Effect of Fc-Glycan Structure on the Conformational Stability of IgG Revealed by Hydrogen/Deuterium Exchange and Limited Proteolysis

Biochemistry
Jing FangZhongqi Zhang

Abstract

Human therapeutic immunoglobulin gamma (IgG) molecules contain an N-glycan on each of their Fc CH2 domains. These glycans include high-mannose, hybrid, and complex types. Recombinant IgG molecules containing high-mannose glycans have been shown to clear faster in human blood, and exhibit decreased thermal stability. The molecular mechanism behind these observations, however, is not well understood. In this work, we used hydrogen/deuterium exchange combined with mass spectrometry (HDX MS), as well as proteolytic degradation under a native-like condition, to assess the impact of different glycoforms on the molecular structure and stability of recombinant IgG1 and IgG2 molecules expressed from Chinese hamster ovary cells. Our HDX MS data indicate that the conformation of these IgG molecules was indeed influenced by the glycan structure. IgG molecules containing high-mannose and hybrid glycans showed more conformational flexibility in the CH2 domain. This conclusion was further supported by the analysis of glycopeptides released from these molecules by trypsin digestion under a native-like condition. The higher CH2 conformational flexibility of IgG molecules with high-mannose and hybrid glycans contributes to their decreased therma...Continue Reading

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Apr 24, 2016·Biochimica Et Biophysica Acta·Ngoc Phuong Lan LeMax Crispin
Mar 8, 2018·MAbs·Alexandre AmbrogellyHongcheng Liu
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