Novel EDTA mediated ethanol protein precipitation method and the application for polysorbate quantification in high protein concentration biopharmaceuticals

Non-ionic surfactants such as Polysorbate 20/ 80 (PS20/ PS80), are commonly used in protein drug formulations to increase protein stability by protecting against interfacial stress and surface absorption. Polysorbate is susceptible to degradation which can impact product stability, leading to the formation of sub-visible and/or visible particles in the drug product during its shelf-life, affecting patient safety and efficacy. Therefore, it is important to monitor polysorbate concentration in drug product formulations of biotherapeutic drugs. The common method for measuring polysorbate concentration in drug product formulations uses mixed mode ion exchange reversed phase HPLC (MAX) coupled to evaporative light scattering detection (ELSD). However, high protein concentration can adversely impact method performance due to high sample viscosity, gel formation, column clogging, interfering peaks and loss of accuracy. To overcome this, a new method was developed based on EDTA mediated ethanol protein precipitation (EDTA/EtOH). This method was successfully implemented for the analysis of polysorbate in antibody formulations with wide range of protein concentration (10–250 mg/mL). •Polysorbates (PS20 and PS80) are excipients used to protect biopharmaceuticals.•Mixed-mode HPLC-ELSD method is commonly used to quantify polysorbates.•Direct analysis of high protein sample causes HPLC blockage and contamination.•Ethanol precipitation solves the issues but it cannot precipitate all products.•With EDTA, ethanol precipitates all evaluated product, improving method performance.