Refined purification of adeno-associated virus vector-engineered strain

Abstract:

[Background] In gene targeted therapy and gene engineering drug development with adeno-associated virus (AAV) as the delivery vector, the challenges of the large-scale preparation of highly purified recombinant AAV (rAAV) should be solved. [Objective] The goal of this study was to obtain highly purified rAAV particles by purification of plasmids and rAAV cell culture medium. [Methods] HiPrep^TM10 Sepharose 6FF and HiTrap PlasmidSelect Xtra gel chromatography columns were used to purify three rAAV plasmids. The study sought to obtain super helical DNA plasmids and optimize the culture of AAV-293 cells in the assembly process. After successful assembly, the virus extract was purified using HiLoad^TM10Q and HiLoad^TM10SP cation exchange columns. HT1080 cells were infected with purified and concentrated rAAV. Flow cytometry was used to enumerate the fluorescent cells to detect the number of live virus and calculate the titer of live virus after concentration. [Results] After HiPrep and HiTrap chromatography columns purification, many copies of highly purified super helical plasmid DNA were obtained. After virus assembly, many highly purified rAAV particles were obtained by cation exchange chromatography. The genome titer of rAAV (TCID₅₀/mL) reached (2.46±0.37)×10¹²(multiplicity of infection of 1.0×10⁴). [Conclusion] A high titer of pure rAAV was prepared by a series of assembly refinements and purification.