Confident and sensitive identification of semaglutide degradation products and impurities using a UHPLC-HRAM MS platform

Importance of the Topic

Therapeutic peptides have become a cornerstone in modern pharmaceutical research, with semaglutide representing one of the most successful GLP-1 receptor agonists for diabetes and weight management. Precise characterization of peptide drugs and their degradation products is essential to ensure safety, efficacy, and regulatory compliance. Advanced analytical approaches are required to detect trace-level impurities and structurally similar degradation species that may arise during manufacturing, storage, or transport.

Goals and Overview

This study demonstrates the combined use of a Vanquish Flex UHPLC system, Hypersil GOLD Peptide column, Orbitrap Exploris 240 high-resolution accurate mass (HRAM) spectrometer, and BioPharma Finder software with the Ardia Platform. The objective is to achieve confident and sensitive identification of semaglutide degradation products and related impurities down to 0.005% relative abundance under forced thermal and oxidative stress conditions.

Methodology and Instrumentation

• Forced degradation: semaglutide (1.0 mg/mL) subjected to 60 °C up to 72 h (thermal) or 0.01% H₂O₂ up to 14 h (oxidative).
• UHPLC: Hypersil GOLD Peptide column (150 × 2.1 mm, 1.9 µm) with 0.1% formic acid in water/acetonitrile gradient; 0.4 mL/min at 50 °C; 5 µL injection.
• MS: Orbitrap Exploris 240 with H-ESI, full MS at 120 000 resolution (m/z 200), ddMS2 at 30 000 resolution; positive mode; mass accuracy <6 ppm.
• Software: BioPharma Finder 5.3 for peptide mapping and modifications; Ardia Platform for reporting and visualization.

Key Results and Discussion

The optimized UHPLC method resolved semaglutide full-length peptide (FLP) from multiple degradation peaks using a formic acid­friendly eluent. Thermal stress generated predominantly truncation and deletion products, while oxidative stress yielded single and double oxidized variants at tryptophan (W25). High-quality full MS and ddMS2 spectra provided unambiguous site assignment of modifications, supported by confidence scores of 100 and ASR ~1.1. In total, 23 species above 0.005% were identified, including isobaric co-eluting impurities (e.g., A2 deletion vs. T5 deletion + oxidation) resolved by characteristic fragment ions. Quantitation based on extracted ion chromatograms and fragment-ion distribution confirmed trace-level impurity levels as low as 0.007% relative to FLP.

Benefits and Practical Applications

• Enhanced sensitivity and resolution facilitate detection of low-abundance impurities.
• High mass accuracy and MS2 data secure confident structural elucidation of modifications.
• Streamlined data processing and reporting accelerate QA/QC workflows and generic peptide development.

Future Trends and Potential Applications

Integration of machine-learning algorithms for automated peak annotation and prediction of degradation pathways will further enhance throughput. Expansion of the HRAM platform to glycopeptide mapping, immunogenicity assessment, and broader biopharmaceutical characterization will support the next generation of peptide therapeutics.

Conclusion

The UHPLC-HRAM MS workflow combining a formic acid-compatible peptide column, Orbitrap Exploris 240, and BioPharma Finder software provides a robust, sensitive, and comprehensive solution for semaglutide degradation product identification and quantitation. This approach meets stringent regulatory requirements and strengthens the analytical toolkit for peptide drug development and quality control.

References

1. Wang L, et al. Signal Transduction and Targeted Therapy. 2022;7(1):48.
2. Buntz B. Drug Discovery & Development. 2024.
3. FDA Guidance: ANDAs for Highly Purified Synthetic Peptides. CDER; 2021.
4. Liu A, Tweed J, Wujcik CE. J Chromatogr B. 2009;877(20-21):1873.
5. Maskos Z, et al. Arch Biochem Biophys. 1992;296(2):514.
6. Badgujar D, et al. J Pept Sci. 2025;31(1):e3652.