Ion-exchange displacement chromatography of proteins Dendritic polymers as novel displacers

Abstract

While the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of biopharmaceuticals, a major obstacle to the implementation of displacement chromatography has been the lack of appropriate displacer compounds. All protein displacement separations reported to date have employed relatively high-molecular-mass (> 2000) polyelectrolyte displacers. In this paper, results are presented on the discovery that low-molecular-mass dendritic polymers can be successfully employed as efficient displacers for protein purification in ion-exchange systems. Pentaerythritol-based dendritic polyelectrolytes ranging in molecular mass from 480 to 5100 were investigated as potential displacers for the purification of proteins in cation-exchange systems. The adsorption properties of these dendrimers were investigated using the steric mass action (SMA) model of non-linear ion-exchange chromatography. An analysis of the resulting SMA parameters using a dynamic affinity plot indicated that these dendrimers should have sufficient affinity to act as protein displacers. Displacement separations of protein mixtures in cation-exchange systems were carried out using zero-, first- and second-generation dendrimers. These experiments demonstrate that this new class of dendritic polyelectrolytes can indeed act as efficient protein displacers. The ability of a low-molecular-mass compound such as the “zero”-generation dendrimer ( M r 480) to displace proteins is very significant in that it represents a major departure from the conventional wisdom that large polyelectrolyte polymers are required to displace proteins in ion-exchange systems. In addition to the fundamental interest generated by low-molecular-mass displacers, it is likely that these displacers will have significant operational advantages as compared to large polyelectrolyte displacers.