Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography.
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 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.
Eglon
Marc N
MN
Regenerative Medicine Institute (REMEDI), National University of Ireland, Galway, Ireland.
Duffy
Aoife M
AM
O'Brien
Timothy
T
Strappe
Padraig M
PM
D000256:Adenoviridae / Q000737:chemistry / Q000302:isolation & purification*; D000837:Anion Exchange Resins / Q000737:chemistry; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D005822:Genetic Vectors / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D014776:Virus Cultivation
D000837:Anion Exchange Resins