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Enrichment of Full AAV2 Using Multicolumn Countercurrent Solvent Gradient Purification (MCSGP)
Julia M Müller1,2, Tereza Müllerová1, Daniela Tobler1
1Institute of Pharma Technology and Biotechnology, University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland.
Biotechnology and Bioengineering
|June 5, 2025
Summary
This study introduces a continuous multicolumn countercurrent solvent gradient purification (MCSGP) process to separate full and empty adeno-associated virus (AAV) capsids. The novel method significantly improves capsid purity and recovery, crucial for gene therapy applications.
Area of Science:
- Biotechnology
- Viral Vector Purification
- Gene Therapy
Background:
- Adeno-associated viruses (AAVs) are key delivery vehicles in gene therapy, but separating full and empty capsids is challenging.
- Empty capsids can reduce therapeutic efficacy and compromise safety in AAV-based products.
Purpose of the Study:
- To develop and demonstrate a continuous multicolumn countercurrent solvent gradient purification (MCSGP) process for enhanced AAV capsid separation.
- To improve the ratio of full to empty viral capsids in purified AAV products.
Main Methods:
- Adaptation of anion exchange chromatography from batch to continuous operation using a twin-column MCSGP system.
- Implementation of six cycles of MCSGP for AAV purification.
- Quantification of full capsid content using droplet digital PCR (ddPCR) and cryo-transmission electron microscopy (cryo-TEM).
Main Results:
- MCSGP increased full capsid content from 30% to 68% (ddPCR) and 27% to 61% (cryo-TEM).
- The process overcame the yield-purity trade-off, enhancing full capsid recovery.
- Productivity improved by up to 23%, and buffer consumption decreased by up to 27% compared to batch methods.
Conclusions:
- The twin-column MCSGP process is a viable proof-of-concept for high-purity AAV purification.
- MCSGP offers economic and environmental benefits through increased productivity and reduced buffer usage.
- This continuous purification method can support the growing demand for high-quality AAV products in gene therapy.
