Enrichment of full AAV2 using multicolumn countercurrent solvent gradient purification (MCSGP)

Adeno-associated viruses (AAVs) are non-pathogenic viruses that have become promising delivery vehicles in cell and gene therapy, with several AAV-based therapeutics receiving approval in recent years. However, a critical challenge is the separation of full and empty viral capsids, as empty capsids lack therapeutic DNA and may compromise product efficacy and safety. This study describes a proof-of-concept of a twin-column multicolumn countercurrent solvent gradient purification (MCSGP) process to enhance the ratio of full to empty capsids during AAV purification. Starting from a batch method with anion exchange chromatography, the process was adapted to continuous operation. The implementation of MCSGP, with six cycles, increased full capsid content in the final product pool from 30% to 68% (ddPCR) and 27% to 61% (cryo-TEM) respectively. Moreover, MCSGP was capable of overcoming the inherent yield-purity trade-off in the polishing step by enhancing full capsid recovery. Additionally, the MCSGP process improved productivity by up to 23% and reduced buffer consumption up to 27% compared to the batch method (ddPCR). The increased productivity and reduced buffer consumption offer both economic and environmental benefits. This study demonstrates the potential of MCSGP to meet the rising demand for high-quality AAV products in gene therapy.

Keywords

AAV, Adeno-associated virus, Anion exchange chromatography, Continuous purification, Full/empty separation, Multicolumn countercurrent solvent gradient purification (MCSGP)

Subject (DDC)

600 - Technik, Medizin, angewandte Wissenschaften

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Date of the last check

ISBN

ISSN

0006-3592
1097-0290

Language

English

Created during FHNW affiliation

Yes

Strategic action fields FHNW

Publication status

Published

Review

Peer review of the complete publication

Open access category

Closed

License

Citation

Müller, J., Müllerová, T., Tobler, D., Hauri, D., Plieninger, R., Higuchi, Y., Takahashi, R., Vogg, S., Müller‐Späth, T., & Villiger, T. (2025). Enrichment of full AAV2 using multicolumn countercurrent solvent gradient purification (MCSGP). Biotechnology & Bioengineering, 122(9), 2420–2432. https://doi.org/10.1002/bit.29036

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