Characterization of Adeno-Associated Virus Vectors Using Ion-Exchange Chromatography and Light Scattering

Begin with a sample that contains empty and nucleic acid-filled adeno-associated virus capsids.

Load the sample onto an anion-exchange chromatography column packed with positively charged resin and connected to multi-angle light scattering (MALS).

The negatively charged capsids bind to the resin through electrostatic interactions.

The nucleic acid adds a stronger negative charge, causing the nucleic acid-filled capsids to bind more strongly to the resin than empty capsids.

Apply a salt gradient to elute the capsids. Lower salt concentrations disrupt weak interactions and release the empty capsids.

Higher salt concentrations release nucleic acid-filled capsids.

As the fractions elute, direct them through a laser beam for MALS analysis.

Early-eluting fractions containing empty capsids scatter less light due to their lower molar mass.

Later-eluting nucleic acid-filled capsids scatter more light, reflecting their higher molar mass.

This charge-based separation, combined with MALS-derived molar mass measurements, allows a quantitative distinction between empty and nucleic acid-filled capsids.