An In‑Depth Analysis of Semaglutide, a Glucagon‑Like Peptide‑1 Receptor Agonist

Importance of the Topic

Peptide-based biotherapeutics have transformed treatment paradigms for metabolic disorders and beyond. Semaglutide, a glucagon-like peptide-1 receptor agonist, is a leading therapy for type II diabetes and obesity. High analytical purity and impurity profiling are essential to ensure safety, efficacy, and regulatory compliance in peptide manufacturing and stability studies.

Objectives and Study Overview

This application note investigates reversed-phase ion-pair (IP-RP) separations of semaglutide acetate on seven different column chemistries under varying gradients, temperatures, and ion-pair reagents (trifluoroacetic acid and formic acid). Goals include optimizing impurity resolution, minimizing peptide carry-over, and achieving confident sequence confirmation by LC/MS.

Methodology

• Seven columns were screened: AdvanceBio Peptide Plus, AdvanceBio Peptide Mapping, PLRP-S (100 Å and 300 Å), Polaris Amide C18, Polaris C18-A, and Pursuit C18.
• Mobile phases: 0.1% TFA or 0.1% FA in water (A) and acetonitrile (B).
• Flow rate: 1.0 mL/min, column temperature: 25 °C, injection volume: 10 µL, UV detection at 220 nm, 30 min gradients.
• Thermal stress: semaglutide at 1 mg/mL in 0.1% TFA heated to 85 °C for 60 min to generate degradation products.

Instrumentation

Agilent 1290 Infinity II UHPLC system, including high-speed pump, multisampler with thermostat, multicolumn thermostat, diode array detector, coupled to Agilent 6545XT AdvanceBio LC/Q-TOF. Data were processed using Agilent OpenLab, MassHunter Data Analysis, and BioConfirm software.

Results and Discussion

• Under 0.1% TFA gradients, all columns produced sharp, symmetrical semaglutide peaks; superficially porous phases (AdvanceBio) gave shorter retention due to surface modification.
• In 0.1% FA, residual silanol interactions on most C18 and PLRP-S columns led to peak tailing; only AdvanceBio Peptide Plus, Polaris Amide C18, and Polaris C18-A maintained good peak shape due to charged bonding chemistries.
• Wider-pore (300 Å) PLRP-S improved mass transfer and peak resolution for degraded peptides relative to 100 Å media.
• AdvanceBio Peptide Plus column under FA enabled optimal impurity separation while maintaining high resolution and minimal carry-over.
• LC/MS analysis on AdvanceBio Peptide Plus confirmed semaglutide identity with characteristic multiply charged ions ([M+3H]3+, [M+4H]4+, [M+5H]5+, [M+6H]6+) matching 4,113.58 Da.

Benefits and Practical Applications

• Selecting appropriate column chemistry and ion-pair reagent enhances impurity detection and quantitation in peptide therapeutics.
• Charged or surface-modified phases mitigate secondary interactions, improving peak symmetry under MS-compatible conditions.
• Method parameters are readily transferable to preparative scales for peptide purification workflows.

Future Trends and Opportunities

Advances in core–shell and hybrid stationary phases promise further gains in resolution and throughput. Integration of high-resolution MS with real-time data analytics and machine learning will accelerate impurity profiling. Expanded pore sizes and novel bonding chemistries tailored to modified peptides can enhance both analytical and preparative separations.

Conclusion

Optimal IP-RP LC methods for semaglutide require careful alignment of column chemistry, pore size, and ion-pair reagent. Agilent AdvanceBio Peptide Plus column delivers superior selectivity and MS compatibility under formic acid conditions, supporting robust impurity profiling and sequence confirmation.

References

Tripodi A. A. P. and Coffey A., An In-Depth Analysis of Semaglutide: Comparative IP-RP Analytical Outcomes Using Different Column Chemistries, Application Note, Agilent Technologies, Inc., 2024.