Migration of an HILIC Method for Released Glycan Analysis from the ACQUITY UPLC H- Class PLUS Bio System to the Arc Premier System

Importance of the Topic

Reliable migration of chromatographic methods is critical in biopharmaceutical development to ensure consistent characterization of glycosylation patterns, a key quality attribute in therapeutic proteins. Hydrophilic interaction liquid chromatography (HILIC) is widely used to analyze released glycans, but differences in liquid chromatography (LC) hardware can affect assay outcomes, particularly for metal‐sensitive species.

Objectives and Study Overview

This application note evaluates method transfer of a HILIC glycan assay from the ACQUITY UPLC H-Class PLUS Bio System to the Arc Premier System. The aim is to assess comparability of peak areas, retention times, and recovery of highly sialylated glycans between platforms, demonstrating the Arc Premier’s suitability for biopharmaceutical workflows.

Methodology and Instrumentation

The original 35-minute HILIC gradient was scaled to 51.4 minutes on the Arc Premier System to maintain equivalent linear velocity. An XBridge Premier BEH Amide column (4.6 mm × 150 mm) and Waters RapiFluor-MS Glycan Standard served as the test analytes. Key parameters compared included peak area percent and relative retention time for glycan species across five injections.

Used Instrumentation

• ACQUITY UPLC H-Class PLUS Bio System
• Arc Premier System with MaxPeak High Performance Surfaces
• XBridge Premier BEH Amide Column, 4.6 × 150 mm

Main Results and Discussion

Peak area percentages between the two systems differed by less than 2% for all glycan species, with low-abundance peaks (<1%) varying by under 0.1%. Relative retention times were within ±2 seconds. The Arc Premier System demonstrated excellent precision (RSD <1% for area, <0.1% for retention time) and exhibited up to a threefold improvement in recovery of highly sialylated glycans. This enhancement is attributed to MaxPeak High Performance Surfaces minimizing metal-induced adsorption of negatively charged analytes.

Benefits and Practical Applications

• Seamless migration of HILIC glycan assays between LC platforms with minimal method re-optimization
• Enhanced recovery and peak shape for metal-sensitive, highly sialylated glycans
• High precision in peak area and retention time supporting robust QC and R&D workflows

Future Trends and Opportunities

Future work may explore application of high performance surface technologies to other metal-sensitive analytes and extend these surfaces to multi-omics LC–MS platforms. Integration with automated method transfer tools and expansion into higher throughput formats could further streamline glycan profiling in biopharmaceutical development.

Conclusion

The Arc Premier System successfully delivered comparable performance to the ACQUITY UPLC H-Class PLUS Bio System for HILIC glycan analysis, while significantly improving recovery of highly sialylated species. This demonstrates its capability to support critical analytical methods across the lifecycle of biopharmaceutical products.

Reference

1. M. Lauber et al. Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces. Waters Application Note 720006930EN, 2021.
2. S. Ippoliti, Y. Q. Yu, W. Chen. Improved Recovery and Peak Shape of Sialylated O-glycopeptides of Erythropoietin (EPO) with ACQUITY Premier CSH Column. Waters Application Note 720007227EN, 2021.