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Multiplexing N-glycan analysis by DNA analyzer.

Hua-Tao Feng1,2, Pingjing Li2, Guo Rui2

  • 1Department of Chemistry, National University of Singapore, Singapore.

Electrophoresis
|April 21, 2017
PubMed
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This study introduces a rapid and accurate method for analyzing N-glycan structures using capillary electrophoresis-laser induced fluorescence (CE-LIF) and magnetic bead purification. The optimized approach enhances throughput and precision for N-glycan profiling in biopharmaceutical applications.

Area of Science:

  • Glycomics and Glycobiology
  • Analytical Chemistry
  • Biopharmaceutical Analysis

Background:

  • N-glycan structure analysis is crucial for biopharmaceutical applications, biosimilar development, and understanding biological roles.
  • Detailed characterization of N-glycosylation is essential due to its impact on bio-safety and bio-activity.
  • Analyzing complex N-glycan mixtures is challenging due to structural complexity and lengthy sample preparation.

Purpose of the Study:

  • To develop a faster and more precise method for N-glycan analysis.
  • To improve the throughput of N-glycan assays.
  • To enable comprehensive N-glycan structure elucidation from a single sample injection.

Main Methods:

  • Coupling a 16-capillary DNA analyzer with magnetic bead glycan purification for accelerated sample preparation.
Keywords:
Capillary electrophoresisDNA analyzerGlycansLaser-induced fluorescenceMagnetic beadsMultiple capillaries

Related Experiment Videos

  • Utilizing three fluorescent labeling dyes for cross-validation and enhanced glycan identification precision.
  • Employing exoglycosidase treatments (neuramididase, galactosidase, fucosidase) for confirmation of glycan structures.

    • Main Results:

    • The optimized method combines multi-capillary separation, magnetic bead purification, and multiple labeling dyes for high-throughput N-glycan analysis.
    • Two new fluorescent dyes were introduced to improve glycan identification precision and simplify sample preparation.
    • Exoglycosidase studies successfully confirmed the results obtained from the three-dye cross-validation.

    Conclusions:

    • The developed method allows for rapid and accurate analysis of N-glycans, significantly increasing assay throughput.
    • The combination of techniques provides sufficient structural information for N-glycan structure elucidation in a single injection.
    • This advancement facilitates more efficient characterization of N-glycans in biopharmaceutical research and development.