(299c) Recovery of Autologous Plasmin from Serum with Affinity Membranes

Plasmin is a protein of the fibrinolityc system with activity against fibronectin and laminin, components of the vitreoretinal interface. It can be used for treatment of diabetic rethinopaty, macular pukers and vitreomacular adhesion since it works by dissolving the proteins that link the vitreous to the macula, resulting in posterior detachment of the vitreous from the retina. Plasmin can be obtained by enzymatic activation of blood plasminogen using urokinase or streptokinase.

The purification of plasminogen from blood is performed with packed column chromatography, by exploiting the affinity between plasminogen and L-lysine. However, due to its low concentration, which is about 0.2 g/L in human blood, with a single step affinity purification from serum or plasma it is not possible to obtain sufficient amount of plasminogen for the subsequent enzymatic activation .

In this work we describe several strategies that have been investigated in order to improve the membrane chromatography step which is performed with L-lysine affinity membranes. The affinity membranes were obtained by immobilization of L-lysine on regenerated cellulose membrane supports and characterized in terms of ligand density, binding capacity and selectivity. The effect of operating parameters like flow rate, type of blood source (serum, plasma or Cohn fractions) and elution conditions on the activity of plasmin conversion have been investigated in detail. The comparison with a chromatography column packed with a commercial lysine affinity beads demonstrated the superior performance of the affinity membranes and the feasibility of the proposed process.