Fu Xiao-Qian, Leong Hui-Yi, Qiao Liang-Zhi, Zhou Jia-Nan, Hu Wei, Yao Shan-Jing, Lin Dong-Qiang
Key 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.
Hangzhou Jiayin Biotech Ltd., Hangzhou, 310018, China.
Biotechnol Lett. 2025 Jan 8;47(1):16. doi: 10.1007/s10529-024-03555-3.
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.
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%.
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.
腺相关病毒（AAV）因其安全性、稳定性和长期表达特性而被广泛用作基因治疗载体。本研究的目的是开发一种双水相系统（ATPS）作为分离和纯化AAV的通用平台。
本研究利用聚乙二醇（PEG）/盐双水相系统分离和纯化了多种AAV血清型，包括AAV5、AAV8和AAV9，重点关注血清型特异性性能和部分空衣壳的去除。结果表明，所有AAV血清型主要富集在双水相系统的相间，回收率高（>95%），杂质去除率高（>95%）。PEG/柠檬酸钠双水相系统与血清型无关，但为了获得最佳性能，需要对每种血清型的组分浓度进行工艺优化。值得注意的是，一步双水相萃取也显示出能够从粗细胞裂解物中去除一定量的空衣壳，去除率在4%至25%之间。
结果证明了PEG/柠檬酸钠双水相系统在分离和纯化不同AAV血清型方面的实际适用性，解决了基因治疗载体生产中的关键挑战。
