Monosaccharide composition analysis is routinely performed on biopharmaceutical products [such as monoclonal antibodies (mAbs) and glycoproteins] throughout their development and production processes to ensure proper function, efficacy, and safety, stability, serum half-life, as well as for quality control and release purposes. As the long-term pioneer as well as market leader in the field of therapeutic antibody development, Creative Biolabs has successfully developed a versatile antibody glycan analysis platform to illustrate antibody monosaccharide composition and screen the changes in glycosylation. As your best partner, Creative Biolabs is very glad to revolutionize your particular project and promote your success based on the values of quality, timing, and price.
Glycosylation represents a major post-translational modification of monoclonal antibodies (mAbs) and glycoproteins that are involved in various biological processes, including transcription, differentiation, apoptosis, cell adhesion, receptor-ligand binding, as well as oncogenic transformation and immune response. Particularly, these glycosylated mAbs and proteins are one of the fastest growing therapeutic drugs and their activity, stability, serum half-life and adverse reactions are largely determined by glycosylation. Glycosylation is, therefore, one of the critical quality attributes that must be rigorously characterized and monitored.
Fig.1 Summary of IgG N-glycan present on the variable region (Fab) and the constant heavy chain tail region (Fc) with associated immunologic functions.1
Monosaccharide composition analysis can screen for changes in glycosylation as well as quantify the amounts of individual monosaccharides, most often fucose, galactosamine, glucosamine, galactose, glucose, and mannose. Most mammalian glycoproteins contain three major types of glycans: N-linked, O-linked and glycosylphosphatidylinositol (GPI) lipid anchors, which consist of one or more monosaccharide units. For therapeutic mAbs, monosaccharide composition analysis is extremely important due to its important role in affecting both antibody characteristics (e.g., antigenicity, stability, folding, serum half-life, and antigen binding affinity) and effector functions [e.g., antibody-dependent-cell mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)]. Especially, beyond profiling changes in total glycan composition, monosaccharide analysis can also be used as an exploratory technology to identify various monosaccharide modifications, including phosphorylation and sulfation.
To support researchers in glycoprotein engineering, we provide a series of technologies for monosaccharide analysis, including but not limited to HPAEC-PAD, HPLC, and MS.
High-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) is a classic procedure used for the analysis of monosaccharides released from a glycoprotein. With high selectivity and specificity, HPAEC-PAD permits direct analysis of nonderivatized monosaccharides, providing not only the total glycosylation status of a protein, but also the amounts of specific monosaccharides. Particularly, HPAEC-PAD analyzes the carbohydrate species without the need for derivatization. Generally, samples are acid hydrolyzed to release the monosaccharides, lyophilized, dissolved in water, and then analyzed by HPAEC-PAD. This specific method can not only provide details about fucosylation, an indication of the presence of O-linked glycans, but also determine mannose content as an indication of high-mannose N-linked glycans.
High-performance liquid chromatography (usually with fluorescence detector) is another common method for the determination of two most abundant sialic acids (Neu5Ac and Neu5Gc) by derivatization. During this analysis process, 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB) is typically used to modify the sialic acids after release from glycoprotein to provide a fluorescent product. HPLC allows separation and detection of a wider variety of modified sialic acids. Highly efficient hydrophilic interaction chromatography (HILIC)-based ultra-performance liquid chromatography (UPLC) is emerging as a powerful tool for monosaccharide composition analysis.
Mass spectrometry is a versatile technique that has increasingly been used in monosaccharide analysis due to its sensitivity, potential for high throughput analysis, and the capability for tolerating diverse biological mixtures. Separation techniques such as liquid chromatography (LC) and ion mobility are typically used in conjunction with mass spectrometry to analyze the antibody monosaccharide with more accuracy.
Taking advantage of these sophisticated methodologies, Creative Biolabs has developed, optimized, and validated a panel of platforms to offer high-quality antibody monosaccharide analysis services. Our impeccable antibody monosaccharide analysis can guarantee our worldwide customers with the most compelling and reproducible outcomes, offering absolute confidence for subsequent investigations. Besides, we can also provide customized services with different formats, endpoints, and parameters to satisfy any specific requirement. If you are interested in our services, please contact us or directly sent us an inquiry.
Reference
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