Detection and quantitation of afucosylated N-linked oligosaccharides in recombinant monoclonal antibodies using enzymatic digestion and LC-MS.

Journal of the American Society for Mass Spectrometry
Yi DuHongcheng Liu

Abstract

The presence of N-linked oligosaccharides in the CH2 domain has a significant impact on the structure, stability, and biological functions of recombinant monoclonal antibodies. The impact is also highly dependent on the specific oligosaccharide structures. The absence of core-fucose has been demonstrated to result in increased binding affinity to Fcγ receptors and, thus, enhanced antibody-dependent cellular cytotoxicity (ADCC). Therefore, a method that can specifically determine the level of oligosaccharides without the core-fucose (afucosylation) is highly desired. In the current study, recombinant monoclonal antibodies and tryptic peptides from the antibodies were digested using endoglycosidases F2 and H, which cleaves the glycosidic bond between the two primary GlcNAc residues. As a result, various oligosaccharides of either complex type or high mannose type that are commonly observed for recombinant monoclonal antibodies are converted to either GlcNAc residue only or GlcNAc with the core-fucose. The level of GlcNAc represents the sum of all afucosylated oligosaccharides, whereas the level of GlcNAc with the core-fucose represents the sum of all fucosylated oligosaccharides. LC-MS analysis of the enzymatically digested antib...Continue Reading

References

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Citations

Mar 16, 2013·Proceedings of the National Academy of Sciences of the United States of America·Nicole M OkeleyPeter D Senter
Apr 2, 2015·Journal of Pharmaceutical and Biomedical Analysis·Cai TieJinlan Zhang
Nov 9, 2012·Analytical Chemistry·Alain BeckSarah Sanglier-Cianférani

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