title: Human hepatoma cell line HuH-7 is an effective cellular system to produce recombinant factor IX with improved post-translational modifications.
abstract: Recombinant factor IX (rFIX) is increasingly used to treat patients with hemophilia B. CHO (Chinese Hamster Ovary) cells are commonly used for the production of rFIX but they have a limited capacity for introducing post-translational modifications (PTM) leading to incomplete γ-carboxylation, low phosphorylation and sulfation profiles as compared with plasma-derived preparations. Imperfect PTM might have an impact on the activity of Factor IX molecule. Several studies in animal models as well as clinical trials have previously reported a lower recovery of rFIX compared to plasma-derived FIX concentrates. In the present study, we aimed to produce a rFIX having a profile of PTM similar to plasma-derived FIX, using human hepatoma cell line HuH-7. We showed that rFIX produced by HuH-7 cells followed the classical intracellular pathway before secretion. In addition, improved PTM were associated with fully active molecule compared to plasma-derived and recombinant control FIX molecules. Secreted rFIX presented as a single band at the correct molecular weight. HuH-7 cellular clones were obtained and they secreted a biologically active human FIX. FIX was then purified for a detailed evaluation of PTM. Glycosylation and sialylation profiles were similar to plasma-derived and rFIX and mass spectrometry analysis demonstrated the presence of phosphorylated and sulfated forms of rFIX. These data strongly support that HuH-7 cells may represent an effective cellular system for production of rFIX exhibiting PTM similar to plasma-derived FIX.
authors.affiliation: Unite Hemostase, Inflammation & Sepsis EA 4174, Universite Lyon1-Hospices Civils de Lyon, Faculte de Medecine Laennec, 69372 Lyon cedex 08, France. nathalie.enjolras@univ-lyon1.fr
authors.forename: Nathalie
authors.initials: N
authors.lastname: Enjolras
authors.forename: Yesim
authors.initials: Y
authors.lastname: Dargaud
authors.forename: Eloïse
authors.initials: E
authors.lastname: Pérot
authors.forename: Florine
authors.initials: F
authors.lastname: Guillaume
authors.forename: Michel
authors.initials: M
authors.lastname: Becchi
authors.forename: Claude
authors.initials: C
authors.lastname: Négrier
mesh_terms: D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D005164:Factor IX / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D005164:Factor IX