Category: Preclinical Development
Purpose: Structural elucidation of degradation impurities in formulated drug product is very important for development and optimization of formulated drugs, and in certain cases a requirement for regulatory submissions. These impurities are products of active pharmaceutical ingredient (API) by different degradation chemistries during formulation processing storage and patient in-use. LC-MS/MS is the most important tool besides NMR for structural identification of degradation products. MS/MS data interpretation is typically carried out by a tedious manual process. A data processing tool that incorporate common degradation chemistries for small peptides was developed to treat the LC-MS/MS data which greatly improves efficiency.
Methods: The LCMS experiments are performed on Waters SynptG1 Stove (Milford, MA) mass spectrometer. An ultra-performance liquid chromatography (UPLC) MSe method was developed with low collision energy (10 eV) as channel one to detect intact molecules and high collision energy ramping from 20 to 40 eV as channel two to obtain pseudo MS/MS data. For data analysis, MassChemSite (Molecular Discovery Ltd.) was used. Inputs to the software were the mol file of API and the LC-MSe chromatogram for API and for the degradation mixture. The software incorporate common degradation chemistries. Extraction of peaks and assignment of chemical structures was performed automatically by the software.
Results: A general HR-MS/MS method was developed to support all degradation analysis. MassChemsite was customized to adopt common degradation chemistries such as hydrolysis, oxidation, esterification and dehydration reactions. The software compared MS/MS data of API and degradation products then used a scoring function to assign the best fit structures to mass fragments and rate the final structures. Finally, degradation product structures were proposed. Well-studied degradation chemistry was chosen to test this methodology: Aspartate Deamidation, degradation of disulfide bond and amide hydrolysis and Oxidation reactions. The structures of major degradation products matches to those reported in the literature.
Conclusion: The data analysis cycle, which is the main bottleneck for the structural elucidation of small peptide degradants by LC-MS/MS, is greatly shortened by using MassChemSite.
Ismael Zamora– Sant Cugat del Valles, Catalonia, Spain
Blanca Serra– Sant Cugat del Valles, Catalonia, Spain
Yuejie Zhao– Kenilworth, New Jersey
Yong Liu– Principal Scientist, Merck & Co., Inc., Kenilworth, New Jersey
Kevin Bateman– Scientific Associate Vice President, Merck & Co., Inc., West Point, Pennsylvania
Blanca Serra– Scientific Developer, Lead Molecular Design, SL, Sant Cugat Del Valles, Catalonia, Spain