Mechanistic Insights into AAV Capsid-Stationary Phase Interactions Governing Native Stability and Chromatographic...

Area of Science:

• Biophysical Chemistry
• Biotechnology
• Analytical Chemistry

Background:

• Adeno-associated viruses (AAVs) are key gene therapy vectors, but their stability and separation depend heavily on solution conditions.
• Effective separation of full, empty, and partially filled AAV capsids is vital for characterization, process development, and product safety.
• Conventional anion exchange (AEX) chromatography uses conditions that compromise AAV capsid stability for better separation.

Purpose of the Study:

• To investigate AAV capsid interactions with strong and weak AEX stationary phases.
• To develop novel AAV separation strategies that balance capsid stability with chromatographic resolution.
• To establish practical design principles for stability-preserving AAV chromatography.

Main Methods:

• Mechanistic investigation of AAV8 capsid interactions with strong AEX.
• Development of novel AAV8 separation strategies using a weak AEX stationary phase.
• Systematic variation of buffer pH and ionic strength.
• Implementation of a two-dimensional (2D) in-line buffer exchange configuration.

Main Results:

• Identification of operational regimes balancing AAV capsid stability and chromatographic separation efficiency.
• Successful separation of AAV capsids in stability-optimized, high-salt matrices using the 2D buffer exchange system.
• Demonstration of robust separation without the need for off-line desalting.

Conclusions:

• Elucidation of factors influencing AAV capsid charge, ligand properties, and microenvironmental buffering in chromatography.
• Establishment of practical design principles for stability-preserving chromatography of AAVs.
• Foundation laid for more reliable analytical and preparative AAV workflows.