title: Detection and quantitation of afucosylated N-linked oligosaccharides in recombinant monoclonal antibodies using enzymatic digestion and LC-MS.
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 antibodies after reduction provided a quick estimate of the levels of afucosylation. An accurate determination of the level of afucosylation was obtained by LC-MS analysis of glycopeptides after trypsin digestion.
authors.affiliation: Merck Research Laboratories, 1011 Morris Ave, Union, NJ 07083, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Du
authors.forename: Kimberly
authors.initials: K
authors.lastname: May
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
authors.forename: Hongcheng
authors.initials: H
authors.lastname: Liu
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005643:Fucose / Q000032:analysis* / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D009844:Oligosaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005643:Fucose; D014357:Trypsin