Immobilized exoglycosidase matrix mediated solid phase glycan sequencing

Area of Science:

• Biochemistry
• Glycobiology
• Biotechnology

Background:

• Characterizing glycoprotein glycans is crucial for biopharmaceutical development and biomedical research.
• Existing methods for glycan analysis can be time-consuming and lack robustness.
• Efficient tools are needed to support rapid and reliable N-glycan sequencing.

Purpose of the Study:

• To design and produce immobilized exoglycosidases for enhanced solid-phase N-glycan sequencing.
• To create a robust and efficient enzymatic system for analyzing complex carbohydrate structures.
• To support the biopharmaceutical industry and biomedical field with advanced glycan analysis tools.

Main Methods:

• Production of three 6HIS-tagged exoglycosidases (neuraminidase, β-galactosidase, hexosaminidase) in bacterial expression systems.
• Oriented immobilization of enzymes via the 6HIS-tag for improved active site accessibility.
• Premixing enzymes in a matrix format and processing in a low-salt buffer for stability and storage.
• Demonstration of digestion efficiency using solid-phase sequencing and capillary electrophoresis.

Main Results:

• High yields of bacterial expression for all three exoglycosidases.
• Successful oriented immobilization leading to high digestion performance.
• Demonstrated digestion efficiencies on a glycoprotein therapeutic (palivizumab) and human serum glycans.
• Validation of the immobilized enzyme system for rapid N-glycan sequencing.

Conclusions:

• The developed immobilized exoglycosidase system offers a robust and efficient method for N-glycan sequencing.
• This enzymatic approach facilitates rapid characterization of carbohydrate moieties in glycoproteins.
• The technology has significant implications for quality control in biopharmaceutical manufacturing and biomarker discovery.