Analytical Characterization of GLP-1 Agonists: Sequence Confirmation by Q-TOF MS/MS

Importance of the topic

Sequence confirmation of synthetic peptide therapeutics is a core quality attribute in biopharmaceutical development and manufacturing. GLP‑1 receptor agonists and related multi‑agonists are rapidly expanding therapeutic classes with sequence length, noncanonical residues, and lipid conjugations that can strongly influence potency, pharmacokinetics, and safety. High‑resolution tandem mass spectrometry provides the molecular specificity and sensitivity needed to verify primary sequence, detect substitutions or deletions, and characterize site‑specific modifications — all requirements for robust identity testing, batch consistency assessment, and regulatory documentation.

Study objectives and overview

This application note evaluated an LC/Q‑TOF MS/MS workflow to achieve comprehensive sequence confirmation of three representative GLP‑1 agonists: retatrutide, tirzepatide, and liraglutide. The aims were to (1) obtain high‑quality MS/MS fragmentation across full peptide backbones, (2) demonstrate sequence coverage including modified and proline‑rich regions, and (3) assess data interpretation using BioConfirm software for confident peptide identification.

Used instrumentation

Key analytical instruments and software used in the study included:

• Agilent 1290 Infinity II LC system (high‑speed pump, multisampler, multicolumn thermostat)
• Altura Peptide Plus column, 2.1 × 150 mm, 2.7 µm particle size
• Agilent Revident LC/Q‑TOF (G6575A) with dual AJS ESI source
• Agilent MassHunter Data Acquisition for LC/TOF and LC/Q‑TOF (v12.0)
• Agilent MassHunter BioConfirm (v12.1) for sequence assignment and scoring

Methodology

Samples of the three peptide standards were prepared at 2 mg/mL in DMSO and diluted to 1 mg/mL in water prior to LC/MS analysis. The LC method used a reversed‑phase peptide column maintained at 55 °C with 0.1% formic acid aqueous (A) and 0.1% formic acid in acetonitrile (B). A 22‑minute gradient (starting ~20% B and ramping to 80% B) and a 0.4 mL/min flow enabled effective separation. Typical injection volume for MS detection was 0.4 µL.

MS acquisition employed positive‑ion ESI with the Revident Q‑TOF. Important acquisition parameters included a broad m/z range (MS up to ~3,200 m/z), MS/MS range to 3,200 m/z, acquisition rates of ~5 spectra/sec for MS and ~3 spectra/sec for MS/MS, a narrow isolation window (~1.3 m/z), dynamic precursor selection (up to 5 precursors/cycle, active exclusion), and a collision‑energy ramp scaled to m/z. An isotope model optimized for peptides and precursor selection biased toward higher charge states (+2, +3, >+3) supported robust fragmentation.

Main results and discussion

The LC/Q‑TOF MS/MS workflow produced high‑quality, reproducible fragmentation for all three GLP‑1 agonists, achieving full sequence coverage in each case. Key observations:

• Retatrutide (39 residues, multiple noncanonical residues including Aib and α‑methyl‑Leu, and a C20 diacid lipid conjugate) yielded strong precursor ions and comprehensive fragmentation across proline‑rich regions. Observed fragment ions included 37 b‑ions and 38 y‑ions (M+H)+ equivalents, producing a complete fragmentation ladder and a BioConfirm score of 25.0.
• Tirzepatide (39 residues, Aib substitutions, C‑terminal amidation, C20 diacid lipid on Lys) also produced extensive MS/MS coverage with 36 b‑ions and 38 y‑ions identified and 100% sequence confirmation; BioConfirm score was 25.0.
• Liraglutide (31 residues, C16 fatty‑acid conjugation) generated clear MS/MS spectra with 29 b‑ions and 29 y‑ions assigned, providing full sequence coverage and a BioConfirm score of 25.0.

Notably, fragmentation extended across regions adjacent to modified residues and dense proline clusters — areas that commonly challenge collision‑induced dissociation. The datasets showed both complete b and y ion ladders with high signal intensity, enabling unambiguous residue‑level assignments. The reproducible performance and the BioConfirm scoring supported automated, confident sequence verification suitable for QC workflows.

Benefits and practical applications of the method

The demonstrated LC/Q‑TOF MS/MS approach offers several practical advantages for peptide therapeutic characterization and quality control:

• Complete primary sequence confirmation, including identification of noncanonical residues and lipid conjugation sites.
• High mass accuracy and resolving power to distinguish close mass variants and modified species.
• Robust fragmentation across proline‑rich and modified regions that are often problematic for standard CID approaches.
• Efficient data interpretation through integrated software (BioConfirm) that provides scoring and annotated fragment ladders helpful for routine QC and batch release testing.

Future trends and opportunities

Analytical requirements for therapeutic peptides will continue to evolve as sequence complexity and modification diversity increase. Expected trends and opportunities include:

• Expanded adoption of high‑resolution MS/MS workflows for regulatory and release testing of modified peptides and multimers.
• Integration of ion‑mobility separation or alternative fragmentation techniques (e.g., ETD/EThcD) to complement CID for labile modifications and larger peptides.
• Improved informatics and automated reporting pipelines to streamline sequence confirmation, impurity profiling, and comparability studies.
• Application of HRMS‑based workflows to characterize next‑generation multi‑agonists, peptide conjugates, and degradants formed during formulation or storage.

Conclusion

The Agilent Revident LC/Q‑TOF MS/MS workflow demonstrated in this study reliably delivers full sequence coverage and high‑confidence peptide identification for complex GLP‑1 agonists, including peptides with noncanonical residues, proline‑rich segments, and fatty‑acid conjugations. The combination of targeted LC separation, optimized Q‑TOF acquisition, and BioConfirm data analysis supports routine sequence confirmation in development and quality control environments.

References

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2. Melson, E.; et al. What Is the Pipeline for Future Medications for Obesity? International Journal of Obesity (London). 2025, 49(3), 433–451. doi: 10.1038/s41366-024-01473-y