Analysis of Polysorbate 80 in Protein Formulations Using 2D LCMS

Significance of the topic

Polysorbate 80 is a nonionic surfactant extensively used in biotherapeutic formulations to prevent protein aggregation and surface adsorption while enhancing solubility. Reliable quantitation and molecular profiling of this excipient are critical for quality control, stability assessment, and regulatory compliance in pharmaceutical development.

Objectives and study overview

This work describes a two-dimensional HPLC-MS method (Co-Sense for BA) for simultaneous quantitation and structural characterization of polysorbate 80 in protein formulations. The method aims to automate protein removal, improve sensitivity for nonchromophoric surfactants, and resolve complex mixtures of by-products.

Methodology and instrumentation

A sample pretreatment column traps polysorbate 80 while excluding high-molecular-weight proteins and polar excipients. Following valve switching, the trapped surfactant is eluted onto an analytical C18 column. Detection is achieved by mass spectrometry due to the lack of UV chromophores.

• Co-Sense for BA 2D HPLC system with two LC-20AD pumps, solvent switching valve, MAYI-ODS trap column, and analytical Kinetex C18 column
• MS detection using Shimadzu LCMS-2020 single quadrupole or LCMS-8050 triple quadrupole in positive ESI mode
• Quantitation in SIM mode monitoring m/z 783 corresponding to the 2NH4+ adduct of polyoxyethylene sorbitan monooleate

Main results and discussion

Quantitative analysis of a model sample (IgG with 100 mg/L polysorbate 80) demonstrated excellent recovery (99 %) and reproducibility (RSD 1.11 % for peak area). Calibration over 10–200 mg/L showed linearity with R2 > 0.999. High-resolution characterization separated multiple surfactant species, including mono-, di-, trio-, and tetraoleates, as well as polyoxyethylene sorbitan and isosorbide derivatives. Protein removal efficiency was confirmed by UV detection of the trap column eluent lacking protein peaks.

Benefits and practical applications of the method

This automated approach eliminates laborious sample pretreatment steps such as derivatization or hydrolysis and offers simultaneous quantitation and profiling of polysorbate variants. It is directly applicable to biopharmaceutical QC/QC laboratories for stability studies, formulation development, and batch release testing.

Future trends and possibilities of use

Advancements may include coupling with high-resolution MS for deeper structural elucidation, extension to other polysorbate grades, and integration into continuous manufacturing workflows. Further automation and data processing tools will enable real-time monitoring of surfactant degradation products and oxidation profiles.

Conclusion

The presented 2D HPLC-MS method using the Co-Sense for BA system provides a robust, sensitive, and streamlined solution for quantifying and characterizing polysorbate 80 in protein formulations, facilitating improved quality control and formulation stability assessment.

Reference

E. Hvattum, W.L. Yip, D. Grace, K. Dyrstad, Characterization of polysorbate 80 with liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy: Specific determination of oxidation products of thermally oxidized polysorbate 80, Journal of Pharmaceutical and Biomedical Analysis 62 (2012) 7–16.