Monoclonal antibodies (mAbs) have emerged as some of the most important treatments on the market. These mAbs prescribed for therapeutic use are usually expressed from eukaryotic cell lines (such as CHO) and, therefore, are N-glycosylated at the two common sites of the Fc portion of their heavy chain asparagine residues. Since glycosylation can measure efficacy, safety, and production conditions, it is often crucial to characterize and routinely monitor the N-glycan profile of mAb drugs.
Creative Biolabs is a forward-looking company and a leading customized service provider in the field of glycoprotein analysis. Based on our rich experience and advanced platform, we have developed novel analytical tools to quantify N-glycosylation site occupancy by labeling/label-free strategies with LC-MS/MS and completed many challenging projects. We are happy to help you achieve a milestone success in your glycoprotein structural analysis project.
Glycosylated proteins usually show both macro-heterogeneity (variable occupancy of glycosylation sites) and micro-heterogeneity (the type of glycans connected to a glycosylation site, the degree of modification and elongation is variable), which increases their complexity. Glycosylation plays an important role in many biological functions, including communication and interaction between cells, development, morphogenesis, embryogenesis, immunity, protein folding, transport, blood protein modification, mucosal development and differentiation. Therefore, glycosylation site mapping helps to identify glycosylation sites, which can indicate the function of the glycan. In addition, site-specific glycosylation analysis and the assignment of specific structures to specific sites can clarify the glycosylation properties, micro-heterogeneity and activity of proteins. Glycosylation is very important in the development of biopharmaceuticals, because their glycan chains significantly affect the stability, activity, antigenicity and pharmacodynamics of the product. Glycosylation site mapping mainly contributes to the micro-heterogeneity of glycans.
Figure 1: A representative schematic structure of monoclonal antibody and N-glycosylation sites on it. (Liu, 2017)
N-glycosylation site occupancy
Protein N-glycosylation plays an important role in physiological and pathological processes. Characterization of site-specific N-glycosylation includes large N-glycan heterogeneity (glycosylation site occupancy) and micro-heterogeneity (site-specific glycan structure) for understanding glycoprotein biosynthesis and Function is very important. The heterogeneity of N-glycan macromolecules is a physiological characteristic of glycoproteins, and technical obstacles limit the research on the regulation and function of this heterogeneity. Quantitative methods based on liquid chromatography (LC)-mass spectrometry (MS) are becoming a powerful tool for glycosylation characterization. Creative Biolabs has developed labeled and label-free quantitative MS methods for quantifying N-glycosylation site occupancy.
Figure 2: Flowchart of our N-glycosylation site occupancy analysis method.
O-glycosylation site occupancy
O-linked glycans are characterized by smaller but highly diverse branched carbohydrate molecules, rather than N-linked glycans, and are usually attached to proteins in any hypothetical area, therefore, this leads to the complexity of the analysis, And it is difficult to ascertain these exact positions of O- glycosylation. We can perform a summary analysis of all glycosylation sites, and we can also analyze site-specific glycosylation.
The general steps of O-glycosylation site occupancy analysis include proteolysis of glycoproteins, precipitation steps, glycopeptide enrichment and separation by hydrophilic interaction liquid chromatography. The enriched O-glycopeptide fraction was analyzed by mass spectrometry using reverse phase liquid chromatography coupled with ion trap mass spectrometer. Other label-free methods based on glycopeptide/peptide ion signal intensity can also be used to analyze O-glycosylation sites.
Figure 3: Schematic of our O-glycosylation site occupancy analysis method.
With extensive experience in glycan characterization, our team can work on custom solutions to meet the unique requirements of each product. With flexible global capabilities, you can provide services where you need it most. Contact our expert scientists today to discuss how our range of glycan analytical services can improve your unique process.
Creative Biolabs provides luciferase-based ADCC assay. This Jurkat cell based assay is pioneered by Creative Biolabs, and the methodology is very well accepted by the field. See attached ADCC Reporter Assay Protocol for further details.
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