Three novel high performance affinity chromatographic media for the separation of antithrombin III from human plasma

Novel monodisperse, non‐porous, cross‐linked poly (glycidyl methacrylate) beads (PGMA) were employed as the support for high performance affinity chromatography. Heparin was covalently attached to PGMA beads by three different coupling methods. Heparin‐PGMA‐I was prepared by directly coupling amino‐groups of heparin with PGMA. Heparin‐PGMA‐II and III were prepared by the coupling of heparin to amino‐PGMA, which was obtained by amination of PGMA. Heparin‐PGMA‐II was prepared by coupling the carboxyl groups of heparin to amino‐PGMA by using water‐soluble carbodiimide as coupling reagent, and heparin‐PGMA‐III was prepared by the reductive amination of heparin and amino‐PGMA with sodium cyanoborohydride. The heparin contents of heparin‐PGMA‐I, II and III were 1.6, 10.2 and 1.0 mg/g beads, respectively. Their affinity capacities for antithrombin III were investigated. Their binding activity to antithrombin III was not proportional to the content of heparin immobilized, and heparin‐PGMA‐I was the most efficient affinity medium for antithrombin III. The resultant affinity media presented minimal non‐specific interaction with other proteins and can be used in a wide pH range. All the three heparin‐PGMA beads were exploited for the separation of antithrombin III from human plasma. The purity of antithrombin III obtained was higher than 90%, which was confirmed by high performance size exclusion chromatography. Copyright © 2001 John Wiley & Sons, Ltd.