Biotherapeutic Peptide Mass Confirmation and Impurity Profiling on a SmartMS Enabled BioAccord LC-MS System

Significance of the Topic

The accurate identification and purity assessment of biotherapeutic peptides is crucial during research, development, manufacturing and quality control. High-resolution mass spectrometry complements conventional optical chromatography by resolving co-eluting impurities and providing structural information on modifications and degradation products.

Objectives and Study Overview

This study demonstrates an integrated workflow on the BioAccord LC-MS System equipped with SmartMS and UNIFI software to deliver:

• Accurate mass confirmation of peptide APIs and known variants
• Impurity profiling based on chromatographic separation and MS detection
• Relative quantification of peptides and their impurities

Methodology and Instrumentation

The workflow employs:

• Sample preparation: Liraglutide at 6 mg/mL (rDNA origin)
• Chromatography: ACQUITY UPLC I-Class PLUS, ACQUITY UPLC Peptide CSH C18 column (1.7 µm, 2.1×150 mm), 45 °C, gradient elution with 0.1% formic acid in water/acetonitrile at 0.12 mL/min
• Detection: ACQUITY RDa Mass Detector in positive mode (full scan 50–2000 m/z, capillary 1.50 kV, desolvation 550 °C, fragmentation cone 95–100 V)
• Auxiliary detector: Tunable UV at 215 nm
• Data management: UNIFI Application on waters_connect platform

Main Results and Discussion

Using the peptide mapping workflow, liraglutide and its impurities were chromatographically separated and identified. Impurities included isomeric species, N-terminal truncations and oxidized forms. Fragmentation spectra confirmed sequence and modification sites, such as the palmitic acid attachment at Lys20. Identified impurities were added to a scientific library and monitored via an accurate mass screening workflow. Extracted ion chromatograms and UV/MS integration provided relative abundance data. Automated reporting generated compliance-ready summaries for rapid decision-making.

Benefits and Practical Applications

This integrated approach offers:

• Compliance-ready workflows combining UV and MS for qualitative and quantitative analyses
• SmartMS-guided operation enabling users without prior LC-MS experience to perform routine assays
• Streamlined method development, transfer and product deviation investigations supported by structural MS data

Future Trends and Potential Applications

Emerging developments include expanded scientific libraries for novel and non-natural amino acids, fully automated sample-to-report workflows, deeper proteoform profiling and increased adoption of compact high-resolution MS platforms in regulated laboratory environments.

Conclusion

The BioAccord LC-MS System, combined with UNIFI workflows, provides a unified, user-friendly platform for peptide mass confirmation and impurity profiling, supporting high regulatory standards and accelerating biotherapeutic development.

References

• Prabhala BK, Mirza O, Hojrup P, Hansen PR. Characterization of Synthetic Peptides by Mass Spectrometry. Methods Mol Biol. 2015;348:77–82.
• Zeng K, Geerlof-Vidavisky I, Gucinski A, Jiang X, Boyne MT 2nd. Liquid Chromatography-High Resolution Mass Spectrometry for Peptide Drug Quality Control. AAPS J. 2015;17(3):643–651.