Simple and Efficient Purification of Semaglutide Using the Agilent 1290 Infinity II Preparative LC System

Importance of the Topic

The development of GLP-1 receptor agonists such as semaglutide has revolutionized the management of type 2 diabetes and obesity by offering once-weekly dosing, significant weight loss, and cardiovascular benefits. As generic versions of peptide drugs advance toward approval, regulatory guidelines demand robust evidence of structural and functional equivalence to reference products. High-purity and high-concentration peptide samples are essential for techniques like circular dichroism, NMR, and X-ray crystallography to assess higher-order structure and ensure therapeutic efficacy.

Objectives and Study Overview

This application note describes a streamlined preparative HPLC workflow for isolating semaglutide from commercial drug products (Ozempic and Wegovy) using the Agilent 1290 Infinity II Preparative LC System. Key goals included: selective separation of semaglutide from excipients, automated peak-triggered fraction collection, reproducible performance over multiple injections, and confirmation of purity under analytical HPLC conditions.

Methodology and Instrumentation

Instrumentation and reagents were:

• Preparative LC: Agilent 1290 Infinity II Preparative Binary Pump, Open-Bed Sampler/Collector, and 1260 III DAD with preparative flow cell.
• Analytical LC: Agilent 1260 Infinity III Prime Bio Flexible Pump, Multisampler with thermostat, Bio-Inert DAD.
• Columns: Prep 100Å C18 (21.2 × 50 mm, 5 µm); AdvanceBio Peptide Plus (2.1 × 150 mm, 2.7 µm).
• Mobile phases (prep): 0.05% formic acid in water (A) and in acetonitrile (B); (analytical): 0.4% TFA in water (A) and in acetonitrile (B).
• Gradient (prep): 80:20 A/B to 50:50 over 4 min, ramp to 100% B by 4.5 min, re-equilibrate to 80:20 by 7 min; flow rate 20 mL/min; injection 1 mL.
• Detection and fractionation: UV 280 nm, peak-based triggering (threshold 3 mAU, up slope 3 mAU/s, down slope 2 mAU/s) during 2–4.5 min window.
• Samples: semaglutide API in 30% ACN (1 mg/mL), Ozempic 1 mg/mL, Wegovy 2.4 mg/mL without dilution.

Main Results and Discussion

Under analytical conditions (0.4% TFA), semaglutide eluted at 11.7 min, while excipients appeared at ~1.6 min. Preparative trials compared 0.1% TFA, no modifier, and 0.05% formic acid; 0.05% FA provided stable, sharp semaglutide peaks and facile acid removal post-collection. Six consecutive 1 mL injections of Ozempic yielded %RSD of retention time 0.12% and peak area 1.08%. Reanalysis of pooled fractions confirmed complete separation of semaglutide and excipients in both products.

Benefits and Practical Applications

• Generates high-purity semaglutide free of excipients, suitable for structural and physicochemical characterization.
• Minimizes TFA-salt formation by using low-level formic acid, enhancing downstream compatibility.
• Automated, reproducible workflow supports generic drug development, quality control, and scale-up.

Future Trends and Potential Applications

• Adaptation of this preparative approach for other peptide therapeutics and biosimilars.
• Integration with LC-MS or high-resolution detectors for impurity profiling.
• Implementation of continuous or semi-automated purification platforms to increase throughput.
• Exploration of alternative acid modifiers and greener solvents for improved sustainability.

Conclusion

The optimized preparative HPLC method employing 0.05% formic acid and automated UV-triggered fractionation on the Agilent 1290 Infinity II system affords rapid, reproducible isolation of semaglutide from drug formulations. This workflow delivers high-purity material for advanced structural analyses and supports regulatory requirements in generic peptide development.

References

1. Lincoff AM, Brown-Frandsen K. Semaglutide and Cardiovascular Outcomes in Obesity without Diabetes. N Engl J Med. 2023;389(24):2221–2232.
2. Anderer S. FDA Approves Generic Liraglutide to Address GLP-1 Drug Shortage. JAMA. 2025;333(9):746–746.
3. Kuril AK, Saravanan K. Analytical Considerations for Characterization of Generic Peptide Product: A Regulatory Insight. Anal Biochem. 2024;694:115633.
4. Kim Y, Bigelow L. High-Throughput Protein Purification for X-Ray Crystallography and NMR. Adv Protein Chem Struct Biol. 2008;75:85–105.
5. Sikora K, Maciej J. The Role of Counter-Ions in Peptides—An Overview. Pharmaceuticals. 2020;13(12):442.
6. Efficient Method Optimization of Semaglutide Analysis Using an Agilent 1260 Infinity II Bio Prime LC System and Blend Assist. Agilent Technologies; publication number 5994-7414EN; 2024.
7. Rapid Confirmation of GLP-1 Analog (Liraglutide) Using Agilent InfinityLab LC/MSD iQ. Agilent Technologies; publication number 5994-7415EN; 2024.