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

被引:34
作者
Eglon, Marc N. [1 ]
Duffy, Aoife M. [1 ]
O'Brien, Timothy [1 ]
Strappe, Padraig M. [2 ]
机构
[1] Natl Univ Ireland, REMEDI, Galway, Ireland
[2] Univ Sydney, Brain & Mind Res Inst, Sydney, NSW 2006, Australia
基金
爱尔兰科学基金会;
关键词
adenovirus; chromatography; gel filtration; HPLC; ion exchange; purification; EXTRACELLULAR NUCLEASE; PROTEIN STABILIZATION; SERRATIA-MARCESCENS; STRUCTURAL PROTEINS; HEXON;
D O I
10.1002/jgm.1383
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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 (TM) 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 10(7) pfu/cm(2) of cell culture harvested compared to 3.3 x 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 (C) 2009 John Wiley & Sons, Ltd.
引用
收藏
页码:978 / 989
页数:12
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