Going with the flow: peptide affinity membranes for high-throughput, high-yield purification of adenovirus and lentiviral vectors.

Membrane chromatography offers a compelling alternative to packed-bed resins for viral vector purification, as its open porosity and convective mass transport eliminate diffusive mass transfer limitations, enabling processing at substantially shorter residence times. Yet, the translation of affinity ligands from resins to membranes is challenged by the low density of reactive groups, which constrains functional ligand loading and limits binding capacity. Seeking to overcome this limitation, this study presents a method for functionalizing cellulose membranes with peptide affinity ligands and demonstrates its application to the purification of adenovirus serotype 5 (AdV5), VSV-G and Cocal pseudotyped lentiviral vectors (LVVs), and adeno-associated virus serotype 9 (AAV9). The membranes were initially grafted with hyperbranched polyethyleneimine to amplify reactive group density, and subsequently functionalized with peptide ligands via thiol- and amine-mediated conjugation. For AdV5, the AEFFIWNAC-cellulose membranes achieved a dynamic binding capacity (DBC10%) of 1.6 × 1011 vg/mL at the residence time of 20 sec, viral genome recovery of 89%, and a host cell protein log reduction value (HCP LRV) of 2.21 - exceeding the cognate peptide-functionalized resin in both capacity and productivity. For VSV-G pseudotyped LVV, the GKEAAFAAC-cellulose membrane delivered a DBC10% of 1.5 × 1011 vp/mL (RT: 20 sec), 87% total particle recovery, 45% transducing particle recovery, and an HCP LRV of 2.18, completing purification in ∼30 min vs. ∼2 hours needed with resins. Purification of Cocal pseudotyped LVV afforded 91% particle recovery, 67% functional recovery, and an HCP LRV of 2.25, confirming the broad applicability of the affinity membrane across LVV pseudotypes. Extension to AAV9 demonstrated feasible ligand immobilization and measurable binding, although the binding capacity was lower than that of the corresponding resin. These results establish hyperbranched polymer-amplified peptide affinity membranes as a high-productivity medium for the purification of large viral vectors.