title: Multi-omic profiling -of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production.
abstract: Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO-K1 cells under growth-limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO-producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)(+) , adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT-PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)(+) and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post-translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time-course analysis of high- and low-producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity.
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark.|Cell Culture Technology, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark.|The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ley
authors.affiliation: Cell Culture Technology, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark.
authors.forename: Ali Kazemi
authors.initials: AK
authors.lastname: Seresht
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark.|Cell Culture Technology, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark.
authors.forename: Mikael
authors.initials: M
authors.lastname: Engmark
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: Olivera
authors.initials: O
authors.lastname: Magdenoska
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: Kristian Fog
authors.initials: KF
authors.lastname: Nielsen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
authors.forename: Helene Faustrup
authors.initials: HF
authors.lastname: Kildegaard
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark. mr@bio.dtu.dk.
authors.forename: Mikael Rørdam
authors.initials: MR
authors.lastname: Andersen
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology*; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D055432:Metabolomics; D020543:Proteome / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D020543:Proteome; D011994:Recombinant Proteins; D004921:Erythropoietin
keywords: Chinese hamster ovary; chemostat; erythropoietin; metabolic adaptation; metabolomics; transcriptomics