(402e) Pure Autologous Plasmin Obtained With Affinity Membranes
Some eye conditions like diabetic rethinopaty, macular pukers, retinal detachment may benefit from vitreoctomy. Enzymatic vitreoctomy with plasmin is envisaged to augment or even replace conventional vitreoctomy by proposed means of less surgical risks, less surgeon time, lower costs. In addition, the use of autologous plasmin is beneficial since it avoids rejection problems and the search of compatible donors. Plasmin has the property to hydrolize a variety of glycoproteins, including laminin and fibronectin, by degrading the links between these components of the vitreoretinal interface and the inner limiting membrane, therapeutic posterior vitreous detachment has become possible.
Plasmin can be obtained by conversion of plasminogen using a variety of enzymes, including tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). The purification of plasminogen from blood is normally performed with bead-based affinity chromatography.
However, for ophthalmology applications affinity membranes are ideally suited for the development of a disposable device to be used directly by the surgeon in the operating theatre, since they can be easily packed in small units providing a fast and economic process.
In this work we prepared affinity membranes for plasminogen purification using L-lysine as affinity ligand. To this aim regenerated cellulose membranes were used as a support for ligand immobilization. L-lysine was coupled by applying different binding protocols, with different buffers and reactants, testing also the effects of different temperatures.
The immobilization yield has been studied as a function of reaction time and L-lysine concentration. Ligand density was measured using a colorimetric assay with Orange 7 as indicator.
The membranes have been characterized in batch and in dynamic experiments using pure plasminogen, bovine and human sera. During chromatographic experiments with serum, fractions have been collected and analyzed with both HPLC and SDS-PAGE electrophoresis. In particular, from SDS-PAGE gel electrophoresis a well-defined plasminogen band can be shown in the eluate sample indicating that the affinity membranes obtained are suitable for the purification of plasminogen.
This work was financially supported by MIUR, Italian Ministry of Education, University and Research (PRIN 2008) and by the University of Bologna, Italy.