Liraglutide Native SEC-MALS Characterization Using Empower™ Chromatography Data System (CDS)

Significance of the topic

Understanding the native oligomeric state and aggregation of therapeutic peptides is critical for product safety, efficacy, and manufacturability. Native SEC coupled with multi-angle light scattering (MALS) and concentration detection (differential refractive index, dRI) delivers absolute molar mass and reliable mass-fraction quantitation under formulation-relevant conditions, avoiding biases introduced by calibration-based SEC or UV-only quantitation when particulate scattering is present.

Objectives and overview of the study

This application note demonstrates a native SEC-MALS workflow for characterizing liraglutide oligomerization using Waters instrumentation controlled and analyzed in Empower CDS 3.10.0. The objectives were to (1) determine absolute molecular weights of species present in a formulation-relevant matrix, (2) resolve higher-order oligomers without exclusion artifacts, and (3) obtain accurate mass-fraction quantitation while minimizing light-scattering bias from large particulate species.

Methodology

• Sample and conditions: Liraglutide drug substance analyzed at 6 mg/mL under non-denaturing conditions to mimic typical drug product formulation.
• Chromatography: XBridge Premier Protein SEC Column (250 Å, 2.5 μm, 7.8 × 300 mm) with mobile phase 10 mM Na2HPO4, pH 8.1 at 0.5 mL/min.
• Detection and analysis: DAWN MALS photometer for angular scattering, Optilab dRI for concentration (dn/dc = 0.185 mL/g), and Arc Premier 2998 PDA UV for orthogonal signal. Data acquisition and automated peak detection/integration used Empower CDS 3.10.0 with the traditional integration algorithm and the MALS analysis option.
• Analytical inputs: UV extinction coefficient for liraglutide reported as 6,990 M−1 cm−1 (1.86 (mg/mL)−1 cm−1) at 280 nm; dn/dc fixed at 0.185 mL/g for native conditions.

Applied instrumentation

• Arc Premier HPLC System (quaternary solvent and sample manager)
• XBridge Premier Protein SEC Column, 250 Å, 7.8 × 300 mm
• Arc Premier 2998 PDA UV detector
• DAWN multi-angle light scattering (MALS) photometer
• Optilab differential refractive index (dRI) detector
• Empower 3.10.0 Chromatography Data System with advanced detector license for MALS

Main results and discussion

• Chromatography and molar mass assignment: Native SEC-MALS dRI chromatograms revealed two distinct peaks. Retention times near 12.3 and 13.6 minutes were associated with higher-order oligomers based on measured weight-average molar mass (Mw).
• Molar mass and mass fractions: The dominant species corresponded to a hexameric assembly (measured Mw ≈ 22.4 kDa) and represented 90.7% of total mass (percent CV 0.4% across triplicate injections). The secondary peak had Mw ≈ 50 kDa, consistent with ~13-mer species. Triplicate injections yielded a %CV of 0.7% for Mw of the main peak, indicating high precision.
• No monomer detected: Under these native conditions no monomeric liraglutide peak was observed, consistent with earlier reports that liraglutide favors oligomeric assemblies in formulation-like buffers.
• Choice of concentration detector: Optilab dRI was selected over UV as primary concentration detector to avoid overestimation of aggregates due to light scattering by particulate species in the 20–30 nm range. dRI operates at wavelengths and detection principles less susceptible to scattering bias, enabling more accurate mass-fraction quantitation for large assemblies and non-aromatic peptides.
• Data processing: Empower CDS traditional automatic integration simplified reproducibility by avoiding fixed retention-time windows and enabled integrated processing of UV, MALS and dRI signals with automated Mw and mass-fraction calculations.

Benefits and practical applications of the method

• Calibration-free absolute molar mass: SEC-MALS provides direct Mw measurement independent of SEC calibration standards, removing a significant source of bias when characterizing heterogeneous or non-globular assemblies.
• Native-state assessment: Method preserves non-covalent assemblies, informing on biologically and formulation-relevant oligomerization and aggregation states.
• Robust quantitation of aggregates: dRI-based concentration measurement mitigates UV scattering artifacts, improving accuracy for samples containing larger particulates or non-UV-active peptides.
• Integrated and compliant workflow: Empower CDS integration with MALS/dRI reduces manual processing, increases reproducibility, and supports reporting in regulated environments.

Future trends and potential applications

• Broader adoption for peptide therapeutics: As lipidated and aggregated peptide drugs become more prevalent, native SEC-MALS will be increasingly important for stability testing, comparability studies, and release testing.
• Hybrid orthogonal workflows: Combining native SEC-MALS with denaturing SEC-MALS, mass spectrometry, and orthogonal particle sizing can deliver comprehensive profiles of covalent versus non-covalent aggregates and particulate content.
• Method automation and high-throughput adaptations: Advances in software integration, autosampler workflows, and faster columns will support higher throughput screening of formulation conditions and stability timepoints.
• Improved characterization of heterogeneous assemblies: Advances in data models and light-scattering analysis will enhance resolution and interpretation of broad or overlapping oligomer distributions.

Conclusion

Native SEC-MALS using DAWN MALS and Optilab dRI, controlled and analyzed in Empower CDS, provides a nondestructive, calibration-free approach to determine absolute molar masses and accurate mass fractions of liraglutide oligomers under formulation-relevant conditions. Careful selection of a high-pore-size SEC column and dRI concentration detection enabled effective separation and unbiased quantitation of hexameric and higher-order species. The workflow demonstrates precision (low %CVs) and practical value for peptide development, stability assessment, and quality control.

References

1. Brichtová E, Přáda E, et al. Glucagon-like peptide 1 aggregates into low-molecular-weight oligomers off-pathway to fibrillation. Biophysical Journal. 2023;122(12):2475–2488.
2. Přáda Brichtová E, et al. Effect of Lipidation on the Structure, Oligomerization, and Aggregation of Glucagon-like Peptide 1. Bioconjugate Chemistry. 2025;36(3):401–414.
3. Zhang X, et al. Identification of GLP-1 analog oligomeric states using SEC-MALS. Application Note. Wyatt Technology Corporation. (Application note referenced by original report).
4. Bothe JR, et al. Peptide oligomerization memory effects and their impact on the physical stability of the GLP-1 agonist liraglutide. Molecular Pharmaceutics. 2019;16(5):2153–2161.