Transferring RapiFluor-MS Labeled N-Glycan HILIC Separations Between UPLC and HPLC

Importance of the Topic

Glycan profiling is critical for the characterization of biotherapeutics, vaccines and diagnostic markers. Hydrophilic interaction liquid chromatography (HILIC) combined with fluorescent and mass-spectrometric detection enables sensitive analysis of released N-glycans. A robust method transfer between ultra-performance liquid chromatography (UPLC) and conventional high-performance liquid chromatography (HPLC) ensures scalability, accessibility and consistent data across laboratories.

Objectives and Study Overview

This work aims to demonstrate seamless transfer of RapiFluor-MS labeled N-glycan separations from UPLC to HPLC platforms. By applying established method-scaling principles, the study evaluates chromatographic performance, quantitation and resource consumption differences between 1.7 μm UPLC columns and 2.5 μm HPLC columns under compatible HILIC conditions.

Methodology and Applied Instrumentation

UPLC system: ACQUITY UPLC H-Class Bio with ACQUITY UPLC Glycan BEH Amide, 130 Å, 1.7 μm, 2.1 × 150 mm column and ACQUITY UPLC FLR detector.
HPLC system: Waters Alliance HPLC with XBridge Glycan BEH Amide XP, 130 Å, 2.5 μm particles configured as 3.0 × 225 mm (150 mm + 75 mm) column and Alliance 2475 FLR detector.
Mobile phase A: 50 mM ammonium formate, pH 4.4; B: acetonitrile. Gradient conditions were calculated using L/dp scaling and adjusted for system dwell volume. RapiFluor-MS Glycan Performance Test Standard at 20 pmol/μL was analyzed with injection volumes of 1.2 μL (UPLC) and 3.7 μL (HPLC). Data were processed using MassLynx 4.1 and Empower Pro 3.0.

Main Results and Discussion

• Chromatographic profiles for 21 major N-glycan peaks were qualitatively comparable when plotted over normalized retention time.
• UPLC achieved a 55 min run with 20 mL mobile phase and 1.2 μL sample, whereas HPLC required 121.3 min, 62 mL mobile phase and 3.7 μL sample.
• Relative retention times matched within 5% for most peaks after dwell volume correction; low-abundance species showed up to 35% deviation.
• Relative peak areas were consistent between platforms, confirming quantitative transferability.

Benefits and Practical Applications

• UPLC offers higher throughput, lower solvent consumption and minimal sample requirements, ideal for high-volume glycan screening.
• HPLC configuration provides a cost-effective option for laboratories lacking UPLC systems and for preparative fraction collection.
• Scalable method transfer maintains data consistency across analytical platforms, supporting regulatory compliance and inter-laboratory studies.

Future Trends and Applications

• Development of advanced HILIC stationary phases optimized for larger-particle HPLC supports narrower peak widths.
• Integration with high-resolution mass spectrometry and automated data analysis pipelines for structural elucidation.
• Application of machine learning models to optimize gradients, minimize run times and predict glycan behavior.
• Expanded use in quality control of biosimilars, vaccine glycoprofiling and clinical biomarker discovery.

Conclusion

Method transfer of RapiFluor-MS labeled glycan separations between UPLC and HPLC platforms is readily achievable by matching stationary phase chemistry and scaling column dimensions (L/dp), flow rates and gradients. UPLC provides marked gains in speed and efficiency, while HPLC compatibility ensures broad accessibility and flexibility for purification workflows.

References

1. Gillece-Castro B, van Tran K, Turner JE, Wheat TE, Diehl DM. N-Linked Glycans of Glycoproteins: A New Column for Improved Resolution. Waters Application Note 720003112en. 2009.
2. Lauber MA, Koza SM, Turner JE, Iraneta PC, Fountain KJ. Amide-Bonded BEH HILIC Columns for High Resolution, HPLC-Compatible Separations of N-Glycans. Waters Application Note 720004857en. 2013.
3. Lauber MA, Brousmiche DW, Hua Z, Koza SM, Guthrie E, Magnelli P, Taron CH, Fountain KJ. Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Novel Fluorescence and MS-Active Labeling Reagent. Waters Application Note 720005275en. 2015.
4. Neue UD, McCabe D, Ramesh V, Pappa H, DeMuth J. Transfer of HPLC procedures to suitable columns of reduced dimensions and particle sizes. Pharmacopeial Forum. 2009;1622–1626.