Workflows for N-Glycan Analysis of Biotherapeutics Using LC/FLD/MS

Importance of N-Glycan Analysis in Biotherapeutics

N-Glycosylation is a critical quality attribute in more than 60 percent of modern biotherapeutic products. Glycan structures on monoclonal antibodies, Fc-fusion proteins and other glycoproteins influence pharmacokinetics, effector functions such as ADCC and CDC, and immunogenicity. Monitoring glycosylation profiles ensures product consistency, safety and efficacy.

Objectives and Overview

This work presents streamlined workflows for released N-glycan analysis of biotherapeutics using liquid chromatography with fluorescence detection and mass spectrometry. Key goals include reducing sample preparation time, improving sensitivity and enabling robust structural confirmation. The study compares three labeling strategies for HILIC-LC/FLD/MS and capillary electrophoresis, and describes a plate-based assay for total sialic acid quantitation.

Methodology and Instrumentation

Sample preparation employs rapid in-solution deglycosylation and labeling with the Gly-X platform. Three dye classes are evaluated:

• InstantPC for HILIC-LC/FLD/MS offering strong fluorescence and MS signal
• 2-AB Express for HILIC-LC/FLD/MS with a well-established fluorescent label
• APTS Express for capillary electrophoresis introducing a negative charge

Separation uses AdvanceBio Glycan Mapping HILIC columns (1.8 and 2.7 μm) on Agilent 1260 and 1290 Infinity II LC systems. Detection combines fluorescence (ex/em settings for each dye) and positive-mode MS on a 6545XT AdvanceBio Q-TOF with MassHunter BioConfirm software. Automation options include the Agilent AssayMAP Bravo liquid handler.

Main Results and Discussion

InstantPC labeling reduced sample prep to under one hour and produced the highest signal in both FLD and MS. HILIC separation achieved baseline resolution of major glycans on monoclonal antibodies such as Rituxan and Fc-fusion proteins like Enbrel. 2-AB Express delivered comparable glycan profiles with a two-hour workflow, suitable for labs maintaining historical data. APTS labeling enabled rapid CE analysis of released glycans. Exoglycosidase sequencing confirmed structural assignments. Total sialic acid quantitation using a plate-based assay provided intra- and inter-operator precision below 5 percent CV across various glycoproteins.

Benefits and Practical Applications

• Reduced preparation time from days to under two hours for multiple samples.
• High sensitivity fluorescence and MS detection for detailed glycan profiling.
• Automation compatibility for high throughput in QC environments.
• Flexible dye selection to match lab infrastructure and historical datasets.
• Plate-based sialic acid assay streamlines monitoring of a key glycan attribute.

Future Trends and Applications

Next-generation glycan analysis will integrate further automation, rapid MS sequencing workflows, expanded glycan libraries including isomer standards for alpha-2,3 and alpha-2,6 sialylation, and enhanced bioinformatics for glycoform quantitation. Growth in biosimilar development will demand even more robust CQA monitoring. Emerging CE-MS coupling and microfluidic platforms may further accelerate glycan characterization.

Conclusion

The presented workflows deliver rapid, reliable and versatile approaches for released N-glycan analysis in biotherapeutic development and QC. Choice of labeling chemistry balances speed, sensitivity and legacy compatibility, while HILIC-LC/FLD/MS and CE provide complementary structural and quantitative data. Plate-based sialic acid quantitation adds a high-throughput tool for monitoring critical glycan features.

Reference

• Jones BioPharm Intl 2017;30:20-25
• Higel et al Eur J Pharm Biopharm 2016;100:94
• Liu J Pharm Sci 2015;104:1866-1884
• ProZyme Technical Note 5994-0999EN
• Agilent Technical Note 5991-8550EN