Comprehensive Aggregate Profiling of Liraglutide and Semaglutide Using an Agilent 1290 Infinity II Bio 2D-LC and Agilent InfinityLab LC/MSD XT

Significance of the Topic

Aggregation is a key quality attribute for peptide therapeutics such as liraglutide and semaglutide. Uncontrolled aggregate formation can compromise drug safety, efficacy, and immunogenicity, making comprehensive aggregate profiling essential in quality control and biopharmaceutical development.

Objectives and Study Overview

The goal of this work was to establish a robust workflow combining size exclusion chromatography (SEC) with two-dimensional LC (2D-LC) and mass spectrometry (MS) to detect, separate, and characterize aggregates of liraglutide and semaglutide. The study focused on optimizing SEC conditions, implementing heart-cutting and active solvent modulation, and determining molecular weights of both reversible and covalent aggregates.

Methodology and Used Instrumentation

A phosphate buffer and an organic solvent mobile phase containing arginine, acetonitrile, and acetic acid were evaluated using Agilent AdvanceBio SEC 130Å and 300Å columns to minimize hydrophobic interactions and peak tailing. High-resolution 2D-LC analysis employed heart-cutting with a Poroshell CS-C18 column under MS-friendly reversed-phase conditions. Active Solvent Modulation and a multi-inject strategy increased sample loading capacity for aggregate peaks. Mass spectra were acquired on an Agilent InfinityLab LC/MSD XT and deconvolved using OpenLab CDS 2.8.

Main Results and Discussion

• SEC optimization with AdvanceBio SEC 300Å and 35% acetonitrile/15% acetic acid improved peak shape and resolution of peptide aggregates.
• Heat stress at 80 °C induced distinct aggregate peaks for liraglutide and semaglutide, with two separate aggregate populations observed in Ozempic.
• Heart-cutting 2D-LC combined with MS revealed reversible aggregates that dissociate under RP conditions and covalent aggregates with molecular weights roughly twice the monomer.
• Multi-inject and ASM functions enhanced detection sensitivity by increasing the injected fraction to 80% of loop volume.

Benefits and Practical Applications

• Improved detection and characterization of peptide aggregates under diverse mobile phase conditions.
• Ability to distinguish reversible and irreversible aggregates informs formulation development and comparability studies.
• Enhanced QC workflows for biopharmaceutical manufacturing and generic peptide drug evaluation.

Future Trends and Potential Applications

• Extending this approach to additional peptide and protein therapeutics for aggregate profiling databases.
• Incorporating AI-driven deconvolution and automated data processing to streamline analysis.
• Exploring novel stationary phases and mobile phase additives to further reduce hydrophobic interactions.
• Adapting microflow or nano-LC interfaces to improve sensitivity for low-abundance aggregates.

Conclusion

The integration of optimized SEC conditions with 2D-LC/MS using Agilent AdvanceBio columns and InfinityLab LC/MSD XT provided a powerful platform for detailed profiling of liraglutide and semaglutide aggregates. This method enables comprehensive characterization of both reversible and covalent aggregates, supporting improved quality control and regulatory compliance for peptide therapeutics.

References

1. U.S. Food and Drug Administration. Immunogenicity Assessment for Therapeutic Protein Products. Guidance for Industry, 2014.
2. U.S. Food and Drug Administration. ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs for rDNA Origin. Guidance for Industry, 2021.
3. European Medicines Agency. Guideline on the Development and Manufacture of Synthetic Peptides, 2023.
4. Zapadka KL, Becher FJ, Gomes dos Santos AL, Jackson SE. Factors Affecting the Physical Stability (Aggregation) of Peptide Therapeutics. Interface Focus. 2017;7(6):20170030.
5. Zapadka KL, et al. A pH-Induced Switch in Human Glucagon-Like Peptide-1 Aggregation Kinetics. J. Am. Chem. Soc. 2016;138(50):16259–16265.
6. Powell T, Knight MJ, Wood A, O'Hara J, Burkitt W. Detection of Isopeptide Bonds in Monoclonal Antibody Aggregates. Pharm. Res. 2021;38(9):1519–1530.