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Affinity Chromatography

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Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

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Published on: April 2, 2012

Selectivity for full AAV capsids in affinity capture with camelid ligands.

Lukas Bongers¹, Linda E Franken², Dominik Hoch¹

¹Gene Therapy Technical Research & Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.

Journal of Chromatography. A

|December 27, 2025

Summary

A new affinity capture method enhances the purification of adeno-associated viral vectors (AAV). This approach significantly enriches full AAV capsids, improving the efficiency of downstream processing for gene therapy applications.

Keywords:

Adeno-associated virus Affinity chromatography Camelid affinity ligands Full capsid selectivity Gene therapy

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Area of Science:

Biotechnology
Bioprocessing
Gene Therapy

Background:

Adeno-associated viral vectors (AAV) are critical for gene therapy.
Current downstream processing relies on affinity capture and anion exchange (AEX) chromatography.
Separating full and empty AAV capsids remains a significant challenge due to similar biophysical properties.

Purpose of the Study:

To develop a novel affinity capture method for enriching full AAV capsids.
To investigate the impact of various conditions and affinity ligands on full capsid enrichment.
To improve the efficiency and purity of AAV purification processes.

Main Methods:

Screening of additives (salts, temperature) and affinity ligands (AAVX, AAV8, AAV9, CaptoAVB, AVIPure AAV8).
Testing the method on AAV8 WT, AAV9 WT, and rAAV2 derivatives.
Analyzing elution fractions to determine full capsid enrichment and purity.

Main Results:

Demonstrated a 2.5-fold enrichment of full capsids using a robotic screening approach.
Identified specific affinity ligands, particularly AAVX, that offer high resolution for full capsid selectivity.
Achieved fractions approaching 100% full capsids by analyzing elution peaks in fine increments.

Conclusions:

The novel affinity capture method effectively enriches full AAV capsids.
This approach improves the full-to-total ratio (FTR) in the final product without additional unit operations.
The findings offer a trade-off between yield and purity, optimizing AAV purification for gene therapy manufacturing.