Reduced Cost Blood Factor Production - Model Molecule, Protein C

降低成本的血液因子生产-模型分子，蛋白质CDuane F。马里兰州巴尔的摩县Bruley大学NSF提案编号0090749 本研究是美国红十字会（ARC）和马里兰州巴尔的摩县大学（UMBC）的共同努力，旨在研究固定化金属亲和色谱（IMAC）作为替代免疫亲和色谱分离高分子量同源血液因子的替代方法。感兴趣的源材料是血浆Cohn Fraction 1V-1，其是血液蛋白的复杂混合物，包括确保血液止血所必需的维生素K依赖性蛋白（VKD）。通过NSF-SGER资助的初步研究证实，IMAC是一种高度选择性的分离技术，可能能够在制备规模上实现所需的分离。IMAC的机制包括使用适当的金属离子/螯合剂组合，其对血液中存在的特定氨基酸具有亲和力。证据表明，匹配的IMAC基板的数量和生物分子组氨酸单元的位置可以导致有效的分离时，使用适当的缓冲液。该研究项目包括对一些关键的VKD血液蛋白的表面组氨酸浓度的调查，在选定的IMAC柱上测定蛋白C的平衡等温线，以及使用实验设计技术来确定最佳色谱操作条件，该条件将允许分离毫克量的蛋白C用于后处理。迫切需要以低成本大量生产用于治疗应用的各种人血液因子。血液因子以一种非常复杂的方式连接在一起，因此在正常情况下，血液会凝结以止血。当凝血剂和抗凝血剂失衡时，会出现两种病理状态：（a）患者是血友病患者，或（B）患者是血栓患者。蛋白C缺乏可导致深静脉血栓形成（DVT），并可能导致肺栓塞，这是危及患者生命的并发症。应该指出的是，凝血功能异常是导致死亡的主要原因，超过了艾滋病和癌症。目前使用肝素或香豆素的治疗是有效的，但可能有危险的副作用。高容量，低成本的蛋白C产品可以为蛋白C缺乏症患者提供廉价，安全的治疗。

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.