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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 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.
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https://hal-normandie-univ.archives-ouvertes.fr/hal-01848420
Contributor : Arnaud Lehner <>
Submitted on : Tuesday, July 24, 2018 - 3:25:31 PM
Last modification on : Friday, April 5, 2019 - 3:24:07 PM

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Ada Triguero, Gleysin Cabrera, Louise Royle, David Harvey, Pauline Rudd, et al.. Chemical and enzymatic N-glycan release comparison for N-glycan profiling of monoclonal antibodies expressed in plants. Analytical Biochemistry, Elsevier Masson, 2010, 400 (2), pp.173 - 183. ⟨10.1016/j.ab.2010.01.027⟩. ⟨hal-01848420⟩

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