title: Post-translational modification of a chimeric EPO-Fc hormone is more important than its molecular size in defining its in vivo hematopoietic activity.
abstract: BACKGROUND
  Recombinant erythropoietin (EPO) has been marketed as biopharmaceutical for anemia and chronic renal failure. Long-acting EPO variants that aimed at achieving less frequent dosing have been generated, either by the addition of glycosylation sites or increasing its molecular weight.
  
  
  METHODS
  The hEPO cDNA linked to the human IgG Fc fragment was cloned as a single codifying gene on the pAdtrack-CMV vector, yielding the recombinant adenoviral genome. For in vitro and in vivo expression assays cervical cancer cell line (SiHa) and nulliparous goats were used, respectively. The hematopoietic activity of EPO-Fc, expressed as the differential increment of hematocrit was evaluated in B6D2F1 mice. NP-HPLC of the 2AB-labeled N-glycan was carried out to profile analysis.
  
  
  RESULTS
  The direct transduction of mammary secretory cells with adenoviral vector is a robust methodology to obtain high levels of EPO of up to 3.5mg/mL in goat's milk. SiHa-derived EPO-Fc showed significant improvement in hematopoietic activity compared to the commercial hEPO counterpart or with the homologous milk-derived EPO-Fc. The role of the molecular weight seemed to be important in enhancing the hematopoietic activity of SiHa-derived EPO-Fc. However, the lack of sialylated multi-antennary glycosylation profile in milk-derived EPO-Fc resulted in lower biological activity.
  
  
  CONCLUSIONS
  The low content of tri- or tetra-antennary sialylated N-glycans linked to the chimeric EPO-Fc hormone, expressed in the goat mammary gland epithelial cells, defined its in vivo hematopoietic activity.
  
  
  CONCLUSIONS
  The sialylated N-glycan content plays a more significant role in the in vivo biological activity of hEPO than its increased molecular weight.
authors.affiliation: Biotechnology and Biopharmaceuticals Laboratory, Department of Physiopathology, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, P.O. Box 160C, Concepción, Chile.
authors.forename: Emilio R
authors.initials: ER
authors.lastname: Salgado
authors.affiliation: Biotechnology and Biopharmaceuticals Laboratory, Department of Physiopathology, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, P.O. Box 160C, Concepción, Chile.
authors.forename: Raquel
authors.initials: R
authors.lastname: Montesino
authors.affiliation: Biotechnology and Biopharmaceuticals Laboratory, Department of Physiopathology, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, P.O. Box 160C, Concepción, Chile.
authors.forename: Sivana P
authors.initials: SP
authors.lastname: Jiménez
authors.affiliation: Biotechnology and Biopharmaceuticals Laboratory, Department of Physiopathology, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, P.O. Box 160C, Concepción, Chile.
authors.forename: Mauricio
authors.initials: M
authors.lastname: González
authors.affiliation: Clinical Sciences Department, School of Veterinary Sciences, Universidad de Concepción, Avenida Vicente Méndez 595, Chillan, Chile.
authors.forename: Florence
authors.initials: F
authors.lastname: Hugues
authors.affiliation: Clinical Sciences Department, School of Veterinary Sciences, Universidad de Concepción, Avenida Vicente Méndez 595, Chillan, Chile.
authors.forename: Oscar I
authors.initials: OI
authors.lastname: Cabezas
authors.affiliation: Biotechnology and Biopharmaceuticals Laboratory, Department of Physiopathology, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, P.O. Box 160C, Concepción, Chile.
authors.forename: Rafael
authors.initials: R
authors.lastname: Maura-Perez
authors.affiliation: Biotechnology and Biopharmaceuticals Laboratory, Department of Physiopathology, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, P.O. Box 160C, Concepción, Chile.
authors.forename: Paulina
authors.initials: P
authors.lastname: Saavedra
authors.affiliation: Biotechnology and Biopharmaceuticals Laboratory, Department of Physiopathology, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, P.O. Box 160C, Concepción, Chile.
authors.forename: Emilio
authors.initials: E
authors.lastname: Lamazares
authors.affiliation: Department of Pharmacology, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, P.O. Box 160C, Concepción, Chile.
authors.forename: Alexis
authors.initials: A
authors.lastname: Salas-Burgos
authors.affiliation: Biotechnology and Biopharmaceuticals Laboratory, Department of Physiopathology, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, P.O. Box 160C, Concepción, Chile.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Vera
authors.affiliation: Department of Pharmacology, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, P.O. Box 160C, Concepción, Chile.
authors.forename: Oliberto
authors.initials: O
authors.lastname: Sánchez
authors.affiliation: Biotechnology and Biopharmaceuticals Laboratory, Department of Physiopathology, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, P.O. Box 160C, Concepción, Chile. Electronic address: jotoledo@udec.cl.
authors.forename: Jorge R
authors.initials: JR
authors.lastname: Toledo
mesh_terms: D000818:Animals; D004305:Dose-Response Relationship, Drug; D004921:Erythropoietin / Q000494:pharmacology*; D057809:HEK293 Cells; D006410:Hematopoiesis / Q000187:drug effects*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000494:pharmacology*; D051379:Mice; D011134:Polysaccharides / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*
chemical_list: D007141:Immunoglobulin Fc Fragments; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin
keywords: Adenovirus vector; Erythropoietin; Hematopoietic activity; Mammary epithelial cell; Milk expression; N-glycans