dSEC and dSEC-HRMS Characterization of GLP-1a and Related Peptide Therapeutics

Significance of the topic

Denaturing size-exclusion chromatography coupled with high-resolution mass spectrometry (dSEC-HRMS) addresses a critical need in peptide therapeutic development: reliable detection and characterization of low-abundance high molecular weight species (HMWS) that can affect safety, efficacy and stability. GLP-1 analogues such as semaglutide and tirzepatide are surface-active and prone to form oligomeric, partially dissociable or non-dissociable assemblies in solution. Standard native SEC methods can underestimate or mischaracterize these forms because of secondary interactions with column hardware and mobile phases. Implementing denaturing mobile phases together with inert, surface-modified SEC hardware improves size-based separation and, when combined with HRMS, enables molecular-level identification of oligomeric impurities down to low abundance levels.

Objectives and overview of the study

• Evaluate denaturing SEC (dSEC) conditions and optimized SEC column chemistries for resolving monomeric and oligomeric species of GLP-1 analogues (tirzepatide and semaglutide).
• Combine dSEC separations with high-resolution MS (dSEC-HRMS) to determine accurate masses of low-abundance HMWS and distinguish distinct noncovalent oligomeric assemblies.
• Compare column performance (ACQUITY Premier 125 Å vs comparative SEC column and MaxPeak Premier 250 Å) for HMWS recovery and suppression of secondary interactions.

Methodology

• Denaturing mobile phase: 1 g/L arginine with acetonitrile and glacial acetic acid (65/20/15, v/v/v) to reduce surface activity and limit secondary interactions while maintaining MS compatibility.
• Chromatography: Low injection volumes (≈3.5 μL), flow rates around 0.4 mL/min, and column temperature maintained at 25 °C. Columns tested included ACQUITY Premier SEC 125 Å (1.7 μm, 4.6 × 150 mm) and MaxPeak Premier SEC 250 Å for broader size range analyses.
• Detection: UV/TUV detection around 276 nm for peptide chromophores coupled with high-resolution Q-ToF MS detection to capture intact noncovalent oligomer masses in the 500–3,500 m/z range.
• MS source settings (representative): source temp ~120 °C, desolvation ~350 °C, cone gas ~35 L/h, desolvation gas ~600 L/h, capillary voltage ~2.2 kV, sample cone ~20 V.
• Performance comparison: Peak area reproducibility (n=3) and HMWS recovery were used to assess column interaction effects; ACQUITY Premier 125 Å column demonstrated reduced secondary interactions and improved HMWS recovery compared to a conventional comparative column.

Used instrumentation

• ACQUITY Premier System with BioSample Manager (FTN) and BioQuaternary Solvent Manager (QSM) for LC handling.
• ACQUITY Premier SEC 125 Å, 1.7 μm, 4.6 × 150 mm column; MaxPeak Premier SEC 250 Å column for extended size range separations.
• Xevo G3 Q-Tof high-resolution mass spectrometer for intact-mass detection and confirming oligomer stoichiometries.
• TUV detector with 5 mm titanium flow cell (wavelength ~276 nm) for UV monitoring.

Main results and discussion

• Separation and detection: Denaturing SEC conditions enabled clear size-based separation of monomeric peptide and discrete HMWS in semaglutide and tirzepatide. Distinct chromatographic peaks corresponding to dimer- and trimer-sized species (and higher-order forms) were observed.
• Mass confirmation: HRMS identified the predominant monomer masses (e.g., ~4113.6 Da for a representative GLP-1 analogue) and dimer/trimer masses consistent with noncovalent oligomers (~8227 Da for dimer, ~12–14 kDa for trimer species depending on variant). Multiple distinct HMWS species were resolved and mass-assigned, indicating heterogeneous oligomer populations rather than a single aggregated form.
• Column and surface effects: The ACQUITY Premier 125 Å column with hydrophilic MaxPeak surface modification reduced spurious secondary interactions, preventing partial coelution of HMWS with monomer seen on a comparative SEC column. This improvement led to approximately 2× better HMWS recovery and narrower peak widths when profiling low-abundance impurities.
• Quantitation and abundance: HMWS comprised low percentages of total peptide signal (reported examples ranged from ~0.2% up to ~0.52% depending on column and peptide), emphasizing the need for sensitive separation and detection to capture these minor species reliably.
• Method robustness: Consistent total peak areas across replicate injections on optimized columns indicated reproducible recovery and minimized artifactual loss due to surface adsorption.

Practical benefits and applications

• Quality control: dSEC-HRMS provides a robust approach for routine QC laboratories to detect and monitor low-level HMWS that can affect batch release decisions and shelf-life assessments.
• Formulation development: The method helps formulation scientists evaluate how excipients, pH, and solvent systems influence oligomer formation and stability, guiding formulation choices to minimize undesirable HMWS.
• Root-cause analysis: Mass-resolved identification of HMWS supports investigation into whether oligomers are covalent (e.g., cross-linked) or noncovalent, informing mitigation strategies (e.g., process changes or stabilizers).
• Method transferability: Use of MS-compatible denaturing eluents and commercially available, inert SEC columns facilitates transfer between analytical labs while maintaining sensitivity to minor species.

Future trends and potential uses

• Integration with orthogonal techniques: Combining dSEC-HRMS with SEC-MALS, native MS, ion-mobility spectrometry or cross-linking MS will provide complementary size, mass and topology information on oligomers.
• Column and surface engineering: Further improvements in column hardware coatings and pore-size portfolios will expand reliable SEC-MS characterization to larger or more labile peptide/protein assemblies.
• Automation and high-throughput QC: Miniaturized injections, faster columns and automated sample handling will enable routine screening during process development and stability studies.
• Advanced data analysis: Improved deconvolution algorithms and spectral libraries for peptide oligomers will streamline identification of heterogeneous HMWS and support regulatory submissions.
• Expanded regulatory acceptance: Demonstrated robustness and orthogonal confirmation could lead to broader adoption of dSEC-HRMS as a release and stability-indicating method for peptide therapeutics.

Conclusion

Denaturing SEC coupled with high-resolution MS offers a sensitive, mass-informative platform to resolve and characterize low-abundance, non-dissociable high molecular weight impurities in GLP-1 analogues such as semaglutide and tirzepatide. Selection of MS-compatible denaturing mobile phases together with inert, surface-modified SEC columns significantly reduces secondary interactions, increases HMWS recovery and enables accurate mass assignment of oligomeric species. The approach is directly applicable in formulation development, QC and stability testing and forms a strong foundation for integrating orthogonal structural methods.

Reference

1. Wuthrich P, Koza SM, Shiner SJ. dSEC and dSEC-HRMS Characterization of GLP-1a and Related Peptide Therapeutics. Waters Corporation, Milford, MA. Poster/literature code 720009452EN. 2026.
2. Waters Corporation. Denaturing SEC-MS Analysis of High Molecular Weight Impurities in the GLP-1a Lipopeptides Semaglutide and Tirzepatide. Literature code 720009256.
3. Waters Corporation. Advancing Analysis of Covalent High Molecular Weight Insulin With ACQUITY Premier SEC 125 Å Columns. Literature code 720008660.
4. Waters Corporation. Development of Separation Methods for GLP-1 Synthetic Peptides Utilizing a Systematic Protocol and MaxPeak High Performance Surface Technology. Literature code 720008267.
5. Zhang X, et al. Identification of GLP-1 analog oligomeric states using SEC-MALS. Wyatt Technology Application Note AN1901. 2025.