title: HPCE monitoring of the N-glycosylation pattern and sialylation of murine erythropoietin produced by CHO cells in batch processes.
abstract: Using capillary electrophoresis coupled to laser-induced fluorescence (HPCE-LIF), it was possible to profile N-linked oligosaccharides from EPO, including species containing sialic acid, during the course of batch cultures performed either in serum-free or serum-containing medium. Although an unusual high heterogeneity of the N-linked oligosaccharides was observed by both SDS-PAGE and HPCE analysis, the patterns of mEPO glycans after desialylation by mild acid hydrolysis were found to be quite constant over the course of the cultures either with or without serum supplementation. In contrast, when the protein was analyzed by HPCE without acidic desialylation, fingerprints of N-linked oligosaccharides changed with time in serum-containing conditions. This phenomenon appeared to be mainly due to the desialylation of mEPO as a result of a sialidase activity released upon cell lysis. These results demonstrate that though a higher EPO titer was obtained in serum supplemented conditions, sialylation of EPO was severely affected by the presence of serum in the culture medium.
authors.affiliation: Laboratoire des Sciences du Génie Chimique-CNRS, Institut National Polytechnique de Lorraine, ENSAIA, 2, avenue de la Forêt-de-Haye, BP 172, F-54505 Vandoeuvre-lès-Nancy, France.
authors.forename: François
authors.initials: F
authors.lastname: Le Floch
authors.forename: Bertrand
authors.initials: B
authors.lastname: Tessier
authors.forename: Sébastien
authors.initials: S
authors.lastname: Chenuet
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Guillaume
authors.forename: Pierre
authors.initials: P
authors.lastname: Cans
authors.forename: Annie
authors.initials: A
authors.lastname: Marc
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Goergen
mesh_terms: D000818:Animals; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods*; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D019075:Electrophoresis, Capillary / Q000379:methods*; D004921:Erythropoietin / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D051379:Mice; D019158:N-Acetylneuraminic Acid / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D004921:Erythropoietin; D019158:N-Acetylneuraminic Acid