Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography

Abstract Background Adenoviral vectors are used extensively in human gene therapy trials and in vaccine development. Large‐scale GMP production requires a downstream purification process, and liquid chromatography is emerging as the most powerful mode of purification, enabling the production of vectors at a clinically relevant scale and quality. The present study describes the development of a two‐step high‐performance liquid chromatography (HPLC) process combining anion exchange (AIEX) and gel filtration (GF) in comparison with the caesium chloride density gradient method. Methods HEK‐293 cells were cultured in ten‐layer CellStacks ™ and infected with 10 pfu/cell of adenoviral vector expressing green fluorescent protein (Ad5‐GFP). Cell‐bound virus was harvested and benzonase added to digest DNA, crude lysate was clarified by centrifugation and filtration prior to HPLC. Chromatography fractions were added to HEK‐293 cells and GFP expression measured using a fluorescent plate reader. Results Using AIEX then GF resulted in an adenoviral vector with purity comparable to Ad5‐GFP purified by CsCl, whereas the reverse process (GF–AIEX) showed a reduced purity by electrophoresis and required further buffer exchange of the product. The optimal process (AIEX–GF) resulted in a vector yield of 2.3 × 10 7 pfu/cm 2 of cell culture harvested compared to 3.3 × 10 7 pfu/cm 2 for CsCl. The process recovery for the HPLC process was 36% compared to 27.5% for CsCl and total virion to infectious particle ratios of 18 and 11, respectively, were measured. Conclusions We present a simple two‐step chromatography process that is capable of producing high‐quality adenovirus at a titre suitable for scale‐up and clinical translation. Copyright © 2009 John Wiley & Sons, Ltd.