Premium
Manipulating adenoviral vector ion‐exchange chromatography: Hexon versus fiber
Author(s) -
Ruščić Jelena,
AmbriovićRistov Andreja,
Majhen Dragomira,
Kolundžija Sandra,
Barut Miloš,
Benihoud Karim,
Krajačić Mladen
Publication year - 2016
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201600829
Subject(s) - ion chromatography , capsid , adenoviridae , chemistry , chromatography , ion exchange , lysis , mastadenovirus , peptide , amino acid , hydrophilic interaction chromatography , fiber , viral vector , high performance liquid chromatography , biochemistry , ion , recombinant dna , gene , organic chemistry
The serotype specificity of adenovirus ion‐exchange chromatography has previously been studied using standard particle‐based columns, and the hexon protein has been reported to determine retention time. In this study, we have submitted Adenovirus type 5 recombinants to anion‐exchange chromatography using methacrylate monolithic supports. Our experiments with hexon‐modified adenoviral vectors show more precisely that the retention time is affected by the substitution of amino acids in hypervariable region 5, which lies within the hexon DE1 loop. By exploring the recombinants modified in the fiber protein, we have proven the previously predicted chromatographic potential of this surface constituent. Modifications that preserve the net charge of the hexon protein, or those that cause only a small charge difference in the fiber protein, in addition to shortening the fiber shaft, did not change the chromatographic behavior of the adenovirus particles. However, modifications that include the deletion of just two negatively charged amino acids in the hexon protein, or the introduction of a heterologous fiber protein, derived from another serotype, revealed recognizable changes in anion‐exchange chromatography. This could be useful in facilitating chromatography‐approach purification by creating targeted capsid modifications, thereby shifting adenovirus particles away from particular interfering substances present in the crude lysate.