Novel high-throughput assay for polysorbate quantification in biopharmaceutical products by using the fluorescent dye DiI.

Martos A, Berger M, Kranz W, Spanopoulou A, Menzen T, Friess W, Wuchner K, Hawe A.

Polysorbates (PSs) are the most common surfactants in therapeutic protein formulations and it is crucial to monitor their concentration along the life cycle of biopharmaceuticals. We developed a simple multi-well plate fluorescence-based assay for the rapid determination of PS20 and PS80 content in biopharmaceutical products. The method is based on the detection of the fluorescence emission intensity of the fluorescent dye DiI in the presence of PS at concentrations below their critical micelle concentration (CMC). This method can be applied for PS content determination in protein formulations (≤100 mg/ml) without the need of a previous protein removal step. The DiI assay implemented in multi-well plate format is suitable for high-throughput concentration screening. It has a linear range from 0.00020 to 0.0025% (w/v) PS20, and the limits of detection (LOD) and quantification (LOQ) were 0.00020 and 0.00055% (w/v), respectively. This assay is markedly more selective and shows no or lower interferences due to hydrophobic components (e.g. silicone oil) potentially present in finished products as compared to the fluorescence micelle assay (FMA) based on N-phenyl-1-naphthylamine (NPN). It also provides comparable results for the PS content as LC-CAD analysis with protein removal, providing a fast alternative.

J Pharm Sci. 2019 OCT

https://jpharmsci.org/article/S0022-3549(19)30648-3/pdf