The AVV particles were generated by transient transfection of HEK293 cells with a triple plasmid. The VPC 2.0 cells were expanded for at least four passages and inoculated into a shake flask (2 L) containing Dynamis cell culture medium prior to transfection with triple-plasmid DNA. Transfection was completed using 10 % FectoVir transfection mixture according to manufactures recommendations. Plasmid DNA was used at 1.0 μg of DNA per 1×10^6 cells for all transfections. Cells were harvested 72 h post transfection and were lysed with Tween-20 buffer containing Benzonase. The crude lysate was centrifuged to remove cellular debris. The clarified lysate was stored frozen at −80 °C in 50 mL aliquots and thawed completely before ATPS experiments.

The adeno-associated virus (AAV) has been widely employed as a popular gene therapy vector. However, current methods for its separation and purification exhibit numerous limitations. In this study, polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS) was utilized as an efficient and environmentally-benign approach to separate AAV. The separation efficiency of AAV particles from other cellular components was investigated using various ATPS compositions. The effects of PEG molecular weight, types of phase-forming salts, and concentrations of different components were tested and compared. The optimal separation conditions were determined to be 17 % (w/w) PEG 600, 16 % (w/w) sodium citrate, and 15 % (w/w) crude cell lysate. The AAV particles were predominantly enriched in the interphase, while the impurities were observed in the top and bottom phases. High AAV recovery (>99 %) and efficient impurity removal (>95 %) were achieved. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transmission electron microscopy were used to characterize and verify the separated AAV particles. The results indicated that aqueous two-phase extraction possesses a robust capability for the enrichment and separation of AAV particles directly from crude cell lysates, and it shows promising potential for the separation of other viral particles.