Advancing Chromatographic Performance in Peptide Mapping

Importance of the topic

Peptide mapping is a cornerstone technique in biopharmaceutical development for verifying protein primary structure and detecting post-translational modifications that can impact safety and efficacy. High-resolution liquid chromatography coupled with mass spectrometry (LC/MS) demands inert hardware to minimize peptide interactions with metal surfaces, ensuring accurate quantitation and robust identification of critical quality attributes such as deamidation and oxidation.

Objectives and overview of the study

This study compares the performance of a new inert Agilent Altura ZORBAX Eclipse Plus C18 column against a conventional stainless-steel (SS) ZORBAX RRHD Eclipse Plus C18 column. The goal was to assess improvements in peak shape, reproducibility, and resolution during peptide mapping of synthetic monoclonal antibody (mAb) peptides and trypsin-digested Herceptin, with a focus on challenging hydrophobic and modified peptides.

Methodology and instrumentation

• Sample preparation Herceptin was digested with trypsin at a 50:1 protein-to-enzyme ratio, followed by C18 spin-column cleanup to remove impurities and protect column life.
• Chromatographic system Agilent 1290 Infinity II Bio LC with a high-speed pump, multisampler with thermostat, and multicolumn thermostat.
• Columns Agilent ZORBAX RRHD Eclipse Plus C18 (2.1×150 mm, 1.8 µm) and Agilent Altura ZORBAX Eclipse Plus C18 inert column (2.1×150 mm, 1.8 µm) featuring Ultra Inert technology.
• Mass spectrometer Agilent 6545XT AdvanceBio LC/Q-TOF operated in positive AJS ESI mode, extended dynamic range for MS and Auto MS/MS with a mass range of m/z 100–3,000, 2 GHz acquisition.
• Data processing Agilent MassHunter BioConfirm software version 12.1 with tolerances of ±5 ppm (MS1) and ±20 ppm (MS2), and allowance for common variable modifications including deamidation, glycoforms, oxidation, and pyroGlu.

Main results and discussion

• Chromatographic comparison The Altura inert column produced sharper peaks and higher signal intensity for hydrophobic peptides like VVSVLTVLHQDWLNGK, which exhibited peak broadening and reduced response on SS hardware.
• Reproducibility and stabilization Over 30 injections of a synthetic mAb peptide mix, the inert column stabilized immediately, whereas the SS column required 10–20 injections to achieve comparable peak area recovery for selected peptides.
• Peak symmetry and resolution Tailing factors for challenging peptides averaged 1.3 on the Altura column versus 1.7 on the SS column. Resolution between deamidated and unmodified variants improved significantly, enabling confident PTM assignment.
• Herceptin peptide mapping The inert column delivered 87.03% sequence coverage. Missing peptides were short, hydrophilic segments likely lost during C18 cleanup. MS/MS spectra of deamidated NTAYLQMDSLR confirmed site-specific modification at position 8.
• Critical separation of deamidated species Chromatographic separation of native (655.8318 m/z) and deamidated (656.3221 m/z) forms was essential due to overlapping isotopic distributions, highlighting the need for high-resolution LC to avoid misidentification.

Benefits and practical applications of the method

The Altura ZORBAX Eclipse Plus C18 inert column delivers:

• Enhanced detection of hydrophobic and modified peptides.
• Improved peak shapes and reduced tailing, critical for PTM profiling.
• Immediate reproducibility without extensive column conditioning.
• Better resolution of closely eluting variants, supporting accurate quantitation.
• Compatibility with high-throughput biopharmaceutical workflows to reduce solvent use and run times.

Future trends and possibilities for application

Ongoing advances in inert column technology may enable even steeper gradients and shorter run times without compromising resolution. Integration with next-generation mass spectrometers offering higher scan speeds and enhanced fragmentation methods will further improve coverage of low-abundance and labile modifications. Automation of sample cleanup and inline desalting may preserve hydrophilic peptides and boost overall sequence coverage in complex digests.

Conclusion

The Agilent Altura ZORBAX Eclipse Plus C18 inert column, paired with the 1290 Infinity II Bio LC and 6545XT AdvanceBio LC/Q-TOF, significantly improves chromatographic performance for peptide mapping. It offers superior peak shape, faster stabilization, and finer resolution of PTMs compared to conventional SS hardware, supporting robust identification and quantitation in biopharmaceutical development.

Reference

• Hsiao J Pushing the Boundaries of Chromatographic Separation with Inert HPLC Column Hardware Agilent Technologies application note 5994-8618EN 2025
• Vanluchene H mRNA Mapping with IO-RPLC-MS on a Low-Adsorption Flow Path Agilent Technologies application note 5994-8055EN 2025
• Feith A Robust and Reliable Peptide Mapping Agilent Technologies application note 5994-2718EN 2024
• Sciuto S Peptide Mapping of Trastuzumab Tryptic Digests on an Agilent 6545XT AdvanceBio LC/Q-TOF Agilent Technologies application note 5994-7969EN 2024