VPB1193 - Easy and efficient method for purification of human thrombin, for use as a component of fibrin sealants.

Background: Thrombin (Tb) is a serine protease generated by activation of its inactive precursor, prothrombin. One of its functions is the conversion of fibrinogen to fibrin. In addition, Tb is used with therapeutic purposes in fibrin sealants, where it acts as a local hemostatic agent.

Aims: To develop a method of purification of Tb, using human plasma as raw material.

Methods: The procedure consists of the activation of prothrombin to Tb by diluting the plasma with distilled water and adjusting the pH to 5.3. The resulting precipitate is separated and dissolved with buffer, the pH is raised to 6.5, CaCl2 is added and the fibrinogen that forms is separated by filtration. The Tb solution is subjected to ultrafiltration with a 10 KD membrane, and diafiltered to bring the sample to the conductivity conditions necessary for chromatography. The second step is cation exchange chromatography using a column with SPXL and connected to an AKTA Purifier equipment. The Tb contained in the solution binds to the matrix and is eluted by increasing the ionic strength of the buffer. The purity of the product obtained is evaluated by SDS-PAGE. The specific activity is assessed by measuring thrombin activity by a coagulometric method using an international standard (NIBSC 01/580) as reference. Total proteins are measured by the bicinchoninic acid method.

Results: The Tb eluates from three independent processes have an average Tb activity of 142 IU/ml, with a yield of 81% and a specific activity of 415 IU/mg. The analysis of the process samples by SDS PAGE shows a band of 37 kDa corresponding to α Thrombin, which represents the most preponderant protein band.

Conclusion(s): The results obtained show that the developed method is reproducible and that purified Tb could be used as a component of fibrin sealants since it meets the quality requirements of the European Pharmacopoeia.