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Updated: Jun 3, 2025
Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications
Published on: February 9, 2024
Application of aqueous two-phase extraction for separation and purification of various adeno-associated viruses
Xiao-Qian Fu1, Hui-Yi Leong1, Liang-Zhi Qiao1
1Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang Key Laboratory of Smart Biomaterials, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310058, China.
Objective:
Adeno-associated viruses (AAVs) are widely used as gene therapy vectors due to their safety, stability, and long-term expression characteristics. The objective of this work is to develop an aqueous two-phase system (ATPS) as a universal platform for the separation and purification of AAVs.
Results:
This study utilized polyethylene glycol (PEG)/salt ATPSs to separate and purify various AAV serotypes, including AAV5, AAV8, and AAV9, which focusing on serotype-specific performance and partial empty capsid removal. The results showed that all the AAV serotypes were mainly enriched in the interphase of ATPS, with achieving high recovery (> 95%) and impurity removal (> 95%). The PEG/sodium citrate ATPS was serotype-independent, but the process optimization of component concentrations for each serotype was necessary to attain the best performance. Notably, a single-step aqueous two-phase extraction also demonstrated the ability to remove some amount of empty capsids from the crude cell lysate, with removal rate ranging from 4 to 25%.
Conclusions:
The results demonstrated the practical applicability of PEG/sodium citrate ATPS in separating and purifying different AAV serotypes, which addressing key challenges in gene therapy vector production.
