Abstract
The success of adeno-associated virus (AAV)-based therapeutics in gene therapy poses the need for rapid and efficient processes that can support the growing clinical demand. Nonwoven membranes represent an ideal tool for the future of virus purification: owing to their small fiber diameters and high porosity, they can operate at high flowrates while allowing full access to target viral particles without diffusional limitations. This study describes the development of nonwoven ion-exchange membrane adsorbents for the purification of AAV2 from an Sf9 cell lysate. A strong anion-exchange (AEX) membrane was developed by UV grafting glycidyl methacrylate on a polybutylene terephthalate nonwoven followed by functionalization with triethylamine (TEA), resulting in a quaternary amine ligand (AEX-TEA membrane). When operated in bind-and-elute mode at a pH higher than the pI of the capsids, this membrane exhibited a high AAV2 binding capacity (9.6 × 10(13) vp·mL(-1)) at the residence time of 1 min, and outperformed commercial cast membranes by isolating AAV2 from an Sf9 lysate with high productivity (2.4 × 10(13) capsids·mL(-1)·min(-1)) and logarithmic reduction value of host cell proteins (HCP LRV ~ 1.8). An iminodiacetic acid cation-exchange nonwoven (CEX-IDA membrane) was also prepared and utilized at a pH lower than the pI of capsids to purify AAV2 in a bind-and-elute mode, affording high capsid recovery and impurity removal by eluting with a salt gradient. To further increase purity, the CEX-IDA and AEX-TEA membranes were utilized in series to purify the AAV2 from the Sf9 cell lysate. This membrane-based chromatography process also achieved excellent DNA clearance and a recovery of infectivity higher that that reported using ion-exchange resin chromatography.