Understanding the effects of column temperature on high resolution glycan separations

Significance of the topic

N-linked glycosylation of therapeutic monoclonal antibodies influences efficacy, biological activity and immunogenicity. Accurate glycan profiling ensures batch-to-batch consistency, supports regulatory compliance and safeguards patient safety.

Goals and overview of study

This application note evaluates the Thermo Scientific Accucore 150 Amide HILIC column for separating 2-AB labeled N-linked glycans released from monoclonal antibodies. It also assesses how column temperature (35 °C, 45 °C, 60 °C) affects resolution and compares performance with a leading 1.7 μm fully porous HILIC column.

Methodology

Glycans were enzymatically released from mAbs and labeled via reductive amination with 2-aminobenzamide (2-AB). Magnetic bead cleanup removed excess dye. UHPLC separation employed a gradient from 80% to 0% acetonitrile in 50 mM ammonium formate (pH 4.4) over a 55 min run, with variable column temperatures to optimize selectivity. Detection combined fluorescence (320 nm ex/420 nm em) and negative-mode high-resolution MS (m/z 600–2000 at 70 000 resolution).

Instrumentation

• Thermo Scientific Vanquish Flex UHPLC system (binary pump, split sampler, fluorescence detector, column compartment)
• Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer
• Applied Biosystems MicroAmp reaction tubes; Invitrogen DynaMag-2 magnet
• Fisher Scientific dry baths; autosampler vial kit

Main results and discussion

Lower column temperatures (35–45 °C) improved resolution of critical isomeric pairs (e.g., FA2(6)G1/FA2(3)G1) achieving R ≈ 1.9 with CV < 2%. Across four antibodies (infliximab, NIST mAb, trastuzumab, hyperglycosylated mAb), the Accucore column consistently resolved hybrid, bisected, fucosylated and afucosylated structures that co-eluted on the competitor column. Minor immunogenic species (α-1,3 galactose, Neu5Gc) were clearly separated and quantified using extracted ion chromatograms with high mass accuracy (± 6 ppm).

Benefits and practical applications

• Enhanced isomer separation enables precise quantitation for quality control and cell line screening.
• Lower operating pressures extend column lifetime and allow flexible temperature control.
• Detailed glycan profiles support regulatory filings and immunogenicity assessment.

Future trends and opportunities

Advances in solid-core HILIC packing and temperature optimization will further refine selectivity. Combining isotopic labeling and automated sample processing can increase throughput. Emerging MS fragmentation methods promise deeper structural insights into complex glycan variants.

Conclusion

The Accucore 150 Amide HILIC column offers superior resolution, reproducibility and lower backpressure compared to fully porous alternatives, making it a powerful tool for high-resolution glycan analysis in therapeutic antibody development and quality control.

Reference

1. Reider B.; Szigeti M.; Guttman A. Evaporative fluorophore labeling of carbohydrates via reductive amination. Talanta. 2018, 185, 365–369.
2. Szabo Z.; Khan S. H.; Widdowson P. J.; Reusch D.; Viner R.; Carillo S.; Huhmer A.; Bones J. A Streamlined Workflow for Twoplexing of N-linked Glycans Using Light (12C6) and Heavy (13C6) Isotopologues of 3-Aminobenzenesulfonic Acid. Anal. Chim. Acta. 2020, 1099, 155–164.
3. Hilliard M.; Alley W.R. Jr; McManus C.A.; Yu Y.Q.; Hallinan S.; Gebler J.; Rudd P.M. Glycan characterization of the NIST RM monoclonal antibody using a total analytical solution: From sample preparation to data analysis. MAbs. 2017, 9(8), 1349–1359.