Protein Adsorption and Transport Kinetics in Novel Stationary Phases for Protein Chromatography Based on the UNOsphere Architecture

This work examines protein adsorption equilibrium and transport kinetics in a new class of stationary phases for protein chromatography based on a hydrophilic macroporous polymer bead, known as UNOsphere (Bio-Rad Laboratories, Hercules, CA), that possesses improved protein adsorption capacity, kinetics, and mechanical strength. Three different materials are considered: UNOsphere SUPrA, a protein A matrix, UNOsphere S, a cation exchanger based on a short ligand chemistry, and Nuvia S, which is based on essentially the same backbone matrix but contains grafted charged polymeric surface extenders.
The results for UNOsphere SUPrA show rapid IgG adsorption kinetics, which results primarily from the large-pore size and small particle size afforded by the relative rigidity of the UNOsphere backbone. A comprehensive model taking into account IgG binding on a distribution of protein A ligands with different accessibility was developed to describe IgG adsorption in batch and column systems. Coupled with a newly developed model describing the pressure-flow relationship in large diameter columns, the binding kinetics model allows rational design of process-scale units that maximize productivity while meeting specified pressure constrains.
The results for Nuvia S show that the backbone pores are essentially completely filled with a gel-phase formed by the grafted charged polymeric surface extenders. This phase is inaccessible by neutral macromolecules but provides readily accessible binding sites for adsorption of both small (e.g. lysozyme) and large (e.g. IgG) positively charged giving very high binding capacities. The adsorption kinetics of these proteins is also fast in Nuvia S, apparently as a result of a solid diffusion mechanism. Compared to UNOsphere S, which has large open pores, Nuvia S exhibits more than twice the protein binding capacity and much faster adsorption rates. For Nuvia S, however, the adsorption kinetics is a strong function of the protein type and charge, and of the nature of the counterion indicating that binding strength, which is strongly affected by these characteristics, is correlated with transport rates. A model was developed to describe protein adsorption kinetics in Nuvia S for single and multicomponent systems based on batch adsorption and confocal microscopy measurements of intraparticle protein concentration profiles. The model, based on a Maxwell-Stefan description of diffusion fluxes, predicts, in agreement with the experimental data, rapid kinetics for the adsorption of a single protein on a clean particle and for the simultaneous adsorption of two or more proteins. Very slow rates are predicted, however, also in agreement with experimental data, when two proteins counterdiffuse within the particle as a result of the apparent inability of adsorbed protein molecules to pass each other in the spatially-constrained network defined by the grafted charged polymers.