High-throughput immunoglobulin G N-glycan characterization using rapid resolution reverse-phase chromatography tandem...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Glycobiology

Background:

• Characterizing N-glycans on recombinant immunoglobulin G (rIgG) is crucial for understanding drug efficacy and stability.
• Traditional glycan analysis methods are often time-consuming and labor-intensive, limiting throughput.
• There is a need for faster, more efficient analytical techniques for N-glycan profiling.

Purpose of the Study:

• To develop and validate an optimized high-throughput method for the characterization of 2-aminobenzamide (2-AB)-labeled N-glycans from rIgG.
• To improve the efficiency and speed of N-glycan analysis compared to conventional protocols.
• To enable sensitive and comprehensive glycan profiling and mass spectrometry-based characterization.

Main Methods:

• Microwave-assisted deglycosylation for rapid release of N-glycans.
• Automated sample cleanup strategy for streamlined processing.
• Rapid resolution reverse-phase high-performance liquid chromatography (RRRP-HPLC) for N-glycan separation and profiling.
• Direct coupling with electrospray ionization mass spectrometry (ESI-MS) for glycan characterization.

Main Results:

• A comprehensive N-glycan profile generated within 45 minutes using RRRP-HPLC with fluorescence detection.
• Successful and sensitive characterization of N-glycans using intact mass spectrometry (MS) and tandem mass spectrometry (MS/MS) fragmentation.
• An estimated fourfold increase in throughput efficiency compared to traditional methods.

Conclusions:

• The optimized high-throughput method significantly accelerates the analysis of 2-AB-labeled N-glycans from rIgG.
• This approach offers a substantial efficiency gain for both single and multiple sample processing.
• The method provides a rapid, sensitive, and comprehensive platform for N-glycan characterization in biopharmaceutical development.