Integrated Structural and Functional Characterization of a GLP-1 Analogue Using LC/MS and SPR

Significance of the topic

Liraglutide and other GLP-1 receptor agonists are central to modern treatment of metabolic diseases (type 2 diabetes, obesity) and are increasingly explored for broader therapeutic benefits. As next‑generation GLP‑1 analogues incorporate diverse chemical modifications (lipidation, PEGylation, amino‑acid substitutions), early integrated characterization of both chemical integrity and receptor function is essential to guide design, support stability and comparability studies, and de-risk development by linking structural variants to biological activity.

Objectives and study overview

This application note demonstrates a combined LC/MS and digital surface plasmon resonance (digital SPR) workflow to (1) map structural changes and degradation products of a GLP‑1 analogue (liraglutide) and (2) measure how those structural changes affect binding kinetics to the GLP‑1 receptor. The study compares native material, H2O2‑oxidized species, and chymotrypsin‑digested fragments to illustrate cases where structural modifications do or do not alter function.

Methodology

The analytical strategy integrated orthogonal assays: high‑resolution LC/Q‑TOF mass spectrometry for structural profiling and digital SPR for label‑free kinetic binding. Key experimental workflows included:

• Oxidative stress: liraglutide (2.0 mg/mL stock in 30% ACN, diluted to 0.5 mg/mL) exposed to 2.5% H2O2 overnight to generate oxidized variants.
• Proteolytic digestion: chymotrypsin digestion (peptide 200 μg/mL in 100 mM Tris pH 8.0 with 10 mM CaCl2) at 37 °C for 2 h, enzyme:substrate 1:30 (w/w), reaction stopped with formic acid.
• LC/MS peptide mapping: reversed‑phase separations (AdvanceBio Peptide Mapping and Altura Peptide Plus columns) with Q‑TOF acquisition (m/z range up to 1700, positive ESI) and MS/MS confirmation; data processed in MassHunter BioConfirm/Qualitative packages.
• SPR kinetics: amine coupling immobilization of liraglutide on carboxyl sensors, multi‑cycle kinetics (five concentrations generated by automated serial dilution of a 900 nM GLP‑1R stock), data referenced and fitted to a 1:1 Langmuir binding model to extract ka, kd and KD.

Instrumentation used

• Agilent 1290 Infinity II Bio LC system (pump, multisampler, multicolumn thermostat)
• Agilent 6545XT AdvanceBio LC/Q‑TOF mass spectrometer
• AdvanceBio Peptide Mapping column and Altura Peptide Plus column (Agilent)
• Nicoya Digital SPR Alto 16‑channel instrument with Nicosystem Pro software and 16‑channel carboxyl cartridge
• Supporting reagents and consumables: sequencing‑grade chymotrypsin, H2O2, DFA/formic acid, buffers for EDC/NHS amine coupling and regeneration (glycine‑HCl)
• Agilent MassHunter suites for MS data processing

Main results and discussion

Structural findings (LC/MS):

• H2O2 treatment produced multiple oxidized liraglutide species resolved by reversed‑phase LC. Deconvoluted monoisotopic masses showed mass additions consistent with mono (+16 Da), di (+32 Da) and tri (+48 Da) oxidation states. A +14 Da shift consistent with carbonylation was also observed.
• Partial chromatographic separation of isomeric oxidation products (notably tryptophan oxidation isomeric forms) was achieved; MS/MS supported assignments.
• Chymotrypsin digestion generated a set of discrete peptide fragments (designated C1–C5, plus a secondary cleavage product C4*) consistent with expected cleavage at aromatic residues; fragment masses matched theoretical values within a few ppm.

Functional findings (SPR):

• Kinetic analysis returned nearly identical equilibrium dissociation constants for native and oxidized liraglutide: KD ≈ 3.51 nM (native) versus 3.68 nM (oxidized), with overlapping association (ka) and dissociation (kd) rates within experimental error. This indicates that the oxidative modifications observed under these stress conditions did not measurably impair GLP‑1R binding.
• In contrast, chymotrypsin‑digested samples showed no detectable binding to GLP‑1R under the assay conditions, demonstrating complete loss of receptor engagement after backbone cleavage.
• These results illustrate a critical point: some chemical modifications (e.g., certain oxidative changes at tryptophan) can produce detectable structural heterogeneity without functional consequence, while proteolytic fragmentation that disrupts N‑terminal or key structural elements abolishes activity.

Benefits and practical applications

This integrated LC/MS + digital SPR workflow provides multiple practical benefits for biopharma analytics:

• Enables direct correlation between molecular-level modifications and receptor function, improving decision‑making during lead optimization and formulation development.
• Supports forced‑degradation and stability studies by linking degradation profiles to potency risk assessment.
• Improves impurity profiling and comparability assessments by determining which variants require control based on functional impact rather than abundance alone.
• Facilitates early screening of analogues with different lipidation or substitution patterns to prioritize candidates with intact receptor engagement.

Future trends and opportunities

Expected developments and opportunities in structural–functional peptide characterization include:

• Expanded use of orthogonal, higher‑throughput SPR platforms coupled to automated LC/MS workflows for faster candidate triage.
• Integration of native/ intact‑mass MS and top‑down approaches to complement peptide mapping for labile modifications and non‑standard conjugates.
• Improved separation strategies and ion‑mobility‑enabled MS to resolve and assign isomeric oxidation products more confidently.
• Application of advanced data analytics and machine learning to predict which structural variants will alter function and to accelerate SAR interpretation.
• Tighter regulatory expectations for integrated structural‑functional evidence in comparability and stability dossiers for peptide therapeutics.

Conclusion

The combined use of high‑resolution LC/Q‑TOF MS and digital SPR provides a robust, complementary approach to characterize GLP‑1 analogues. The study shows that oxidative variants of liraglutide produced under the tested conditions do not necessarily reduce receptor binding, whereas proteolytic cleavage can abolish activity. Such orthogonal characterization is therefore essential to prioritize control strategies, guide formulation and storage decisions, and support development and comparability activities.

References

1. Müller T D, et al. Glucagon‑Like Peptide 1 (GLP‑1). Mol Metab. 2019;30:72–130.
2. Přáda Brichtová E, Edu I A, Li X, Becher F, Gomes Dos Santos AL, Jackson SE. Effect of Lipidation on the Structure, Oligomerization, and Aggregation of Glucagon‑like Peptide 1. Bioconjug Chem. 2025;36(3):401–414.
3. Suresh Babu CV. Characterization of Forced Degradation Impurities of Glucagon‑Like Peptide‑1 Agonists by LC/Q‑TOF Mass Spectrometry. Agilent Technologies application note 5994‑7794EN. 2025.
4. Minkoff BB, Bruckbauer ST, Sabat G, Cox MM, Sussman MR. Covalent Modification of Amino Acids and Peptides Induced by Ionizing Radiation from an Electron Beam Linear Accelerator Used in Radiotherapy. Radiat Res. 2019;191(5):447–459.
5. Bellmaine S, Schnellbaecher A, Zimmer A. Reactivity and Degradation Products of Tryptophan in Solution and Proteins. Free Radic Biol Med. 2020;160:696–718.
6. Lu X, et al. Effects of Tryptophan‑Selective Lipidated Glucagon‑Like Peptide 1 (GLP‑1) Peptides on the GLP‑1 Receptor. J Endocrinol. 2025;264(3):e240026.
7. Donnelly D. The Structure and Function of the Glucagon‑Like Peptide‑1 Receptor and Its Ligands. Br J Pharmacol. 2012;166(1):27–41.
8. Adelhorst K, Hedegaard BB, Knudsen LB, Kirk O. Structure‑Activity Studies of Glucagon‑Like Peptide‑1. J Biol Chem. 1994;269(9):6275–6278.
9. Babu SCV. Enhanced Peptide Characterization and Stability Assessment Using UV‑Visible Second‑Derivative Spectroscopy on an Agilent Cary 3500 Multicell UV‑Vis Spectrophotometer. Agilent Technologies application note 5994‑8551EN. 2025.