Monitoring polysorbate 80 degradation in protein solutions using Total Holographic Characterization

[Display omitted] •Detect distinguish and quantify oleic acid from lipase induced degradation of PS80.•Simultaneously detect and distinguish oleic acid, protein aggregates and silicone oil.•Detection of oleic acid at concentrations less than 100 ng/mL.•Protein solutions in which PS80 has been degraded contain significantly higher concentrations of aggregates.•The experimental results demonstrate the accuracy, sensitivity and precision of THC as a tool to monitor PS80 degradation in biopharmaceuticals. The degradation of polysorbate surfactants can limit the shelf life of biologic pharmaceutical products. Polysorbate is susceptible to degradation via either oxidation or hydrolysis pathways which releases free fatty acids (FFA) and other complex polymers. Degradants from Polysorbate 80 (PS80) can form particles and impact drug product quality. PS80 degradation products appear at low concentrations, and their refractive indexes are similar to that of the buffer, making them very challenging to detect. Furthermore, aggregates of FFA are similar in size and refractive index to protein aggregates adding complexity to characterizing these particles in protein solutions. Total Holographic Characterization (THC) is used in this work to characterize FFA particles of oleic acid and linoleic acid, the two most common degradation products of PS80. We demonstrate that the characteristic THC profile of the FFA oleic acid emulsion droplets can be used to monitor the degradation of PS80. THC can detect oleic acid at a concentration down to less than 100 ng/mL. Using the characteristic THC signal of oleic acid as a marker, the degradation of PS80 in protein solutions can be monitored quantitatively even in the presence of other contaminants of the same size, including silicone oil emulsion droplets and protein aggregates.