Denaturing SEC-MS Analysis of High Molecular Weight Impurities in the GLP-1a Lipopeptides Semaglutide and Tirzepatide

Significance of the topic

The development and quality control of therapeutic lipopeptide GLP-1 analogs such as semaglutide and tirzepatide require sensitive analytical methods to detect and characterize high molecular weight species (HMWS). HMWS can arise from non-covalent self-association, truncation, oxidation or covalent crosslinking and may impact safety, efficacy and formulation stability. Denaturing size-exclusion chromatography coupled to high-resolution mass spectrometry (dSEC-HRMS) delivers both separation of size variants under denaturing conditions and intact mass information, making it a valuable complementary tool to SEC-MALS and other orthogonal techniques for early-stage and release testing of peptide therapeutics.

Objectives and study overview

This application note describes a dSEC-HRMS workflow for detecting and characterizing low-abundance, non-dissociable HMWS in semaglutide and tirzepatide formulations. Key aims were to:

• Resolve HMWS under denaturing SEC conditions,
• Obtain intact high-resolution masses for monomer and HMWS peaks, and
• Use UV A280 detection to provide relative abundance estimates and to evaluate monomer linearity for concentration assessment.

Samples analyzed were commercially formulated products (semaglutide 1.34 mg/mL and tirzepatide 10 mg/mL) tested past expiry to ensure detection of impurity species.

Methodology

The separation used a highly denaturing mobile phase and a small-pore SEC column optimized for peptide/lipopeptide analysis. Major experimental conditions:

• Column: ACQUITY Premier SEC 125 Å, 1.7 µm, 4.6 x 150 mm,
• Mobile phase: 0.05% trifluoroacetic acid (TFA) in 55% acetonitrile,
• Flow rate: 0.20 mL/min, column temperature: 45 °C,
• UV detection: TUV at 280 nm monitored at 5 Hz for relative quantitation,
• MS detection: electrospray ionization (ESI) to a quadrupole time-of-flight (Q-ToF) mass analyzer, scanning nominal m/z 500–3500 with high-resolution deconvolution of intact masses.

Denaturing conditions are intended to disrupt non-covalent assemblies; species that remain as discrete HMWS under these conditions were described as non-dissociable but not definitively assigned as covalent without further structural work.

Instrumentation used

The analyses were performed on an ACQUITY UPLC system with an ACQUITY Premier SEC 125 Å, 1.7 µm 4.6 x 150 mm column coupled to a Xevo G3 Q-ToF mass spectrometer using ESI. UV detection at 280 nm (TUV) provided simultaneous optical quantitation. Data acquisition and processing used UNIFI and waters_connect informatics.

Main results and discussion

Summary of key findings:

• Monomer masses measured for semaglutide and tirzepatide matched predicted intact masses, confirming method suitability for intact peptide analysis.
• Semaglutide displayed two HMWS peaks interpreted as a dimer (HMWS1) and a trimer (HMWS2) by retention time. Average relative abundances by A280 were ~2.6% for HMWS1 and ~0.17% for HMWS2 across tested injection loads.
• Observed intact masses for semaglutide HMWS peaks were generally consistent with multimer assignments but showed notable mass deltas. One trimer-related species (HMWS2-1) was ~691.5 Da lighter than the expected trimer mass, indicating a significantly truncated peptide rather than a simple non-covalent trimer of full-length monomer.
• Tirzepatide exhibited two HMWS peaks (HMWS1a and HMWS1b) eluting in the dimer region with very low relative abundances (~0.06–0.07% each). The deconvoluted masses suggested dimeric forms but again with substantial mass differences from the predicted intact dimer.
• A prominent truncated tirzepatide-related species (HMWS1a-1, ~7903.16 Da) was ~1723.9 Da smaller than the expected dimer mass, implying cleavage or processing to form a truncated peptide that participates in higher molecular weight species.

Interpretation and limitations:

• The use of denaturing mobile phase (high organic, TFA, elevated temperature) reduces non-covalent interactions; HMWS that persist are considered non-dissociable under the applied conditions, but this does not prove covalent linkage.
• Small mass shifts consistent with simple crosslinks between aromatic residues (e.g., W–W or W–Y) would produce very small mass differences (~−2 Da), which were not observed. Some observed mass differences are consistent with truncation and/or oxidative modifications but definitive structural assignment requires orthogonal experiments (MS/MS, bottom-up proteolysis, targeted fragmentation, or other chemical analyses).
• The method delivered high-quality HRMS spectra for low-abundance HMWS, but additional characterization is necessary to determine the precise chemical nature of the variants.

Benefits and practical applications

This dSEC-HRMS approach offers several practical advantages for peptide therapeutic development and QC:

• Sensitive detection and intact-mass characterization of low-abundance HMWS (sub-percent levels),
• Simultaneous optical quantitation (A280) enabling rapid relative abundance estimates and potential monomer concentration assessment when UV response is linear,
• Compatibility with formulated, high-concentration samples without the need for extensive sample cleanup,
• Complementary information to SEC-MALS and native MS approaches—allowing discrimination between size-based separation and intact-mass identification.

Use cases include stability and forced-degradation studies, formulation screening, lot release impurity profiling, and troubleshooting of aggregation or truncation pathways during manufacturing.

Future trends and potential applications

To extend and refine HMWS characterization for lipopeptides and similar therapeutics, likely developments include:

• Integration of targeted MS/MS and top-down fragmentation to localize truncation sites and modification chemistries,
• Combined workflows with SEC-MALS or native MS to resolve stoichiometry and molecular weight under non-denaturing conditions,
• Use of ion-mobility separation to add conformational resolution of co-eluting species,
• Higher-resolution mass analyzers and improved deconvolution algorithms for complex heterogeneous species,
• Standardized stability-indicating dSEC-HRMS methods for regulatory submissions and comparability studies, and automated informatics pipelines to integrate UV, MS intact-mass and MS/MS data for streamlined characterization.

Conclusion

The reported dSEC-HRMS method using an ACQUITY Premier SEC 125 Å column and a Xevo G3 Q-ToF produced high-quality intact-mass data and UV-based relative quantitation for low-abundance, non-dissociable HMWS in semaglutide and tirzepatide formulations. The method resolved putative dimeric and trimeric species and revealed truncated peptide species contributing to the observed HMWS. While the approach cannot alone determine covalent linkage or precise structural modifications, it is a powerful screening and characterization tool that complements SEC-MALS and targeted MS/MS workflows for comprehensive impurity profiling of lipopeptide therapeutics.

References

1. Zhang X, Kenrick S, Chen M. Identification of GLP-1 analog oligomeric states using SEC-MALS. Waters Application AN1901, 2025.

2. Nugrahadi PP, Hinrichs WL, Frijlink HW, Schöneich C, Avanti C. Designing Formulation Strategies for Enhanced Stability of Therapeutic Peptides in Aqueous Solutions: A Review. Pharmaceutics. 2023;15(3):935.

3. Engelund DK, Jensen SS, Lundqvist J. Stable Semaglutide Compositions and Uses Thereof. U.S. Patent Application 17/288,128, 2021.

4. Wuthrich P, Koza SM, Shiner S. Advancing Analysis of Covalent High Molecular Weight Insulin With ACQUITY Premier SEC 125 Å Columns. Waters Application Note. 2025.

5. Bellmaine S, Schnellbaecher A, Zimmer A. Reactivity and Degradation Products of Tryptophan in Solution and Proteins. Free Radical Biology and Medicine. 2020;160:696-718.