Characterization of polysorbate 80 in (bio)pharmaceuticals using HPLC with charged aerosol detection

Significance of the Topic

Polysorbate 80 (PS80) is a non-ionic surfactant commonly used in pharmaceutical and biopharmaceutical formulations to stabilize proteins and prevent aggregation. Its chemical complexity, stemming from variable degrees of esterification and fatty acid composition, necessitates robust analytical methods for routine quality control and lot-to-lot comparisons.

Objectives and Study Overview

This study aimed to develop and validate an RP-HPLC method with charged aerosol detection (CAD) for rapid profiling of PS80 formulations. The key objectives were:

• Achieve uniform detector response across PS80 species with differing esterification levels.
• Optimize CAD signal linearization in a single run, reducing analysis time and solvent use.
• Divert non-retained matrix components (e.g., histidine, sucrose) to waste to protect the detector and streamline data processing.

Methodology and Instrumentation

A model PS80 formulation containing histidine and sucrose was prepared in aqueous solution and injected onto a Thermo Scientific Accucore C18 column (150 x 2.1 mm, 2.6 µm) using a Vanquish Flex Inverse Gradient LC system. The method employed:

• Mobile phases: 5 mM ammonium formate (pH 4.8) and 50/50 isopropanol/acetonitrile.
• Inverse gradient delivery with a diverter valve switching early peaks (0–1.3 min) to waste.
• Charged Aerosol Detector P series with multiple power values (PVs 1.5, 1.8, 2.25, 2.4) recorded as separate channels.
• Calibration standards from 0.5 to 2.5 mg/mL PS80, processed in triplicate.
• Data acquisition and processing via Chromeleon CDS 7.3.2.

Results and Discussion

The inverse gradient approach effectively separated four PS80 peak groups corresponding to different esterification species. Matrix peak diversion prevented early-eluting histidine and sucrose from contaminating the detector, enhancing robustness and integration accuracy. Key findings:

• Optimal CAD linearization at PV 1.8, achieving a coefficient of determination (R2) of 0.9997 and residual error below 4% across the calibration range.
• Relative peak areas for the four PS80 groups were reproducible (RSD ≤ 2.8%), with group abundances ranging from 12.5% to 34.5%.
• Overall PS80 purity calculated by summing all peak areas was 96.2%.

Benefits and Practical Applications

• Streamlined sample throughput by integrating matrix diversion and multi-channel CAD linearization in a single run.
• Improved detector longevity and reduced downtime by eliminating non-target compounds before detection.
• Reliable lot-to-lot comparison enabling consistent quality control of PS80 excipients.

Future Trends and Opportunities

• Extension of inverse gradient CAD methods to other polysorbates and surfactants in biopharma formulations.
• Integration with mass spectrometric detectors for structural confirmation of PS80 species.
• Automation and online data processing enhancements for real-time quality monitoring.
• Application of advanced detector functions and calibration protocols to broaden linear dynamic range.

Conclusion

The developed RP-HPLC inverse gradient method with CAD detection provides a robust, efficient, and accurate approach for profiling PS80 formulations. By optimizing detector linearization and employing matrix diversion, the method supports reliable quality control and formulation development in pharmaceutical and biopharmaceutical workflows.

References

1. De Pra M.; Ispan D.A.; Meding S.; Müllner T.; Lovejoy K.S.; Grosse S.; Cook K.; Carillo S.; Steiner F.; Bones J. Degradation of polysorbate investigated by a high-performance liquid chromatography multi-detector system with charged aerosol and mass detection. Journal of Chromatography A, 2023, 1710, 464405.
2. Thermo Fisher Scientific. Application Note 003506: Characterization of polysorbate 80 in (bio)pharmaceuticals using HPLC-CAD, 2025.
3. Thermo Fisher Scientific. Technical Note 003816: Method transfer and optimization of deoxycholic acid analysis using HPLC-CAD, 2025.
4. Thermo Fisher Scientific. Technical Note 73299: Charged Aerosol Detection – Use of the power function and robust calibration practices, 2019, Appendix B.
5. Thermo Fisher Scientific. Application Note 73979: Polysorbate-80 profiling by HPLC with charged aerosol and mass detection, 2021.