Over the past few years, recombinant monoclonal antibodies (mAbs) have gained significant importance in diagnostic and therapeutic applications. The key properties of antibodies require thorough characterization to verify the correctness of the overall molecule and ensure the identification of a reproducible, safe, and effective biological drug. Armed with extensive experience and professional scientists, Creative Biolabs is able to supply various antibody glycan analysis services for our global customers.
Glycosylation is one of the most important post-translational modifications of eukaryotic cell proteins. Glycan-modified proteins are involved in a wide range of biological and physiological processes including recognition and regulatory functions, cellular communication, gene expression, cellular immunity, growth, and development. Glycan functions are often dependent on the structure of the oligosaccharide. Many analytical approaches have been employed in the area of glycan and glycoproteomics research, such as monosaccharide analysis, glycan analysis, glycosylation site profiling, and intact glycoprotein profiling. These analyses are performed to determine the identity and quantity of the carbohydrates present, the post-translational modification site, and the multiple glycoforms of a protein that may be present. Choosing the appropriate analysis technique depends on instrumental capabilities and experimental demands.
Fig.1 Glycoprotein analysis.
Monosaccharide composition analysis can screen for changes in glycosylation relative to a reference protein as well as quantify the amounts of individual monosaccharides, most often galactosamine, galactose, fucose, glucosamine, mannose, and glucose. Monosaccharides are weak acids, and under basic conditions, pH 12-14. The carbohydrates are either partially or completely ionized and therefore can be separated by anion-exchange chromatography.
Glycan analysis enables monitoring of relative quantities of a particular set of glycans. Besides, it can provide valuable information regarding changes in glycan patterns or identify aberrant glycans. Because of the complexity of a glycan structure, structural elucidation is performed after the release of glycans from a protein. The preferred form of release for N-linked glycans is by enzymatic means. Once released, these oligosaccharides, no matter neutral or charged, can be analyzed by several methods, such as mass spectrometry (MS) and high-performance liquid chromatography (HPLC).
Glycans can be profiled at the peptide level, such that relative quantification of specific glycosylation sites is achieved. A combination of higher-energy collisional dissociation (HCD) or collision-induced dissociation (CID) with electron transfer dissociation (ETD) is used. ETD provides extensive fragmentation along the peptide backbone to enable peptide sequencing while preserving glycan attachments, allowing glycosylation site localization. CID or HCD provides information about the glycan composition.
Intact glycoprotein profiling by MS is a good way to ascertain the expected pattern and degree of glycosylation. Due to the inherent heterogeneity and variation of the attached glycan moieties, intact glycoprotein profiling is best performed on a high-resolution accurate-mass hybrid Orbitrap MS with electrospray ionization (ESI). One of the intrinsic advantages of ESI-MS is its ability to place multiple charges on particular species. Intact glycoprotein profiling can also be performed by either direct infusion or liquid chromatography-mass spectrometry (LC/MS).
Fig.2 The glycoprotein analysis work-flow. (Joenväärä, 2008)
Besides, we have developed our powerful Antibody GlycoOpitimize™ Platform for optimization of antibody glycosylation, including fucosylation, sialylation, galactosylation, etc.
Based on years of extensive experience in antibody characterization and Fc engineering, Creative Biolabs can offer a full range of antibody glycosylation analysis services. With the help of our professional scientists, we are confident in providing the most comprehensive antibody functional detection services to meet every customer's requirements. If you are interested in our services, please feel free to contact us.
Reference
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