Immunoaffinity Isolation of Vitamin K-Dependent Proteins

点击翻译按钮获取中文摘要

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.