(433d) Affinity Membranes for Plasminogen Purification

Some eye conditions like diabetic rethinopaty, macular pukers, retinal detachment may benefit from vitreoctomy. Enzymatic vitrectomy with plasmin is envisaged to augment or even replace conventional vitrectomy 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 properties 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 by the surgeon in the operating theatre, since they can be easily packed in a device to be used in 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, Sartobind epoxy™, were used as the support for ligand immobilization. L-lysine was coupled by soaking the membranes in an aqueous solution of Na₂B₄O₇/NaOH and 1.4 dioxane at pH 10 and 80°C at different reaction times and L-lysine concentrations. 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 both pure plasminogen and human serum. Pure plasminogen was obtained, in house, by purification from human serum with a commercial affinity chromatography resin, Pall Lysine HyperD, packed in 1 mL chromatography column and used to characterise the affinity membranes.

During experiments with human serum, fractions have been collected in all chromatographic steps, and analysed with both HPLC and SDS-PAGE electrophoresis. In particular, from the HPLC SEC analysis of the eluate a well defined plasminogen peak can be shown indicating that the lysine affinity membranes are suitable for the purification of plasminogen.

Aknowledgements

This work was financially supported by MIUR, Italian Ministry of Education, University and Research (PRIN 2008) and by the University of Bologna, Italy.