AN EFFICIENT METHOD FOR THE DETERMINATION OF TRACE EXCIPIENT IMPURITIES IN BIOTHERAPEUTIC DRUG PRODUCTS CONTAINING POLYSORBATE

Význam tématu

Monitoring of polysorbate excipient purity and degradation products is critical in biotherapeutic formulations to ensure drug safety and efficacy. Polysorbate 80 (PS-80) prevents protein aggregation and surface adsorption but can degrade under residual esterase activity. Trace free fatty acids like oleic acid serve as sensitive indicators of polysorbate breakdown, requiring analytical methods with high sensitivity, specificity and throughput for quality control throughout the product lifecycle.

Cíle a přehled studie

The study aimed to develop and validate a rapid, robust UPLC-UV assay for quantifying trace levels of free oleic acid as a surrogate marker of PS-80 degradation in formulated monoclonal antibody products. Key objectives included:

• Establishing dynamic range and linearity for oleic acid detection down to sub-ppm levels.
• Selecting and evaluating an internal standard for accurate quantitation.
• Optimizing a low-volume liquid–liquid extraction (LLE) for sample preparation compatible with direct injection.
• Demonstrating assay specificity and applicability to real biopharmaceutical samples.

Použitá metodika a instrumentace

• Chromatography on Waters ACQUITY UPLC H-Class Bio System with TUV detector (200 nm) and Protein BEH C4 1.7 µm, 2.1×100 mm column at 30 °C.
• Isocratic elution (65% acetonitrile/35% water, both containing 0.1% formic acid) at 0.2 mL/min, 5 min run time.
• Internal standard: cis-10-nonadecenoic acid, baseline-resolved from oleic acid with no carry-over.
• Sample preparation via low-volume LLE using NaOH hydrolysis at 65 °C followed by acetonitrile extraction and phase separation.
• Injection volume: 1 µL; sample temperature: 10 °C.

Hlavní výsledky a diskuse

The method exhibited excellent performance:

• Dynamic range from 0.24 ppm to 1000 ppm oleic acid with linearity R²≥0.9999 over six orders of magnitude.
• Recovery of oleic acid post-LLE exceeded 90% across concentrations, with average internal standard correction factors between 1.07 and 1.29.
• Limit of detection around 60 ppb via mass spectrometry ion monitoring corroborated UV findings.
• Assay specificity confirmed by absence of free oleic acid in PS-20-formulated trastuzumab, and detection of <1% free oleic acid relative to internal standard in PS-80 infliximab after base hydrolysis.

Přínosy a praktické využití metody

• High-throughput 5-minute isocratic assay reduces run times and improves laboratory productivity.
• Minimal sample preparation enables streamlined workflows in regulated QC environments.
• UV-based detection offers a cost-effective alternative to MS-only methods while maintaining sensitivity.
• Robust performance supports lifecycle monitoring of polysorbate stability in biopharmaceutical products.

Budoucí trendy a možnosti využití

• Integration with online solid‐phase extraction or automated robotic sample handling to further boost throughput.
• Adaptation of the method to other nonionic surfactants and degradation markers beyond oleic acid.
• Coupling to high‐resolution MS for structural elucidation of additional degradation species.
• Implementation in process analytical technology (PAT) for real‐time monitoring during drug manufacturing.

Závěr

A rapid, sensitive and robust UPLC-UV assay was established for trace free oleic acid quantitation as a proxy for PS-80 degradation in biotherapeutic formulations. The method’s excellent linearity, recovery, and specificity make it a valuable tool for routine quality control and stability studies.