Protein Adsorption and Transport in Novel Chromatographic Anion Exchange and Multimodal Resins

Understanding the mechanism of protein adsorption and transport and the effects of the stationary phase architecture are critical for the optimum design of chromatographic processes. This work studies the properties of novel stationary phases that are based on a rigid macroporous backbone matrix synthesized with hydrophilic polymers (UNOsphere), which can be functionalized with a variety of ligands. Cationic ligands introduced with a range of grafted polymeric surface extenders as well as multimodal anionic ligands are considered. Both macroscopic and microscopic studies of particle properties, as well as orthogonal measurements of protein adsorption isotherms and kinetics, are used to understand these materials.
Characterizations of anion exchangers with no polymer grafts (UNO Q), moderate graft content (Nuvia HR Q), and high graft content (Nuvia Q) show the pore volume accessible by neutral macromolecules decreases greatly with the grafts content. Higher binding capacities are found for BSA and PEGylated BSA (with 10 kD and 30 kD PEG chain) in polymer grafted adsorbents. The adsorption kinetics of these proteins are also faster in Nuvia HR Q and Nuvia Q, apparently as a result of a solid diffusion mechanism. However, this enhancement in adsorption kinetics depends on the relative size of protein and grafted polymer as well as ionic strength. The adsorption of thyroglobulin on Nuvia HR Q is found to be highly hindered at low ionic strength, but faster kinetics are observed at higher ionic strength.
Two resins with multimodal ligands are also studied: Nuvia cPrime, based on the UNOsphere matrix, and Capto MMC, based on an agarose matrix. The two resins have similar multimodal ligand but the pore structures are distinctly different. Equilibrium binding capacities for lysozyme and (mAb) are similar for both resins at comparable pH and salt concentration, although Capto MMC shows a weaker dependence on salt concentration as a result of its more hydrophobic character. The main difference is the binding kinetics of the mAb. The rate of mass transfer is much smaller in Capto MMC at pH values of 5 or 6, as a result of the smaller pores and much smaller column dynamic binding capacities are obtained for this resin.