Fast and Efficient Peptide Mapping of a Monoclonal Antibody (mAb): UHPLC Performance with Superficially Porous Particles

Importance of the Topic

The comprehensive mapping of peptides in monoclonal antibodies is essential for the characterization of biotherapeutic products. Reversed-phase peptide mapping coupled with mass spectrometry provides critical information on sequence variants, post‐translational modifications, and overall product quality.

Objectives and Study Overview

This application note evaluates the performance of a 2.7 μm superficially porous AdvanceBio Peptide Mapping column on traditional LC systems. The study aims to reduce peptide mapping analysis time, maintain high resolution, and compare performance against a sub-2 μm UHPLC peptide mapping column.

Methodology and Used Instrumentation

The workflow included denaturation, reduction, alkylation, and tryptic digestion of an IgG1 mAb. Key steps:

• Denaturation and reduction using DTT and trifluoroethanol at 60 °C
• Alkylation with iodoacetamide and subsequent quenching
• Trypsin digestion at 37 °C for 20 h
• Quenching with formic acid

Liquid chromatography was performed on an Agilent 1290 Infinity LC System with AdvanceBio Peptide Mapping columns (2.1×100 mm and 2.1×150 mm), using water/0.1% formic acid (A) and acetonitrile/0.1% formic acid (B). Detection by UV (215/220 nm) and MS using an Agilent 6530 Accurate-Mass Q-TOF with dual jet stream ESI.

Main Results and Discussion

The superficially porous column achieved a full peptide map in 14 minutes at 433 bar, compared to a 75-minute run at 211 bar, without loss of peak capacity (57 vs. 59 peaks). LC/MS comparisons showed sequence coverages of 99.63% (14 min) vs. 99.84% (40 min). Deamidated forms of a key heavy chain peptide were fully resolved in both runs, and MS/MS spectra confirmed PTMs. Direct comparison to a sub-2 μm UHPLC column demonstrated similar peptide identifications (76 peptides) but required higher pressure (700 bar) to match performance.

Benefits and Practical Applications

• Rapid peptide mapping on standard HPLC instruments
• High resolution and sensitivity for PTM profiling
• Reduced analysis time improves laboratory throughput
• Lower pressure operation enhances system flexibility

Future Trends and Opportunities

Superficially porous particle technology may extend to other biotherapeutic workflows, enabling even faster separations. Integration with automated sample preparation and high-throughput screening, as well as coupling to next-generation mass spectrometers, will further advance peptide mapping capabilities.

Conclusion

The AdvanceBio Peptide Mapping column delivers UHPLC-like speed and resolution at conventional LC pressures, providing robust and efficient peptide mapping for monoclonal antibodies. It supports high sequence coverage and confident PTM characterization while increasing laboratory productivity.

References

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