title: Human cell lines: A promising alternative for recombinant FIX production.
abstract: Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins.
authors.affiliation: Department of Clinical, Toxicological and Food Science Analysis, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil; Center for Cell-based Therapy and Regional Blood Center, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil. Electronic address: alinesb@fcfrp.usp.br.
authors.forename: Aline
authors.initials: A
authors.lastname: de Sousa Bomfim
authors.affiliation: Center for Cell-based Therapy and Regional Blood Center, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil; Department of Medical Clinic, Faculty of Medicine of Ribeirão Preto, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil. Electronic address: marcelafreitas@usp.br.
authors.forename: Marcela
authors.initials: M
authors.lastname: Cristina Corrêa de Freitas
authors.affiliation: Center for Cell-based Therapy and Regional Blood Center, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil. Electronic address: virginia.picanco@gmail.com.
authors.forename: Virgínia
authors.initials: V
authors.lastname: Picanço-Castro
authors.affiliation: Center for Cell-based Therapy and Regional Blood Center, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil. Electronic address: mario.neto@hemocentro.fmrp.usp.br.
authors.forename: Mário
authors.initials: M
authors.lastname: de Abreu Soares Neto
authors.affiliation: Center for Cell-based Therapy and Regional Blood Center, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil; Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil. Electronic address: kamilla@fcfrp.usp.br.
authors.forename: Kamilla
authors.initials: K
authors.lastname: Swiech
authors.affiliation: Center for Cell-based Therapy and Regional Blood Center, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil; Department of Medical Clinic, Faculty of Medicine of Ribeirão Preto, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil. Electronic address: dimas@fmrp.usp.br.
authors.forename: Dimas
authors.initials: D
authors.lastname: Tadeu Covas
authors.affiliation: Department of Clinical, Toxicological and Food Science Analysis, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil; Center for Cell-based Therapy and Regional Blood Center, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil. Electronic address: elisa@fcfrp.usp.br.
authors.forename: Elisa
authors.initials: E
authors.lastname: Maria de Sousa Russo
mesh_terms: D001709:Biotechnology; D005164:Factor IX / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D005164:Factor IX
keywords: Coagulation factor IX; Gamma-carboxylation; Hemophilia B; Human cell lines; Lentiviral vector