Towards continuous mAb purification: Clearance of host cell proteins from CHO cell culture harvests via "flow-through affinity chromatography" using peptide-based adsorbents

Sobhana Alekhya Sripada; Wenning Chu; Taufika Islam Williams; Matthew A Teten; Brian J Mosley; Ruben G Carbonell; Abraham M Lenhoff; Steven M Cramer; Jerome Bill; Yinges Yigzaw; David J Roush; Stefano Menegatti

doi:10.1002/bit.28096

Towards continuous mAb purification: Clearance of host cell proteins from CHO cell culture harvests via "flow-through affinity chromatography" using peptide-based adsorbents

Biotechnol Bioeng. 2022 Jul;119(7):1873-1889. doi: 10.1002/bit.28096. Epub 2022 Apr 22.

Authors

Sobhana Alekhya Sripada¹, Wenning Chu¹, Taufika Islam Williams^{2

3}, Matthew A Teten⁴, Brian J Mosley⁴, Ruben G Carbonell^{1

4}, Abraham M Lenhoff⁵, Steven M Cramer⁶, Jerome Bill⁷, Yinges Yigzaw⁷, David J Roush⁸, Stefano Menegatti^{1

4}

Affiliations

¹ Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, USA.
² Molecular Education, Technology, and Research Innovation Center (METRIC), North Carolina State University, Raleigh, North Carolina, USA.
³ Department of Chemistry, North Carolina State University, Raleigh, North Carolina, USA.
⁴ Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Raleigh, North Carolina, USA.
⁵ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, USA.
⁶ The Howard P. Isermann Department of Chemical and Biological Engineering and the Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, USA.
⁷ Genentech, South San Francisco, California, USA.
⁸ Merck & Co., Kenilworth, New Jersey, USA.

PMID: 35377460
DOI: 10.1002/bit.28096

Abstract

The growth of advanced analytics in manufacturing monoclonal antibodies (mAbs) has highlighted the challenges associated with the clearance of host cell proteins (HCPs). Of special concern is the removal of "persistent" HCPs, including immunogenic and mAb-degrading proteins, that co-elute from the Protein A resin and can escape the polishing steps. Responding to this challenge, we introduced an ensemble of peptide ligands that target the HCPs in Chinese hamster ovary (CHO) cell culture fluids and enable mAb purification via flow-through affinity chromatography. This study describes their integration into LigaGuard™, an affinity adsorbent featuring an equilibrium binding capacity of ~30 mg of HCPs per mL of resin as well as dynamic capacities up to 16 and 22 mg/ml at 1- and 2-min residence times, respectively. When evaluated against cell culture harvests with different mAb and HCP titers and properties, LigaGuard™ afforded high HCP clearance, with logarithmic removal values (LRVs) up to 1.5, and mAb yield above 90%. Proteomic analysis of the effluents confirmed the removal of high-risk HCPs, including cathepsins, histones, glutathione-S transferase, and lipoprotein lipases. Finally, combining LigaGuard™ for HCP removal with affinity adsorbents for product capture afforded a global mAb yield of 85%, and HCP and DNA LRVs > 4.

Keywords: CHO; flow-through chromatography; host cell proteins; monoclonal antibodies; peptide-based adsorbents.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Antibodies, Monoclonal* / chemistry
CHO Cells
Cell Culture Techniques
Chromatography, Affinity / methods
Cricetinae
Cricetulus
Peptides / chemistry
Proteomics* / methods

Substances

Antibodies, Monoclonal
Peptides