title: Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics.
abstract: Chinese hamster ovary (CHO) cells are the preferred host cell line for manufacturing a variety of complex biotherapeutic drugs including monoclonal antibodies. We performed a proteomics and bioinformatics analysis on the spent medium from adherent CHO cells. Supernatant from CHO-K1 culture was collected and subjected to in-solution digestion followed by LC/LC-MS/MS analysis, which allowed the identification of 3281 different host cell proteins (HCPs). To functionally categorize them, we applied multiple bioinformatics tools to the proteins identified in our study including SignalP, TargetP, SecretomeP, TMHMM, WoLF PSORT, and Phobius. This analysis provided information on the presence of signal peptides, transmembrane domains, and cellular localization and showed that both secreted and intracellular proteins were constituents of the supernatant. Identified proteins were shown to be localized to the secretory pathway including ones playing roles in cell growth, proliferation, and folding as well as those involved in protein degradation and removal. After combining proteins predicted to be secreted or having a signal peptide, we identified 1015 proteins, which we termed as CHO supernatant-ome (CHO-SO), or superome. As a part of this effort, we created a publically accessible web-based tool called GO-CHO to functionally categorize proteins found in CHO-SO and identify enriched molecular functions, biological processes, and cellular components. We also used a tool to evaluate the immunogenicity potential of high-abundance HCPs. Among enriched functions were catalytic activity and structural constituents of the cytoskeleton. Various transport related biological processes, such as vesicle mediated transport, were found to be highly enriched. Extracellular space and vesicular exosome associated proteins were found to be the most enriched cellular components. The superome also contained proteins secreted from both classical and nonclassical secretory pathways. The work and database described in our study will enable the CHO community to rapidly identify high-abundance HCPs in their cultures and therefore help assess process and purification methods used in the production of biologic drugs.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University , Baltimore, Maryland 21218, United States.|Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases , National Institute of Health, Building 14A, Bethesda, Maryland 20892, United States.
authors.forename: Amit
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune LLC , 1 MedImmune Way, Gaithersburg, Maryland 20878, United States.
authors.forename: Deniz
authors.initials: D
authors.lastname: Baycin-Hizal
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University , Baltimore, Maryland 21218, United States.
authors.forename: Daniel
authors.initials: D
authors.lastname: Wolozny
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK-2970 Hørsholm, Denmark.
authors.forename: Lasse Ebdrup
authors.initials: LE
authors.lastname: Pedersen
authors.affiliation: Department of Biology, Brigham Young University , Provo, Utah 84602, United States.|Department of Pediatrics, University of California , San Diego, California 92093, United States.
authors.forename: Nathan E
authors.initials: NE
authors.lastname: Lewis
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University , Baltimore, Maryland 21218, United States.
authors.forename: Kelley
authors.initials: K
authors.lastname: Heffner
authors.affiliation: Institute of Basic Biomedical Sciences, Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine , 733 North Broadway Street, Baltimore, Maryland 21205, United States.
authors.forename: Raghothama
authors.initials: R
authors.lastname: Chaerkady
authors.affiliation: Institute of Basic Biomedical Sciences, Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine , 733 North Broadway Street, Baltimore, Maryland 21205, United States.
authors.forename: Robert N
authors.initials: RN
authors.lastname: Cole
authors.affiliation: Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases , National Institute of Health, Building 14A, Bethesda, Maryland 20892, United States.
authors.forename: Joseph
authors.initials: J
authors.lastname: Shiloach
authors.affiliation: Department of Pathology, Johns Hopkins School of Medicine , 400 North Broadway Street, Baltimore, Maryland 21287, United States.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune LLC , 1 MedImmune Way, Gaithersburg, Maryland 20878, United States.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Bowen
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University , Baltimore, Maryland 21218, United States.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Betenbaugh
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics; D001692:Biological Transport; D016466:CHO Cells / Q000166:cytology / Q000378:metabolism*; D049109:Cell Proliferation; D002853:Chromatography, Liquid; D019295:Computational Biology / Q000379:methods*; D003412:Cricetulus; D017077:Culture Media, Conditioned / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D003599:Cytoskeleton / Q000737:chemistry; D015870:Gene Expression; D058977:Molecular Sequence Annotation; D025941:Protein Interaction Mapping; D021382:Protein Sorting Signals / Q000235:genetics; D059748:Proteolysis; D020543:Proteome / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D055571:Secretory Pathway / Q000235:genetics; D053719:Tandem Mass Spectrometry
chemical_list: D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D021382:Protein Sorting Signals; D020543:Proteome; D011994:Recombinant Proteins
keywords: CHO; host cell proteins; immunogenicity; ontology; proteomics; secretome; signal peptides