Glycosylation significantly affects the effector functions of the immune system, including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). This is especially important when one or more of these effector functions are the main mechanism of action. In addition, the carbohydrate structure attached to the monoclonal antibody crystallizable fragment (Fc) affects efficacy and safety, so they are usually classified as key quality attributes. Therefore, the analysis and characterization of glycosylation is an important part of protein structure analysis in the entire development process.
The complex branching and heterogeneous nature of glycans pose significant analytical challenges to their identification and characterization. The coupling of liquid chromatography (LC) and mass spectrometry (MS) has emerged as one of the most powerful tools for the characterization of glycan structures. At Creative Biolabs, we present a streamlined end-to-end analytical platform used for the characterization of glycosylation profile for a therapeutic mAb (IgG1). In this platform, we use new column technology and high-resolution mass spectrometer to simplify the sample preparation program where N-linked glycans were prepared in less than one hour. The analytes were then subjected to HILIC-UPLC/FLR/MS investigations and the resulting data were analyzed to obtain glycosylation profile information of the Fc region for further characterization of IgG1 heterogeneity.
Broadly speaking, our therapeutic protein glycosylation analysis service can be performed on three levels:
At Creative Biolabs, we can provide a series of methods to perform glycosylation site profiling, including but not limited to the followings.
RP-HPLC-MS - analysis of separated peptides and glycopeptide fragments. This method is semi-quantitative because different glycopeptides have different degrees of ionization in the mass spectrometer.
LC-ESI-MS - combines the resolving power of HPLC separation with the high-quality accuracy of a mass spectrometer. It provides unparalleled sensitivity and selectivity for detecting and quantifying site-specific glycan structures.
HILIC-SPE-MS- more selective for glycosylated species and does not favor specific glycan types. In addition, the use of this technique does not require chemical modification of glycans.
ESI/ETD-CID-MS/MS- allows the fragmentation of glycan species to be observed mainly at the glycosidic bond, while the peptide backbone remains intact. CID provides information about the composition of glycans.
Fig.1 Glycan profiles of human serum IgG and trastuzumab (Herceptin®) analyzed by HILIC–UPLC.
Fig.2 Determining monosaccharide compositions and linkages using exoglycosidase array digestion of N-glycans.
For more detailed information about our glycan characterization services, please explore:
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.
All products and services are for Research Use Only. Do Not use in humans.
ADCC Assay WT vs AfucoTM Mabs Visit
Antibody Fc Engineering: Towards Better Therapeutics Visit
Creative Biolabs has established a team of customer support scientists ready to discuss ADCC/CDC optimization strategies, antibody production, bioinformatics analysis and other molecular biology/biotechnology issues.