Chemical and enzymatic N-glycan release comparison for N-glycan profiling of monoclonal antibodies expressed in plants

详细信息    查看全文

• 作者：Ada Triguero ; Gleysin Cabrera ; Louise Royle ; David J. Harvey ; Pauline M. Rudd ; Raymond A. Dwek ; Muriel Bardor ; Patrice Lerouge ; José ; A. Cremata
• 关键词：Plantibodies ; Chemical deglycosylation ; Enzymatic N-glycan release ; Exoglycosidases digestions ; Negative-mode ESI-MS
• 刊名：Analytical Biochemistry
• 出版年：2010
• 出版时间：15 May 2010
• 年：2010
• 卷：400
• 期：2
• 页码：173-183
• 全文大小：2089 K

文摘

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.