Comprehensive Investigation of Polymer Oxidation using Interactive Visualizations and User Modified Libraries in PolyMatchSuite

Significance of the Topic

Polymeric surfactants such as polysorbate 20, polysorbate 80 and poloxamers are integral additives in protein therapeutics and viral vectors. Their susceptibility to oxidation at ambient temperatures, driven by trace metal ions or peroxides, generates a complex array of degradation products that can compromise drug efficacy and safety. Despite numerous studies, comprehensive analytical workflows for non-targeted detection and visualization of oxidized polymer species remain limited.

Objectives and Study Overview

This investigation aimed to develop an end-to-end workflow leveraging an open-source software platform (PolyMatch Suite) and advanced LC/Q-TOF instrumentation to detect, annotate and visualize oxidized polymer series. Key goals included:

• Adapting PolyMatch for non-targeted polymer oxidation screening
• Establishing chromatographic separation of isomeric oxidation products
• Demonstrating interactive visualization techniques for rapid data interpretation

Methodology and Instrumentation

Sample Preparation:

• Polysorbate 80 diluted to 0.02% w/v in aqueous mobile phase
• Oxidation induced by 3% hydrogen peroxide at 40 °C overnight

Chromatography and Mass Spectrometry:

• Agilent AdvanceBio Surfactant Profiling column (2.1 × 100 mm, 15 min gradient)
• 1290 Infinity II UHPLC coupled to 6546 LC/Q-TOF
• Auto MS/MS acquisition via MassHunter Workstation

Data Analysis with PolyMatch Suite:

• Homologous series detection, mass defect filtering and fragment screening
• Generalized library incorporating double bond equivalents, functional groups and adduct patterns
• Automated peak picking, blank subtraction, annotation and interactive visualization

Main Results and Discussion

The combined workflow enabled identification of 261 high-confidence oxidized polymer species (score A). Key findings included:

• Distinct mass defect series corresponding to PEG/POE chains, showing oxidation-induced shifts
• Chromatographic resolution of isomeric oxidation products within the same mass defect series
• Diagnostic MS/MS fragment (m/z 323.2576) characteristic of keto-oleic acid confirming oxidation
• Visualization plots (mass defect vs. retention time, Kendrick mass defect) streamlined false positive exclusion and structural grouping

Benefits and Practical Applications

This integrated approach offers:

• Rapid, non-targeted screening of polymer oxidation products in drug formulations
• Interactive visuals to facilitate decision-making in QA/QC and stability studies
• Open-source extensibility, allowing library expansion to novel polymer chemistries

Future Trends and Opportunities

Next steps include:

• Incorporating additional oxidized species into the PolyMatch library for broader coverage
• Extending the workflow to other therapeutic polymers and excipients
• Leveraging machine learning for predictive identification of degradation pathways

Conclusion

The developed LC/MS/MS method, coupled with the PolyMatch Suite, establishes a robust platform for comprehensive detection and visualization of polymer oxidation by-products. This workflow enhances formulation stability assessment, supports regulatory compliance, and can be adapted for ongoing surveillance of biopharmaceutical integrity.

References

1. Weber J., Buske J., Mäder K., Garidel P., Diederichs T. Oxidation of Polysorbates: An Underestimated Degradation Pathway? Int. J. Pharm.: X 2023, 6, 100202.
2. Koelmel J.P., Stelben P., Oranzi N., Kummer M., Godri D., Qi J., Rennie E.E., Lin E., Weil D., Pollitt K.J.G. PolyMatch: Novel Libraries, Algorithms, and Visualizations for Discovering Polymers and Chemical Series. J. Am. Soc. Mass Spectrom. 2024, 35, 413–420.