Rapid Confirmation of GLP-1 Analog (Liraglutide) Using Agilent InfinityLab LC/MSD iQ

Significance of the topic

Peptide therapeutics such as liraglutide are key in managing type 2 diabetes and related metabolic disorders. Ensuring the integrity and purity of these biopharmaceuticals through rapid molecular weight confirmation is critical for regulatory approval, quality control, and effective drug development.

Objectives and study overview

This study aims to develop a rapid method for intact mass confirmation of liraglutide using the Agilent InfinityLab LC/MSD iQ system. It evaluates different reversed-phase columns and acidic modifiers to optimize sensitivity, resolution, and compatibility with mass spectrometry.

Methodology

Analyses were performed on liraglutide solutions at 1 mg/mL and 100 µg/mL, using a flexible liquid chromatography setup to screen three column chemistries (AdvanceBio Peptide Mapping, Peptide Plus, Poroshell Aq-C18) and three acidic modifiers (0.1% formic acid, trifluoroacetic acid, difluoroacetic acid). Gradient conditions were optimized for each combination to assess peak shape, resolution, and MS response.

Instrumentation used

• Agilent 1260 Infinity II Bio Prime LC system with flexible pump, multisampler, multicolumn thermostat, and diode array detector
• Agilent InfinityLab Quick Change Valve for column switching
• Agilent InfinityLab LC/MSD iQ with electrospray ionization (ESI) in positive mode
• Agilent OpenLab CDS 2.8 software for data acquisition and deconvolution

Key results and discussion

The AdvanceBio Peptide Mapping column yielded the best overall performance. TFA provided superior UV peak shape but suppressed MS signals. DFA offered a balance between chromatographic behavior and MS sensitivity, detecting low-concentration impurities at 10 µg/mL that were not observed under TFA. FA delivered the highest MS sensitivity but exhibited poor peak symmetry. Deconvolution of multiple charge states (+3 to +6) using OpenLab CDS confirmed the intact mass of liraglutide at 3750.23 Da, matching theoretical values.

Benefits and practical applications

This approach enables fast screening of chromatographic conditions and reliable mass confirmation in a single workflow, supporting quality control of peptide therapeutics and accelerating late-stage development. The combination of versatile valve switching and sensitive mass detection ensures high data integrity.

Future trends and potential applications

Advances may include integration of microflow and high-throughput systems, novel ion-pairing reagents to enhance MS compatibility, and automated data analysis using artificial intelligence. Expanding mass range capabilities will allow application to larger proteins and complex biologics.

Conclusion

The Agilent InfinityLab LC/MSD iQ platform, combined with flexible chromatography and deconvolution software, provides a robust solution for rapid intact mass confirmation of liraglutide, balancing chromatographic performance and MS sensitivity for routine biopharmaceutical analysis.

References

1. US FDA Guidance for Industry on synthetic peptide drug products referencing rDNA biologics (2021).
2. EMA Guideline on the Development and Manufacture of Synthetic Peptides (2023).
3. Unit Mass Spectral Deconvolution for Molecular Weight Confirmation of Large Molecules, Agilent Tech (2024).
4. Simplify Method Development Using Agilent 1260 Infinity II Prime LC System and InfinityLab LC/MSD iQ, Agilent Tech (2020).