Advanced Applications

Quantifying Glycosylation

Analysis of large polypeptides from enzymatically digested intact antibodies has been reported to enable the quantification of N-linked glycosylation and in particular the levels of afucosylation [113]. These middle-up approaches are summarized in the workflow in Figure 10.4.

By incorporating endoglycosidases into an analysis, the oligosaccharide population can be simplified into two groups: endoglycosidases cleave between the two GlcNAc residues of the chitobiose core, thus generating mAbs or fragments with either GlcNAc or GlcNAc + Fuc moieties still attached. LC-MS analysis of reduced mAbs enzymatically digested with EndoF2 and EndoH has been reported, which enabled an estimation of both fucosylation and afucosylation [142]. A similar approach was employed by Goetze et al. [86]. However instead of reduction, the immunodegrading enzyme (IdeS) was used to generate Fab and glycosylated Fc/2 subunits prior to analysis. The IdeS cleavage site is located in the hinge region (-PELLG/G- in IgG1) [143, 144] and simplifies the complexity of the analysis by concentrating on the glycosylated Fc/2 fragment. Determination of afucosylation and high mannose levels have also been demonstrated using an IdeS and endoglycosidase strategy by Firth et al. (paper submitted) [145] via the application of EndoS2. EndoS has a lower affinity for high mannose compared with EndoS2, and therefore a sequential application of the two enabled the quantification of the high-mannose species based upon the change in relative MS signals observed.

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