title: Chemical and enzymatic N-glycan release comparison for N-glycan profiling of monoclonal antibodies expressed in plants.
abstract: Plants synthesize N-glycans containing the antigenic sugars alpha(1,3)-fucose and beta(1,2)-xylose. Therefore it is important to monitor these N-glycans in monoclonal antibodies produced in plants (plantibodies). We evaluated several techniques to characterize the N-glycosylation of a plantibody produced in tobacco plants with and without the KDEL tetrapeptide endoplasmic reticulum retention signal which should inhibit or drastically reduce the addition of alpha(1,3)-fucose and beta(1,2)-xylose. Ammonium hydroxide/carbonate-based chemical deglycosylation and PNGase A enzymatic release were investigated giving similar 2-aminobenzamide-labeled N-glycan HPLC profiles. The chemical release does not generate peptides which is convenient for MS analysis of unlabeled pool but its main drawback is that it induces degradation of alpha1,3-fucosylated N-glycan reducing terminal sugar. Three analytical methods for N-glycan characterization were evaluated: (i) MALDI-MS of glycopeptides from tryptic digestion; (ii) negative-ion ESI-MS/MS of released N-glycans; (iii) normal-phase HPLC of fluorescently labeled glycans in combination with exoglycosidase sequencing. The MS methods identified the major glycans, but the HPLC method was best for identification and relative quantitation of N-glycans. Negative-mode ESI-MS/MS permitted also the correct identification of the linkage position of the fucose residue linked to the inner core N-acteylglucosamine (GlcNAc) in complex N-glycans.
authors.affiliation: Department of Carbohydrate Chemistry, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: Ada
authors.initials: A
authors.lastname: Triguero
authors.forename: Gleysin
authors.initials: G
authors.lastname: Cabrera
authors.forename: Louise
authors.initials: L
authors.lastname: Royle
authors.forename: David J
authors.initials: DJ
authors.lastname: Harvey
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Rudd
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Dwek
authors.forename: Muriel
authors.initials: M
authors.lastname: Bardor
authors.forename: Patrice
authors.initials: P
authors.lastname: Lerouge
authors.forename: José A
authors.initials: JA
authors.lastname: Cremata
mesh_terms: D064753:Ammonium Hydroxide; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006031:Glycosylation; D006878:Hydroxides / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D046549:Plantibodies / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014026:Nicotiana / Q000378:metabolism*; D062367:ortho-Aminobenzoates / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D006878:Hydroxides; D046549:Plantibodies; D011134:Polysaccharides; D062367:ortho-Aminobenzoates; D064753:Ammonium Hydroxide; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C000219:anthranilamide