Investigation of excipient-related artifacts observed in the leachables study of tiragolumab by LC-HRMS and GC-MS.

Leachables studies of protein formulations are faced with many analytical challenges arising from relatively high levels of complex formulation components. In the stressed sample of the monoclonal antibody drug tiragolumab with the main excipients methionine, histidine, sucrose, and polysorbate, two unknown compounds were detected in the screening for non-volatile leachables by reversed phase (RP) liquid chromatography high-resolution mass spectrometry (LC-HRMS). Unknown 1 was proven to be a gas-phase mass spectral adduct between methionine and the sucrose-derived hexose. Separation of methionine and hexose by hydrophilic interaction chromatography (HILIC) resulted in the disappearance of the unknown peak. It was also observed that non-covalent amino acid-sugar adducts in the gas phase were prevalent in both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) modes. Unknown 2 was identified as a Maillard reaction product 1-(3H-imidazo[4,5-c]pyridin-4-yl)ethan-1-one by LC-HRMS and gas chromatography mass spectrometry (GC-MS). The formation mechanism was confirmed by the reaction between histidine and the sucrose-derived methylglyoxal (MGO) under hydrothermal conditions. This study exemplifies false positive leachable results and delves into the chemical reactions and interactions that generated such artifacts.