Mechanistic Insights into AAV Capsid–Stationary Phase Interactions Governing Native Stability and Chromatographic Separation Using AAV8 as a Model System

<b>Background/Objectives:</b> Adeno-associated viruses (AAVs) are widely used gene therapy vectors; yet their physicochemical stability and chromatographic behavior are highly sensitive to the solution conditions they are in. Effective separation of full (F), empty (E), and partially filled (P) capsids-most commonly achieved by anion exchange (AEX) chromatography-is essential for standard analytical characterization, process development, and product safety. However, conventional AEX methods rely on low-conductivity alkaline mobile phases with low salt, which promote capsid binding and therefore higher resolution, at the expense of structural stability. Conversely, formulations such as near-neutral buffers might preserve capsid integrity but often impair AEX retention and separation resolution. <b>Methods:</b> Here, we extend a mechanistic investigation using AAV8 capsids as a model system, focusing on detailed capsid interactions with strong AEX, and present novel AAV8 separation strategies on a weak AEX stationary phase. <b>Results:</b> By systematically varying buffer pH and ionic strength, we identify operational regimes that balance capsid stability with chromatographic separation efficiency. In parallel, we introduce an integrated two-dimensional (2D) in-line buffer exchange configuration that decouples AEX performance from sample formulation, enabling robust separation of stability-optimized, high-salt matrices without off-line desalting. <b>Conclusions:</b> By elucidating the roles of capsid charge modulation, ligand physicochemical properties, and local microenvironmental buffering, this study establishes practical design principles for stability-preserving chromatography. It lays a foundation for more reliable analytical and future preparative AAV workflows.