Research Project
2004057
A project is proposed to continue development of reagents and methods for immobilizing ligands onto membranes for affinity purifications, by photoimmobilizing hydrophilic polymeric coupling reagents to which the ligands will be coupled. During the Phase I project and since that time, important advances have been made in reagents and methods for immobilizing antibodies and protein A onto membranes. Reagents and methods were developed that greatly increased the amount of active receptor immobilized. This method uses hydrophilic polymers having photoactivatable groups and multiple reactive groups for coupling to affinity ligands. The polymeric coupling reagents being developed are designed to form very open polymeric layers when immobilized, to allow penetration of the ligands to be immobilized into the layer and thereby achieve efficient coupling also having very low nonspecific adsorption of proteins from the solutions being purified. Although a small amount of further development of Protein A immobilization will be carried out during the Phase II project, the primary focus of this proposed project will be immobilization of heparin for affinity purification of a wide variety of proteins, including clotting factors, protease inhibitors, lipoproteins, growth factors, lipolytic enzymes, extracellular matrix proteins and viral coat proteins. Use of these methods for preparing affinity membranes is expected to increase the efficiency of the purification process, to improve the quality of such purified products and to reduce the costs of a variety of bioseparations. PROPOSED COMMERCIAL APPLICATION: This project is expected to result in techniques for producing improved affinity membranes for purifying proteins from plasma, genetically engineered cells or other sources. This is a rapidly growing market and membrane-based affinity purification is expected to become an increasingly important purification method.
