Affinity membranes for the purification of autologous plasmin from serum

Plasmin is protein of the fibrinolityc system, obtained by activation of plasminogen, which is of interest for ophthalmology applications. It can be used as a treatment for diabetic rethinopaty, macular pukers, but also as a facilitator for vitreoctomy since it has the properties to hydrolize a variety of glycoproteins by degrading the links between these components of the vitreoretinal interface and the inner limiting membrane. The purification of plasminogen from blood is conventionally performed with bead-based affinity 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 high levels of purity and a sufficient amount of protein. In this work we describe several strategies that have been investigated in order to improve the affinity chromatography step which is performed with 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.