Therapeutic Peptide Analysis of GLP‑1 Agonists

Significance of the Topic

The analysis of therapeutic peptides, such as GLP-1 agonists, is critical for ensuring safety, efficacy, and regulatory compliance in biopharmaceutical development. Robust methods to confirm peptide identity, assess purity, and detect synthesis- or degradation-related impurities are essential for consistent manufacturing, supplier qualification, and quality control.

Objectives and Study Overview

This study evaluates the performance of an Agilent Altura Peptide Plus column coupled to the InfinityLab Pro iQ Plus mass spectrometer for characterizing four GLP-1 analogs—semaglutide, liraglutide, retatrutide, and tirzepatide—sourced from multiple suppliers. Key goals include molecular weight confirmation and comparative impurity profiling to support supplier selection and quality consistency.

Methodology and Instrumentation

Sample Preparation:

• Liraglutide, semaglutide, retatrutide, tirzepatide prepared at 1.0 mg/mL in DMSO or 30% acetonitrile.

Chromatography:

• Column: Agilent Altura Peptide Plus, 2.1×150 mm, 2.7 μm
• Gradient: 0.1% formic acid in water (A) and acetonitrile (B) over 15 min at 0.4 mL/min, 60 °C
• Injection: 5 μL, column oven 10 °C

Mass Spectrometry:

• InfintyLab Pro iQ Plus with Jet Stream ESI, positive mode
• Gas temps: 300 °C (drying), 250 °C (sheath); flows: 11 L/min (drying), 12 L/min (sheath)
• Capillary voltage: 3000 V; scan range m/z 200–3000; scan time 500 ms

Main Results and Discussion

Each peptide yielded a clear total ion chromatogram and deconvoluted mass spectrum.

• Semaglutide: single chromatographic peak and mass match, indicating high purity.
• Liraglutide: main peak plus five low-level impurities separated chromatographically.
• Tirzepatide: distinct main peak with three coeluting impurities detectable in deconvolution.
• Retatrutide: single peak obscuring two coeluting impurity species in the mass spectrum.

Supplier comparison for liraglutide:

• Supplier 2 showed the highest purity with no detectable impurities.
• Supplier 3 exhibited moderate impurity levels.
• Supplier 1 had multiple significant impurity peaks, indicating lower quality.

Tirzepatide sources also differed, with Supplier 1 displaying higher purity than Supplier 2.

Benefits and Practical Applications of the Method

The Altura Peptide Plus column and Pro iQ Plus platform deliver rapid, high-resolution separations and accurate mass confirmation. The workflow supports routine quality control, supplier qualification, and regulatory documentation for peptide therapeutics.

Future Trends and Potential Applications

Emerging directions include integrating high-resolution MS for deeper impurity profiling, automating sample preparation, coupling peptide analytics with biological assays, and employing machine-learning algorithms to predict impurity formation and stability.

Conclusion

This application demonstrates a robust LC–MS approach for comprehensive analysis of GLP-1 agonists. The method reliably confirms molecular weight, distinguishes impurity patterns among peptides and suppliers, and supports quality consistency in biopharmaceutical production.

References

Colalto C. Aspects of Complexity in Quality and Safety Assessment of Peptide Therapeutics and Peptide-Related Impurities: A Regulatory Perspective. Regul. Toxicol. Pharmacol. 2024, 153, 105699.