Heparin is the most widely used anticoagulant drug in modern medicine. It is a highly sulfated polysaccharide (glycosaminoglycan) found covalently attached to a core protein stored in intracellular granules of mast cells that are found in large numbers in the intestines and lungs of many animals. The heparan sulfate glycosaminoglycan is a less sulfated version of heparin that is attached to core proteins of proteoglycans, ubiquitously found on the external cell membrane of all animal tissues. In response to a health crisis that took place in early 2008, we propose to develop a metabolically engineered heparin from a non-animal source. This health crisis involved the adulteration of heparin produced from hogs in China with an oversulfated chondroitin sulfate, leading to the death of nearly 100 Americans. The proposed 3-year project is a translational and multi-disciplinary research effort aimed at producing heparin in metabolically engineered Chinese hamster ovary (CHO) cells. By engineering the CHO cell glycosylation pathway, which normally affords the related polysaccharide heparan sulfate, a metabolically engineered heparin will be prepared with a structure identical to the pharmaceutical heparin prepared from animals. Chemical and in vitro bioequivalence studies will provide the necessary pre-clinical data required to carry metabolically engineered heparin forward as a generic heparin. CHO cells are widely used in the biotechnology/biopharmaceutical industry for the production of recombinant therapeutic proteins. They are accepted by the FDA as a host for the production of human therapeutics, and protocols exist for removal of host cell proteins, nucleic acids, and viral contaminants. Despite the widespread use of CHO cells for production of recombinant proteins, there are no reports of CHO cells used to produce therapeutic carbohydrates. Hence, the intellectual merit of the proposed work is to demonstrate that the biosynthetic pathway for heparan sulfate can be modified to produce heparin, and that this heparin can be secreted by the CHO cells on a heparin sulfated core protein into the culture medium for collection. To achieve this goal, we will perform the following four tasks: 1. Engineer the CHO cell pathway of heparan sulfate biosynthesis to afford the more highly sulfated glycosaminoglycan, heparin 2. Engineer the CHO cell so that it makes this highly sulfated glycosaminoglycan attached to a core protein normally carrying heparan sulfate and export this proteoglycan to the external cell surface and then shed it into the medium 3. Recover and confirm chemical and in vitro bioequivalence of CHO cell metabolically engineered heparin with USP heparin 4. Scale-up the production and recovery of CHO cell metabolically engineered heparin while maintaining chemical and bioequivalence.
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