Performance of large virus removal filters during AAV processing: Influence of flux and process disruptions

As adeno-associated viral vectors (AAV) continue to advance through the clinical pipeline, effective downstream purification strategies must be developed to ensure bulk drug purity and safety. AAV are produced within mammalian cells, bringing forth risks associated with viral contamination. Although existing downstream operations provide some degree of viral inactivation and removal, regulatory agencies have recommended the incorporation of a dedicated virus removal filtration step to ensure robust viral clearance. Recently published studies have demonstrated that membrane filters with nominal pore sizes between 35 and 50 nm can provide effective AAV transmission while removing larger viruses, although these results were obtained over a limited range of conditions. This study represents the first investigation into the effects of filtrate flux and process disruptions on virus reduction filtration for AAV. Experiments were performed using purified AAV capsids and carboxylate-modified polymeric nanoparticles with a nominal diameter of 20 nm. Initial results confirmed that both systems exhibited nearly identical transient transmission profiles during virus filtration. Virus filtration performed at various filtrate fluxes (between 20 and 185 L/m²/h) revealed that moderately higher AAV yield may be obtained at lower fluxes. The data were analyzed using a modified internal polarization model, which was extended to account for the effects of process disruptions on transient particle transmission and recovery. Process disruptions were employed to increase AAV yield beyond 99% without compromising overall clearance of large viruses. At least a 4-log reduction in xenotropic murine leukemia virus (XMuLV) was observed under all conditions tested, even following multiple process pauses.