N-Linked Glycans Analysis of Monoclonal Antibodies, Biosimilars, and Glycoproteins with High Resolution Mass Spectrometry and Fluorescence Detection using Restek’s Raptor Polar X Column and Protein Metrics’ Software Suite

Importance of Topic

Glycosylation of therapeutic antibodies plays a critical role in determining their stability, solubility, and immunogenicity. As a key Critical Quality Attribute (CQA) under Quality by Design (QbD) guidelines, consistent profiling of N-linked glycans is essential for ensuring safety and efficacy in biopharmaceutical development.

Objectives and Study Overview

This study evaluates a comprehensive workflow for releasing, labeling, separating, and characterizing N-linked glycans from monoclonal antibodies, biosimilars, and standard glycoproteins. The goals include achieving baseline chromatographic resolution, high mass accuracy, and automated data processing using Protein Metrics’ Detached Glycan workflow.

Methodology and Instrumentation

Sample Preparation and Labeling:

• Deglycosylation: PNGase F treatment released N-linked glycans from NIST mAb, bevacizumab biosimilar, and RNase B.
• Fluorescent Tagging: Procainamide labeling enhanced detection sensitivity and quantitation.

Chromatography and Detection:

• Column: Restek Raptor Polar X (2.1×100 mm, 2.7 µm) combining ion-exchange and HILIC retention modes.
• Mobile Phases: 0.5% formic acid in water (A) and 90:10 ACN:water with 20 mM ammonium formate (pH 3) (B).
• Gradient: 88–75% B over 30 min, followed by column wash and re-equilibration at 10 °C.
• Fluorescence: Excitation 310 nm, emission 370 nm, cell at 40 °C.

Applied Instrumentation

The analytical platform consisted of a Shimadzu UHPLC coupled to a Shimadzu LCMS-9030 Q-ToF mass spectrometer and an RF-20AXS fluorescence detector. MS data were acquired in data-dependent acquisition (DDA) mode over m/z 100–2200, achieving mass accuracy below 6 ppm. Data processing was performed with LabSolutions and the Detached Glycan (N-Linked) Workflow from Protein Metrics.

Main Results and Discussion

Baseline separation of high-mannose (Man5–Man9), hybrid, and complex glycans was achieved for RNase B, bevacizumab, and NIST mAb. Extracted ion chromatograms and fluorescence traces confirmed resolution of isomeric species. All labeled glycans exhibited mass errors below 5 ppm. MS/MS fragmentation spectra provided characteristic ions for structural confirmation. Normalized glycan distribution profiles highlighted distinct glycosylation patterns across sample types.

Benefits and Practical Applications

This integrated HILIC/ion-exchange column and high-resolution MS workflow supports:

• Routine quality control of biotherapeutics
• Biosimilar comparability assessments
• Research into glycoprotein heterogeneity and functionality

Future Trends and Opportunities

Advancements in chromatographic stationary phases, enhanced MS sensitivity, and higher‐order fragmentation techniques will drive deeper glycan structural elucidation. Combining ion mobility separation, label‐free quantitation, and automated data pipelines promises more comprehensive and high‐throughput glycoanalysis workflows.

Conclusion

The combination of Restek’s Raptor Polar X column, Shimadzu LCMS-9030 Q-ToF, and Protein Metrics software delivers a robust platform for accurate N-linked glycan profiling. This workflow meets the stringent requirements of biopharmaceutical development and quality assurance, ensuring reliable monitoring of glycosylation as a key CQA.

Reference

1. Li H, et al. J Biomed Sci. 2019;27:59.
2. Smith J, et al. GXP-CC News. 2014;July 15.
3. Shimadzu Scientific Instruments. NIST mAb Workflow on QToF, Application Note LCMS-103; 2020.
4. Ludger. Procainamide Glycan Labeling Kit Guide; 2020.
5. Sigma-Aldrich. Glycan SPE Tube Datasheet; 2020.