LC/MS Characterization of GLP-1 Receptor Agonist Oligomers and Other Elusive Impurities

LC/MS Characterization of GLP-1 Receptor Agonist Oligomers and Other Elusive Impurities — Summary

Significance of the topic:
The physicochemical integrity of peptide therapeutics such as GLP-1 receptor agonists (GLP-1 RAs) is critical for safety, efficacy, and manufacturability. Impurities produced during synthesis, formulation, storage, or forced degradation (e.g., oxidation, oligomerization, adduction) can alter product quality attributes and must be identified and localized. The poster demonstrates a complementary LC/MS workflow that balances rapid impurity prescreening with detailed structural assignment to accelerate impurity characterization during biotherapeutic development.

Objectives and study overview:

• Develop a fast prescreening strategy for peptide impurities using a single-quadrupole detector, then perform targeted in-depth MS/MS characterization on a Q-TOF instrument.
• Characterize oligomerization, isomer-specific behavior, oxidation products, and unexpected modifications (e.g., a +12 Da adduct) in liraglutide samples subjected to forced oxidation and prolonged storage.
• Demonstrate instrument and acquisition strategies (mixed-mode scan, high m/z filtering, all-ions CID, guided de novo sequencing) that reveal structural details otherwise obscured by complex spectra.

Methodology and experimental design:

• Sample preparation: Liraglutide prepared at 10 µM in 15% acetonitrile/0.1% formic acid. Forced oxidation conducted with 0.75% H2O2; aged samples stored at 3 °C for 15 months; fresh controls analyzed same day.
• Screening: Rapid impurity prescreening using mixed-mode acquisition (full scan + SIM) on the InfinityLab Pro iQ Plus single quadrupole to flag oxidation products and other mass variants.
• Detailed characterization: Targeted MS/MS on the 6545XT AdvanceBio LC/Q-TOF. High m/z filtering (quadrupole set to transmit > 1,900 m/z) combined with all-ions CID was used to monitor monomer ejection patterns from oligomers and to minimize overlapping charge-state/isotopic interferences.
• Data processing: OpenLab and MassHunter suites for acquisition and visualization; MassHunter BioConfirm and ExDViewer used for guided de novo sequencing and localization of unexpected mass shifts.

Instrumentation used:

• LC column: Altura PeptidePlus.
• LC system: Agilent 1290 Infinity III Bio LC System.
• Mass detectors: InfinityLab Pro iQ Plus single quadrupole mass detector for prescreening; Agilent 6545XT AdvanceBio LC/Q-TOF for MS/MS and high m/z filtering/all-ions CID.
• Software: OpenLab Acquisition 2.8; MassHunter Acquisition 11.0; MassHunter Qualitative Analysis 12.0; MassHunter BioConfirm 12.1; ExDViewer.

Main results and discussion:

• Isomer- and age-dependent oligomerization: Fresh liraglutide showed rapid oligomer formation for the main form while an isomeric form exhibited minimal oligomerization; after extended storage both forms produced abundant oligomers. Overlapping dimer/tetramer signals complicated direct interpretation without spectral filtering.
• High m/z filter and all-ions CID strategy: Applying a quadrupole high-pass filter (>1,900 m/z) reduced interference from non-oligomeric signals and improved monitoring of monomer ejection into low m/z regions during CID. Monomer ejection kinetics (intensities of 3+ and 2+ monomer charge states vs. collision energy) were similar between isomers, indicating comparable dissociation pathways per charge state.
• Sodium adduction differences: Significant differences in sodiation of ejected monomers were observed between the two forms. At 60 V collision energy, only ~1.8% of liraglutide monomers were sodiated versus ~49% for the isomer, implicating structural differences that influence sodium binding and potentially oligomer stability/pathways.
• Identification of a +12 Da N‑terminal modification: An abundant impurity in aged samples was detected at +12 Da relative to the parent peptide and did not match predicted database entries. Non-targeted deconvolution and generation of amino-acid mass tags localized this addition to the N-terminus, consistent with literature reports linking N-terminal histidine reactivity with excipient-derived carbon additions.
• Oxidation mapping: Forced oxidation produced multiple oxidation isomers with distinct retention times. Mixed-mode SQ prescreening rapidly resolved oxidation complexity; subsequent Q-TOF CID localized a dioxygenation modification to tryptophan at position 25, supported by b/y ion fragments and sequence coverage mapping.

Benefits and practical applications of the method:

• Efficient two-tier workflow: Fast, accessible prescreening with single-quadrupole mixed-mode acquisition reduces the number of samples requiring high-resolution MS/MS, saving time and resources during development and QC investigations.
• Improved oligomer analysis: High m/z filtering and all-ions CID permit clearer observation of monomer ejection and adduction patterns from high-mass oligomers, addressing challenges from overlapping isotope/charge distributions.
• Capability to localize unexpected modifications: Guided de novo sequencing and amino-acid mass tagging allow structural localization of modifications not present in predicted impurity lists—important for risk assessment and formulation troubleshooting.
• Actionable impurity mapping: Combining prescreen and targeted MS/MS supports faster identification of oxidation hotspots and other degradants, informing formulation, storage, and stability strategies.

Future trends and potential applications:

• Broader adoption of mixed acquisition modes on accessible single-quadrupole detectors for routine impurity surveillance in peptide therapeutics.
• Expanded use of high m/z filtering and orthogonal dissociation strategies to dissect complex oligomeric assemblies, including noncovalent aggregates and covalent multimers.
• Integration of advanced de novo sequencing tools and MSn approaches to increase confidence in locating novel or excipient-derived modifications without prior knowledge.
• Application of these workflows to other biotherapeutic classes (e.g., constrained peptides, small proteins) and to regulatory stability studies where comprehensive impurity characterization is required.

Conclusion:
A complementary LC/MS workflow that pairs rapid mixed-mode prescreening on a single-quadrupole detector with targeted, high-resolution Q-TOF MS/MS enables timely and confident characterization of complex peptide impurities. High m/z filtering and all-ions CID improve oligomer analysis, while guided de novo sequencing localizes unexpected modifications such as a +12 Da N-terminal addition. The approach accelerates impurity assignment and provides actionable structural insight for formulation and development decisions.

References:

1. Přáda Brichtová, E.; Edu, I. A.; Li, X.; Becher, F.; Gomes dos Santos, A. L.; Jackson, S. E. Effect of lipidation on the structure, oligomerization, and aggregation of glucagon-like peptide-1. Bioconjugate Chem. 2025, 36, 401–414.
2. Sheikh, A. R.; Vitore, J. G.; Bhalekar, V. S.; Jain, S.; Kukreja, D.; Giri, T.; Sharma, N.; Benival, D.; Shah, R. P. Reactivity of N-terminal histidine of peptides toward excipients and excipient-related impurities: A case study of liraglutide excipient compatibility. J. Pharm. Sci. 2024, 113, 3246–3254.

Additional notes:

• Data processing and sequence localization relied on MassHunter and ExDViewer tools as described in the study.
• Conflict of interest: the author is an employee of the instrumentation vendor; this commercial relationship is disclosed and should be considered when interpreting instrument-specific performance claims.