Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography

Abstract Adeno-associated virus (AAV) empty capsids typically co-purify with genome containing AAV2 vectors purified by column chromatography. This study describes a method to remove empty capsids from genome containing vector particles by anion exchange chromatography. The separation is based on the slightly less anionic character of empty particles compared to vectors. Detailed methods to achieve AAV2 vector purification and particle separation using cation exchange resin POROS 50HS followed by anion exchange resin Q-Sepharose xl are described. Chromatographic separation of AAV2 particles was achieved using gradients based on sodium acetate and ammonium acetate, and was optimal at pH 8.5. Efficient removal of particle surface nucleic acid impurities was found to be important to achieve good particle separation. In a large scale experiment performed using partially purified vector containing a mixture of 1.56 × 10 14  vg and 2.52 × 10 15 empty capsids as a starting material, the optimized anion exchange chromatography method resulted in a vector peak of 1.15 × 10 14  vg containing 0.25 × 10 14 empty capsids, corresponding to 74% vector yield and 86-fold reduction in empty capsids in the vector product.