title: Metabolic shifts do not influence the glycosylation patterns of a recombinant fusion protein expressed in BHK cells.
abstract: BHK-21 cells expressing a human IgG-IL2 fusion protein, with potential application in tumor-targeted therapy, were grown under different nutrient conditions in a continuous system for a time period of 80 days. At very low-glucose (< 0.5 mM) or glutamine (< 0. 2 mM) concentrations, a shift toward an energetically more efficient metabolism was observed. Cell-specific productivity was maintained under metabolically shifted growth conditions and at the same time an almost identical intracellular ATP content, obtained by in vivo (31)P NMR experiments, was observed. No significant differences in the oligosaccharide structures were detected from the IgG-IL2 fusion protein preparations obtained by growing cells under the different metabolic states. By using oligosaccharide mapping and MALDI/TOF-MS, only neutral diantennary oligosaccharides with or without core alpha1-6-linked fucose were detected that carried no, one or two beta1-4-linked galactose. Although the O-linked oligosaccharide structures that are present in the IL2 moiety of the protein were studied with less detail, the data obtained from the hydrazinolysis procedure point to the presence of the classical NeuAcalpha2-3Galbeta1-3GalNAc structure. Here, it is shown that under different defined cellular metabolic states, the quality of a recombinant product in terms of O- and N-linked oligosaccharides is stable, even after a prolonged cultivation period. Moreover, unaffected intracellular ATP levels under the different metabolic states were observed.
authors.affiliation: IBET/ITQB- Instituto de Biologia Experimental e Tecnológica / Instituto de Tecnologia Química e Biológica, Apartado 12, P-2781-901 Oeiras, Portugal.
authors.forename: H J
authors.initials: HJ
authors.lastname: Cruz
authors.forename: C M
authors.initials: CM
authors.lastname: Peixoto
authors.forename: M
authors.initials: M
authors.lastname: Nimtz
authors.forename: P M
authors.initials: PM
authors.lastname: Alves
authors.forename: E M
authors.initials: EM
authors.lastname: Dias
authors.forename: J L
authors.initials: JL
authors.lastname: Moreira
authors.forename: M J
authors.initials: MJ
authors.lastname: Carrondo
mesh_terms: D000818:Animals; D001709:Biotechnology; D002240:Carbohydrate Sequence; D002460:Cell Line; D006224:Cricetinae; D004734:Energy Metabolism; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007376:Interleukin-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D007074:Immunoglobulin G; D007376:Interleukin-2; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins