Size Exclusion Chromatography- Based Identification of Liraglutide Oligomeric Forms

Importance of the Topic

Liraglutide is a lipidated analog of the GLP-1 peptide hormone used in type 2 diabetes therapy. Its amphiphilic nature drives self-assembly into oligomers, impacting drug stability, efficacy, and safety. Accurate characterization of these oligomeric forms is critical for formulation development, quality control, and process optimization in biopharmaceutical manufacturing.

Objectives and Study Overview

This study aimed to establish a robust size exclusion chromatography (SEC) workflow combined with UV detection and high-resolution mass spectrometry (LC/Q-TOF) to identify and quantify the oligomeric states of liraglutide under varying pH conditions. The method was optimized to reveal pH-dependent transitions and to monitor time-dependent changes in oligomer populations.

Methodology

Sample Preparation

• Liraglutide stock at 2.0 mg/mL prepared in sodium phosphate buffers (pH 6.4, pH 7.0, pH 8.0) or 100 mM ammonium acetate for MS experiments.
• Incubation studies conducted over three days at controlled temperature with daily sampling.

Chromatographic Conditions

• SEC/UV: Agilent AdvanceBio SEC 130 Å, 7.8 × 300 mm, 2.7 µm; mobile phase of 150 mM sodium phosphate or phosphate:acetonitrile:0.1 % arginine (9:25:65), flow rate 0.8 mL/min, 30 °C, detection at 280 nm.
• SEC/MS: Agilent AdvanceBio SEC 120 Å, 2.1 × 150 mm, 1.9 µm PEEK; 20 mM ammonium acetate, 0.07 mL/min, column at 30 °C; MS in positive ESI mode, m/z 100–10 000.

Used Instrumentation

• Agilent 1290 Infinity II bio LC system with high-speed pumps, multisampler with cooling, multicolumn thermostat, and diode array detector.
• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Jet Stream ESI source.
• Agilent OpenLab CDS v2.8, MassHunter LC/MS v11.0, and MassHunter BioConfirm v12.1 software suites.

Key Results and Discussion

SEC/UV analysis revealed that at pH 6.4 liraglutide predominantly forms higher-order oligomers (~12-mer), while at pH 8.0 lower-order oligomers (~3-mer) dominate. Calibration with protein standards confirmed column performance and allowed approximate molecular weight estimation. SEC/MS data showed freshly prepared samples rich in low-order species; prolonged incubation produced detectable high-order oligomer peaks in the mass spectra. Deconvoluted mass data from the LC/Q-TOF enabled unambiguous assignment of oligomer masses, demonstrating a mixture of 2- to 15-mer assemblies.

Benefits and Practical Applications

This combined SEC/UV and SEC/MS approach provides:

• High-resolution separation and quantitation of peptide oligomers under biocompatible conditions.
• Accurate mass identification of complex oligomeric assemblies.
• A workflow suitable for formulation screening, stability studies, and process monitoring in peptide drug development.

Future Trends and Opportunities

Further developments may include integration of multi-angle light scattering or fluorescence detection for absolute molecular weight determination, real-time monitoring of oligomer dynamics during manufacturing, high-throughput screening of buffer and excipient effects, and application to other lipidated or self-assembling therapeutic peptides.

Conclusion

A robust SEC workflow employing the Agilent 1290 Infinity II bio LC and AdvanceBio SEC columns, coupled with LC/Q-TOF mass spectrometry, effectively characterizes pH-dependent oligomeric states of liraglutide. The method offers valuable insights for formulation design, stability assessment, and quality control in biopharmaceutical peptide development.

References

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