Workflows for GLP-1 Receptor Agonists and Therapeutic Peptides: Identity, Impurity, Bioanalysis, and Stability Testing

Significance of the topic

Peptide therapeutics — notably GLP-1 receptor agonists (GLP-1 RAs) — are a rapidly growing drug class combining the advantages of small molecules and biologics: tunable PK/PD, high specificity, generally low immunogenicity, and predictable catabolism to amino acids. Their success in metabolic disease and obesity has driven high demand and increasingly complex molecule designs (lipidation, spacers, PEGylation, co-agonists). These advances impose stringent analytical requirements across the product lifecycle: nondestructive raw-material identity checks, orthogonal impurity profiling, sensitive bioanalysis for PK/PD, and stability/forced-degradation characterization that links chemical changes to biological function. The compendium of Agilent application notes presents integrated workflows addressing these needs with practical, QC‑compatible solutions.

Objectives and study overview

• Provide practical, orthogonal analytical workflows to verify identity of peptide raw materials, resolve and identify product-related impurities, quantify therapeutic peptides in biological matrices, and assess stability/degradation.
• Demonstrate specific implementations: through-container Raman identification of Fmoc-protected amino acids; HILIC-MS impurity profiling for Exenatide, Semaglutide, and Tirzepatide; HILIC with DAD/ELSD to profile excipients and actives; automated LC/MS bioanalysis of semaglutide and tirzepatide using AssayMAP cleanup and triple-quadrupole detection; UV-Vis second-derivative for oxidation monitoring; single-quadrupole and Q-TOF workflows for stability/impurity mapping; and an integrated LC/MS + SPR workflow to relate structural changes to receptor binding for liraglutide.

Methodology

• Raw-material ID: Spatially offset Raman spectroscopy (SORS) handheld system (Agilent Vaya) to identify Fmoc-protected amino acids through amber and HDPE containers without opening, preserving sensitive reagents.
• Impurity profiling (purity and formulation analysis): Hydrophilic interaction liquid chromatography (HILIC-Z stationary phases) coupled to UV/DAD, ELSD and MS (single-quadrupole InfinityLab LC/MSD iQ and Pro iQ Plus). Conditions used ammonium formate mobile phases, gradients tailored for peptide retention, and low-adsorption (Ultra Inert) flow paths to avoid metal-related adsorption and improve phosphate/excipient recovery.
• Bioanalysis: Automated sample cleanup using AssayMAP Bravo with RP-S cartridges following protein precipitation (ACN:MeOH 1:1); LC separation on peptide columns (AdvanceBio Peptide Mapping); quantitation by Agilent 6495D triple-quadrupole in MRM mode. LLOQs and calibration strategies were validated with QC panels and inter/intraday runs.
• Stability and structural characterization: Forced-oxidation (H2O2), pH storage studies, and chymotrypsin digestion. Detection by high-resolution LC/Q-TOF (6545XT) for deconvoluted masses and site-specific MS/MS; second-derivative UV-Vis spectroscopy on Cary 3500 Multicell to enhance spectral resolution of aromatic residues and detect subtle oxidation; single-quadrupole Pro iQ Plus for sensitive routine impurity surveillance and OpenLab CDS deconvolution.
• Functionality testing: Digital surface plasmon resonance (Nicoya digital SPR) to measure GLP-1 receptor (GLP-1R) binding kinetics (multi-cycle kinetics) and directly correlate structural modifications with receptor engagement.

Instrumentation used

• Agilent Vaya handheld Raman (SORS) for through-container raw-material verification.
• Agilent 1290 Infinity III / Infinity II Bio LC systems; Altura and Poroshell HILIC-Z columns (Ultra Inert) and AdvanceBio peptide columns for LC separations.
• Mass spectrometers: Agilent InfinityLab LC/MSD iQ (single quadrupole), Agilent Pro iQ Plus single-quadrupole LC/MS, Agilent 6545XT AdvanceBio LC/Q‑TOF, and Agilent 6495D triple-quadrupole (LC/TQ) for quantitative MRM assays.
• AssayMAP Bravo automated sample-prep platform with RP‑S cartridges for high-throughput cleanup and enrichment.
• Detectors: Diode array (DAD), evaporative light scattering detector (ELSD), Agilent Cary 3500 Multicell UV‑Vis spectrophotometer for second-derivative spectra.
• Nicoya Digital SPR (16-channel Alto cartridge) for label-free kinetic binding assays.

Main results and discussion

• Raw-material verification: The Vaya SORS system reliably identified and discriminated Fmoc‑protected amino acids through HDPE and amber containers in under 40 seconds, leveraging Fmoc-specific Raman features and eliminating the need to open light- or air-sensitive inventories.
• HILIC as orthogonal separation: HILIC-Z provided complementary selectivity to RPLC, separating GLP‑1 RAs and revealing impurities not easily resolved by RPLC. When coupled to single‑quadrupole MS it enabled rapid MW confirmation of eluting peaks and helped detect low‑level species.
• Impurity identification examples: Tirzepatide contained a +163 Da impurity (identified as Tyr addition) at ~1.1% UV level; Semaglutide exhibited low‑level stereoisomeric (isomer) impurities and species corresponding to His-cyclization (+12 Da) and truncations (–222 Da); Exenatide oxidation (+16 Da) produced partially resolved Met sulfoxide diastereomers on HILIC.
• Importance of low-adsorption flow paths: Replacing stainless‑steel flow components with Ultra Inert/biocompatible hardware markedly improved peak shape, recovery and precision for phosphate and several peptides, and enabled detection of minor oxidized species that were obscured on metal flow paths.
• Automated bioanalysis: AssayMAP RP‑S cartridges enhanced semaglutide assay sensitivity ~5× over solvent precipitation alone; semaglutide quantitation achieved LLOQ 0.2 ng/mL (100 μL plasma) and linearity to 1,000 ng/mL. Tirzepatide quantitation reached LLOQ 0.05 ng/mL with the same platform, both meeting regulatory precision/accuracy criteria across QC levels.
• Stability mapping and spectral tools: Second‑derivative UV-Vis on Cary 3500 resolved overlapping aromatic bands and sensitively reported tryptophan oxidation. LC/Q-TOF and Pro iQ Plus deconvolution detected mono-, di-, and tri‑oxidized products (+16, +32 etc.), unknown mass shifts, and traced their evolution under different pH/storage conditions — tirzepatide showed greater oxidation at pH 5 even at 5 °C.
• Structure–function linkage: Integrated LC/MS and digital SPR for liraglutide revealed that oxidative modifications did not measurably change GLP‑1R affinity (KD ~3.5 nM native vs oxidized), whereas proteolytic cleavage (chymotrypsin) abolished binding. This underscores that some chemical modifications preserve function while backbone integrity is critical.

Benefits and practical applications

• Nondestructive raw-material verification reduces contamination risk and preserves shelf life of light/air-sensitive reagents used in SPPS.
• HILIC-MS workflows add orthogonal selectivity to RPLC, improving impurity coverage especially for polar, charged, and fatty-acid-modified peptides; DAD+ELSD permits simultaneous monitoring of active ingredient and excipients in drug products.
• Automated AssayMAP sample cleanup combined with LC/TQ MRM enables antibody-free, high-sensitivity PK assays suitable for discovery and clinical support, with fast method development and high throughput.
• Low-adsorption hardware (Ultra Inert) and appropriate surface chemistry reduce analyte losses and improve detection of labile or metal-interacting species—important for robust QC assays.
• Combining high-resolution MS, spectral deconvolution, derivative UV spectroscopy, and label-free SPR provides a comprehensive characterization toolbox that links chemical changes to biological activity, informing formulation, comparability, and stability decisions.

Future trends and possibilities

• Broader adoption of orthogonal and multimodal analytics (HILIC + RPLC, LC‑MS, UV‑derivative, SPR) in regulated QC to meet increasingly complex peptide modifications.
• Greater automation and sample‑prep standardization (robotic platforms, cartridge-based enrichment) to support higher throughput PK/TK studies and early candidate triage.
• Integration of compact, robust MS platforms (single-quadrupole and stacked modules) into routine QC labs for cost-effective impurity surveillance and in-process control.
• Use of label-free, multiplexed binding platforms (digital SPR) for comparability and functional QC, combined with MS mapping to identify which modifications affect potency or safety.
• Application of advanced data processing (automated deconvolution, machine learning) to accelerate spectral interpretation and impurity annotation across large datasets.

Conclusion

The provided Agilent workflows illustrate a coherent, practical analytical strategy for modern peptide therapeutics: preserve material integrity via through-container Raman screening; expand impurity coverage using orthogonal HILIC separations on low-adsorption hardware; employ targeted, automated LC/MS quantitation for high-sensitivity bioanalysis without reliance on antibodies; and combine high-resolution MS, UV-derivative spectroscopy, and label-free SPR to relate chemical modifications to biological function. Together these methods strengthen development, QC/QA and regulatory readiness for complex GLP‑1 analogs and next-generation peptide medicines.

Reference

1. Prullière F.; Welsby C. Differentiating Biopharmaceutical Raw Materials Using Spatially Offset Raman Spectroscopy. Agilent Technologies application note, publication 5991-2013EN, 2021.
2. Alvarez P.; Lecluyse C.; Vandendriessche I.; Sandra P.; Sandra K.; Schneider S.; Huber U. HILIC Analysis of GLP‑1 Receptor Agonists Using an Agilent 1290 Infinity III Bio LC with DAD and ELSD. Agilent Technologies application note 5994-8308EN, 2025.
3. McCalley D. V. Understanding and Manipulating the Separation in Hydrophilic Interaction Liquid Chromatography. J. Chromatogr. A 2017, 1523, 49–71.
4. Guimaraes G. J.; Bartlett M. G. Managing Nonspecific Adsorption to Liquid Chromatography Hardware: a Review. Anal. Chim. Acta 2023, 1250, 340994.
5. Agilent Application Note: Rapid Testing of Biopharmaceutical Solvents; Vaya Handheld Raman Spectrometer resources (Agilent literature).
6. Ozempic (semaglutide) prescribing information and product labeling; DailyMed 2021; Rybelsus & Wegovy labeling 2021–2022.
7. Mounjaro / Zepbound (tirzepatide) product labeling; DailyMed 2022–2023.
8. Badgujar D.; Bawake S.; Sharma N. Identification and Characterization of Major Degradation Products of Liraglutide Using LC‑HRMS. J. Pept. Sci. 2025.
9. Davidson M. H. Cardiovascular Effects of GLP‑1 Agonists. Am. J. Cardiol. 2011, 108 (3 Suppl), 33B–41B.
10. Additional Agilent application notes cited within the compendium (Cary 3500 UV-Vis second-derivative, Pro iQ Plus application notes, LC/MS workflows) and journal references cited therein.