Separation of Deamidated Peptides with an Agilent AdvanceBio Peptide Plus Column

Importance of the Topic

Deamidation of asparagine and glutamine residues is a prevalent post-translational modification that can compromise the stability and efficacy of biopharmaceutical proteins. Accurate detection and quantification of deamidated peptides by LC/MS is complicated by a minimal mass shift (0.984 Da) and potential chromatographic coelution with nondeamidated forms. Optimizing separation is essential for reliable proteomic analysis, quality control, and drug development workflows.

Goals and Overview of the Study

The application note evaluates the performance of a charged-surface C18 column (Agilent AdvanceBio Peptide Plus) in separating site-specific deamidated peptides from their native counterparts, compared to a conventional end-capped C18 peptide mapping column. The study also examines the influence of mobile phase acid modifiers and concentrations on chromatographic resolution.

Methodology and Instrumentation

Sample Preparation and Digestion:

• A monoclonal antibody (mAb) was expressed in CHO cells, purified, and trypsin-digested at pH ~11 for 4 hours at 60 °C to accelerate deamidation.
• Peptides were desalted and prepared in LC/MS-grade solvents.

Liquid Chromatography-Mass Spectrometry:

• LC System: Agilent 1290 Infinity II with binary pump, autosampler, and column compartment.
• Columns Compared:
  
  • AdvanceBio Peptide Mapping, 2.1×150 mm, 2.7 µm superficially porous C18.
  • AdvanceBio Peptide Plus, 2.1×150 mm, 2.7 µm superficially porous C18 with positively charged surface.
• Mobile Phases: A—water + 0.05–0.3% formic acid or 0.1% TFA; B—acetonitrile + matching modifier.
• Gradient: 3% to 40% B over 40 min at 0.4 mL/min, 60 °C column temperature.

Mass Spectrometry:

• Agilent 6546 Q-TOF in positive mode (m/z 300–1700, 5 spectra/s), Jet Stream source.
• Data processed with Agilent MassHunter BioConfirm and Qualitative Analysis software.

Main Results and Discussion

Comparison of Column Selectivity:

• Conventional C18 column showed partial coelution of deamidated variants for three of five test peptides, leading to overlapping mass spectra.
• Charged-surface C18 column fully resolved all deamidated variants later than their unmodified forms, enhancing confidence in identification and quantitation.

Effect of Mobile Phase Modifier Concentration:

• Lowering formic acid from 0.1% to 0.05% increased resolution of deamidated species; raising to 0.3% reduced selectivity.
• Replacing 0.1% formic acid with 0.1% TFA diminished separation performance and increased coelution risk due to stronger ion pairing and suppressed ionization.

Mechanistic Insight:

• The positively charged C18 surface reduces retention of positively charged peptides, and deamidation adds an acidic group, increasing retention differentially for deamidated forms.

Benefits and Practical Applications

• Enhanced chromatographic resolution simplifies manual and automated detection of deamidation by LC/MS.
• Improved quantitation accuracy for quality control in biopharmaceutical development.
• Applicable to other protein modifications where subtle mass shifts and hydrophobicity changes complicate analysis.

Future Trends and Opportunities

• Development of mixed-mode and charged stationary phases for broader PTM separations.
• Integration of optimized columns with high-throughput and automated sample preparation workflows.
• Exploration of alternative volatile modifiers and pH regimes to tune selectivity for challenging peptide variants.

Conclusion

The Agilent AdvanceBio Peptide Plus charged-surface C18 column markedly improves the separation of deamidated peptides from their native forms compared to a standard C18 column. Mobile phase optimization—particularly formic acid concentration—further refines resolution. Adoption of this approach enhances confidence in deamidation analysis for proteomic research and biopharmaceutical quality control.

References

1. Gervais D. Protein Deamidation in Biopharmaceutical Manufacture: Understanding, Control and Impact. J Chem Technol Biotechnol, 2015;91:569–575.
2. Wu L. Quantitation of Chemical Induced Deamidation and Oxidation on Monoclonal Antibodies. Agilent Technologies, 2018.
3. Wang W et al. Quantification and Characterization of Antibody Deamidation by Peptide Mapping with Mass Spectrometry. Int J Mass Spectrom, 2011;312:107–113.
4. Nogueira R, Lämmerhofer M, Lindner W. Alternative HPLC Peptide Separation and Purification Using a Mixed-Mode Reversed-Phase/Weak Anion-Exchange Phase. J Chromatogr A, 2005;1089:158–169.
5. Apffel A et al. Enhanced Sensitivity for Peptide Mapping with ESI LC-MS in the Presence of Signal Suppression by TFA. J Chromatogr A, 1995;712:177–190.