaker binding interactions help maintain baculovirus infectivity. These findings highlight the potential of non-woven fiber materials for the purification of enveloped viruses like baculovirus and provide strategies to further enhance recovery and preserve viral infectivity.