An Efficient UV-Based Method for the Assessment of Oleic Acid Content in Biotherapeutic Drug Products Containing Polysorbate-80

Importance of the Topic

Surfactants such as polysorbate 80 are critical stabilizers in biotherapeutic formulations, preventing protein aggregation and surface adsorption. Their degradation can compromise drug efficacy and safety, highlighting the need for reliable analytical methods to quantify degradation products such as oleic acid.

Objectives and Study Overview

The study aimed to develop a rapid, sensitive ultraviolet (UV)–based assay for the quantification of oleic acid released from polysorbate 80 in formulated drug products. The focus was on minimal sample preparation, high throughput, and suitability for regulated environments.

Methodology and Instrumentation

Sample preparation involved liquid–liquid extraction of both free and esterified fatty acids with base hydrolysis followed by acidification and salting‐out. Separation was achieved isocratically on a low‐retentivity C4 column using an ACQUITY UPLC H-Class Bio System with a TUV detector at 200 nm. An optional ACQUITY QDa mass detector provided orthogonal confirmation.

Instrumentation Used

• ACQUITY UPLC H-Class Bio System
• ACQUITY UPLC Autosampler with FTN
• ACQUITY UPLC Protein BEH C4 Column (300 Å, 1.7 µm, 2.1 mm × 100 mm)
• ACQUITY UPLC TUV Detector
• ACQUITY QDa Mass Detector (optional)

Main Results and Discussion

Dynamic range experiments demonstrated linear UV response for oleic acid from 0.24 ppm to 1000 ppm with an LOD of 0.24 ppm. The optimized C4 column minimized dispersion and achieved baseline separation in under five minutes. Recovery studies using an internal standard showed efficiencies up to 98% across a broad concentration range. Analysis of infliximab (PS-80) and trastuzumab (PS-20) formulations revealed no free oleic acid, while hydrolyzed infliximab samples contained trace oleic acid (<1% relative to internal standard).

Benefits and Practical Applications

• High sensitivity and broad dynamic range suitable for trace analysis.
• Minimal sample preparation and direct injection reduce turnaround time.
• Compatibility with widely available UV detectors enhances laboratory deployability.
• Optional MS detection increases specificity when required.

Future Trends and Applications

Continued evolution may include automation of extraction workflows for real-time process monitoring, expansion to other fatty acid degradants, integration with high-resolution mass spectrometry for structural elucidation, and adaptation for online quality control in manufacturing.

Conclusion

The developed UV-based UPLC method provides a robust, sensitive, and high-throughput approach for quantifying oleic acid as an indicator of polysorbate 80 degradation in biotherapeutics. Its simplicity and compatibility with standard laboratory equipment make it well suited for routine quality and stability testing.

References

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