Analysis of Polysorbate 80 in IgG Aqueous Solution by Online SPE Using a Shim-pack MAYI Column – Part 2

Significance of the Topic

Polysorbate 80 is widely used to stabilize protein formulations by preventing denaturation and aggregation. Its complex mixture of polyoxyethylene sorbitan esters and related by-products can affect therapeutic efficacy and shelf life. Effective monitoring of its purity and degradation products is crucial for quality control in biopharmaceuticals.

Objectives and Study Overview

This study aims to apply an online solid phase extraction (SPE) workflow combined with high-resolution liquid chromatography–mass spectrometry (LC-MS) to profile polysorbate 80 and its minor impurities in an antibody model solution. Building on a prior quantitative method, the focus here is on detecting and characterizing by-products that may impact formulation stability.

Methodology

An IgG solution (20 mg/mL) spiked with 100 µg/mL polysorbate 80 underwent automated protein removal via online SPE. The trap column was a Shim-pack MAYI-ODS (5 mm×2.0 mm ID, 50 µm), and the analytical column was a Kinetex 5 µm C18 (100 mm×2.1 mm ID). Mobile phases consisted of 10 mmol/L ammonium formate in water and 2-propanol. A stepped gradient from 3 % to 100 % organic over 100 minutes enabled separation of both major and minor components. UV detection at 280 nm guided elution timing; mass spectral data were collected in positive ESI mode over m/z 300–2000.

Instrument Used

• Shim-pack MAYI-ODS trap column
• Kinetex C18 analytical column
• Shimadzu LCMS-8050 mass spectrometer
• UV detector (280 nm)
• Autosampler and quaternary pumps for three solvent lines

Main Results and Discussion

The total ion chromatogram revealed seven distinct peaks (A–G). Peak D corresponded to the main polysorbate 80 series, matching the previously quantified monooleate species. Peak A was assigned to polyoxyethylene isosorbide and related oligomers, while peak E represented its esterified form. Peak B indicated polyoxyethylene sorbitan likely formed by hydrolysis of the primary ester. Later eluting peaks C, F, and G showed similar repeating polyoxyethylene patterns with variations in fatty acid chain length and unsaturation. High-resolution mass spectra and charge states confirmed these structural assignments.

Benefits and Practical Applications

This online SPE–LC-MS approach delivers rapid, high-resolution profiling of polysorbate 80 and its impurities without extensive manual sample prep. It enables:

• Comprehensive quality assessment of polysorbate excipients in biopharma formulations
• Detection of degradation products from hydrolysis or oxidation
• Improved throughput and reproducibility in QA/QC labs

Future Trends and Potential Uses

Future developments may include automation of data interpretation for impurity profiling, expansion to other nonionic surfactants, and coupling with stability studies under stress conditions. Integration with machine learning tools could further accelerate quality assessments and predictive shelf-life modeling.

Conclusion

The online SPE coupled with high-resolution LC-MS platform effectively separates and identifies polysorbate 80 by-products in protein solutions. This method enhances monitoring capabilities for formulation quality and supports robust QC workflows in biopharmaceutical development.

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, J Pharm Biomed Anal 62 (2012) 7–16