A universal method for the determination of polysorbate 80 in monoclonal antibodies and novel protein therapeutic formulations

Abstract

An evaporative light scattering detector (ELSD) based HPLC method that requires minimal sample preparation has been developed to quantitate polysorbate 80 (PS80) in therapeutic proteins. The method utilizes a mixed-mode chromatography column and a step gradient of formic acid and acetonitrile to separate PS80 from proteins and formulation excipients. Proteins and hydrophilic formulation excipients are not retained in the column and are eluted in the void volume. The retained hydrophobic, heterogenous PS80 species can be quantified as a single peak using the ELSD. A universal standard curve for PS80 in HPLC grade water was utilized in this method. The method accurately quantified PS80 in 18 different sample formulations including monoclonal antibodies, bispecific antibodies, antibody drug conjugates (ADCs), and bispecific ADCs, thereby establishing a truly selective method free of matrix interference. The assay performance evaluated for precision, specificity, accuracy, and linearity demonstrated the method qualification in the range of 0.05–0.6 mg ml⁻¹ PS80 concentrations and up to 150 mg ml⁻¹ protein concentration. The method also accurately monitored the reduction of PS80 in samples subjected to thermal and long-term storage stresses. The methodology was found to be applicable for the quantitation of polymers PLGA and Pluronic F127. The method was further modified to create a 2-dimensional liquid chromatography ELSD system and quantitated other formulation excipients histidine, arginine, and sucrose in the presence of PS80.