A Holistic Workflow for Acquisition, Processing, and Reporting Fluorescent-Labeled Glycans Using the Biopharmaceutical Platform Solution With UNIFI

Importance of Glycan Profiling

Accurate characterization of N-linked glycans on biotherapeutic proteins is essential for ensuring safety, efficacy, and regulatory compliance. Fluorescent labeling combined with HILIC UPLC separation and standardized calibration improves reproducibility across instruments, operators, and laboratories.

Study Objectives and Overview

This application demonstrates a holistic workflow for acquisition, processing, and automated reporting of 2-AB labeled released glycans using the Waters Biopharmaceutical Platform Solution with UNIFI Software. The goal is to streamline routine glycan profiling by integrating chromatography, detection, and informatics into a single compliant-ready environment.

Methodology and Instrumentation

The workflow employs hydrophilic interaction chromatography with fluorescent detection and a dextran calibration ladder to assign Glucose Unit values. Key components include

• ACQUITY UPLC H-Class System
• ACQUITY UPLC BEH Glycan Amide Column (1.7 µm, 2.1×150 mm)
• ACQUITY UPLC FLR Detector
• UNIFI Scientific Information System v1.6
• 2-AB Dextran Calibration Ladder
• GlycoWorks Reductive Amination Single Use Sample Preparation Kit

Mobile phases consist of ammonium formate buffer and acetonitrile. The dextran ladder generates a fifth order polynomial calibration curve for retention time normalization, enabling assignment of Glycan Unit values for unknown samples.

Main Results and Discussion

Repeated injections of the dextran ladder demonstrate high retention time precision and calibration reproducibility. Triplicate analysis of a monoclonal antibody glycan mixture yields consistent GU assignments when searched against the integrated Glycobase database. UNIFI review panels allow rapid assessment of calibration curves, peak integration results, and component summary tables. The unified reporting template consolidates sample lists, calibration details, and relative abundance statistics within a single document.

Benefits and Practical Applications

The integrated UNIFI solution reduces manual data handling and method variation, delivering faster turnaround and higher confidence in routine glycan profiling. Centralized instrument control and audit-trail metadata support regulated laboratory environments. Automated reporting templates simplify documentation for quality control, process development, and comparability studies.

Future Trends and Opportunities

Advances in database integration and algorithmic glycan identification will enhance structural annotation and enable high-throughput screening. Deployment of cloud-based informatics and machine learning models may further improve peak assignment accuracy and predictive analytics for bioprocess monitoring. Expansion to additional labeling chemistries and multilevel structural workflows will broaden applicability across emerging biotherapeutics.

Conclusion

The Waters Biopharmaceutical Platform Solution with UNIFI provides a comprehensive, end-to-end workflow for fluorescent-labeled glycan analysis. By unifying separation, detection, calibration, database searching, and reporting in one software environment, the platform supports robust, compliant, and efficient glycan profiling for routine biopharmaceutical applications.

Reference

• Campbell MP; Roylez L; Radcliffe CM; Swek RA; Rudd PM. GlycoBase and autoGU tools for HPLC-based glycan analysis. Bioinformatics. 2008;24(9):1214–1216.