High Resolution and Rapid Peptide Mapping of Monoclonal Antibody Using an Agilent 1290 Infi nity UHPLC and an Agilent 6550 iFunnel Q-TOF LC/MS System

Significance of the Topic

Peptide mapping is a cornerstone technique in the characterization of biotherapeutic proteins. By enzymatically digesting a monoclonal antibody into smaller peptides and analyzing them via liquid chromatography coupled with mass spectrometry, researchers gain detailed insights into protein sequence integrity and post-translational modifications (PTMs). Rapid, high-resolution peptide maps are essential for quality control, comparability studies, stability monitoring and regulatory compliance in biopharmaceutical development.

Objectives and Study Overview

This study aimed to demonstrate a fast, high-performance workflow for peptide mapping of an IgG1 monoclonal antibody using:

• An Agilent AdvanceBio superficially porous peptide mapping column on a 1290 Infinity UHPLC
• An Agilent 6550 iFunnel Q-TOF mass spectrometer with MS/MS capability

The goal was to achieve deep sequence coverage and confident PTM identification within a 15-minute LC gradient, comparing performance to a conventional 60-minute run.

Methodology and Instrumentation

Sample Preparation:

• Denaturation with trifluoroethanol and reduction with DTT
• Alkylation using iodoacetamide followed by quenching with additional DTT
• Trypsin digestion overnight at 37 °C

LC Conditions (Agilent 1290 Infinity):

• Column: AdvanceBio Peptide Mapping, 2.1×250 mm, 2.7 µm at 60 °C
• Mobile phases: 0.1 % formic acid in water (A), 90 % acetonitrile + 0.1 % formic acid (B)
• Gradient: 3 %→35 % B in 10 min, ramp to 90 % B by 12 min, re-equilibrate to 3 % B by 15 min
• Flow rate: 0.5 mL/min

MS Conditions (Agilent 6550 iFunnel Q-TOF):

• Positive ion mode with Dual JetStream source
• Mass range: m/z 300–1,700 (MS) and 50–1,700 (MS/MS)
• Collision energies ramped by charge state for optimized fragmentation
• Data processing: Agilent MassHunter Qualitative and BioConfirm software for automated feature extraction, sequence matching, PTM assignment and coverage reporting

Main Results and Discussion

The 15-minute gradient delivered 92.1 % sequence coverage of the IgG1 heavy and light chains, identifying 77 unique peptides including key PTMs (glycosylation, oxidation and deamidation). Although a 60-minute run yielded 95.1 % coverage (82 peptides), the shorter method achieved comparable resolution and peak capacity at a fraction of the analysis time and solvent consumption. A critical example was the light chain peptide spanning residues 48–63, where native, isoaspartic and aspartic forms were baseline-resolved, each showing a +0.98 Da mass shift for the deamidated species. Automated extraction consolidated isotopes, adducts and charge states to quantify modification levels (3.7 % and 13 % for the two deamidated forms).

Benefits and Practical Applications

Implementing this rapid peptide mapping approach offers multiple advantages:

• High throughput: fourfold reduction in gradient time boosts sample throughput
• Robust PTM detection: MS/MS confirmation enhances confidence in modification assignments
• Reduced resource use: lower solvent consumption and instrument occupancy
• Scalable QC: suitable for routine lot release, comparability and stability testing in biopharma labs

Future Trends and Potential Applications

Advances likely to further improve peptide mapping include:

• Next-generation superficially porous and core-shell column chemistries for even faster separations
• Enhanced ion-focusing technologies and higher-resolution mass analyzers to detect low-abundance variants
• Automated multi-attribute monitoring workflows integrating targeted quantitation of multiple PTMs
• Machine learning–driven data processing for accelerated feature detection and annotation

Conclusion

The integration of an Agilent 1290 Infinity UHPLC with a superficially porous peptide mapping column and an Agilent 6550 iFunnel Q-TOF mass spectrometer achieves rapid, high-coverage peptide maps of monoclonal antibodies. This workflow balances speed, resolution and sensitivity, enabling confident PTM identification within a 15-minute analysis—supporting efficient quality control and in-depth characterization of biotherapeutics.