ROUTINE MONITORING OF N-GLYCANS USING A NOVEL LABELING REAGENT WITH FLUORESCENCEAND MASS DETECTION

Significance of the Topic

Glycan profiling of biotherapeutics is a critical quality attribute that influences safety, efficacy and consistency of protein drugs. Traditional workflows for released N-glycan analysis are labor-intensive and require skilled operators to generate both fluorescence and mass spectrometric data. The development of an integrated, high-throughput approach addresses the need for rapid, unambiguous glycan monitoring in process development and routine quality control.

Aims and Overview of the Study

This study evaluates a novel fluorescent and mass-sensitive labeling reagent, RapiFluor-MS, combined with ultra-performance liquid chromatography and QDa single-quadrupole mass detection. The objectives were to demonstrate:

• Streamlined sample preparation in 30 minutes
• Enhanced fluorescence and MS response versus conventional labels
• Reliable detection and identification of neutral and charged N-glycans across a broad mass range
• High throughput capabilities through method scaling and selected ion recording

Methodology and Instrumentation

Sample Preparation:
Released glycans from model glycoproteins (human IgG, RNase B, bovine fetuin) and trastuzumab were labeled with RapiFluor-MS following PNGase F digestion.

Chromatography and Detection:

• Column: ACQUITY UPLC Glycan BEH Amide, 130 Å, 1.7 µm, 60 °C
• Mobile phases: ACN (A) and 50 mM ammonium formate pH 4.4 (B)
• Gradient adapted for high resolution and a scaled method reducing analysis time six-fold
• Fluorescence detector: excitation 265 nm, emission 425 nm, 5 Hz data rate
• Mass detector: ACQUITY QDa, cone 15 V, capillary 1.5 kV, m/z 500–1250

Main Results and Discussion

Enhanced Sensitivity:
RapiFluor-MS achieved a 14-fold increase in fluorescence and 160-fold gain in MS signal compared to 2-AB, enabling detection of low-abundance glycans (<0.5% RPA).

Comprehensive Glycan Detection:
All bi-antennary to tetra-sialylated structures (m/z ~1774–3482) were resolved by HILIC and simultaneously detected by fluorescence and QDa MS. Spectral quality supported unambiguous glycan composition assignment.

High Throughput and Co-elution Resolution:
A geometrically scaled HILIC method delivered a six-fold reduction in run time. Selected ion recording (SIR) differentiated co-eluting isomers (e.g., M5 vs A2G1) in trastuzumab samples, facilitating rapid assessment of glycosylation changes during bioprocess development.

Benefits and Practical Applications of the Method

• Significantly reduced sample preparation and analysis time
• Integrated fluorescence and mass detection for orthogonal confirmation without specialized MS expertise
• High-sensitivity detection of glycan variants down to low-abundance species
• Scalable workflow suitable for routine QC or high-throughput process monitoring
• Rapid decision support in biopharmaceutical development and manufacturing

Future Trends and Applications

Advances in glycan analysis will include further automation of labeling and separation workflows, integration with high-resolution mass spectrometers for deeper structural characterization, and real-time online monitoring of glycosylation during cell culture. Miniaturized and multiplexed platforms may emerge to support continuous bioprocessing and real-time quality assurance.

Conclusion

The combination of RapiFluor-MS labeling with UPLC-FLR-QDa detection provides a robust, high-throughput solution for routine N-glycan profiling. Enhanced sensitivity and integrated orthogonal data enable faster and more confident decision-making in biopharmaceutical research and quality control.

Reference

Cosgrave EFJ, Lauber MA, Koza SM, McCarthy SM, Chakraborty A. Routine monitoring of N-glycans using a novel labeling reagent with fluorescence and mass detection. Waters Corporation; 2015.