A case study showing the role of hydrophobicity variants and other enriched mAb proteoforms on filterability through...

A rapid assessment of manufacturability for drug candidates is crucial for advancing a prospective biotherapeutic from a candidate to a bulk drug substance. A lot-to-lot approach to manufacturability is adopted where each biologic batch is assessed for manufacturability as a bulk, unfractionated pool. Manufacturers may explore a more granular approach, independently enriching and evaluating the filterability of antibody variants within each lot, especially within the confines of relative hydrophobicity and surface charge. This study examined the use of bind-and-elute chromatography to alter the proportions of monoclonal antibody (mAb) proteoforms in eluate sub-pools from a mixed-mode chromatography resin-packed column. Filterability of each sub-pool through a virus-retaining filter was subsequently examined. Circular dichroism and Fourier transform infrared spectroscopy were performed for each sub-pool to probe for higher-order structure differences between mAb variants enriched therein. Bioanalytical techniques were also used to assess colloidal stability, surface hydrophobicity, surface charge, and size differences. Results showed that basic charge variants, high-mannose glycovariants, high relative hydrophobicity proteoforms, and high-molecular-weight species were enriched in the last-eluting (terminal) sub-pools. The first sub-pool and the final sub-pool showed the most fouling propensity on VPro virus filters. Circular dichroism showed that enriched proteoforms in the last sub-pool possessed a higher percentage of bends. Most secondary structures did not vary significantly between sub-pools. Diffusion interaction parameter was highly negative across all sub-pools and the bulk unfractionated pool. These results provide a design space for identifying and depleting problematic mAb variants before the crucial virus filtration step.