Mixed-Beds of Strong and Weak Anion Exchange Resins for Protein Separations with Step-Induced pH Gradients

Abstract

A new way of performing pH gradient-based protein separations using a mixed-bed column comprising a small-pore weak base resin and a large-pore strong base resin and a step change in pH at the column entrance is introduced. The approach effectively decouples intracolumn generation of pH gradients, which ensues from ionic interactions with the weak base resin, from protein separation, which ensues from interactions with the strong base resin ligands, resulting in a process that is more easily predictable, less prone to the effects of protein loading on the pH gradient, less susceptible to hydrophobic interactions between protein and resin surface, and more flexible. The approach is demonstrated successfully using a tertiary amine resin as the weak base component together with a range of commercial strong anion exchangers with quaternary amine functionality. A predictive model, based on the potentiometric titration of the weak base resin, is in excellent agreement with experimentally determined pH profiles obtained using mixed-bed columns and amine buffers with pH steps from 9 to 7. The separation of protein charge variants with the mixed-bed concept is demonstrated using these columns for a deamidated monoclonal antibody mixture, which yielded highly homogeneous fractions.