Peptide Drug Stability Analysis Using Agilent InfinityLab LC/MSD and OpenLab CDS Deconvolution

Significance of the Topic

Therapeutic peptides such as GLP-1 agonists play an increasingly important role in biopharmaceuticals. Due to their complex structure and susceptibility to chemical modifications, peptide drugs can form impurities that may affect safety and efficacy. Regulatory agencies require detailed impurity profiling to minimize immunogenic risks and ensure consistent product quality.

Objectives and Study Overview

This study aims to evaluate a forced degradation workflow for two GLP-1 agonist peptides, liraglutide and semaglutide, under acidic, basic, and oxidative stress. Key goals include separation of degradation products, determination of their nominal masses, and demonstration of a streamlined data analysis using single quadrupole mass spectrometry combined with software deconvolution.

Methodology and Instrumentation

Samples of liraglutide and semaglutide were subjected to the following stress conditions at elevated temperature:

• Acidic hydrolysis (0.1 M HCl)
• Basic hydrolysis (0.1 M NaOH)
• Oxidative degradation (0.2% hydrogen peroxide)

Aliquots were taken at defined time points, neutralized, and analyzed by reversed-phase liquid chromatography in gradient mode. An Agilent 1260 Infinity II Prime Bio LC system with AdvanceBio Peptide Mapping and InfinityLab Poroshell EC-C18 columns provided chromatographic separation. Detection was carried out using Agilent InfinityLab LC/MSD iQ and XT single quadrupole mass spectrometers in positive electrospray mode. Spectral deconvolution for neutral mass assignment was performed with Agilent OpenLab CDS version 2.8.

Main Results and Discussion

Initial chromatograms revealed coelution of some impurities with the main peptide peak. Resolution was enhanced by extending the column length through series connection of 100 and 150 mm Poroshell columns and employing a shallow gradient slope. Forced degradation profiles showed distinct impurity patterns for each stress condition, with acid and oxidative treatments generating the most diverse peaks. Deconvolution successfully converted multiply charged ion envelopes into nominal masses, identifying major degradation products of liraglutide and semaglutide and revealing specific mass differences relative to the parent compounds.

Practical Benefits and Applications

This workflow offers several advantages for peptide drug development and quality control:

• Use of single quadrupole MS reduces operational complexity and cost
• Software deconvolution simplifies mass confirmation without high-resolution instruments
• Flexible column configuration allows rapid optimization of separation
• Applicable for early formulation screening and routine stability testing

Future Trends and Opportunities

Emerging directions include integration of high-resolution MS for detailed structural elucidation, advanced data processing algorithms for automated impurity classification, and online coupling with peptide mapping techniques. Expansion to more complex peptide conjugates and multi peptide formulations will further strengthen the robustness of forced degradation analysis.

Conclusion

The combination of Agilent InfinityLab LC/MSD single quadrupole systems, versatile column configurations, and OpenLab CDS deconvolution provides a user friendly and efficient platform for forced degradation studies of peptide therapeutics. This approach meets regulatory requirements for impurity profiling while maintaining ease of use for late-stage development and routine quality control.

Reference

• FDA Guidance ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs for rDNA Origin, 2021
• Ame S et al Influence of Production Process and Scale on Quality of Polypeptide Drugs A Case Study on GLP-1 Analogs Pharm Res 2020 37 120
• Brian R Jarod G Unit Mass Spectral Deconvolution for Molecular Weight Confirmation of Large Molecules Agilent Technologies Technical Overview 5994-6928EN 2024