Comprehensive Characterization of Multiple GLP-1 Analogs

Significance of the topic

Glucagon‐like peptide‐1 (GLP-1) analogs represent an important class of therapeutic peptides used in managing type 2 diabetes and obesity. These molecules feature custom modifications—such as fatty acid attachments and noncanonical amino acids—that impact their stability, pharmacokinetics, and efficacy. Accurate structural characterization of GLP-1 analogs is essential for quality control, impurity profiling, and counterfeit detection in biopharmaceutical development.

Objectives and study overview

This study aims to demonstrate a comprehensive workflow for sequence confirmation and modification mapping of three GLP-1 analogs—liraglutide, semaglutide, and tirzepatide—using an Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer equipped with an ExD cell for electron capture dissociation (ECD). Agilent ExDViewer software is evaluated for its abilities in MS1 deconvolution, targeted MS/MS analysis, and data visualization to streamline peptide characterization.

Methodology and instrumentation

Sample preparation involved diluting each peptide to 10 µM in 15 % acetonitrile with 0.1 % formic acid, followed by mixing equal volumes. Liquid chromatography was performed on an Agilent 1290 Infinity II Bio LC system with an AdvanceBio Peptide Mapping column (2.1 × 150 mm, 2.7 µm) at 60 °C, using a water–acetonitrile gradient (0.1 % formic acid) at 0.4 mL/min. Mass spectrometry employed the Agilent 6545XT LC/Q-TOF with Dual Jet Stream ESI source and an ExD cell for ECD. Parameters included a 2 GHz acquisition range (m/z 120–3200), positive polarity, and optional supplemental collision energy for combined ECD/CID experiments. Data processing used Agilent MassHunter for acquisition and ExDViewer (v4.6.12) for MS1 deconvolution and targeted MS/MS fragment matching, with custom amino acids and modifications defined in the software’s editors.

Main results and discussion

• MS1 deconvolution produced monoisotopic masses for each analog (e.g., semaglutide 4112.12 Da, liraglutide 3751.93 Da, tirzepatide 4837.50 Da), resolving minor salt adducts.
• Electron capture dissociation yielded extensive backbone cleavage, delivering 100 % sequence coverage for the 4+ precursors of all peptides at 2 Hz.
• Side chain fragmentation (w-ions) provided evidence for distinguishing isomeric residues such as aspartate vs. isoaspartate.
• Combining ECD with 35 V supplemental collision energy improved sequence coverage of the less-charged (3+) semaglutide precursor from 57 % to 93 %, without a net loss in fragment ion abundance.
• High-quality modification‐containing fragments (ion score ≥10) were more abundant in ECD spectra than in CID spectra, enhancing confidence in mapping fatty acid linkers on lysine residues.

Benefits and practical applications

• ECD provides a gentle fragmentation approach that preserves labile modifications while delivering rich sequence information.
• ExDViewer’s integrated deconvolution and targeted workflows simplify identification of nonstandard amino acids and custom modifications.
• The combined hardware/software solution accelerates method development by minimizing collision energy optimization.
• High sequence coverage and modification localization support robust quality control in peptide drug development and counter‐fraud analysis.

Future trends and potential applications

• Extending this platform to novel GLP-1 derivative libraries and impurity screening.
• Integration with ion mobility spectrometry for conformer-specific characterization.
• Automation and AI‐driven spectral interpretation to further accelerate peptide analytics.
• Application to other therapeutic peptides and small proteins with complex chemical modifications.

Conclusion

This study establishes a streamlined LC–MS/MS workflow combining ECD on an Agilent 6545XT LC/Q-TOF and ExDViewer software for in-depth structural characterization of GLP-1 analogs. The approach achieves complete sequence coverage, distinguishes isomeric residues, and pinpoints modification sites with high confidence, supporting advanced quality assessment in biopharmaceutical research.

References

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