Enhancing The Quality Of Peptide Mapping Separation For The Analysis Of PTM

Significance of the Topic

Post-translational modifications (PTMs) of therapeutic proteins are critical quality attributes affecting efficacy and safety. Peptide mapping coupled with liquid chromatography and mass spectrometry (LC/MS) is a key method for identifying PTMs but faces separation challenges due to complex peptide mixtures. High-resolution reversed-phase separations are essential for confident PTM identification and quantification.

Study Objectives and Overview

This study evaluated an Agilent AdvanceBio Peptide Plus column, featuring a positively charged superficially porous C18 stationary phase, for enhanced separation of tryptic peptides from a monoclonal antibody (mAb) digest and for improved analysis of PTMs including oxidation, deamidation, and glycosylation.

Methodology

The mAb (trastuzumab) was reduced (DTT), alkylated (IAA), and digested with sequence-grade trypsin overnight. Liquid chromatography employed an Agilent 1290 Infinity II system with 0.1% formic acid mobile phases and a gradient from 3% to 95% acetonitrile over 34 minutes at 0.5 mL/min, column temperature 55 °C. Mass spectrometry used an Agilent 6545XT AdvanceBio LC/Q-TOF in positive ESI mode with extended dynamic range (100–1700 m/z) and data processed by Agilent BioConfirm software.

Instrumentation

• Agilent 1290 Infinity II multisampler (G7167B)
• Agilent 1290 Infinity II high speed pump (G7120A)
• Agilent 1290 Infinity II multicolumn thermostat (G7116B)
• Agilent 6545XT AdvanceBio LC/Q-TOF

Main Results and Discussion

The AdvanceBio Peptide Plus column delivered >99% sequence coverage and high reproducibility across five replicates. Oxidation: The DLTMISR peptide and its oxidized variant were baseline-separated, enabling quantification of 1.6% oxidation. Deamidation: Multiple deamidated forms of NTAYLQMNSLR (isoAsp, Asp) were resolved, allowing precise determination of site-specific deamidation levels. Glycosylation: Glycoforms of the EEEQYNSTR peptide (G0, G1F, G0F, G2F) were separated and quantified (G0 6.1%, G1F 47%, G0F 39%, G2F 7.3%).

Benefits and Practical Applications

Enhanced separation efficiency and peak shape improved PTM detection sensitivity and quantification accuracy. The approach supports robust peptide mapping workflows for quality control of biotherapeutics, facilitating detailed CQA assessment in development and manufacturing.

Future Trends and Potential Applications

Advances may include optimized superficially porous phases, novel ion-pairing agents, microflow LC, and integration with high-throughput MS and data analytics (AI/ML) to further improve PTM profiling and biotherapeutic characterization workflows. Expanding applications to other protein modalities and real-time process monitoring are anticipated.

Conclusion

The Agilent AdvanceBio Peptide Plus column combined with accurate mass LC/Q-TOF analysis offers robust, high-resolution peptide mapping and reliable PTM characterization. This method achieves exceptional sequence coverage and reproducibility, addressing key challenges in biotherapeutic quality assessment.

Reference

• Suresh Babu C.V. LC/MS analysis of peptide mapping with formic acid ion-pairing agent; Application note, Agilent Technologies, Inc. Publication 5991-7979EN, 2017.