Resolution of Protein Charge Variants in Mixed-Bed Chromatography Columns with Step-Induced pH Gradients at High Protein Loadings

Abstract

A mixed-bed column comprising of weak and strong base resins is used for the chromatographic separation of the charge variants of a monoclonal antibody with step-induced pH gradients at protein loadings up to 21.5 mg/mL. A model taking into account competitive binding is developed to predict separation for these conditions. In agreement with the experiments, the model predicts increasingly skewed elution peaks and lower resolution as the protein load increases. However, even at loadings as high as 50% of the column protein binding capacity, high-purity fractions could be collected along the induced pH gradient demonstrating the practical effectiveness of the mixed-bed system.