Sample Displacement Batch Chromatography of Proteins

Abstract

In downstream processing large scale chromatography plays an important role. For its development screening experiments followed by pilot plant chromatography are mandatory steps. Here we describe fast, simple, and inexpensive methods for establishing a preparative chromatography for the separation of complex protein mixtures, based on sample displacement batch chromatography. The methods are demonstrated by anion-exchange chromatography of a human plasma protein fraction (Cohn IV-4), including the screening step and scaling up of the chromatography by a factor of 100. The results of the screening experiments and the preparative chromatography are monitored by SDS-PAGE electrophoresis. In summary we provide a protocol which should be easily adaptable for the chromatographic large scale purification of other proteins, in the laboratory as well as in industry for commercial manufacturing. For the latter these protocols cover the initial piloting steps for establishing a sample batch chromatography based on packed columns rather than batch chromatography.

Application of sample displacement batch chromatography for fractionation of proteoforms

“…SDBC was performed following the procedure described by Kotasinska et al [24,30]. Prior to SDBC, several parameters such as resin, the type of ion-exchange chromatography, and pH were screened to obtain a set of parameters that yielded the largest number of identifiable ovalbumin proteoforms.…”

Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry

“…Within a band a high purity of the component is achieved [72]. DE has been shown to be suitable for separation of complex mixture of tryptic peptides [73][74][75] and proteins [76][77][78][79]. One of the characteristics of DE is that DE has a different selectivity compared with GE [77].…”

“…Thiemann et al published a similar approach termed proteinpurification parameter screening system (PPS), which was not focusing on the identification of appropriate displacers but more general on any kind of parameters for adsorption chromatography, independent of the elution mode [87]. The PPS was successfully applied for purification and identification of an angiotensin-II generating enzyme [88], and for screening for parameters for optimal displacement chromatography of proteins [78,79]. Rational screening was also used for developing a displacement chromatography of proteoforms of a recombinant protein with HIC [89].…”

Application of Displacement Chromatography to Online Two-Dimensional Liquid Chromatography Coupled to Tandem Mass Spectrometry Improves Peptide Separation Efficiency and Detectability for the Analysis of Complex Proteomes

“… 14 During sample loading, the analytes arrange themselves according to their affinity in a process described as sample self-displacement. 15 , 16 The analyte with the highest affinity toward the stationary phase is binding to the chromatographic material at the top of the column, displacing analytes with lower affinities from their binding sites. Compared to conventionally used gradient chromatography (GC), 50–100% of the column binding capacity is used for sample loading in DC.…”

“…The carrier solution must support a high binding affinity for the analytes toward the stationary phase . During sample loading, the analytes arrange themselves according to their affinity in a process described as sample self-displacement. , The analyte with the highest affinity toward the stationary phase is binding to the chromatographic material at the top of the column, displacing analytes with lower affinities from their binding sites. Compared to conventionally used gradient chromatography (GC), 50–100% of the column binding capacity is used for sample loading in DC. , The analytes are eluted in DC by loading a molecule onto the column called “displacer”, which is usually dissolved in the carrier solution.…”