Sequence Confirmation of Cagrilintide Using the Agilent 1290 Infinity II Bio LC and 6545XT AdvanceBio LC/Q‑TOF

Significance of the topic

Cagrilintide is a next‑generation peptide therapeutic (≈38 amino acids) developed as a long‑acting amylin analogue for metabolic disease and weight management. Accurate structural characterization of such modified peptides — including intact mass, site‑specific post‑synthetic modifications, and disulfide connectivity — is essential throughout development and quality control to ensure identity, potency, and safety. High‑resolution LC‑MS/MS workflows provide the orthogonal analytical evidence required for confident sequence verification and monitoring of critical quality attributes.

Objectives and study overview

This application study demonstrates a practical LC/Q‑TOF workflow to (1) measure intact mass of native (oxidized) and reduced cagrilintide, (2) confirm the presence and location of a native disulfide bond by comparison of oxidized and reduced forms, and (3) achieve unambiguous peptide sequence confirmation and localization of a lipophilic eicosanedioic acid–γ‑Glu conjugate using high‑resolution MS/MS fragmentation. The goal is to illustrate analytical performance and suitability of the Agilent 1290 Infinity II bio LC coupled to the 6545XT AdvanceBio LC/Q‑TOF for peptide therapeutics characterization.

Methodology

Sample preparation: cagrilintide was supplied at 1.0 mg/mL in water and diluted to 0.2 mg/mL in 0.1% formic acid (mobile phase A) prior to LC‑MS injection.

Chromatography summary (key parameters):

• Column: Agilent Altura Peptide Plus, 2.1 × 150 mm, 2.7 µm.
• Mobile phases: A = 0.1% formic acid in water; B = 0.1% formic acid in acetonitrile.
• Gradient: 30 min total runtime with typical peptide gradient from 20% to 60% B, column temperature 40 °C, flow 0.4 mL/min.
• Injection volume: 1 µL; sample thermostat 10 °C.

MS acquisition summary (key parameters):

• Instrument: Agilent 6545XT AdvanceBio LC/Q‑TOF with dual AJS ESI in positive ion mode.
• Ion source: drying gas 270 °C at 11 L/min; sheath gas 375 °C at 11 L/min; nebulizer 35 psi.
• Voltages: capillary 3,500 V; nozzle 500 V; fragmentor 175 V; skimmer 65 V; Oct RF Vpp 750 V.
• Mass ranges: MS 300–3,200 m/z; MS/MS 50–3,200 m/z. Acquisition rates: 2 spectra/s (MS), 3 spectra/s (MS/MS).
• MS/MS settings: narrow isolation (~1.3 m/z), up to 3 precursors per cycle, precursor threshold 2,000 counts, active exclusion after 2 spectra with release after 0.2 min; isotope model = peptides.

Data processing: Agilent MassHunter (acquisition) and MassHunter BioConfirm were used for deconvolution, fragment assignment, and sequence confirmation.

Used Instrumentation

Key hardware used in the workflow:

• Agilent 1290 Infinity II bio LC system (high‑speed pump G7120A, multisampler G7137A, multicolumn thermostat G7116B).
• Agilent 6545XT AdvanceBio LC/Q‑TOF system (G6549AA) with dual AJS ESI source.
• Altura Peptide Plus analytical column (part number 227215‑903).

Main results and discussion

Intact mass analysis: Both native (oxidized) and chemically reduced forms of cagrilintide produced single, well‑resolved chromatographic peaks and clean charge envelopes (observed charge states: (M+3H)3+, (M+4H)4+, (M+5H)5+). Deconvoluted average masses matched expected values: native ~4,409.0993 Da and reduced ~4,411.1152 Da — a mass increase of approximately 2 Da consistent with reduction of one disulfide bond (addition of two hydrogen atoms). High mass accuracy and narrow isotopic distributions demonstrated instrument performance for intact mass confirmation.

MS/MS sequence confirmation: High‑resolution tandem MS generated extensive b‑ and y‑ion series across the peptide backbone for both oxidized and reduced forms. Fragmentation ladders enabled assignment of contiguous sequence segments and localization of the fatty‑acid conjugate on the peptide sequence. Diagnostic comparison of fragment ions such as b7 and y36 showed the expected +2 Da shift in reduced material, directly supporting disulfide bond assignment and site‑specific connectivity. Fragment ion quality (signal‑to‑noise, mass accuracy) allowed unambiguous identification of key backbone cleavages and conserved fragmentation patterns between states, indicating preserved primary sequence and localized structural change at the disulfide linkage.

Interpretation: Combining intact mass shifts with targeted MS/MS differences provides orthogonal confirmation of disulfide presence and location while preserving full sequence coverage. The workflow distinguishes backbone sequence information from modification‑related mass shifts, enabling confident characterization of complex peptide therapeutics.

Benefits and practical applications

Practical advantages of the described approach include:

• Rapid and accurate intact mass verification for lot release or incoming material testing.
• Site‑specific evidence for disulfide connectivity via comparative oxidized/reduced MS/MS.
• High‑confidence sequence confirmation including localization of lipophilic conjugates or other modifications.
• Compatibility with routine QC labs through standardized LC gradients and robust data processing (MassHunter/BioConfirm) for automated reporting.

Future trends and applications

Expected developments and opportunities building on this workflow:

• Higher throughput and automation: integration with autosamplers, scheduled MS/MS, and automated BioConfirm pipelines for routine QC and stability studies.
• Native and top‑down MS approaches: retaining higher‑order structure information to complement disulfide mapping and conformational studies.
• Ion mobility separation: adding an orthogonal dimension to resolve isobaric or conformer populations and improve characterization of complex PTMs.
• Expanded PTM mapping: workflows tailored to more diverse lipidations, glycosylation, or nonstandard amino acids used in next‑generation peptides.
• Regulatory alignment: validated LC‑MS/MS methods and acceptance criteria supporting CMC documentation and batch release.

Conclusion

The Agilent 1290 Infinity II bio LC coupled to the 6545XT AdvanceBio LC/Q‑TOF delivers a robust, high‑resolution platform for comprehensive characterization of peptide therapeutics such as cagrilintide. By combining accurate intact mass measurement, targeted reduction experiments, and high‑quality MS/MS fragmentation, the workflow enables unambiguous sequence confirmation, disulfide bond mapping, and localization of lipophilic conjugates — capabilities essential for discovery, development, and QC of complex biopharmaceutical peptides.

Reference

1. Lau, D. C. W.; Erichsen, L.; Francisco, A. M.; et al. A Randomized, Controlled Trial of Cagrilintide for Weight Management. New England Journal of Medicine 2021, 385, 228–239.
2. Lau, J.; Bloch, P.; Schaffer, L.; et al. Discovery of the Once‑Weekly Glucagon‑Like Peptide‑1 (GLP‑1) Analogue Semaglutide and Related Amylin Analogues. Journal of Medicinal Chemistry 2015, 58, 7370–7380.
3. Agilent Technologies. Workflow Solutions for Peptide Therapeutics; Application Compendium, publication 5994‑8771EN, 2025.