Detection and differentiation of active and inactive isoforms of coagulation factors II, VII, IX, and X in prothrombin complex concentrate by mass spectrometry.

PURPOSE: Prothrombin complex concentrates (PCCs) are plasma products containing a mixture of four inactive/proactive coagulation factors. The activated forms of human coagulation factors, like Thrombin (FIIa), Convertin (FVIIa), activated Christmas factor (FIXa) and the activated Stuart-Prower factor (FXa), are impurities in PCCs. Until now no valid assay exists to differentiate the non activated proform (inactive) from active coagulation factor isoforms in PCCs in one measurement. Therefore, the aim of this study was to establish a mass spectrometry (LC-MS/MS)-based assay to address this issue in the ready to use medicinal product. METHODS: Bottom-up proteomics combining double digestion (Glu-C & Lys-C) and LC-MS/MS, was used to differentiate the inactive and active forms of the coagulation factors Prothrombin (FII), Proconvertin (FVII), Christmas factor (FIX) and the Stuart-Prower-factor (FX) in PCCs. RESULTS AND CONCLUSIONS: A targeted pseudo-multiple reaction monitoring (pMRM-LC-MS/MS)-assay was developed for the specific detection of four different coagulation factors in PCCs. Proteotypic peptides for the inactive/active isoforms (zymogen) of the four coagulation factors were identified and validated by the investigation of six investigational and one commercially available PCCs. In conclusion, the semi-quantitative determination and the distinction between the active and the inactive isoform of the respective coagulation factors were possible in one liquid chromatography tandem mass spectrometry (LC-MS/MS) run.