Abstract:The N-glycosylation on therapeutic monoclonal antibodies (mAbs) plays important biological roles such as antibody-dependent cellular cytotoxicity (ADCC) or circulatory half-time in vivo. Although the attached N-glycans are mostly core a1-6-fucosylated biantennary glycans, minor nonhuman N-glycans having N-glycolylneuraminic acid (NeuGc) and Gala1-3Gal epitopes (aGal) have been reported to be present in therapeutic mAbs. Determination of nonhuman N-glycans is important in the evaluation of the mAb characteristi… Show more
“…N-glycan G0F (Man 3 GlcNAc 4 Fuc) comprises >70% of N-glycans in Bevacizumab and Adalimumab, two recombinant humanized mAbs. This highly abundant structure may hinder detection of nonhuman N-glycans that induce antibody-dependent cellular cytotoxicity (ADCC) [12].…”
Glycomics provides an increasingly useful research tool as the genomes and proteomes of more and more animal species are elucidated. In view of the general complexity and heterogeneity of glycans, improved depth-of-coverage and sensitivity are required for glycosylation analysis. In this study, we established the lectin-based isolation/enrichment strategy for total glycomic information. Specific lectins are added onto the filter to capture corresponding glycans prior to release of N-glycans by peptide N-glycosidase F (PNGase F). Non-bound glycans and bound glycans are released and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Application of the strategy to chicken ovalbumin, normal mouse mammary epithelial cells (NMuMG), and human serum resulted in detection of 5, 6, and 11 additional N-glycan structures, respectively. The strategy facilitates identification of intact N-glycans in biological samples, and can be extended to detailed analysis of O-glycome or glycoproteome.
“…N-glycan G0F (Man 3 GlcNAc 4 Fuc) comprises >70% of N-glycans in Bevacizumab and Adalimumab, two recombinant humanized mAbs. This highly abundant structure may hinder detection of nonhuman N-glycans that induce antibody-dependent cellular cytotoxicity (ADCC) [12].…”
Glycomics provides an increasingly useful research tool as the genomes and proteomes of more and more animal species are elucidated. In view of the general complexity and heterogeneity of glycans, improved depth-of-coverage and sensitivity are required for glycosylation analysis. In this study, we established the lectin-based isolation/enrichment strategy for total glycomic information. Specific lectins are added onto the filter to capture corresponding glycans prior to release of N-glycans by peptide N-glycosidase F (PNGase F). Non-bound glycans and bound glycans are released and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Application of the strategy to chicken ovalbumin, normal mouse mammary epithelial cells (NMuMG), and human serum resulted in detection of 5, 6, and 11 additional N-glycan structures, respectively. The strategy facilitates identification of intact N-glycans in biological samples, and can be extended to detailed analysis of O-glycome or glycoproteome.
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