The removal of host cell proteins (HCPs) poses a significant challenge in biomanufacturing, given their diversity in composition, structure, and abundance, and their potential structural homology with target products. Traditional methods for HCP clearance rely on affinity or ion exchange-based product capture steps in bind-and-elute mode and adsorption of HCPs in flow-through polishing steps. This approach has been extensively utilized in the manufacturing of monoclonal antibodies (mAb) for decades, relying on Protein A, and ion exchange or mixed-mode chromatography for product capture and polishing, respectively. Recent studies, however, have highlighted the presence of “problematic HCP” species, which can degrade the mAb product or produce toxic metabolites, or are immunogenic, and, in some cases, co-elute with the mAb product from Protein A resins and are not efficiently removed by commercial polishing media.<br/><br/>To improve HCP clearance, we have identified an ensemble of synthetic peptides as novel mixed-mode ligands for HCP removal from CHO clarified cell culture fluid in mAb polishing steps, and may enable integrated mAb purification processes relying on a sequence of flow-through HCP removal steps. This was done by high throughput screening of solid phase combinatorial libraries peptides for ligands that bind to HCPs in an industrial CHO harvest but do not bind to the mAb product. Proteomic analysis was performed to identify HCPs that bind to the different ligands. A novel method for screening was implemented that relies on the capabilities of a ClonePix device to identify resin particles that bind HCPs and do not bind mAb. The resulting HCP-binding peptides were conjugated on chromatographic resins and evaluated for HCP clearance and mAb yield under different conditions of pH and salt concentration. To gather detailed knowledge on the %-removal of individual HCPs, the flow-through fractions were subjected to proteomic analysis. By global HCP removal, we observed equivalent or higher binding of HCPs compared to commercial polishing media, with minimal co-adsorption of IgG. Notably, we obseverd effective capture of “problematic HCPs”, including those that are not effectively captured by commercial media. Collectively, these results indicate that our mixed-mode peptides show great promise towards developing next-generation media for safer and cost effective mAb manufacturing.

Mixed-mode peptide ligands for improved clearance of CHO host cell proteins

2019 Spring National Meeting

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