MaxPeak High Performance Surfaces Technology Improves HILIC Profiling of Released N-Glycans

Significance of the Topic

Glycosylation of biotherapeutics critically influences drug stability, efficacy, and immunogenicity. Released N-glycan profiling via HILIC-MS is widely used to monitor these critical quality attributes. However, acidic species such as sialylated or phosphorylated glycans often exhibit peak asymmetry and loss due to interactions with metal column hardware under low-ionic strength conditions.

Objectives and Study Overview

This study assesses the performance of the novel ACQUITY Premier Glycan BEH Amide Column featuring MaxPeak High Performance Surfaces Technology. The goals are to compare glycan recovery, resolution, and reproducibility against a conventional stainless steel column, and to evaluate the need for column conditioning for both sialylated and phosphorylated N-glycans.

Methodology and Instrumentation

Sample preparation involved RapiFluor-MS labeled glycan standards and released N-glycans from fetuin and agalsidase. Each column was tested with sequential injections: initial glycan standard runs, fetuin-based conditioning, and post-conditioning runs. Recovery was calculated from fluorescence peak areas.

Used Instrumentation

• LC system: ACQUITY Premier UPLC System
• Columns: ACQUITY Premier Glycan BEH Amide (1.7 µm, 130 Å, 2.1×150 mm) and conventional Glycan BEH Amide
• MS system: Xevo G2-XS QTof (ESI+)
• Data software: MassLynx v4.1 and UNIFI v1.8

Key Results and Discussion

■ Conventional steel columns required extensive conditioning to detect only 13 of 19 glycan species initially, with recovery below 40% for some sialylated glycans.
■ MaxPeak HPS-equipped columns resolved all 19 glycans, including low-abundance sialylated species, from the first injection without conditioning.
■ Effective peak capacity improved from 50 to 66 for conditioned steel columns, while Premier columns maintained capacity consistently above 65.
■ Phosphorylated glycan (Man7-1P) showed ~70% recovery on passivated steel versus full recovery on Premier columns.
■ Retention time reproducibility (%RSD) for six sialylated glycans was 0.12–0.22% on Premier versus 1.15–1.96% on steel columns.

Benefits and Practical Applications

• Out-of-the-box performance eliminates time-consuming column conditioning.
• Enhanced recovery and resolution of acidic glycans streamline QC workflows.
• Improved reproducibility supports robust method transfer across laboratories.

Future Trends and Potential Applications

Integration of high-performance surface technologies across diverse chromatographic modes may extend to other metal-sensitive analytes. Adapting these surfaces for glycoproteomic profiling and high-throughput screening will further enhance biopharmaceutical development and quality control.

Conclusion

MaxPeak High Performance Surfaces on ACQUITY Premier Glycan BEH Amide Columns significantly enhance HILIC-MS analysis of released N-glycans by preventing analyte–metal interactions. This results in superior recovery, resolution, and reproducibility of acidic and phosphorylated glycans without the need for column conditioning, thereby improving robustness of glycan profiling assays.

Reference

1. Lauber MA et al. Rapid Preparation of Released N-glycans for HILIC Analysis. Anal Chem. 2015;87(10):5401–5409.
2. DeLano M et al. Hybrid Organic-Inorganic Surfaces in UHPLC. Anal Chem. 2021;93(14):5773–5781.
3. Lauber MA et al. Low Adsorption HPLC Columns Based on MaxPeak HPS. Waters White Paper 2020.
4. Lauber MA et al. Robustness of Glycan BEH Amide HILIC Separations. Waters Appl Note 2015.
5. Bones J et al. Identification of Mannose-6-Phosphate Glycans on Therapeutic Enzymes. Anal Chem. 2011;83(13):5344–5352.