An Analysis of the Interactions of BSA with an Anion-Exchange Surface Under Linear and Non-Linear Conditions

Abstract

The interactions of BSA with an anion-exchange adsorbent have been studied to aid in the understanding of protein adsorption in ion-exchange chromatography. Linear chromatography, flow microcalorimetry and isotherm measurements were used to analyze adsorption energetics in the linear and overloaded regions of the equilibrium isotherm. The effects of salt type, salt and protein concentration, and temperature are reported. It was observed that under all conditions studied the adsorption process was entropically driven. This was contrary to expectations, since at the pH selected ion exchange is expected to dominate. A major driving force for the adsorption of BSA on the anion exchanger was concluded to be the increase in entropy from the release of water due to interactions between hydrophobic regions on the protein and adsorbent. The data further suggest that the conformational entropy change accompanying protein adsorption on the ion exchanger may also be significant.

Methods for characterization of biochromatography media

“…A microcolumn with ∼100–200 μL of resin can be integrated in a flow calorimeter. This system has been used for direct measurement of the heat of adsorption enabling thermodynamic analysis of protein adsorption on various resins .…”

Mobile‐Phase Modulators as Salt Tolerance Enhancers in Phenylboronate Chromatography: Thermodynamic Evaluation of the Mechanisms Underlying the Adsorption of Monoclonal Antibodies

“…Since at pH 8.5 and 9.0, the tetrahedral boronate is the predominant conformation of the ligand, other nonspecific interactions such as electrostatic interactions, are also possible to occur. The increased intensities of heat signals, as the higher contribution of entropic events to the adsorption process, could be as well related to the release of water molecules and ions from the surfaces of the adsorbent and protein, necessary for the establishment of the electrostatic interactions . Nevertheless, it is interesting to observe that the deconvoluted peaks, especially the first exothermic peak, do not differ in what concerns heat signal between pH values of 8.5 and 9.0 (Figure and Table ), suggesting that no major differences exist in the adsorption process within these conditions.…”

“…The profile changes are probably related to the increased presence of the boronate anion in the ligand structure (mainly due to the shift in the acid–base equilibrium caused by the increase in the pH), which is able to promote electrostatic interactions with the anti‐IL8 mAb (Figure A). For this type of interactions to occur, water and ions have to be released from the surface of the adsorbent and protein, which leads to a consumption of energy and to an increase of entropy . At pH 9.0, the ligand will be predominantly present in its tetrahedral forms — the ones obtained by hydroxylation of the boronic acid or by B–F coordination (Figure A,B, respectively).…”

Chromatographic determination of impurity binding affinities on biomineral crystals

“…Coupled with the trend of increasing retention with increasing temperature, it appears that adsorption on calcite (and perhaps COM as well) occurs via an ion-exchange mechanism. 26,27…”