Chemically Selective Displacers for High-Resolution Protein Separations in Ion-Exchange Systems: Effect of Displacer−Protein Interactions

Abstract

Displacer lead compounds were selected from a commercially available database to identify potential selective displacers for a binary protein mixture in ion exchange chromatography. Parallel batch screening experiments were carried out with these lead compounds to study the effect of displacer concentration on the relative amounts of the proteins displaced. Experiments were conducted with a mixture containing ribonuclease A and alpha-chymo-trypsinogen A which exhibited very similar retention behavior under linear gradient conditions. The batch displacement results indicated that most of these lead compounds were indeed selective for displacing ribonuclease A. In fact, one of these displacers exhibited extremely high selectivity, displacing essentially all of the ribonuclease A while displacing minimal alpha-chymotrypsinogen A at a displacer concentration of 10 mM. These results were validated under column conditions, with the ribonuclease A being displaced and the alpha-chymotrypsinogen A remaining on the column after the displacer breakthrough. In order to examine whether this was mass action or chemically selective displacement, an affinity ranking plot based on the Steric Mass Action (SMA) model was generated, and the results confirmed that this was not a mass action displacement. In order to test the hypothesis that displacer protein binding was playing a role in these separations, Surface Plasmon Resonance (SPR) was carried out. The results suggest that while the chemically selective displacer interacted with alpha-chymo-trypsinogen A, it had no interaction with ribonuclease A. The ability to exploit protein displacer binding in concert with appropriate displacer resin affinities opens up new possibilities for creating selective displacement systems.