Analysis of Polysorbate 80 in IgG Aqueous Solution by Online SPE Using Shim-pack MAYI Column – Part 1

Importance of the subject

Polysorbate 80 is widely employed as a non ionic surfactant in biopharmaceutical formulations to prevent protein aggregation and enhance solubility. Its accurate quantitation in protein containing solutions is critical for quality control and ensuring patient safety. Traditional reversed phase HPLC analysis of such samples can suffer from column degradation due to high protein loads, necessitating robust sample cleanup strategies before analysis.

Objectives and overview of the study

This study demonstrates an online sample pretreatment approach using Shim pack MAYI restricted access media columns within a column switching HPLC system to remove immunoglobulin G matrix components and selectively quantify polysorbate 80. The method aims to integrate deproteinization and surfactant analysis in a seamless automated workflow, improving throughput and reproducibility.

Applied methodology

The system employed a two stage column switching configuration. Firstly, macromolecular proteins are excluded by the hydrophilic coating of the Shim pack MAYI column and directed to waste. Subsequently, polysorbate 80 is retained on a Shim pack MAYI ODS C18 pretreatment column. After protein removal, the valve switches to elute and transfer the retained surfactant to the analytical column while simultaneously rinsing the pretreatment path.

Used instrumentation

• Shimadzu HPLC system with column switching manifold
• Shim pack MAYI restricted access media columns (hydrophilic polymer coated, 2.0 mm ID)
• Shim pack MAYI ODS C18 column (50 mm x 2.0 mm ID, 50 µm)
• UV detector set at 280 nm for protein elution confirmation
• LCMS 2020 with ESI positive mode for polysorbate 80 detection

Main results and discussion

Online deproteinization was confirmed by UV monitoring at 280 nm, showing rapid discharge of immunoglobulin G from the pretreatment column. Polysorbate 80 standards (10 to 200 µg/mL) yielded a total ion chromatogram with strong retention and a selected ion monitoring (SIM) target at m/z 783 (2NH4 adduct of polyoxyethylene sorbitan monooleate). Calibration exhibited excellent linearity (R2 above 0.999). In a protein containing model sample (20 mg/mL IgG, 100 µg/mL polysorbate 80), recovery averaged 99% with retention time RSD of 0.034% and peak area RSD of 1.11%.

Benefits and practical applications

• Fully automated online deproteinization and analysis reduces manual sample preparation.
• Minimized column packing degradation by preventing protein exposure to analytical column.
• High sensitivity and reproducibility suitable for quality control in biopharmaceutical development.
• Adaptable to various protein based formulations and surfactant quantification tasks.

Future trends and potential applications

Integrating high resolution mass spectrometry could enable detailed profiling of polysorbate degradation by products. Further miniaturization and multiplexed SPE columns may allow simultaneous cleanup of multiple analyte classes. Automation developments will support higher throughput bioanalysis, particularly in biosimilar and novel biologics manufacturing.

Conclusion

The online SPE HPLC method using Shim pack MAYI columns provides a robust, reproducible, and efficient workflow for polysorbate 80 determination in protein rich solutions. It streamlines deproteinization and quantitation, offering significant advantages for biopharmaceutical quality control laboratories.

Reference

Shimadzu Application News No L486