A NEW LC-MS APPROACH FOR SYNTHETIC PEPTIDE CHARACTERIZATION AND IMPURITY PROFILING

Importance of the Topic

Peptide therapeutics offer targeted treatment with low toxicity and high biological activity. The chemical synthesis of these compounds can introduce trace impurities that compromise safety and efficacy. Comprehensive impurity characterization is essential for process development and regulatory compliance.

Objectives and Study Overview

This work presents a unified LC-HRMS analytical workflow to characterize active pharmaceutical ingredients (APIs) and impurity profiles in synthetic peptides. Two model peptides, Eledoisin and Salmon Calcitonin, were analyzed to demonstrate the method’s capability in impurity detection, identification, and quantification.

Methodology and Instrumentation

• Sample preparation: Stock solutions of Eledoisin and Salmon Calcitonin were prepared at 0.2–2.0 µg/µL.
• Chromatography: ACQUITY UPLC H-Class Bio System with CSH C18 column (1.7 µm, 2.1×100 mm) at 65 °C;
  Mobile phases: 0.1% formic acid in water (A) and acetonitrile (B);
  Gradients tailored for each peptide over 20–30 min.
• Mass spectrometry: Vion IMS QTof with 2.8 kV capillary voltage, cone temperature 50 °C, desolvation at 300 °C, and high-resolution MS/MS acquisition.
• Informatics: UNIFI Scientific Information System 1.9.2 with peptide mapping and screening workflows for automated processing and reporting.

Main Results and Discussion

The peptide mapping workflow identified API sequences and common modifications such as N-terminal pyroglutamate, oxidation, amino acid insertions/deletions, and deprotection-related adducts. High-resolution MS and MS/MS spectra enabled unambiguous impurity assignment. The screening workflow provided relative abundance measurements, with color-flagged pass/fail criteria at 0.05% levels and repeatability (%RSD ~6%).

Benefits and Practical Applications

• Enhanced sensitivity and specificity for low-level impurities compared to optical assays.
• Regulatory compliance ready: validated platform for QA/QC in pharmaceutical environments.
• Automated library building and impurity screening for both linear and cyclic peptide therapeutics.

Future Trends and Applications

Emerging directions include expanding custom impurity libraries, integrating high-throughput screening, leveraging machine learning for spectrum interpretation, and adopting this workflow in broader regulatory frameworks for peptide drug development.

Conclusion

The described LC-HRMS workflow, coupled with UNIFI informatics, offers a robust, compliant-ready solution for comprehensive peptide API and impurity profiling. It improves detection, identification, and quantification of trace impurities, streamlining quality control and process development.

Used Instrumentation

• ACQUITY UPLC H-Class Bio System with Peptide CSH C18 column (1.7 µm, 2.1×100 mm).
• Vion IMS QTof mass spectrometer.
• UNIFI Scientific Information System 1.9.2.

References

• Mason JM. Design and development of peptides and peptide mimetics as antagonists for therapeutic intervention. Future Medicinal Chemistry. 2010;2(12):1813–1822.
• Pernot M, Vanderesse R, Frochot C, Guillemin F, Barberi-Heyob M. Stability of peptides and therapeutic success in cancer. Expert Opinion on Drug Metabolism & Toxicology. 2011;7(7):793–802.