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Related Experiment Video

Updated: Sep 19, 2025

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production
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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
PubMed
Summary
This summary is machine-generated.

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.

Keywords:
AAVadeno‐associated virusanion exchange chromatographycontinuous purificationfull/empty separationmulticolumn countercurrent solvent gradient purification (MCSGP)

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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.