Characterization of Polysorbate 80 by Liquid Chromatography-Mass Spectrometry to Understand Its Susceptibility to Degradation and Its Oxidative Degradation Pathway

A liquid chromatography-mass spectrometry (LC–MS) method was developed to provide a fingerprint of polysorbate 80 (PS80) subspecies that enables identification of PS80 degradation pathway. The developed method demonstrates unique monoester peak profile of PS80 from different vendors, attributed by differences in relative abundance of the fatty acid monoesters. The LC–MS method was also applied to examine the susceptibility of PS80, at different grades, to auto-oxidation and hydrolysis. PS80 oxidative degradation induced by iron or occurred in open bottle without nitrogen overlay was found to follow the same pathway, but at a much faster rate in the former scenario. The oxidation preferentially occurs at the double bond of fatty acid chains, thus providing explanation on the faster degradation observed in PS80 at Chinese Pharmacopia (ChP) grade than at multi-compendial (MC) grade. In contrast, the difference in susceptibility of MC and ChP grade PS80 against esterase-induced hydrolysis in placebo was not pronounced. The method was also able to provide a fingerprint to identify both PS80 hydrolysis and oxidation in mAb drug product stability samples, but it required a solid phase extraction step to remove protein prior to the analysis.