Impact of Citrate on Mitigating Iron Mediated Polysorbate 80 Degradation in Biotherapeutic Formulation Placebos.

Abstract

Polysorbate 80 (PS80), a widely used polymeric surfactant in biotherapeutic formulation, possesses a unique structural composition that effectively prevents protein aggregation in highly concentrated protein drug formulations. However, PS80 is susceptible to hydrolysis, due to the presence of fatty acid esters that can be enzymatically hydrolyzed, The unsaturated bonds in the fatty acids are prone to oxidative degradation when exposed to air, especially in the presence of transition metals such as iron and copper, which may be introduced during production and purification processes or from contamination in raw materials used in drug formulation. The degradation of PS80, particularly through metal-mediated oxidative degradation, poses a significant challenge for the industry. Among the identified trace metals, iron plays a crucial role as the redox reaction between ferrous ion (Fe(II)) and ferric ion (Fe(III)) generates radicals that initiate the degradation process. In order to investigate the impact of iron on PS80 degradation and understand the mechanism of iron-catalyzed oxidation, we utilized charge-reduction mass spectrometry and two-dimensional ion density mapping technologies to characterize the degradation of PS80. This method has proven to be a convenient and effective tool for the quick and detailed profiling of PS80, allowing for visual monitoring and examination of the changes that reflect the difficult-to-identify and easy-to-miss oxidized species of PS80. Additionally, a high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry method was developed for the separation and measurement of Fe(II) and Fe(III). Through this investigation, we determined that the involvement of Fe(II)/Fe(III) in PS80 degradation is a temperature dependent process. Furthermore, we found citrate not only promotes the conversion of Fe(II) to Fe(III), but it also chelates Fe(III) and prevents its reduction to Fe(II), thus inhibiting the initiation of the PS80 degradation. Therefore, the addition of citrate can be a crucial ingredient for controlling the degradation of PS80 in biologic drug substances and products. Overall, this investigation has provided valuable insights to enhance product stability, optimize processes, and ensure the quality of formulations containing PS80.