Protein variant separations by cation-exchange chromatography on tentacle-type polymeric stationary phases

• Published in: Journal of Chromatography A 1998-12, Vol.828 (1), p.365-372
• Main Authors: Weitzhandler, Michael, Farnan, Dell, Horvath, Judit, Rohrer, Jeffrey S., Slingsby, Rosanne W., Avdalovic, Nebojsa, Pohl, Chris
• Format: Article
• Language: English
• Subjects: Amides - chemistry; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cattle; Chromatography, Ion Exchange - methods; Cytochrome c Group - chemistry; Cytochrome c Group - isolation & purification; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Hemoglobins; Hemoglobins - chemistry; Hemoglobins - isolation & purification; Horses; Isoelectric Point; Monoclonal antibodies; Polymers; Proteins; Rabbits; Ribonuclease, Pancreatic - chemistry; Ribonuclease, Pancreatic - isolation & purification; Species Specificity
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(98)00521-4

We developed a set of prototype cation-exchange column packings that are based on a hydrophilic coated, pellicular polymeric support with a grafted tentacular surface chemistry that is 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 Asp 67 and isoAsp 67 forms. Humanized monoclonal antibody variants differing in the presence of lysine at the C terminus of the heavy chains were baseline-resolved. Finally, the separation of hemoglobin variants found in a sample containing elevated levels of glycated hemoglobin was also demonstrated.