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Objective <p>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.</p> Results <p>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 (&gt; 95%) and impurity removal (&gt; 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%.</p> Conclusions <p>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.</p>

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Application of aqueous two-phase extraction for separation and purification of various adeno-associated viruses

  • 期刊论文

  • 发表日期：

    2025年1月8日

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  • Xiao-Qian Fu,
  • Hui-Yi Leong,
  • Liang-Zhi Qiao,
  • Jia-Nan Zhou,
  • Wei Hu,
  • Shan-Jing Yao,
  • Dong-Qiang Lin

摘要

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.