Peptide mapping of challenging monoclonal antibodies

Significance of the Topic

Peptide mapping of biotherapeutic monoclonal antibodies supports mandatory characterization of primary structure and critical quality attributes including post translational modifications. Regions containing multiple hydrophobic amino acids often evade traditional tryptic digestion leading to incomplete coverage of complementarity determining regions and risking undetected modifications that may affect product quality.

Objectives and Study Overview

This work evaluates automated sample preparation workflows using magnetic SMART Digest enzyme kits to address gaps in peptide detection for challenging mAb regions. The study compares trypsin and pepsin digest strategies on mAbs with known hydrophobic CDR sequences and tracks critical modifications such as oxidation over a degradation time course.

Methodology

An automated KingFisher Duo Prime system performed protein digestion in 96 well plates with the BindIt 4 software.
Enzymes tested included SMART Digest Trypsin and SMART Digest Pepsin magnetic bulk resin formats.
Post digestion samples were acidified and denatured to preserve hydrophobic peptides prior to analysis.
Peptides were separated on a Vanquish Horizon UHPLC system with an Acclaim VANQUISH C18 column under a 60 min gradient.
Detection and fragmentation employed an Orbitrap Fusion Tribrid high resolution accurate mass spectrometer in positive mode with data dependent EThcD and stepped HCD acquisitions.

Instrumentation Used

• Thermo Scientific Vanquish Horizon UHPLC system with Binary Pump H Split Sampler HT and Column Compartment
• Acclaim VANQUISH C18 2.1 by 250 mm 2.2 micrometer column
• Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer with HESI II source
• Thermo Scientific KingFisher Duo Prime purification system controlled by BindIt software

Main Results and Discussion

Pepsin digestion produced multiple shorter peptides in hydrophobic CDR3 regions where trypsin yielded no detectable fragments. Chromatographic retention and mass spectral quality for pepsin peptides were sufficient for confident identification and quantitation. Comparative analysis on a model mAb demonstrated similar oxidation trends at tryptophan residues using both enzymes despite differing peptide complexity. Automated magnetic bead workflows delivered consistent digestion performance and reduced manual handling variability.

Benefits and Practical Applications

• Enhanced coverage of hydrophobic mAb regions enabling detection of critical CDR modifications
• Automated sample preparation improves reproducibility and throughput
• Pepsin offers an alternative protease when trypsin fails to generate detectable peptides
• Compatible with existing LC MS platforms for high confidence peptide mapping and PTM monitoring

Future Trends and Opportunities

The integration of alternative proteases and advanced automation will drive comprehensive biotherapeutic characterization. Emerging software tools can streamline interpretation of complex peptic peptide maps. Continued development of immobilized enzyme kits and reduced reaction times will support high throughput process development and real time quality control of mAb products.

Conclusion

Combining magnetic SMART Digest pepsin kits with automated KingFisher workflows overcomes limitations of tryptic digestion for hydrophobic antibody regions. The approach delivers improved sequence coverage, reliable PTM quantitation, and consistent sample preparation for robust peptide mapping of challenging monoclonal antibodies.

References

1. U.S. FDA Guidance for Industry Q8 R2 Pharmaceutical Development
2. Haberger et al Assessment of chemical modifications of CDRs in recombinant antibodies mAbs 2014 6 2 327 339
3. Millán Martín et al Automated high throughput workflow for peptide mapping PTM monitoring of monoclonal antibodies Thermo Scientific Application Note 21835 2018
4. Sakamoto Kawakami Sasagawa Prediction of peptide retention times Journal of Chromatography 1988 442 69 79