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

Importance of the Topic

Monoclonal antibodies represent a rapidly growing class of biotherapeutics requiring rigorous structural characterization throughout development and production. High-throughput peptide mapping by liquid chromatography–mass spectrometry (LC–MS) provides detailed information on amino acid sequence coverage and site-specific modifications such as glycosylation, oxidation, and deamidation. Accelerating such analyses supports efficient quality control, clone selection, and process optimization.

Objectives and Study Overview

This study evaluated a combined UHPLC–Orbitrap platform for fast, sensitive peptide mapping of two commercial mAbs (rituximab and denosumab). Key aims were to compare sequence coverage and modification quantification across gradient lengths ranging from 5 to 30 minutes and to demonstrate the system’s suitability for high-throughput biopharma workflows.

Methodology and Instrumentation

Sample Preparation:

• Denaturation: 7 M urea, 50 mM Tris-HCl pH 8.0 for 30 min
• Reduction: 5 mM DTT, 37 °C, 30 min
• Alkylation: 10 mM IAA, room temperature, 30 min; quenched with 10 mM DTT
• Digestion: Trypsin protease (MS grade), overnight at 37 °C; quench with TFA to pH 3.0

Chromatography and Mass Spectrometry:

• UHPLC System: Thermo Scientific Vanquish with Acclaim RSLC 120 C18 column (2.1 × 250 mm, 2.2 µm)
• Mobile Phases: A – 0.1% FA in water; B – 0.1% FA in 80/20 ACN/water
• Gradient Programs: Five linear ramps (4→55% B) over 5, 8, 13, 20, and 30 min; corresponding flow rates 1.1, 1.0, 0.6, 0.4, and 0.4 mL/min
• Column Temperature: 60 °C; Injection Volume: 2 µL; UV Detection at 214 nm
• Mass Spectrometer: Q Exactive HF Hybrid Quadrupole-Orbitrap with HESI-II source; full MS at 60,000 resolution; Top5 ddMS2 at 15,000 resolution
• Data Acquisition: Xcalibur 3.0 with SII integration; Data Analysis: PepFinder 2.0 and FreeStyle 1.0

Main Results and Discussion

Sequence Coverage:

• Achieved 100% coverage for both heavy and light chains at all gradient lengths, including the 5 min ramp.

Modification Analysis:

• Consistent relative quantification of key PTMs (e.g., N-terminal pyroglutamate, C-terminal lysine clipping, oxidation, deamidation, N-glycosylation variants)
• Standard deviation across gradient lengths averaged 0.19%, indicating robust performance even under ultra-short gradients.

Chromatographic Performance:

• Peak widths at half-maximum averaged 1.4 s in the 5 min gradient, supporting sharp, well-resolved peaks.
• The Orbitrap’s scan speed yielded ~31 data points per 2.4 s peak (6 full MS + 25 MS/MS), ensuring confident identification of low-abundance species.

Benefits and Practical Applications

The demonstrated UHPLC–Orbitrap workflow delivers:

• High throughput: Up to fivefold reduction in analysis time without loss of data quality.
• Comprehensive mapping: Reliable detection and quantification of a broad range of modifications.
• Operational robustness: Stable pressure and reproducibility over extended use.
• Relevance for biopharma: Suitable for routine quality control, clone screening, and comparability studies.

Future Trends and Opportunities

Advances in column technology and mass spectrometer acquisition rates will further reduce analysis times and enhance sensitivity. Integration of automated sample preparation and real-time data processing using machine learning could enable fully automated high-throughput peptide mapping. Expanding workflows to include other biotherapeutic modalities (e.g., ADCs, bispecifics) will broaden the impact of this approach.

Conclusion

This work demonstrates that coupling a biocompatible UHPLC system with a fast-scanning Orbitrap mass spectrometer enables reliable high-throughput peptide mapping of monoclonal antibodies across gradient lengths as short as 5 min. The platform achieves complete sequence coverage and accurate PTM quantification, making it a powerful tool for biopharmaceutical research and quality control.

Reference

1. Zhang Z. Large-scale identification and quantification of covalent modifications in therapeutic proteins. Anal. Chem. 2009, 81, 8354–8364.
2. Shah B., Jiang X.G., Chen L., 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.