Integrating the affinity and anion-exchange purification of AAV8: how the selective enrichment of full capsids at the affinity capture step enhances the downstream processing of gene therapies.

Adeno-associated virus (AAV) vectors are revolutionizing gene therapy with remarkable therapeutic potential. Yet, their clinical translation is limited by inadequate biomanufacturing technologies that compromise product quality and accessibility. Addressing these issues, this study systematically investigates the conditions used for midstream and downstream AAV processing - namely, HEK293 cell lysis, tangential flow filtration (TFF), and chromatographic purification - and their impact on product yield and quality using AAV8 as a model serotype. We evaluated a panel of detergents (Triton X-100, sodium deoxycholate, Deviron C16/S9-13, CTAB, AAV-MAX) and salts (KCl, NaCl), and found that, while the capsid yields were similar across the various lysates, the subsequent process steps exhibited a strong dependence on lysis conditions. In particular, residual detergents in the lysates affected the efficiency of the TFF step by reducing permeate flux and necessitating the use of membranes with higher molecular weight cut-offs or additional buffer exchange prior to affinity capture. Triton X-100 and sodium deoxycholate afforded efficient cell lysis and high vector yields, but compromised TFF throughput. Conversely, lysates produced using salts or CTAB enabled robust TFF, with high permeate flux, and maintained capsid recoveries above 75 %. The subsequent affinity capture of AAV8 from selected lysates using AvXcel resin afforded yields of 45 - 70 %, >100-fold reduction in host cell impurities, and a 1.9-fold enrichment of cell-transducing units. Product polishing by anion-exchange chromatography reached final titers of 5.0 - 7.7 × 1011 vg/mL, full capsid ratios up to 85 %, and transduction activities of 3.2 × 105 TU/mL, while reducing host cell protein and DNA levels to < 2 ng/mL and < 100 pg/mL, meeting current regulatory guidelines for gene therapy. These results delineate the interplay between midstream and downstream operation conditions, providing practical guidance for AAV purification workflows suitable for large-scale manufacturing.