Increased Resolving Power for Acidic Glycans with an MS-Compatible Anion Exchange Reversed Phase Separation

Significance of the Topic

Acidic N-glycans play a pivotal role in the safety and efficacy of biotherapeutics. Variations in sialylation and phosphate modifications can alter immunogenicity, clearance rates, and therapeutic potency. Reliable analytical methods that resolve charge variants and isomeric structures of glycans are therefore essential for quality control and regulatory compliance in biopharmaceutical development.

Objectives and Study Overview

This study introduces and evaluates a novel mixed-mode anion-exchange reversed-phase liquid chromatography (AX-RPLC) column, the ACQUITY PREMIER Glycan BEH C18 AX, designed to overcome challenges in recovering and separating acidic N-glycans labeled for mass spectrometry. The objectives were to achieve charge-based grouping, improve recovery of acidic species, and enhance resolution of isomeric glycans compared to existing HILIC and mixed-mode columns.

Methodology and Instrumentation

The workflow combined enzymatic release and rapid labeling of N-glycans with RapiFluor-MS and 2-AB tags, followed by AX-RPLC‐MS analysis. Key experimental parameters included:

• Chromatography: ACQUITY UPLC H-Class Bio system, 2.1 × 150 mm, 1.7 µm ACQUITY PREMIER Glycan BEH C18 AX column, 60 °C.
• Mobile phases: water (A) and 10% IonHance Glycan C18 AX concentrate in 40/60 water/acetonitrile (B) with 0–22 mM ammonium formate gradient.
• Detection: fluorescence (RFMS: 265 nm Ex/425 nm Em; 2-AB: 330 nm Ex/420 nm Em) and Xevo G2-XS QTof MS in positive ESI mode (700–3000 Da).

Main Results and Discussion

The ACQUITY PREMIER Glycan BEH C18 AX column achieved clear grouping of glycans by net charge, with retention increasing alongside charge state. More than 20 RFMS-labeled species from human IgG and fetuin were resolved, reaching femtomole sensitivity. The mixed-mode RP functionality enabled separation of NeuGc-containing isomers from NeuAc counterparts, a challenge for HILIC. Comparative trials with a commercial AX-RPLC column showed superior resolution (Rs of 3.1 vs. 1.1 for key tetra-sialylated species) and lower salt requirements for elution.

Benefits and Practical Applications

This method offers:

• Charge-based separation optimized for MS-enhancing labels (RapiFluor-MS, 2-AB).
• High recovery of acidic glycans via MaxPeak High Performance Surface technology.
• Enhanced resolution of isomeric and highly sialylated structures with minimal buffer strength.
• Compatibility with standard sample prep used in HILIC workflows, facilitating adoption in QC labs.

Future Trends and Opportunities

Ongoing work includes extending this mixed-mode approach to phosphorylated glycans and enzyme replacement therapies. Integration with high-throughput automation and coupling to advanced ion-mobility spectrometry may further enhance structural characterization and throughput in glycan profiling.

Conclusion

The ACQUITY PREMIER Glycan BEH C18 AX column delivers an orthogonal, charge-based separation for acidic N-glycans labeled for MS. Its optimized mixed-mode chemistry and inert HPS hardware overcome recovery issues, improve isomer resolution, and offer a robust platform for biotherapeutic quality control.

Used Instrumentation

• ACQUITY UPLC H-Class Bio system
• ACQUITY PREMIER Glycan BEH C18 AX column, 1.7 µm, 2.1 × 150 mm
• Xevo G2-XS QTof mass spectrometer
• MassLynx v4.1 and UNIFI v.1.8 software

References

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