Serotype‐Tailored Midstream and Downstream Operations to Advance the Manufacturing of Adeno‐Associated Viral Vectors

ABSTRACT Adeno‐associated viruses (AAVs) are the leading vectors for in vivo gene therapy, fueling a growing demand for scalable and robust biomanufacturing processes. Yet, the impact of cell lysis conditions on the performance of midstream and downstream unit operations across serotypes remains poorly characterized. This study presents a serotype‐resolved investigation of cell lysis and its cascading effects on AAV2, AAV5, AAV8, and AAV9 processing. A panel of lysis methods including inorganic agents, non‐ionic and ionic detergents, and mechanical disruption was evaluated using an analytical framework of at‐ and off‐line assays to quantify AAV yield, purity, and activity at each stage. Our results reveal pronounced serotype‐specific differences: cetrimonium bromide (CTAB) delivered the highest transduction output of any condition evaluated for AAV8 (2.25 × 10 9 TU/mL) at 55.0% capture step yield, while sodium deoxycholate achieved ~ 98% host cell protein (HCP) reduction for AAV9 (HCP log reduction value 1.60) and the highest functional enrichment for AAV5 (6.73‐fold) and AAV8 (5.80‐fold). AAV2 proved uniquely challenging, with CTAB enabling the highest tangential flow filtration (TFF) yield. Acid precipitation paired with high salt addition reduced aggregation and improved permeate flux for AAV5 and AAV9. TFF performance was governed by the detergents' physicochemical properties: micelle‐forming agents generated multicomponent complexes that fouled membranes in condition‐ and serotype‐specific patterns. Rheological characterization revealed that all clarified lysates converged to the viscosity of the media after filtration, supporting transferable TFF parameters. Mixed‐mode chromatography using a peptide‐functionalized resin amplified lysis‐imprinted differences, decoupling step yield from functional enrichment. Bayesian optimization of the chromatographic step improved purification outcomes: for AAV8 residual HCPs in the eluate decreased > 300‐fold, from 0.91 mg/mL to 3 μg/mL, while for AAV9 the yield rose to ~95% at residual HCP as low as 14 μg/mL, demonstrating that purity and recovery are co‐optimizable when lysis, midstream, and downstream conditions are rationally paired.