Understanding Process Performance Parameters in a Two-Step Monoclonal Antibody Chromatographic Purification Process

Chromatography is used extensively to purify monoclonal antibodies (mAbs) at process scale. This work examines key performance parameters that affect the separation of such process using a two-step purification approach including Protein A (PA) capture followed by weak partitioning chromatography (WPC) with anion-exchange (AEX) resins.
The first part of this work focuses on understanding the fouling mechanism of the Protein A MabSelect resin. A fouling mechanism was proposed based on both macroscopic and microscopic studies. The structural and performance characterization of the cycled resin was studied by chromatographic and batch adsorption/desorption experiments. Microscopic studies including transmission electron microscopy (TEM) and confocal scanning laser microscopy (CLSM) was used to determine the nature and spatial distribution of the foulants and to understand protein conformational change when bound to PA resin. The second part of this work focuses on understanding the relationship between AEX resin structure and protein properties in WPC. Chromatographic experiments were used to determine protein partition and diffusion and to obtain the pore size distribution of AEX resin under WPC conditions. Microscopic experiments were performed with both TEM and CLSM. TEM was used to understand the architecture and pore structure of the AEX resin and CLSM was used to observe mAb and impurity adsorption profile and to understand the mass transfer mechanism under WPC conditions.