Abstract: A purification method was established for effectively increasing the proportion between vector capsids of rAAV5-a viral product containing the therapeutic DNA sequence and empty capsids, viral vectors lacking the therapeutic gene. For the rAAV5 samples obtained from affinity chromatography, several anion exchange chromatography and related gradient elution methods were tested, in which CIMmultus QA (CIMQ) can effectively remove empty capsids from vector capsids. For the CIMQ purification, the buffer matrix and the collection of rAAV5 fractions were optimized. Subsequently, qPCR was used to quantify the recovery rate of the virus genome (Vg) content, and high-performance liquid chromatography (HPLC) or size exclusion chromatography-multi-angle static light scattering (SEC-MALS) was used to determine the vector capsids proportion of rAAV5 samples. In the CIMQ purification process, the proportion of vector capsids in the collected fraction (at UV absorbance UV260/UV280 greater than 1) eluted with Tris-Na2HPO4buffer at pH 8.0 reached 80.3%. A simple and convenient method using anion exchange chromatography was successfully established, which can effectively eliminate empty capsids of rAAV5 from vector capsids so that vector capsids proportion can meet the requirements of clinical application of gene therapy.

Key words: rAAV5, anion exchange chromatography, vector capsids proportion, recovery, purification