Protein variant separations using cation exchange chromatography on grafted, polymeric stationary phases

Abstract

We developed a set of cation exchange column packings (ProPac WCX-10 and ProPac SCX-10) that are based on a hydrophilic coated, pellicular polymeric support grafted with polymer chains bearing ion exchange functionalities. The supports are highly suited to resolving closely related protein variants. These column packings (1) afford minimal band spreading in conjunction with extremely high selectivity, (2) exhibit a very hydrophilic character, and (3) have moderate loading capacity. Cytochrome C variants (bovine, horse, rabbit) were baseline-separated, as was native ribonuclease A and its two deamidation products, the Asp67 and isoAsp67 forms. Humanized monoclonal antibody variants differing in the number of lysine residues at the C terminus of their heavy chains were baseline-resolved. Finally, the separation of hemoglobin variants found in a sample containing elevated levels of glycated hemoglobin was also demonstrated.