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

We developed a set of cation exchange column packings (ProPac (TM) WCX-10 a cid ProPac (TM) SCX-10) that are based on a hydrophilic coated, pellicular polymeric support grafted with polymer chains bearing ion exchange function alities. The supports are highly suited to resolving closely related protei n variants. These column packings (1) afford minimal band spreading in conj unction with extremely high selectivity, (2) exhibit a very hydrophilic cha racter, and (3) have moderate loading capacity. Cytochrome C variants (bovi ne, horse, rabbit) were baseline-separated, as was native ribonuclease A an d its two deamidation products, the Asp(67) and isoAsp(67) 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 lev els of glycated hemoglobin was also demonstrated.