Metabolic Engineering of Chinese Hamster Ovary Cells for Production of Heparin

描述(由申请人提供)： 肝素是现代医学中使用最广泛的抗凝药物。它是一种高度硫酸盐化的多糖(糖胺聚糖)，发现与肥大细胞内颗粒中储存的核心蛋白共价结合，肥大细胞颗粒在许多动物的肠道和肺中大量存在。硫酸肝素糖胺聚糖是一种较少硫酸盐化的肝素，它附着在蛋白多糖的核心蛋白上，广泛存在于所有动物组织的外细胞膜上。 为了应对2008年初发生的一场健康危机，我们提议从非动物来源开发一种代谢工程肝素。这场健康危机涉及中国用过量硫酸软骨素掺入从猪身上生产的肝素，导致近百名美国人死亡。拟议的为期3年的项目是一项翻译和多学科研究努力，旨在通过代谢工程技术在中国仓鼠卵巢(CHO)细胞中生产肝素。通过改造CHO细胞的糖基化途径，通常提供相关的多糖硫酸乙酰肝素，代谢工程肝素将被制备成具有与动物制备的药物肝素相同的结构的肝素。化学和体外生物等效性研究将提供必要的临床前数据，以推动代谢工程肝素作为仿制肝素的发展。CHO细胞在生物技术/生物制药工业中被广泛用于生产重组治疗性蛋白。它们被FDA接受为生产人类治疗药物的宿主，并存在去除宿主细胞蛋白质、核酸和病毒污染物的方案。尽管CHO细胞被广泛用于生产重组蛋白，但还没有关于CHO细胞用于生产治疗性碳水化合物的报道。因此，所提出的工作的智力价值在于证明硫酸肝素的生物合成途径可以被修改以产生肝素，并且这种肝素可以由CHO细胞在肝素硫化的核心蛋白上分泌到培养基中以供收集。为实现这一目标，我们将开展以下四项工作： 1.改造CHO细胞的硫酸肝素生物合成途径，以获得更高硫酸盐化的糖胺聚糖-肝素 2.对CHO细胞进行改造，使其使这种高度硫酸盐化的糖胺聚糖附着在通常携带硫酸乙酰肝素的核心蛋白上，并将这种蛋白多糖输出到细胞外表面，然后将其排放到培养基中 3.恢复和确认CHO细胞代谢工程肝素与USP肝素的化学和体外生物等效性 4.扩大CHO细胞代谢工程肝素的生产和回收，同时保持化学和生物等效性。

DESCRIPTION (provided by applicant): 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.