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Martos, Ariadna; Berger, Michelle; Kranz, Wendelin; Spanopoulou, Anna; Menzen, Tim; Friess, Wolfgang; Wuchner, Klaus; Hawe, Andrea (2020): Novel High-Throughput Assay for Polysorbate Quantification in Biopharmaceutical Products by Using the Fluorescent Dye DiI. In: Journal of Pharmaceutical Sciences, Vol. 109, No. 1: pp. 646-655
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Abstract

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 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate in the presence of PSs at concentrations below their critical micelle concentration. 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 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate 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 and quantification 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 than the fluorescence micelle assay based on N-phenyl-1-naphthylamine. It also provides comparable results for the PS content in liquid chromatography with charged aerosol detection analysis with protein removal, providing a fast alternative. (C) 2020 American Pharmacists Association.