Characterization of polysorbate 80 in (bio)pharmaceuticals using HPLC-CAD

Significance of the Topic

Polysorbate 80 is a vital non-ionic surfactant widely used to stabilize protein therapeutics and other biopharmaceutical formulations. Its complex composition demands detailed profiling to ensure batch consistency, product efficacy and safety. Accurate characterization of degradation pathways such as hydrolysis and oxidation is essential for quality control and formulation development.

Study Objectives and Overview

This study aimed to evaluate the use of an automated diverter valve to remove unretained matrix components and to apply the power value (PV) concept for detector response linearization. The goal was to establish a robust group-based quantitation method for polysorbate 80 (PS80) profiling using an inverse gradient HPLC-CAD workflow.

Methodology and Instrumentation

PS80 samples and model formulations containing histidine and sucrose were prepared in water and analyzed by reversed-phase inverse gradient UHPLC coupled to a charged aerosol detector (CAD). An Accucore C18 column operated at 50 °C was used with a 5 mM ammonium formate buffer (pH 4.8) and an isopropanol/acetonitrile mobile phase. The inverse gradient maintained constant organic content entering the detector, ensuring a true mass balance across PS80 species. Power values of 1.5, 1.8, 2.25 and 2.4 were acquired simultaneously to determine the optimal linear range between 0.5 mg/mL and 2.5 mg/mL PS80.

Used Instrumentation

• Thermo Scientific Vanquish Flex Inverse Gradient LC system (System Base Horizon/Flex, Dual Pump F, Split Sampler FT, Column Compartment H)
• Thermo Scientific Vanquish Charged Aerosol Detector HP
• Thermo Scientific Accucore C18 column 150×2.1 mm, 2.6 µm
• Chromeleon Chromatography Data System 7.3.2
• Additional equipment: vortexer, pH meter, precision pipettes and autosampler

Main Results and Discussion

Redirection of early-eluting histidine and sucrose to waste via the diverter valve eliminated baseline disturbances and improved precision for the first PS80 group by three-fold. The inverse gradient enabled consistent detector response across free polyoxyethylene species (group 1) and increasingly esterified species (groups 2–4). Simultaneous acquisition of four PV channels revealed that PV 1.8 provided the best linearity (R2 > 0.9997) and residual error below 4% across the calibration range. Summed group peak areas yielded a PS80 purity of 96.2% and allowed reliable comparison of batch-to-batch variation.

Benefits and Practical Applications

• Enhanced robustness by diverting unretained excipients away from the detector
• Automated and precise group-based integration of PS80 components
• Rapid identification of optimal linearization settings via simultaneous PV acquisition
• Efficient quality control tool for formulation development and manufacturing release testing

Future Trends and Potential Applications

Emerging opportunities include coupling this workflow with mass spectrometry for structural confirmation of PS80 species, automating sample pretreatment and leveraging machine learning to predict degradation behavior. The power value approach could be extended to other complex surfactants and polymers, while real-time diverter valve control may support inline monitoring in continuous biomanufacturing.

Conclusion

The combination of inverse gradient UHPLC, diverter valve matrix removal and power value optimization on the Vanquish Flex system with CAD delivers a reliable, precise and automated method for polysorbate 80 profiling. This approach supports full mass balance, robust quality assessment and accelerated formulation screening for (bio)pharmaceutical applications.

References

• Thermo Fisher Scientific Application Note 73979 Polysorbate-80 profiling by HPLC with charged aerosol and mass detection 2021
• De Pra M Zspan D A Meding S et al 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
• Carnes K A Oliver L D Brown T A et al A platform analytical method for intact polysorbates in protein-containing biopharmaceutical products via HPLC-CAD Journal of Liquid Chromatography & Related Technologies 2022 45 17–20 259–270
• Thermo Fisher Scientific Technical Note 73299 Charged Aerosol Detection – use of the power function and robust calibration practices to achieve the best quantitative results 2019