Strategic characterization of active pharmaceutical ingredients co-eluting impurities: Identification, enrichment and structural elucidation of an oxidized impurity from mobocertinib drug substance.

Mobocertinib (TAK-788) is a potent, synthetic molecule tyrosine kinase inhibitor specifically designed to selectively target epidermal growth factor receptor and HER2 exon 20 insertion mutations. Previously, it was a drug candidate developed by Takeda Pharmaceuticals used to treat patients with metastatic non-small cell lung cancer. During process development, a new oxidative degradant was identified as a co-eluting peak with the active pharmaceutical ingredient (API) in the release method. To understand the significance of this unknown co-eluting impurity, full structural characterization was performed by following a newly established workflow. In brief, preliminary two-dimensional LC-MS (2D-LC-MS) analysis identified the targeted degradant as a mono-oxygenated product of TAK-788. With the purpose of facile isolation and purification, various forced degradation conditions were screened to enrich the desired degradant, among which hydrogen peroxide-treatment effectively afforded a yield of 65% for this unknown impurity. Following isolation by prep-HPLC, 1D and 2D NMR studies and HRMS characterization assigned the isolate as Mobocertinib-N-oxide. In addition, NMR-based reaction monitoring was conducted to trace its formation, allowing a plausible mechanism of formation to be proposed.