Streamlining the polishing step development process via physicochemical characterization of monoclonal antibody...

When developing purification processes for monoclonal antibodies (mAbs), ensuring the effective removal of high molecular weight (HMW) species is often challenging and labor intensive. In this work, we present a bottom-up characterization approach to achieve streamlined polishing step development as well as a more fundamental understanding of the protein of interest. Prior to physicochemical characterization, in-process HMW species of two IgG4 mAbs (mAb A and mAb B) were isolated via semi-preparative size exclusion chromatography (SEC). Key differences in approximate molecular weight, net charge, and native surface hydrophobicity were then identified using multi-angle light scattering (SEC-MALS), analytical-scale chromatographic screening, isoelectric focusing, and structural aggregation propensity modeling. SEC-MALS revealed two main HMW isoforms for each mAb: dimers and 1.7-mers for mAb A, and tetramers and dimers for mAb B. Analytical-scale chromatographic screening showed promising trends in charge-based separation for mAb A, and hydrophobic-based separation for mAb B. Isoelectric focusing data detected a 30% increase in acidic variants for mAb A HMW species relative to monomer, and a 20% increase in basic variants for mAb B HMW species. Lastly, analytical-scale characterization data was successfully applied to preparative scale purification conditions, producing results highly similar to those observed during analytical characterization of the isolated species. By using this high-throughput approach as a template for preparative-scale process development, key physicochemical differences between aggregate and monomer species were utilized to determine optimal polishing steps for HMW removal.