Automated Glycan Assignment Using Mass Measurement with a Calibrated Retention Time in Glucose Unit

Significance of the Topic

Glycan profiling plays a pivotal role in biopharmaceutical development by ensuring product consistency, safety, and efficacy. Characterizing N-linked glycans on therapeutic antibodies informs on immunogenic risks and supports comparability assessments between innovator and biosimilar products.

Objectives and Study Overview

This work compares the N-glycan profiles of innovator Infliximab and a candidate biosimilar, evaluates batch-to-batch variability, and demonstrates automated glycan assignment using calibrated retention times expressed in Glucose Units (GU) combined with accurate mass measurements.

Methodology and Instrumentation

The analysis was performed on the Waters UNIFI Biopharmaceutical Platform, integrating UPLC and QTof MS workflows.

• Sample Preparation
  • Deglycosylation of antibody samples with PNGase F.
  • Purification of released glycans via HILIC 96-well micro-elution plate.
  • Labeling with 2-AB and removal of excess tag.
• Chromatography and Calibration
  • ACQUITY UPLC H-Class Bio with BEH Glycan column (2.1 x 150 mm, 40 °C).
  • Mobile phases: 50 mM ammonium formate pH 4.4 (A) and acetonitrile (B).
  • Retention time calibration using a 2-AB-labeled dextran ladder to convert into GU values.
• Detection
  • FLR detector (λex 330 nm, λem 420 nm, 10 points/s, gain 10).
  • Xevo G2-S QTof MS in ESI+ mode (capillary 2.75 kV, cone 80 V, source 100 °C, desolvation 300 °C).

Main Results and Discussion

Overlay of FLR chromatograms revealed distinct glycan profiles between innovator and biosimilar Infliximab, attributable to different expression cell lines (murine SP2/0 vs. CHO). Key findings include:

• Presence of alpha(1,3)-Gal and NeuGc-containing glycans in the innovator batches, absent in the biosimilar.
• Batch-to-batch consistency within each product, with relative quantitation indicating low intra-product variability.
• Accurate GU-based retention matching combined with mass confirmation provided reliable glycan assignment from the Waters Glycan GU library.

Benefits and Practical Applications

The integrated UNIFI workflow streamlines both qualitative and relative quantitative glycan analysis, facilitating:

• Rapid comparability assessment for biosimilar development.
• Enhanced quality control in regulated laboratories.
• Timely identification of potentially immunogenic glycoforms.

Future Trends and Opportunities

Emerging developments are likely to include:

• Advanced bioinformatics for automated library expansion and structural elucidation.
• Integration of ion mobility and higher-throughput microflow LC-MS for deeper glycomic coverage.
• Application of glycan profiling in personalized medicine and real-time process monitoring.

Conclusion

The use of calibrated retention times in GU combined with accurate mass measurement on an integrated UPLC-MS platform offers a robust, high-confidence approach for automated glycan assignment and comparability studies. This workflow effectively distinguished glycan differences between innovator and biosimilar Infliximab and confirmed consistent batch performance.

Reference

1. Waters Corporation. Application Note 720004202en. Automated glycan assignment using GU and mass measurement. 2013.
2. Waters Corporation. Application Note 720004203en. GlycoWorks sample preparation for N-glycan profiling. 2013.