Comparison of Common Fluorescent Labels for LC/MS Analysis of Released N-Linked Glycans

Importance of the topic

Glycosylation plays a pivotal role in the safety and efficacy of biotherapeutic proteins by influencing their immunogenicity, pharmacokinetics and pharmacodynamics. Precise profiling of N glycan structures is essential for ensuring product consistency and biological performance. However, native glycans exhibit low UV absorption and poor ionization efficiency, necessitating derivatization with fluorescent or MS active tags to enhance detection sensitivity and quantification accuracy.

Objectives and study overview

This study benchmarks five common labeling reagents for released N glycans from the monoclonal antibody MabThera and the Fc fusion protein Enbrel. Key goals are to compare fluorescence intensity, MS signal strength, chromatographic behavior and workflow efficiency of:

• InstantPC (rapid glycosylamine reactive dye)
• Procainamide (reductive amination)
• InstantAB (rapid labeling dye)
• 2 aminobenzamide (2 AB, traditional reductive amination)
• 2 aminobenzoic acid (2 AA, traditional reductive amination)

Methodology and instrumentation

Sample preparation and labeling followed the Agilent GlykoPrep Rapid N glycan protocol on 50 micrograms of each glycoprotein. Excess dye was removed by cleanup cartridges and samples were adjusted to 50 microliters prior to analysis.

• Chromatography: UHPLC HILIC on a 2.1 x 150 mm, 1.7 micrometer Amide column with a gradient of 25 to 38 percent 50 mM ammonium formate (pH 4.4) over 2.5 to 50 minutes at 0.4 mL per minute.
• Fluorescence detection: UHPLC fluorescence detector with appropriate excitation and emission wavelengths for each label.
• Mass spectrometry: Quadrupole time of flight MS in positive electrospray mode, capillary voltage 2.8 kilovolt, cone voltage 30 volt, source 120 °C, desolvation 350 °C, scan range m/z 400 to 2000.

Main results and discussion

InstantPC delivered the highest fluorescence and MS responses, nearly doubling the signal of procainamide and far exceeding that of 2 AB and 2 AA. Labeling order had minimal impact on elution sequence but influenced peak resolution. Notable findings include:

• InstantPC and procainamide provided baseline separation of critical glycan isomers (Man5, G1F variants, A1F isomers) for both MabThera and Enbrel.
• Traditional dyes 2 AB and 2 AA showed lower intensity, poor resolution of low abundance species and required lengthy reductive amination steps.
• InstantPC enabled detection of trace glycans (eg A1F[3] and A1F[6]) by both fluorescence and MS, with strong correlation between relative percent areas across detection modes.
• Procainamide offered improved MS ionization compared to 2 AB and 2 AA but did not resolve all isomeric regions and exhibited minor artifacts.

Benefits and practical applications

InstantPC accelerates sample preparation and enhances glycan detection sensitivity, delivering robust quantitation for quality control and research laboratories. Practical advantages:

• Rapid one minute labeling without drying steps.
• Higher fluorescence signal permits analysis of low abundance and sialylated species.
• Strong MS response supports confident glycoform identification and relative quantification.
• Simplified workflow reduces time to result and labor requirements.

Future trends and opportunities

Advances in glycan labeling and detection will continue to drive deeper characterization of biotherapeutics. Anticipated developments include:

• Integration of high throughput automated sample prep with instant labeling chemistries.
• Enhanced MS fragmentation techniques for detailed structural assignment.
• Use of machine learning to predict glycosylation patterns and quality attributes.
• Expansion to multiomic platforms linking glycan profiles with proteomic and metabolomic data.

Conclusion

Comparative evaluation confirms InstantPC as the superior labeling reagent for HILIC UHPLC FLD MS analysis of released N glycans. It combines rapid reaction kinetics, high sensitivity and effective chromatographic resolution, thus streamlining workflows and enabling detection of low abundance species. This approach supports rigorous glycoanalytics for biopharmaceutical development and quality control.

Reference

1. Liu L Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc fusion proteins Journal of Pharmaceutical Sciences 2015 104 6 1866 1884
2. Anumula KR Advances in fluorescence derivatization methods for high performance liquid chromatographic analysis of glycoprotein carbohydrates Analytical Biochemistry 2006 350 1 1 23
3. Kimzey et al A streamlined workflow for characterization of low abundance glycans on therapeutic proteins ProZyme poster ASMS 2014
4. Aich U et al State of the art technologies for rapid and high throughput sample preparation and analysis of N glycans from antibodies Electrophoresis 2016 37 11 1468 1488
5. Kimzey et al Development of an instant glycan labeling dye for high throughput analysis by mass spectrometry ProZyme poster ASMS 2015
6. US Patent 8445292