Human fibrinogen, as a blood product of special origin, is relatively simple to prepare and purify. Therefore, completely isolating and removing the relevant impurity proteins is difficult. Further, which impurity protein components are present is not clear. In this study, human fibrinogen products from seven enterprises were collected from the market, and the presence of impurity proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, the major 12 impurity proteins were identified and screened by in-gel enzymolysis mass spectrometry, and 7 major impurity proteins with different peptide coverage were identified by enzyme-linked immunosorbent assay, in agreement with the mass spectrometry results. The seven major impurity proteins included fibronectin, plasminogen, F-XIII, F-VIII, complement factor H, cystatin-A, and α-2-macroglobulin. The final test results were in the range of undetectable to 50.94 µg/mL, with correspondingly low levels of impurity proteins between different companies and a manageable risk. Moreover, we found that these impurity proteins existed in the form of polymers, which might also be an important cause of adverse reactions. This study established a protein identification technique applicable to fibrinogen products, which provided new ideas for studying the protein composition of blood products. In addition, it provided a new means of testing for companies to monitor the flow of proteomic fractions and improve the purification yield and product quality. It laid the foundation for reducing the risk of clinical adverse reactions.
Keywords: Human fibrinogen; Impurity proteins; In-gel enzymolysis; Mass spectrometry.
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