Characterization of Forced Degradation Impurities of Glucagon-Like Peptide-1 Agonists by LC/Q-TOF Mass Spectrometry

Significance of the topic

Peptide biotherapeutics such as glucagon-like peptide-1 (GLP-1) agonists play a critical role in diabetes and obesity management. Understanding their stability under stress conditions is essential to ensure safety, efficacy, and consistent quality during development and manufacturing.

Objectives and study overview

This study aimed to characterize forced degradation impurities of three GLP-1 agonists (liraglutide, semaglutide, tirzepatide) under oxidative and alkaline stress using liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF MS). The goal was to identify structural changes and assess method suitability for quality control applications.

Methodology and instrumentation

The analysis employed an Agilent 1290 Infinity II Bio LC system with an AdvanceBio Peptide Mapping column and an Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer. MassHunter data acquisition and BioConfirm software enabled spectral deconvolution. Forced oxidation was induced by incubating peptides at 0.05–2% H2O2 overnight; deamidation stress used pH 8.9 buffer at 37 °C for up to 24 days.

Main results and discussion

Oxidative stress generated mono-, di-, and tri-oxidized species with mass shifts of +16, +32, and +48 Da, respectively. Chromatographic separation resolved isomeric oxidation products, highlighting tryptophan as the primary oxidation site. Extracted ion chromatograms revealed mono-oxidation as the predominant pathway. Alkaline stress showed negligible deamidation over 24 days, reflecting the lower reactivity of glutamine residues.

Benefits and practical applications

• High-resolution identification of peptide impurities supports robust drug characterization.
• Fast method suitable for routine quality control and stability testing.
• Enhanced ability to resolve and quantify isomeric degradation products.

Future trends and applications

Integration of higher-resolution MS instruments and advanced data-processing algorithms may further improve impurity profiling. Expanding stress conditions to include photodegradation and enzymatic cleavage can deepen understanding of biotherapeutic stability.

Conclusion

The LC/Q-TOF MS workflow proved effective for detailed mapping of GLP-1 agonist degradation products under stress. It offers a reliable platform for ensuring the quality and safety of peptide-based therapies.

References

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3. ICH Expert Working Group. Quality of Biotechnological Products: Stability Testing Q5C. ICH, 1995.
4. Bellmaine S. et al. Reactivity and Degradation Products of Tryptophan. Free Radic. Biol. Med. 2020, 160, 696–718.