CHO细胞表达rhFSH的大规模无血清培养工艺的研究
发布时间：2019-06-15 06:26
【摘要】：中国仓鼠卵巢细胞(Chinese Hamster Ovary,CHO)是动物细胞表达系统中最具代表性的细胞。是用来表达外源目的蛋白最多也最成功的一类细胞。CHO细胞是目前重组糖基蛋白生产的首选体系,因为与其他表达系统相比,CHO细胞具有更多的优点：准确的转录后修饰功能,表达的糖基化药物蛋白在分子结构、理化性质和生物学功能方面最接近于天然蛋白分子；具有产物胞外分泌功能,便于下游产物分离纯化；具有重组基因的高效扩增和表达能力；可以悬浮培养,表达水平较高,可以大规模生产；属于成纤维细胞,很少分泌自身的内源蛋白,利于外源目的蛋白的分泌。 对于女性来说,FSH主要功能是卵巢细胞和雌激素生成必须的一种生长因子、促分泌剂和细胞生长调节剂；对于男性来说,其主要功能是与黄体生成素(LH)控制的睾丸素结合,从而激活和保持正常精子的数目和质量,是不孕不育的诊断和治疗中发挥着重要作用的一类药物。 动物细胞大规模培养工艺是当前生产具有糖基化活性蛋白产品的重要发展趋势。从当前发展趋势来看,在规模化生产上简化生产工艺、提高细胞密度、提高单位产量、降低生产成本是保证大规模生产的关键。通过细胞扩培实现悬浮的无血清培养,通过细胞鉴定确证细胞的安全性,通过大规模培养工艺的研究实现真正的放大生产。 本课题通过细胞生物反应器大规模培养工艺从小试到中试放大的研究,确定可实现放大生产的CHO细胞培养表达目的蛋白rhFSH的工艺,并通过一系列质量研究证明所表达目的蛋白具有与同类已上市销售产品相同的质量特性。
[Abstract]:Chinese hamster ovarian cell (Chinese Hamster Ovary,CHO is the most representative cell in animal cell expression system. Cho cells are the first choice for the production of recombinant glycosylproteins, because CHO cells have more advantages than other expression systems: accurate post-transcriptional modification function, the expressed glycosylation drug proteins are closest to natural protein molecules in molecular structure, physical and chemical properties and biological functions; It has the function of exocrine secretion, which is convenient for the isolation and purification of downstream products, has the ability of high amplification and expression of recombinant genes, can be cultured in suspension culture, the expression level is high, and can be produced on a large scale. Belonging to fibroblasts, it rarely secretes its own endogenous proteins, which is conducive to the secretion of exogenous target proteins. For women, the main function of FSH is a necessary growth factor, secretory promoting agent and cell growth regulator for ovarian cells and estrogen production, while for men, its main function is to bind to testosterone controlled by luteinizing hormone (LH), so as to activate and maintain the number and quality of normal spermatozoa, which plays an important role in the diagnosis and treatment of infertile sterility. Large-scale culture of animal cells is an important development trend in the production of glycosylated active protein products. From the current development trend, the key to ensure large-scale production is to simplify the production process, increase the cell density, increase the unit output and reduce the production cost. The suspension serum-free culture was realized by cell expansion, the safety of cells was confirmed by cell identification, and the real amplification production was realized by the study of large-scale culture technology. In this paper, through the study of large-scale culture process of cell bioreactor from small scale to pilot scale, the process of expressing target protein rhFSH in CHO cell culture was determined, and a series of quality studies showed that the expressed target protein had the same quality characteristics as the same kind of products that had been sold on the market.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
本文编号：2500034
[Abstract]:Chinese hamster ovarian cell (Chinese Hamster Ovary,CHO is the most representative cell in animal cell expression system. Cho cells are the first choice for the production of recombinant glycosylproteins, because CHO cells have more advantages than other expression systems: accurate post-transcriptional modification function, the expressed glycosylation drug proteins are closest to natural protein molecules in molecular structure, physical and chemical properties and biological functions; It has the function of exocrine secretion, which is convenient for the isolation and purification of downstream products, has the ability of high amplification and expression of recombinant genes, can be cultured in suspension culture, the expression level is high, and can be produced on a large scale. Belonging to fibroblasts, it rarely secretes its own endogenous proteins, which is conducive to the secretion of exogenous target proteins. For women, the main function of FSH is a necessary growth factor, secretory promoting agent and cell growth regulator for ovarian cells and estrogen production, while for men, its main function is to bind to testosterone controlled by luteinizing hormone (LH), so as to activate and maintain the number and quality of normal spermatozoa, which plays an important role in the diagnosis and treatment of infertile sterility. Large-scale culture of animal cells is an important development trend in the production of glycosylated active protein products. From the current development trend, the key to ensure large-scale production is to simplify the production process, increase the cell density, increase the unit output and reduce the production cost. The suspension serum-free culture was realized by cell expansion, the safety of cells was confirmed by cell identification, and the real amplification production was realized by the study of large-scale culture technology. In this paper, through the study of large-scale culture process of cell bioreactor from small scale to pilot scale, the process of expressing target protein rhFSH in CHO cell culture was determined, and a series of quality studies showed that the expressed target protein had the same quality characteristics as the same kind of products that had been sold on the market.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
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