1. Application of aqueous two-phase extraction for separation and purification of various adeno-associated viruses.
- Author
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Fu, Xiao-Qian, Leong, Hui-Yi, Qiao, Liang-Zhi, Zhou, Jia-Nan, Hu, Wei, Yao, Shan-Jing, and Lin, Dong-Qiang
- Abstract
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. [ABSTRACT FROM AUTHOR]
- Published
- 2025
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