Reduced Cost Blood Factor Production - Model Molecule, Protein C

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AbstractREDUCED COST BLOOD FACTOR PRODUCTION -MODEL MOLECULE, PROTEIN CDuane F. Bruley University of Maryland Baltimore CountyNSF Proposal Number 0090749 This research is a joint effort between The American Red Cross(ARC) and the University of Maryland Baltimore County(UMBC) to study Immobilized Metal Affinity Chromatography(IMAC) as an alternative process to replace Immuno Affinity Chromatography for the separation of high-molecular-weight, homologous blood factors. The source material of interest is blood plasma Cohn Fraction 1V-1, which is a complex mixture of blood proteins including the Vitamin K Dependent Proteins (VKD) that are essential to insure blood hemostasis. Initial studies via an NSF-SGER grant verified that IMAC is a highly selective separation technology that might be able to achieve the desired separations on a preparative scale. The mechanism of IMAC includes the use of appropriate metal ion/chelating agent combinations that have affinity for specific amino acids that occur in blood. Evidence shows that matching the IMAC substrate with the number and the placement of biomolecule histidine units can lead to efficient separations when the appropriate buffers are used. This research project includes investigations of the surface histidine concentration on some of the critical VKD blood proteins, the determination of equilibrium isotherms for Protein C on selected IMAC columns, and the use of experimental-design techniques to determine optimal chromatographic operating conditions that will allow the separation of milligram quantities of Protein C for post-award animal studies.There is an urgent need for the production of a variety of human blood factors in large quantities at low cost for therapeutic applications . The blood factors are linked together in a very complex way so that under normal conditions the blood will clot to stop bleeding. When an imbalance of coagulants and anti-coagulants occurs, two pathologic states can develop: (a) the patient is a hemophilic, or (b) the patient is a clotter.Our model blood factor is Protein C, which is the pivotal anti-coagulant in the human coagulation cascade. Protein C deficiency can result in deep vein thrombosis (DVT) with the possibility of pulmonary embolus ,which is a life-threatening complication for the patient. It should be noted that abnormal clotting disorders are the leading cause of death, over AIDS and cancer. Present treatments with Heparin or Coumadin are effective but with dangerous side effects possible. High-volume, low-cost Protein C products could provide inexpensive, safe treatment for patients with Protein C deficiency.