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Carbohydrate Analysis

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Creative Biolabs has established a platform to analyze the carbohydrates of therapeutic drug candidates, strictly following the ICH Topic Q6b guideline. For glycoproteins, the carbohydrate content (amino sugars, neutral sugars, and sialic acid), should be identified. Besides, the detailed structure of the carbohydrate chains, the oligosaccharide pattern (antennary profile) and the glycosylation sites of the polypeptide chain should be determined, to the extent possible.

Carbohydrate analysis

Glycosylation is an effective approach of developing diversity in proteins and modulating their bioactivities due to the inherent alteration of glycans. Glycosylation significantly influences protein yield, solubility, stability against proteolysis, immunogenicity, and clearance rate in vivo. The most dominant glycosylation sites on human endogenous and therapeutic proteins locate at asparagine residues (N-linked glycosylation through Asn-X-Thr/Ser sequence motif) and at serine or threonine residues (O-linked glycosylation) with monosaccharides, e.g. fucose, galactose, mannose, N -acetylglucosamine (Glc-NAc), N -acetylgalactosamine, and sialic acid. For therapeutic antibodies, glycosylation of Fab leads to a decrease in renal accumulation related to the level of glycosylation. This decrease is caused by decrease in both, a renal filtration (evidenced by higher blood retention of the sa-glyco-Fab) as well as in retention of the filtered fraction. In addition, glycosylation has also proven to be a powerful approach to enhance bio-distribution in the brain.

Creative Biolabs provides a series of protein carbohydrate analysis:

Monosaccharide composition analysis
Gas Chromatography with Mass Spectrometry (GC-MS) will be utilized to analyze TMS derived neutral and amino monosaccharides, while Liquid Chromatography will serve to analyze sialic acids with DMB-labelled sugars. The analyses allow detect and quantitate the desired monosaccharides, e.g. Fucose, Mannose, Galactose and N-Acetylglucosamine in a single experiment and sialic acids (N-Acetylneuraminic acid and N-Glycolylneuraminic acid) in two experiments.

Oligosaccharide population analysis
On-line Liquid Chromatography with Electrospray-Mass Spectrometric (LC/ES-MS, Q-TOF) serves to analyze intact glycoproteins, glycopeptides and 2-AB labeled oligosaccharides, N-linked glycans following permethylation (plus MSe and MS/MS as required). Linkage (by methylation) analysis is also available if required.

Glycosylation site analysis (data from peptide mapping)
The occupancy of glycosylation site will be assessed by intact molecular weight analysis and peptide mapping, compared with the expected N-linked glycosylation site.

All the glycoproteins are characterized in house in 2-3 weeks.




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