Comprehensive analysis of peptide pharmaceutical: Development of three orthogonal chromatographic methods and characterization of bivalirudin degradation products by high-resolution mass spectrometry.

Bivalirudin is a synthetic oligopeptide used as an anticoagulant for the treatment of acute coronary syndromes and for the prevention of thrombosis in patients undergoing percutaneous coronary intervention. As peptides are complex molecules that may exhibit inherently complex impurity profiles, three orthogonal ultra-performance liquid chromatographic methods were developed in this work, in accordance with Analytical Quality-by-Design principles, to ensure the safety and efficacy of the bivalirudin drug product. Starting from a structure-driven theoretical assessment of bivalirudin, which anticipates the formation of peptide fragments, deamidation and oxidation products, as well as covalent oligomers under stress conditions, method development was directed into three orthogonal chromatographic modes-size-exclusion (SEC), reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) - enabling a comprehensive evaluation of its degradation pathways. For the first time, bivalirudin oligomers (dimers and trimers) were detected and characterized using size-exclusion chromatography. The combination of HILIC and RP chromatography methods ensured separation of 10 non-oligomeric bivalirudin impurities. The developed methods employed MS-compatible mobile phases to facilitate structural elucidation of bivalirudin degradation products by high-resolution mass spectrometry. Stress conditions included acid and base hydrolysis, oxidation with H₂O₂, thermal decomposition, and light exposure, as regulated by ICH guidelines. Extensive risk assessment was conducted using Ishikawa diagrams and heat maps. Method development was carried out using Design of Experiments methodology, supported by Fusion AE software. The developed ultra-performance liquid chromatography (UPLC) methods were validated, confirming their suitability for analyzing bivalirudin and its related impurities.