High Throughput Peptide Mapping with the Vanquish UHPLC System and the Q Exactive HF Mass Spectrometer

Importance of the Topic

Monoclonal antibodies (mAbs) are central to biopharmaceutical development and quality control. Fast and accurate peptide mapping is essential to characterize sequence integrity, post-translational modifications (PTMs) and glycosylation patterns across multiple samples in development and production stages.

Objectives and Study Overview

This study evaluates a high-throughput workflow combining Vanquish UHPLC with Q Exactive HF mass spectrometry for rapid peptide mapping of two therapeutic mAbs, rituximab and denosumab. The aim is to achieve full sequence coverage and quantify key PTMs within analysis times ranging from 5 to 30 minutes.

Methodology and Instrumentation

Sample preparation:

• Denaturation in 7 M urea, 50 mM Tris-HCl (pH 8.0).
• Reduction with 5 mM DTT, alkylation with 10 mM IAA.
• Overnight tryptic digestion at 37 °C.

Liquid chromatography–mass spectrometry:

• Thermo Scientific Vanquish UHPLC with 2.1 × 250 mm Acclaim RSLC 120 C18 column.
• Gradient times of 5, 8, 13, 20 and 30 min; flow rates 1.1–0.4 mL/min.
• Q Exactive HF Orbitrap MS with HESI-II source; Full MS/dd-MS2 (Top 5) acquisition.
• Data analysis using Xcalibur, PepFinder and FreeStyle software.

Results and Discussion

All gradients (5–30 min) yielded >99 % sequence coverage for both light and heavy chains of rituximab and denosumab. Ultra-short 5 min runs produced sharp peaks (< 1 s FWHM) and sufficient data points (> 30 scans per peak). Key PTMs—oxidation, deamidation, glycosylation variants and N-terminal pyroglutamate formation—were consistently identified and relatively quantified with low variability across gradients.

Benefits and Practical Applications

• High throughput enables rapid screening during clone selection and process optimization.
• Short gradients reduce solvent consumption and increase sample throughput.
• Robust and reproducible platform supports routine QA/QC workflows.

Future Trends and Possibilities

Advances may include integration of additional proteases, automated sample handling, real-time data interpretation with machine learning, and further miniaturization of LC columns to push analysis times below 5 minutes without loss of information.

Conclusion

The combined Vanquish UHPLC–Q Exactive HF system offers a robust, high-speed peptide mapping solution, delivering complete sequence coverage and reliable PTM quantification in as little as 5 minutes. This platform meets the demands of biopharmaceutical development and quality control.

References

1. Zhang Z. Large-scale identification and quantification of covalent modifications in therapeutic proteins. Anal. Chem. 2009;81:8354–8364.
2. Shah XG, Jiang LG, Chen Z, Zhang Z. LC-MS/MS peptide mapping with automated data processing for routine profiling of N-glycans in immunoglobulins. J. Am. Soc. Mass Spectrom. 2014;25:999–1011.