LC-UV-MS Peptide Mapping Development for Easy Transfer to LC-UV QA/QC

Significance of the Topic

Peptide mapping is a critical analytical tool in biopharmaceutical development and quality control. It enables confirmation of the primary sequence of monoclonal antibodies, detection and localization of post translational modifications such as deamidation, oxidation and glycosylation, and supports stability and identity testing in research and QA/QC laboratories.

Objectives and Study Overview

This study demonstrates the development of a reliable LC UV MS peptide mapping workflow using the Thermo Scientific Vanquish Flex UHPLC system coupled to a Q Exactive HF mass spectrometer. The goal is to show efficient method transfer from high resolution LC MS setups to UV based quality control environments without loss of performance.

Methodology and Instrumentation

The monoclonal antibody rituximab was digested with the SMART Digest Kit, applying heat stable immobilized trypsin at 70 C for 75 minutes. Disulfide reduction used TCEP. Peptides were separated on an Acclaim RSLC C18 column (2.1 x 250 mm, 2.2 µm) with a 30 minute gradient at 0.3 mL per minute and 50 C, followed by column wash and re equilibration for a total cycle time of 56 minutes. UV detection at 214 nm (10 Hz) was recorded simultaneously with MS acquisition on a Q Exactive HF using a HESI II source. Detailed HPLC and MS parameters were controlled by Xcalibur software and data were processed with PepFinder version 2.0 for sequence coverage analysis.

Instrumentation Used

• Vanquish Flex UHPLC system with quaternary pump and biocompatible flow path
• Active pre heater and column compartment
• Diode array detector with 10 mm LightPipe cell
• Q Exactive HF Hybrid Quadrupole Orbitrap mass spectrometer
• SMART Digest Kit for rapid tryptic digestion

Key Results and Discussion

Simultaneous UV and total ion chromatograms showed aligned peptide profiles, supporting confident peak assignment. Sequence coverage of 99.2% for both light and heavy chains was achieved, with peptides of varying abundance and missed cleavages mapped across the primary sequence. The SMART Digest workflow provided reproducible digestion. A subset of common modifications including oxidation, deamidation, pyroGln formation and multiple glycoforms at N301 was identified and relatively quantified. Recovery rates were classified as good for abundant peptides and fair for lower level variants, confirming the sensitivity and robustness of the setup.

Benefits and Practical Applications

• The biocompatible flow path and pre pressurization in the Vanquish Flex system ensure exceptional retention time precision for UV based assays
• Simultaneous LC UV MS allows straightforward method translation from discovery environments to routine QC labs
• SMART Digest simplifies sample preparation with high reproducibility and throughput
• High sequence coverage and reliable modification quantification support critical biotherapeutic characterization tasks

Future Trends and Applications

As biotherapeutics grow in complexity, demand will increase for rapid, high precision peptide mapping workflows. Integration of automated sample preparation, more agile column technologies and AI driven data analysis will further streamline QC methods. The combination of robust UHPLC systems with high resolution MS and UV detectors will remain key for advanced protein characterization and regulatory compliance.

Conclusion

The Vanquish Flex UHPLC system paired with the Q Exactive HF mass spectrometer and SMART Digest Kit offers a robust, transferable peptide mapping platform. It combines high sequence coverage, precise retention times and reliable quantification of modifications, facilitating method transfer to UV based QA/QC settings without compromising data quality.

References

1. Thermo Scientific Technical Note 108 Reliable Solvent Mixing in UHPLC Sunnyvale CA 2011
2. Thermo Scientific Oral Technical Presentation 71659 High Throughput Peptide Mapping with the Vanquish UHPLC System and the Q Exactive HF Mass Spectrometer Germering Germany 2015
3. Thermo Scientific Application Note 1132 Reliable Results in Peptide Mapping Using the Vanquish Flex UHPLC System Germering Germany 2015