Selective displacement chromatography of proteins

Abstract

In contrast to high molecular weight polyelectrolyte displacers, the efficacy of low molecular weight displacers are dependent on both mobile phase salt and displacer concentration. This sensitivity to the operating conditions opens up the possibility of carrying out selective displacement where the product(s) of interest can be selectively displaced while the low affinity impurities can be desorbed in the induced salt gradient ahead of the displacement train, and the high affinity impurities either retained or desorbed in the displacer zone. This type of displacement combines the operational advantages of step gradient and the high resolution inherent in a true displacement process, in a single operation. Theoretical expressions are presented for establishing selective displacement operating conditions (initial salt concentration, displacer concentration) based on the Steric Mass Action parameters of the displacer and the linear Steric Mass Action parameters of the feed proteins. Experimental results are presented to elucidate the concept of selective displacement in both cation and anion exchange systems. A mixture of alpha-lactalbumin and beta-lactoglobulin A and B has been used for anion-exchange systems; a four-protein mixture consisting of ribonuclease B, bovine and horse heart cytochrome c, and lysozyme has been employed in cation exchange systems. This article also demonstrates that on-line monitoring can be readily employed for the selective displacement process, thus facilitating the scale-up and control of the process. This work sets the stage for the development of robust large scale high resolution separations using selective displacement chromatography. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 119-129, 1997.