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

Marc N. Eglon, Aoide M. Duffy, Timothy O'Brien, Padraig Strappe

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37 Citations (Scopus)

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 CellStacksTM 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 x 107 pfu/cm2 of cell culture harvested compared to 3.3 x 107 pfu/cm2 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.
Original languageEnglish
Pages (from-to)978-989
Number of pages12
JournalJournal of Gene Medicine
Volume11
Issue number11
DOIs
Publication statusPublished - 2009

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