Sample displacement chromatography as a method for purification of proteins and peptides from complex mixtures

Abstract

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed.

Comparison of displacement versus gradient mode for separation of a complex protein mixture by anion-exchange chromatography

“…Furthermore these instruments offer a broad dynamic range for most of the analytes [20]. For analyzing the success of chromatographic separations analysis of the fractions with SDS-PAGE is common [22]. However, qualitative and semi-quantitative analysis of target proteins by SDS-PAGE is possible only by western blots.…”

Polymethacrylate‐based monoliths as stationary phases for separation of biopolymers and immobilization of enzymes

“…Under overloading conditions, the molecules of the sample that have higher affinity to the binding sites of the chromatographic support compete with sample components of lower affinity. As a result of displacement, the components with lower affinity are concentrated, and replaced by components of higher affinity . If a complex biological mixture, like human plasma diluted with buffer with low ionic strength, is continuously loaded onto a system that contains three anion‐exchange columns that are successively ordered, the sample displacement effect occurs, and different plasma proteins, mostly the ones with lower abundance, are enriched.…”

“…Already in 1988, Hodge's and Mant's group described SDC as “a novel approach to preparative liquid chromatography which takes advantage of the differential relative hydrophobicities of components of a sample mixture, so that the column is optimally loaded with an aqueous solution of the sample mixture, the more hydrophobic components compete more successfully for these sites than more hydrophilic components, which are displaced and immediately eluted from the column” . If extended to every ligand such as ion‐exchange, affinity and corresponding, additional modes of interactions, it is still the best definition for SDC . Only two years later, the same group developed a multi column system for preparative purification of peptides that was optimized about ten years later .…”

“…ELISA plate with miniaturized monolithic discs mounted on the bottom for high‐throughput sample preparation by use of laboratory robots. Reprinted from with permission.…”

Sample Displacement Batch Chromatography of Proteins

“…The publication of Veeraragavan et al [ 6 ] is one of the earliest reports about the separation of proteins by SDC. A comprehensive review about SDC of proteins appeared recently [ 7 ]. This protocol focuses on the fi rst steps in the development of a downstream processing procedure, including small-scale scouting trials and a scaling from laboratory to pilot plant involving a scale-up factor of 100-fold.…”