Analysis of Monoclonal Antibody N-Glycans by Fluorescence Detection and Robust Mass Selective Detection Using the Agilent LC/MSD XT

Importance of the topic

The glycosylation profile of therapeutic monoclonal antibodies (mAbs) critically affects their biological activity, immunogenicity, and pharmacokinetics. Reliable characterization of N-glycans is therefore essential during biopharmaceutical development, process optimization, and quality control to ensure product safety and efficacy.

Aim and overview

This work describes a streamlined workflow that combines hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLD) and a compact Agilent LC/MSD XT single quadrupole mass spectrometer. The goal is to achieve rapid, high-resolution separation and confident mass-based identification of mAb N-glycans with minimal sample handling.

Methodology and instrumentation

• Sample preparation: Enzymatic release of N-glycans from CHO-expressed and commercial mAb standards, followed by labeling with the Agilent InstantPC fluorescent tag.
• Automation: High-throughput processing on the Agilent AssayMAP Bravo liquid handling platform, eliminating centrifugation and drying steps.
• Chromatography: Agilent 1260 Infinity II UHPLC with AdvanceBio Glycan Mapping HILIC columns (formats: 2.1×100 mm, 1.8 µm; 2.1×150 mm, 2.7 µm; 2.1×150 mm, 1.8 µm), operated below 600 bar.
• Detection:
  • Fluorescence: Agilent 1260 FLD (λEx = 285 nm, λEm = 345 nm).
  • Mass spectrometry: Agilent LC/MSD XT single quadrupole with Jet Stream ion source in positive mode (m/z 500–1400).
• Software: OpenLab CDS ChemStation for data acquisition and analysis.

Main results and discussion

• Separation optimization: Three gradient methods were evaluated for different cycle times; Method C (2.1×150 mm, 1.8 µm) delivered the best resolution and speed across CHO mAb 1, NIST mAb, and commercial mAb samples.
• Enhanced sensitivity: The Jet Stream source combined with InstantPC labeling enabled detection of glycan species down to 0.1 % relative abundance without additional concentration steps.
• Comprehensive identification: Thirty-two N-glycan species—including neutral, fucosylated, sialylated, and galactosylated variants—were assigned based on doubly charged ions ([M+2H]²⁺, [M+NH₄+H]²⁺, [M+H+Na]²⁺) and known HILIC retention behavior.
• Accurate quantitation: Relative peak areas from FLD were supplemented by MS integration to resolve coeluting species and ensure correct assignment of minor components.

Benefits and practical applications

• Routine compatibility: Operates under 600 bar, making it compatible with existing LC infrastructure.
• Time and cost savings: Rapid sample prep (< 1 hour manual; high-throughput automation) and elimination of drying steps reduce analysis time and resources.
• Robust data: Fluorescence detection ensures reliable quantitation while single-quad MS provides structural confirmation for every peak.
• Quality control: Supports comprehensive glycan profiling for biopharmaceutical development, process monitoring, and batch release testing.

Future trends and potential applications

Key developments will include integration of advanced data analytics and artificial intelligence for automated glycan annotation, expansion of the workflow to diverse glycoprotein formats, enhanced labeling chemistries for even higher ionization efficiency, and full automation for real-time monitoring in biomanufacturing environments.

Conclusion

The combination of HILIC-FLD with the Agilent LC/MSD XT single quadrupole detector and InstantPC labeling provides a sensitive, robust, and efficient platform for in-depth profiling of mAb N-glycans. This approach enables confident identification and quantitation of both major and trace glycan species, streamlining analytical workflows and strengthening quality control capabilities in biotherapeutic development.