The removal of host cell protein (HCP) impurities represents a major challenge to purification processes in biomanufacturing given their diverse composition, structure, abundance, and potential similarity to the target product. Currently, HCP clearance relies on chromatographic adsorbents designed for either direct product binding or capture of HCPs in flow-through mode with broad specificity (e.g., ion exchange or mixed-mode). While this approach has proven effective at attaining high purity biotherapeutics, product-bound HCPs and HCPs that affect product stability continue to pose significant challenges. We propose a new approach for removing HCPs from cell culture supernatants in flow-through mode using new-generation multimodal adsorbents developed by our group on Tosoh resins. Our proteomic study of the HCPs present in the cell culture fluid and chromatographic fractions indicated that, while many were found to bind to commercial resins, a group of 24 HCPs are not captured effectively by any. With the growing interest in flow-through approaches to HCP removal, we resolved to improve upon commercial media by developing new small synthetic peptides that specifically capture CHO HCPs. First, (1) HCP-binding candidates were identified by screening solid-phase peptide libraries to select leads that bind HCP, yet not IgG. HCP-binding candidates showed enrichment towards hydrophobic-positive and zwitterionic sequences. We (2) initially evaluated these peptides synthesized on Toyopearl resin by measuring their static binding of HCPs from a CHO culture harvest containing IgG. We observed equivalent or higher binding of HCPs, with minimal IgG binding, on our peptide-Toyopearl adsorbents as compared to commercial resins. We finally (3) selected ligands to be conjugated to chromatographic resins at optimal density, and be evaluated by dynamic HCP removal. The results of this work can transform HCP removal in flow-through mode for downstream processing by addressing “problematic” HCPs and making downstream more robust in clearing HCPs from different cell line sources.

Peptide-toyopearl adsorbents for removing CHO host cell proteins from cell culture supernatants

256th National Meeting (2018)

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