Removal of host cell proteins from cell culture fluids by weak partitioning chromatography using peptide-based adsorbents

• Published in: Separation and purification technology 2021-02, Vol.257, p.117890, Article 117890
• Main Authors: Lavoie, R. Ashton, Chu, Wenning, Lavoie, Joseph H., Hetzler, Zachary, Williams, Taufika Islam, Carbonell, Ruben, Menegatti, Stefano
• Format: Article
• Language: English
• Subjects: Flow-through chromatography; Host cell proteins; Monoclonal antibodies; Peptide ligands; Polishing; Proteomics; Weak partitioning mode
• ISSN: 1383-5866; 1873-3794
• DOI: 10.1016/j.seppur.2020.117890

•Purification of monoclonal antibodies (mAb) by weak partitioning chromatography (WPC).•Peptide ligands 4MP and 6HP bind CHO host cell proteins (HCPs) in flow-through mode.•4MP+6HP-Toyopearl resin affords mAb yield >80% and purity ~90% upon optimal loading.•4MP+6HP-Toyopearl resin captures highly immunogenic and difficult-to-remove HCPs.•Potential of 4MP+6HP-Toyopearl resin as HCP scrubber in mAb purification processes. This work presents the removal of host cell proteins (HCPs) from a Chinese Hamster Ovary clarified cell culture fluid (CHO CCCF) containing a therapeutic monoclonal antibody (mAb) by weak partitioning chromatography (WPC). The chromatographic adsorbents were produced by functionalizing Toyopearl resin with HCP-binding tetrameric multipolar (4MP) or hexameric hydrophobic/cationic (6HP) peptides. The CCCF was loaded on columns packed with either 4MP-Toyopearl or 6HP-Toyopearl resin only, or a 4MP and 6HP resin mixture at different values of residence time (RT: 0.5, 1, 2, and 5 min). The temporal profiles of concentration of HCPs and mAb in the effluents confirmed the binding mechanism by WPC, where both HCPs and mAb are initially bound by the peptide ligands, but, as more CCCF is fed to the column, the incoming HCPs displace the bound mAbs. In particular, 4MP was shown to capture more selectively high molecular weight HCPs, while 6HP was more effective in binding low molecular weight HCPs. Under optimal loading conditions (~60–80 g of proteins per L of adsorbent; RT of 5 min), the 6HP+4MP-Toyopearl adsorbent provided mAb yield and purity of >80% and up to 90%, respectively. Conversely, the control resin Toyopearl SuperQ-650 M resulted in 70% yield and 75% purity under the same conditions. Proteomic analysis of the effluents demonstrated that 6HP+4MP-Toyopearl adsorbent removes HCPs known for their immunogenicity or IgG co-elution or degradation, demonstrating the potential of these peptide-based resins as HCP scrubbers in mAb purification processes.