Evaluation of chemically selective displacer analogues for protein purification.

A library of molecular analogues to the selective displacer, N'1'-(4-methylquinolin-2-yl)ethane-1,2-diamine dinitrate, was employed to study the effects of changes in displacer chemistry on their efficacy for selective separations. High throughput screens were carried out using a robotic liquid handling system to examine the ability of these compounds to selectively displace proteins in batch adsorption systems. Experiments were conducted using the model protein pairs ribonuclease A/alpha-chymotrypsinogen A and cytochrome C/lysozyme on a strong cation exchanger. Selectivity pathway and DC-50 plots were constructed from the analogue screen data, and results indicated that minor changes in the molecular design of the displacer can have a significant impact on the separation behavior. Specifically, charge density and spacing of resin and protein interaction moieties were found to be important. The screen also identified a new displacer, 4-methyl-2-piperazin-1-yl-quinoline, which produced a more selective displacement than previously reported with the original compound. A steric mass action dynamic affinity plot was constructed to validate that this new displacer was acting as a chemically selective, rather than a steric mass action selective displacer. Finally, saturation transfer difference NMR experiments were conducted to examine protein-displacer interactions with these displacers and protein pairs. These results demonstrate how subtle changes in displacer design can be employed to fine-tune the separation performance of chemically selective displacers.