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Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications
Published on: February 9, 2024
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Characterization of difficult-to-remove host cell proteins in adeno-associated virus downstream processing
Thomas M Leibiger1, Lie Min1, Kelvin H Lee1
1Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19713, USA.
Molecular Therapy. Methods & Clinical Development
|December 1, 2025
Summary
A new purification method for adeno-associated virus (AAV) was developed using chromatography. This scalable process effectively removes host cell proteins (HCPs), improving AAV purity for therapeutic applications.
Area of Science:
- Biotechnology
- Biopharmaceutical Manufacturing
- Viral Vector Purification
Background:
- Adeno-associated virus (AAV) is a promising vector for gene therapy.
- Efficient purification of AAV is critical for clinical applications.
- Existing purification methods face challenges in scalability and impurity removal.
Purpose of the Study:
- To develop a scalable, serotype-agnostic downstream purification platform for adeno-associated virus (AAV).
- To characterize residual host cell protein (HCP) clearance across different chromatographic steps.
- To understand the mechanisms of HCP retention during AAV purification.
Main Methods:
- Development of a three-step chromatographic platform: affinity chromatography (AC), anion-exchange chromatography (AEX), and multi-modal polishing chromatography (MMC).
- Quantitative proteomic profiling using sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) to identify and quantify HCPs.
- Analysis of four AAV serotypes (AAV2, -5, -8, and -9) produced in suspension HEK293 cells.
Main Results:
- The downstream process achieved cumulative vector genome (VG) yields between 44.6% and 69.2%.
- Full capsid enrichment ranged from 2.62- to 5.93-fold across all AAV samples.
- Significant reduction in HCPs, with 880, 99, and 21 HCPs remaining after AC, AEX, and MMC, respectively.
Conclusions:
- The developed chromatographic platform is effective for scalable, serotype-agnostic AAV purification.
- Vector-mediated mechanisms, such as capsid or genome association, contribute to the retention of difficult-to-remove HCPs.
- This study enhances the understanding of impurity clearance in AAV downstream processing, aiding future purification process design.
