Performance evaluation of a new reversed-phase column for peptide mapping and monitoring of monoclonal antibodies

Significance of the Topic

Peptide mapping and targeted peptide monitoring are fundamental techniques in the characterization and quality control of monoclonal antibody therapeutics. They enable confirmation of primary amino acid sequence, localization and quantification of post-translational modifications such as deamidation, oxidation and glycosylation, and support regulatory compliance in biopharmaceutical development and manufacturing.

Objectives and Study Overview

This work assesses the performance of the newly introduced Thermo Scientific Hypersil GOLD Peptide C18 column (150 × 2.1 mm, 1.9 µm, 175 Å) for peptide mapping and monitoring of monoclonal antibodies. The study encompasses evaluation of chromatographic reproducibility, resolution of hydrophilic and hydrophobic peptides, and robustness across column lots and individual columns.

Methodology

Sample preparation involved reduction, alkylation and tryptic digestion of: 1) BSA for system performance evaluation (SET), 2) NISTmAb RM 8671 for system suitability testing (SST), 3) rituximab for peptide mapping replicates, and 4) adalimumab for column reproducibility studies. UHPLC-MS/MS peptide mapping employed a 115 min gradient (1 % to 90 % acetonitrile + 0.1 % formic acid) on the Hypersil GOLD Peptide column. UHPLC-UV-MS peptide monitoring used a 45 min gradient (2 % to 90 % B) under similar mobile phases. Data were processed with Thermo Scientific BioPharma Finder and Chromeleon CDS.

Instrument Used

• Thermo Scientific Vanquish Horizon UHPLC system
• Thermo Scientific Vanquish Flex UHPLC system
• Thermo Scientific Orbitrap Exploris 240 mass spectrometer
• Thermo Scientific ISQ EM single quadrupole mass spectrometer
• Hypersil GOLD Peptide C18 column (150 × 2.1 mm, 1.9 µm, 175 Å)

Main Results and Discussion

System performance evaluation and suitability tests demonstrated retention time precision with %RSD between 0.04 % and 0.63 %, and peak area precision below 10 %. Chromatographic resolution for critical deamidation variants of the PENNYK peptide ranged from 1.4 to 4.3, ensuring clear separation of non-modified and modified isoforms. Column lot-to-lot reproducibility showed RT %RSD ≤ 0.6 % and area %RSD ≤ 6.2 % across five injections, while column-to-column precision for glycopeptide quantification remained below 3 % RSD.

Benefits and Practical Applications

The Hypersil GOLD Peptide column delivers high chromatographic resolution and reproducibility required for routine multi-attribute method workflows. Its robust performance in both MS/MS mapping and UV-MS monitoring facilitates reliable detection of low-abundance variants and supports streamlined quality control in biopharmaceutical laboratories.

Future Trends and Potential Applications

Advances in ultra-high resolution columns and integration with automated data analysis will further accelerate peptide mapping and multi-attribute testing for complex biologics. The column’s performance suggests applicability to other therapeutic modalities, including fusion proteins and antibody-drug conjugates, and to high-throughput process monitoring.

Conclusion

The Thermo Scientific Hypersil GOLD Peptide C18 column demonstrates excellent chromatographic performance for peptide mapping and monitoring of monoclonal antibodies, meeting stringent reproducibility and resolution requirements across diverse mAb digests.

Reference

1. Millán-Martín S., et al. Comprehensive multi-attribute method workflow for biotherapeutic characterization and current good manufacturing practices testing. Nat. Protoc. 2023, 18(4), 1056–1089.
2. Thermo Fisher Scientific Application Note 002735. Performance comparison of reversed-phase C18 columns for peptide mapping of monoclonal antibodies, 2024.
3. Thermo Fisher Scientific Application Note 002776. Hypersil GOLD Peptide column for reliable monitoring of glycopeptide variants in monoclonal antibodies by LC-UV-MS analysis, 2024.
4. EDQM, Ph. Eur. Commission adopts harmonised general chapter 2.2.46. Chromatographic separation techniques.