Reducing the Complexity of Polysorbate Oxidation By-Product Screening by LC/MS/MS

Significance of the Topic

Biopharmaceutical formulations commonly include polysorbate surfactants to stabilize protein therapeutics. Oxidative degradation of these surfactants—driven by residual peroxides, light exposure, and trace metals—poses a significant risk to product stability, efficacy, and safety. Rapid and unambiguous screening methods for polysorbate oxidation by-products are essential for developing mitigation strategies and ensuring long-term formulation integrity.

Objectives and Overview of the Study

This application note describes a comprehensive LC/MS/MS workflow to screen and identify oxidative degradation products of polysorbate 80. Key goals include:

• Developing a targeted screening approach using a custom suspect library and the Find by Formula algorithm.
• Implementing an untargeted suspect screening strategy with PolyMatch software to discover novel oxidation markers.
• Demonstrating the performance of an Agilent AdvanceBio Surfactant Profiling HPLC column coupled to an Agilent 6546 LC/Q-TOF system for high-resolution separations and accurate mass measurements.

Methodology and Instrumentation

Sample Preparation

• Polysorbate 80 diluted to 0.02% w/v in 10 mM ammonium acetate (mobile phase A).
• Oxidation induced by incubating with 3% hydrogen peroxide at 40 °C overnight.

Chromatography

• Agilent AdvanceBio Surfactant Profiling 300 Å, 3.5 μm, 2.1 × 100 mm column with guard.
• Mobile phase A: 10 mM ammonium acetate; B: methanol.
• Gradient from 5% B to 91% B over 10 minutes, total run time 18 minutes; flow rate 0.3 mL/min; column temperature 50 °C; injection volume 2 μL.

Mass Spectrometry

• Agilent 6546 LC/Q-TOF with Dual Agilent Jet Stream source.
• Gas temperature 200 °C; nebulizer 30 psig; sheath gas 350 °C at 11 L/min; capillary voltage 3,500 V; fragmentor 145 V.
• Data acquired in Auto MS/MS (top five precursors per cycle) at 5 spectra/sec for MS and 3 spectra/sec for MS/MS; mass range m/z 50–3,000; collision energies at 10, 20, 40 V.

Data Processing

• Targeted screening with Agilent MassHunter Qualitative Analysis 12.0 using the Find by Formula (FBF) algorithm and a Personal Compound Database and Library (PCDL) of >4,800 known polysorbate degradants.
• Suspect screening and untargeted polymer annotation with Agilent PolyMatch Flow and Visualizer software, leveraging Kendrick mass defect plots, homologous series detection, and MS/MS fragment screening.

Main Results and Discussion

Targeted Analysis

• POE1 monohydroperoxy oleate identified with FBF scores of 82.5 in control and 99.5 in oxidized samples; abundance increased by an order of magnitude upon oxidation.
• Series of POE11–POE15 monohydroperoxy oleates showed high FBF scores (96.3–99.7) and sub-1 ppm mass errors; levels increased consistently in oxidized samples with peak area RSDs below 6%.

Suspect Screening Analysis

• PolyMatch detected 261 high-confidence (A-score) polymer species, including oxidized polysorbate isomers separated chromatographically.
• Kendrick mass defect plots normalized to CH2CH2O and retention time versus m/z projections facilitated clear visualization of homologous oxidation series.
• MS/MS spectra confirmed diagnostic ions (e.g., m/z 323.2576 for keto oleic acid) that guided future library expansion.

Benefits and Practical Applications of the Method

• Rapid screening workflow enabling both targeted identification of known degradants and discovery of novel oxidation products.
• High-resolution separations achieved in under 20 minutes, improving throughput.
• Accurate mass and isotopic fidelity support unambiguous compound identification.
• Software tools streamline data processing: FBF for fast targeted analysis, PolyMatch for comprehensive polymer annotation.

Future Trends and Applications

• Expansion of PCDL and PolyMatch libraries to include newly discovered oxidation markers and fragmentation patterns.
• Integration of iterative exclusion MS/MS strategies to increase coverage of low-abundance species.
• Application of the workflow to other surfactants and complex polymer formulations.
• Use of advanced data analytics and machine learning to predict and mitigate oxidation pathways in real time.

Conclusion

The presented LC/MS/MS screening workflow combines the high-resolution separations of the Agilent AdvanceBio Surfactant Profiling column with targeted and untargeted data analysis strategies. This approach effectively profiles polysorbate oxidation by-products, offering robust tools for formulation monitoring and mitigation strategy development in biopharmaceutical research.

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