Shining Light on Polysorbate Hydrolysis by LC/ELSD

Importance of the Topic

Protein therapeutics require nonionic surfactants such as polysorbate 20 and 80 to prevent aggregation and surface adsorption. Enzymatic hydrolysis by residual host cell proteins can compromise surfactant efficiency and product stability. A rapid, reliable assay to monitor polysorbate degradation is critical for ensuring biopharmaceutical quality, especially in high-concentration formulations.

Objectives and Study Overview

This application note describes the development of a 10-minute LC/ELSD method using an Agilent AdvanceBio Surfactant Profiling column. The goals are to resolve free fatty acids from polysorbate monoesters, assess method robustness and carryover, and demonstrate additional applications including free fatty acid profiling and semi-quantitative polysorbate measurement.

Methodology and Instrumentation

• Agilent 1290 Infinity II UHPLC system with high-speed pump, multisampler, column compartment, and 1290 Infinity II ELSD
• AdvanceBio Surfactant Profiling column, 300 Å pore size, 3.5 µm, 2.1 × 50 mm with guard cartridge
• Mobile phases: 10 mM ammonium acetate (A) and methanol (B)
• Optimized 10-minute gradient for hydrolysis monitoring and free fatty acid separation
• ELSD settings: evaporator and nebulizer at 30 °C, gas flow 1.20 SLM, needle wash 20:80 methanol:water
• Sample buffer: phosphate-citrate saline at pH 6.0; polysorbates spiked at 100 ppm, fatty acids at 5–10 ppm

Key Results and Discussion

• Oleic acid and PS 80 monoester baseline separation in under 10 minutes; average resolution ~5.0 (RSD 3.1%) across four column batches
• Retention time precision RSD <1.5% and peak area RSD 7.9%–12.4% for PS 80 components
• PS 20 method yielded resolution ~8.7 between lauric acid and monoester, with injection-to-injection RSD ≤1.4% for retention and 1.3% for peak area
• No carryover detected for polysorbates or free fatty acids using the defined needle wash protocol
• Free fatty acid profiling of six common acids achieved with modified gradient; saturated species produced stronger ELSD responses
• Semi-quantitative PS 80 assay over 0.0005%–0.5% range showed log-log linearity (R2 = 0.9985) and LOQ at 5 ppm (RSD 20.9%)
• Protein samples spiked with 0.02% and 0.05% PS 80 returned accuracies of 94%–110% and RSD ≤7.1%

Benefits and Practical Applications

• Rapid screening of polysorbate hydrolysis without derivatization or lengthy run times
• High reproducibility and minimal carryover support high-throughput QC workflows
• Dual functionality for degradation monitoring and semi-quantitative surfactant measurement
• Free fatty acid analysis as an alternative to GC-based assays

Future Trends and Potential Applications

• Incorporation of stable isotope-labeled standards for absolute quantitation
• Automation within continuous manufacturing quality control
• Extension to other nonionic surfactants and lipid excipients
• Integration with multi-attribute methods for comprehensive protein stability assessment

Conclusion

The described LC/ELSD approach using the AdvanceBio Surfactant Profiling column delivers a fast, robust, and reproducible solution for monitoring polysorbate hydrolysis, free fatty acid profiling, and semi-quantitative surfactant analysis, meeting critical needs in biopharmaceutical development and quality control.

References

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