Detailed profiling of polysorbate 80 oxidative degradation products and hydrolysates using liquid chromatography-tandem mass spectrometry analysis.

RATIONALE: Polysorbate 80 (PS80) is an amphipathic, nonionic surfactant that is commonly used to stabilize proteins in biopharmaceutical formulations. PS80 undergoes oxidative and/or enzymatic degradation. However, because PS80 is a complex mixture consisting of many constituents, comprehensive evaluations of its oxidative degradation products are difficult and insufficient.

METHODS: Our previously reported comprehensive liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based method for PS80 effectively provides an overall profile of PS80 components under simple LC conditions. In this study, we attempted to shorten the analysis time. Furthermore, PS80 was oxidatively degraded in a solution containing histidine and iron, and the oxidative degradation products were evaluated using a modified LC/MS/MS method. In addition, enzymatically hydrolyzed PS80 samples were analyzed.

RESULTS: We succeeded in shortening the analysis time from 70 to 20 min while maintaining the resolution of the PS80 components of the same selected reaction monitoring transition. Both the previously reported oxidative degradation products and the newly discovered products were successfully detected, and their composition ratios and changes over time were observed. Changes in the hydrolysates over time are shown in the analysis of the hydrolyzed PS80 samples.

CONCLUSIONS: This study clearly showed the presence of changes in PS80 oxidative and/or enzymatic degradation products, including those previously unreported. These results demonstrate that a detailed profiling of PS80 degradation products can be performed using LC/MS/MS, which is less expensive and more generally adopted than high-resolution MS.