Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer

Significance of the Topic

Polysorbate 80 is a nonionic surfactant widely used in biotherapeutic formulations to prevent protein aggregation, reduce surface adsorption, and enhance solubility. Consistent quantitation and characterization of polysorbate 80 are critical for ensuring product quality, stability, and safety in both research and manufacturing environments.

Aims and Study Overview

This work presents a two-dimensional HPLC approach coupled with mass spectrometry to enable reliable quantification and comprehensive profiling of polysorbate 80 in biotherapeutic products without derivatization or extensive sample pretreatment.

Instrumental Setup

The analysis was performed on a Shimadzu Co-Sense for BA system featuring dual LC-20AD pumps, a solvent-switching valve, DGU-20A5R degasser, SIL-20AC autosampler, CTO-20AC column oven with a 6-port 2-position valve, and CBM-20A controller. Detection was carried out using an LCMS-2020 single quadrupole and an LCMS-8050 triple quadrupole mass spectrometer with ESI in positive mode.

Methodology

Sample pretreatment employs a Shim-pack MAYI-ODS trap column to selectively retain polysorbate 80 while excluding proteins and polar excipients. After protein removal, valve switching transfers the trapped analyte to an analytical C18 column for separation. Quantitation uses a short Kinetex C18 column (50×2.1 mm, 5 μm) with a 0.3 mL/min gradient, monitoring m/z 783 in selected ion mode. Characterization employs a longer C18 column (100×2.1 mm, 5 μm) and a shallow gradient over 100 minutes in full scan mode to resolve monooleate and various by-products.

Main Results and Discussion

• Quantitative performance demonstrated linearity from 10 to 200 mg/L (R2 > 0.999), recovery of 99%, and reproducibility with RSDs of 0.034% (retention time) and 1.11% (peak area).
• Characterization separated polyoxyethylene, polyoxyethylene isosorbide, polyoxyethylene sorbitan, and higher oleate species, facilitating detection of degradation products from oxidation and hydrolysis.
• Protein removal efficacy was confirmed by UV monitoring of trap column eluent, showing negligible antibody breakthrough.

Benefits and Practical Applications

The automated two-dimensional workflow offers minimal sample handling, no derivatization, and direct analysis of polysorbate 80 in complex biotherapeutic matrices. It supports QC labs for formulation screening, stability studies, and lot release testing.

Future Trends and Applications

Future developments may integrate high-resolution MS for accurate mass profiling, expand multi-attribute monitoring of other excipients, and enable real-time process analytics in biomanufacturing.

Conclusion

The presented HPLC-MS method with online protein removal provides sensitive, robust quantitation and detailed characterization of polysorbate 80, streamlining analytical workflows in biotherapeutic development and quality control.

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.