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Recombinant adenoviruses continue to be a leading vector choice for gene transfer applications, with growing interest in the use of less prevalent serotypes, and of chimeras. As a result, the development of scaleable purification processes for alternative serotypes is needed. Anion-exchange chromatography is routinely used for scaleable adenovirus type 5 purification; however, retention varies for other serotypes because of differences in the exposed capsid proteins. Understanding how the viral surface influences retention behavior can provide a rational basis for chromatography development and optimization. In this work, chimeric vectors were used to show that the hexon protein is responsible for retention differences in anion-exchange chromatography. Next, the relative retention of eight serotypes from three subgroups was studied. Although all serotypes bound to the anion-exchange resin, the sodium chloride required to elute the virus varied over a 2- fold range, from 270 to 490 mM. Retention was accurately correlated to the electrostatic properties of the hexon protein, with an average error in sodium chloride concentration required to elute of only 14 mM. This correlation enables preparative chromatography gradients for alternative serotypes to be established with minimal effort.