Polysorbate Degradation Analysis with the AdvanceBio Surfactant Profiling HPLC Column

Meaning of the Topic

Polysorbates serve as critical surfactants, emulsifiers and stabilizers in healthcare products, especially in protein-based therapeutics. Their degradation via hydrolysis or oxidation can compromise product stability, making sensitive analytical control essential for ensuring safety and efficacy.

Study Objectives and Overview

This guide outlines an end-to-end workflow for analyzing polysorbate degradation using the Agilent AdvanceBio Surfactant Profiling HPLC column. It aims to enable early detection of degradants, support regulatory compliance and prevent downstream protein stability issues.

Methodology and Instrumentation

Sample preparation focuses on protein removal, typically via precipitation using a 50/50 methanol–ethanol mixture, followed by dilution to match the initial mobile phase. Chromatographic separation employs a 2.1×50 mm AdvanceBio Surfactant Profiling column at 25 °C with a 10 mM ammonium acetate aqueous phase and methanol organic phase. A gradient from 0 % to 95 % organic over 5.1 min, followed by re-equilibration to 0 % at 10 min, ensures resolution between free fatty acids, monoesters and higher esters. Needle washes with methanol–water minimize carryover. Method optimization includes controlling mobile phase pH, limiting pump pressure ramp rates and using reproducible salt batches.

Main Results and Discussion

The method delivers baseline separation of oleic acid and polysorbate 80 monoester within a 10 min run. Batch-to-batch reproducibility across four column batches showed retention time RSDs below 2 % and peak area RSDs under 13 %. Resolution values for critical peak pairs ranged from 4.7 to 5.2. The workflow demonstrated robustness in ELSD detection and compatibility with MS-based confirmation when diverting high-organic streams to waste.

Benefits and Practical Applications

• Rapid, high-resolution profiling of polysorbate degradation products.
• Early incorporation into biotherapeutic development reduces risk of stability failures.
• Compatibility with ELSD and high-resolution MS enables both quantitation and structural elucidation.
• Scalable for high-throughput quality control in biopharmaceutical labs.

Future Trends and Potential Applications

• Integration with automated sample preparation and inline protein removal.
• Expansion to other surfactant classes and complex matrices.
• Enhanced MS workflows for deeper oxidation product characterization.
• Development of regulatory guidelines for surfactant impurity profiling.

Conclusion

The AdvanceBio Surfactant Profiling HPLC method provides a robust, reproducible approach for monitoring polysorbate degradation, supporting quality assurance in therapeutic protein development.

Instrumentation Used

• Agilent AdvanceBio Surfactant Profiling HPLC columns (2.1×50 mm, 100 mm, 150 mm).
• Agilent 1290 Infinity II UPLC system.
• Agilent InfinityLab LC Series Evaporative Light Scattering Detector.
• High-resolution accurate mass spectrometer (ESI-TOF).
• Guard columns, inline filters and Quick Connect fittings for UHPLC.

References

1. Host Cell Protein Contaminants in mAb and Protein Therapy Manufacturing. USP Biologics.
2. Aryal B., Lehtimaki M., Rao V.A. Stress Mediated Polysorbate 20 Degradation and Its Impact on Therapeutic Proteins. Pharm. Res. 2024, 41, 1217–1232.
3. European Medicines Agency. Information for the Package Leaflet Regarding Polysorbates as Excipients. EMA/CHMP/190743/2016; 2023.
4. Wuchner K. et al. Industry Perspective on Polysorbates in Biopharma Part 1: Survey Report. J. Pharm. Sci. 2022, 111, 1280–1291.
5. Wuchner K. et al. Industry Perspective on Polysorbates in Biopharma Part 2: Control Strategy. J. Pharm. Sci. 2022, 111, 2955–2967.
6. He C., Hale W.A., Coffey A., Tripodi A.P., McQuay A. Shining Light on Polysorbate Hydrolysis by LC/ELSD Featuring the AdvanceBio Column. Agilent Tech. 2025.
7. Agilent InfinityLab LC Series Evaporative Light Scattering Detectors User Manual. 2022.
8. In-Depth Characterization of Surfactant Degradation in Biotherapeutics: Agilent AdvanceBio Columns. Agilent Tech. 2025.