Immunoaffinity Isolation of Vitamin K-Dependent Proteins

在抗凝治疗中使用高纯度血浆来源的维生素k依赖蛋白需要这些蛋白产品缺乏促凝级联蛋白。在促凝和抗凝维生素k依赖蛋白中发现的同源序列使得传统的分离过程不足以生产高纯度的维生素k依赖蛋白。本研究采用经典分离和免疫亲和分离相结合的步骤，从cryopoor血浆和Cohn分数沉淀物中生产高纯度的人蛋白C。预纯化体积缩减步骤是通过阴离子交换或小麦胚芽凝集素亲和层析实现的。从这一步洗脱，然后进行新的单克隆抗体分离步骤。选择单克隆抗体是因为它们能够在存在或不存在钙的情况下结合蛋白C。因此，正在评估的免疫吸附材料形成的Ab-Protein C复合物是钙依赖的，因此使用钙或EDTA缓冲溶液的温和洗脱条件。这延长了免疫吸附培养基的使用寿命。几种交联、珠状琼脂糖和纤维素被评价为支持基质。根据所开发的免疫吸附材料的可回收性程度来评估工艺可行性。希望从这些研究中发展出一种维生素k依赖性血浆蛋白的模型免疫亲和分离工艺技术。最近显示的蛋白质C以及其他抗凝血维生素k依赖物如蛋白质S的临床适应症表明，开发大规模生产这些蛋白质的分离技术迫在眉睫。目前，血浆是唯一可以满足生化和临床研究建议的每年kg或更多需求的来源。即使这些蛋白质具有成本效益的基因工程版本的出现（可能在未来5年内可用），由于这些蛋白质的合成版本可能会产生免疫原性，因此需要替代和天然来源。血浆来源的抗凝蛋白的可用性将有助于为那些基因工程版本的免疫原性存在问题的病例提供一种替代疗法。毫无疑问，用于血浆衍生蛋白的相同纯化技术将用于基因工程蛋白的生产。

The use of highly pure plasma-derived vitamin K-dependent protein in anticoagulant therapies necessitates that these protein products be devoid of procoagulant cascade proteins. The homologous sequences found in both pro-and anticoagulant vitamin K-dependent proteins render classical separation processes inadequate for production of highly pure vitamin K-dependent proteins. This study employs a combination of classical separation and immunoaffinity isolation steps to produce highly pure human Protein C from cryopoor plasma and Cohn fraction precipitates. A prepurification-volume reduction step is achieved via anion exchange or wheat germ lectin affinity chromatography. Eluate from this step is then subjected to a novel monoclonal antibody isolation step. The monoclonal antibodies employed are selected for their ability to bind Protein C either in the presence or absence of calcium. Thus, the immunosorbent materials being evaluated form Ab-Protein C complexes that are calcium dependent such that gentle elution conditions of either calcium or EDTA buffer solutions are employed. This provides for extended longevity of the immunosorbent media. Several crosslinked, beaded-agaroses and celluloses are being evaluated as support matrices. Process feasibility is evaluated based upon the degree of recyclability of the immunosorbent materials developed. It is hoped that a model immunoaffinity isolation process technology for vitamin K-dependent plasma proteins will evolve from these studies. The recently demonstrated clinical indications for Protein C as well as other anticoagulant vitamin K-dependents such as Protein S, signals the urgency for the development of isolation technolgy for producing these proteins at large scale. Currently, plasma is the only source which can meet the kg per annum or greater needs suggested by biochemical and clinical studies. Even with the advent of cost-effective genetically engineered versions of these proteins (which could become available in the next 5 years), the need for an alternative and natural source of these proteins is necessitated by the immunogenicity that will likely occur with synthetic versions of these same proteins. The availability of plasma- derived anticoagulant proteins would help provide an alternative therapy needed for those cases where immunogenicity for the genetically engineered version is a problem. There is no doubt that the same purification technology developed for plasma derived proteins will be useful in genetically engineered protein production.