Harnessing the power of electrophoresis and chromatography: Offline coupling of reverse phase liquid chromatography‐capillary zone electrophoresis‐tandem mass spectrometry for analysis of host cell proteins in monoclonal antibody producing CHO cell line

• Published in: Electrophoresis Vol. 42; no. 6; pp. 735 - 741
• Main Authors: Kumar, Ramesh, Shah, Rohan L., Ahmad, Shadab, Rathore, Anurag S.
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 01.03.2021
• Subjects: Animals; Antibodies, Monoclonal - metabolism; Biological activity; CHO Cells; Chromatography; Chromatography, Liquid; Chromatography, Reverse-Phase; Coupling (molecular); Cricetinae; Cricetulus; Electrophoresis; Electrophoresis, Capillary; Host cell proteins; Hydrophobicity; Liquid chromatography; Mass spectrometry; Monoclonal antibodies; Ovaries; Peptidases; Peptides; Proteins; Quality management; Secretome; Tandem Mass Spectrometry; Monoclonal antibodies; Host cell proteins; CE; Liquid chromatography; Mass spectrometry
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.202000252

Host cell proteins (HCPs) are widely regarded as a critical quality attribute for a biotherapeutic product. Bottom up MS is the present gold standard for HCP analysis but suffers from incomplete protein identification due to complex nature of the HCP mixture and limited separation efficiency of the preceding LC‐based systems. In this paper, we present for the first time an application involving use of LC‐CE‐MS/MS platform for analysis of HCPs. It has been demonstrated that the proposed platform has been able to successfully identify 397 HCPs from the supernatants of recombinant Chinese hamster ovary cells, twice and thrice the number of proteins identified by the state‐of‐the‐art LC‐MS/MS (189 HCPs) and CE‐MS/MS (128 HCPs) analyses, respectively. Of these, 225 HCPs were unique to the LC‐CE‐MS/MS approach and were not identified by either LC‐MS/MS or CE‐MS/MS. It is observed that the LC‐CE‐MS/MS platform combines the benefits of LC‐MS/MS and CE‐MS/MS techniques and identifies peptides in a wider range of size, pI, and hydrophobicity. Additionally, LC‐CE‐MS/MS also identified more HCPs associated with cellular components, molecular functions, biological processes, peptidases, and secretory proteins. The proposed approach would thus be a useful addition in HCP analysis and secretome studies of mAb‐producing Chinese hamster ovary cells.