Selectivity of monolithic supports under overloading conditions and their use for separation of human plasma and isolation of low abundance proteins

Abstract

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These two proteins frequently interfere with detection, determination and purification of low abundance proteins that can be potential biomarkers and biomarker candidates for various diseases. Some low abundance plasma proteins such as clotting factors and inhibitors are also important therapeutic agents. In this paper, the characterization of ion-exchange monolithic supports under overloading conditions was performed by use of sample displacement chromatography (SDC). If these supports were used for separation of human plasma, the composition of bound and eluted proteins in both anion- and cation-exchange mode is dependent on column loading. Under overloading conditions, the weakly bound proteins such as HSA in anion-exchange and IgG in cation-exchange mode are displaced by stronger binding proteins, and this phenomenon was not dependent on column size. Consequently, small monolithic columns with a column volume of 100 and 200 μL are ideal supports for high-throughput screening in order to develop new methods for separation of complex mixtures, and for sample preparation in proteomic technology.

Sample displacement chromatography of plasmid DNA isoforms

“…Due to convective mass transfer which leads to diffusionindependent chromatographic properties, the use of convective chromatographic supports, such as monoliths, is advantageous for performing DC and SDC purification of biomacromolecules over particulate chromatographic supports [10,13]. For successful SDC on particulate chromatographic supports, flow rates must be five times lower to achieve optimal sample displacement compared to standard gradient elution separation [14].…”

“…SDC approach should optimally be performed under overloading conditions, since the main separation occurs already during sample loading and column capacity is thus optimally used [8]. SDC was introduced for preparative purification of peptides in reverse-phase mode [9] and has subsequently been successfully applied for purification of proteins in ion-exchange [10], affinity [11] and HIC [12] modes, using traditional porous particle chromatographic media, http operating under diffusion mass transfer conditions. SDC approach is most useful for samples where the product binds to a chromatographic support more strongly than the impurities, because this enables fast and efficient purification of complex samples [8,11].…”

Therapeutic plasma proteins – application of proteomics in process optimization, validation, and analysis of the final product

“…Both target proteins and harmful impurities were identified, and chromatographic conditions were optimized in order to achieve their separation. Additionally, if monolithic disks are used, a simple scaling up of the separation process is possible [34].…”

“…Recently, we used columns packed with 0.2 mL of anion-or cationexchange or affinity resin for high-throughput separation of plasma proteins. In parallel experiments, we also used monolithic supports (CIM s disks) carrying the same ligands [33,34]. Both columns and monolithic discs were installed into a 96-well microplate, and fast and reproducible separation could be achieved.…”

Medicinal Chemistry Meets Proteomics: Fractionation of the Human Plasma Proteome

“…They applied monolithic ion exchange columns under overloading conditions with the consideration that HSA, IgG and other weakly bound plasma proteins were displaced by stronger binding ones in anion and cation exchange modes [74].…”