Simultaneous analysis of polysorbate 80 and poloxamer 188 in biopharmaceutical formulations using charged aerosol detector and single quadrupole mass spectrometer

Importance of the Topic

Surfactants such as polysorbate 80 and poloxamer 188 are widely used excipients in protein-based biopharmaceutical formulations to maintain stability during manufacturing, storage, and transport. Accurate quantification of these complex mixtures is critical for quality control, regulatory compliance, and ensuring product safety.

Objectives and Study Overview

This study aimed to develop and validate a sensitive, robust, and reproducible method for the simultaneous analysis of polysorbate 80 and poloxamer 188 in recombinant protein formulations. Key goals included:

• Achieving a broad linear dynamic range with low limits of detection and quantification.
• Identifying potential degradation products and impurities.
• Demonstrating method suitability for routine biopharmaceutical quality control.

Methodology and Instrumentation

A combined HPLC approach with charged aerosol detection (CAD) and single quadrupole mass spectrometry (LC–CAD–ISQ) was applied.

• Chromatographic separation on a C18 surfactant plus column with a gradient of ammonium formate buffer and organic solvents.
• Charged aerosol detector parameters optimized for linearity using a power function value.
• ISQ EM single quadrupole mass spectrometer in positive HESI mode for impurity identification and peak confirmation.
• Sample clean up using column switching to divert high protein content to waste.

Used Instrumentation

• Thermo Scientific Vanquish Core HPLC with quaternary pump, split sampler, column compartment, charged aerosol detector, and switching valve.
• Accucore C18 and Acclaim Surfactant Plus columns.
• Thermo Scientific ISQ EM single quadrupole mass spectrometer.
• Chromeleon CDS software for data acquisition and baseline correction.

Main Results and Discussion

• Linearity: 5 to 100 mg/L (R2 > 0.9997) and nonlinear fit up to 500 mg/L (R2 = 1.0000) for both surfactants.
• Limits: LOD of 1.0 mg/L for polysorbate 80 and 2.0 mg/L for poloxamer 188; LOQ of 2.0 and 5.0 mg/L, respectively.
• Precision: Repeatability RSD < 4.2% across low, medium, and high concentration levels.
• Accuracy: Recoveries between 95.7% and 110.0% for both analytes.
• Stability: Poloxamer 188 remained stable under 60 C for 5 days, whereas polysorbate 80 showed approximately 10% degradation.

Benefits and Practical Applications

The validated method provides:

• Simultaneous quantification of two key surfactants in a single run, reducing analysis time and resources.
• Enhanced sensitivity without reliance on chromophores.
• Ability to detect and identify degradation products, supporting formulation stability studies.

Future Trends and Potential Applications

Emerging directions include:

• Extension of CAD calibration strategies to other nonchromophoric excipients.
• Integration with high-resolution mass spectrometry for deeper impurity profiling.
• Automation of sample preparation for high-throughput screening in QC environments.

Conclusion

A single HPLC–CAD–ISQ method has been successfully developed for reliable and reproducible analysis of polysorbate 80 and poloxamer 188 in biopharmaceutical formulations. The approach meets stringent regulatory requirements and supports robust quality control workflows.

References

1. Gervasi V et al Parenteral protein formulations an overview of approved products within the European Union European Journal of Pharmaceutics and Biopharmaceutics 131 2018 8 24
2. Roy I et al Polysorbate degradation and particle formation in a high concentration mAb formulation strategies to minimize effect of enzymatic polysorbate degradation Journal of Pharmaceutical Sciences 110 9 2021 3313 3323
3. Graf T et al Identification and characterization of polysorbate degrading enzymes in a monoclonal antibody formulation Journal of Pharmaceutical Sciences 110 11 2021 3558 3567
4. Bollenbach L et al Poloxamer 188 as Surfactant in Biological Formulations An Alternative for Polysorbate 20 80 International Journal of Pharmaceutics 2022 121706
5. Fox CB Squalene emulsions for parenteral vaccine and drug delivery Molecules 14 9 2009 3286 3312
6. Thermo Fisher Scientific Application Note 73979 Polysorbate 80 profiling by HPLC with mass and charged aerosol detection 2023