Work

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

Year: 2026

Type: article

Abstract: Background/Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.

Source: Pharmaceutics

DOI: 10.3390/pharmaceutics18020263

Authors: Timotej Žvanut, Mitja Martelanc, Aleš Štrancar, Andreja Gramc Livk

Institutions: University of Nova Gorica, BIA Separations (Slovenia)

Language: English

Topic: Protein purification and stability

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,499

Funders: Univerza v Ljubljani, Univerza v Mariboru