Chemical and enzymatic N-glycan release comparison for N-glycan profiling of monoclonal antibodies expressed in plants |
| |
Authors: | Ada Triguero Louise Royle David J Harvey Raymond A Dwek Patrice Lerouge |
| |
Institution: | a Department of Carbohydrate Chemistry, Center for Genetic Engineering and Biotechnology, Havana, Cuba b Dublin-Oxford Glycobiology Laboratory, National Institute for Bioprocessing Research and Training, Conway Institute University College Dublin Belfield, Dublin 4, Ireland c Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford, UK d CNRS-UMR 6037, IFRMP 23, Université de Rouen, 76821 Mont Saint Aignan Cédex, France |
| |
Abstract: | Plants synthesize N-glycans containing the antigenic sugars α(1,3)-fucose and β(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 α(1,3)-fucose and β(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 α1,3-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. |
| |
Keywords: | Plantibodies Chemical deglycosylation Enzymatic N-glycan release Exoglycosidases digestions Negative-mode ESI-MS |
本文献已被 ScienceDirect 等数据库收录! |
|