CHO細胞表達rhFSH的大規(guī)模無血清培養(yǎng)工藝的研究
發(fā)布時間：2019-06-15 06:26
【摘要】：中國倉鼠卵巢細胞(Chinese Hamster Ovary,CHO)是動物細胞表達系統(tǒng)中最具代表性的細胞。是用來表達外源目的蛋白最多也最成功的一類細胞。CHO細胞是目前重組糖基蛋白生產的首選體系,因為與其他表達系統(tǒng)相比,CHO細胞具有更多的優(yōu)點：準確的轉錄后修飾功能,表達的糖基化藥物蛋白在分子結構、理化性質和生物學功能方面最接近于天然蛋白分子；具有產物胞外分泌功能,便于下游產物分離純化；具有重組基因的高效擴增和表達能力；可以懸浮培養(yǎng),表達水平較高,可以大規(guī)模生產；屬于成纖維細胞,很少分泌自身的內源蛋白,利于外源目的蛋白的分泌。 對于女性來說,FSH主要功能是卵巢細胞和雌激素生成必須的一種生長因子、促分泌劑和細胞生長調節(jié)劑；對于男性來說,其主要功能是與黃體生成素(LH)控制的睪丸素結合,從而激活和保持正常精子的數(shù)目和質量,是不孕不育的診斷和治療中發(fā)揮著重要作用的一類藥物。 動物細胞大規(guī)模培養(yǎng)工藝是當前生產具有糖基化活性蛋白產品的重要發(fā)展趨勢。從當前發(fā)展趨勢來看,在規(guī)�；a上簡化生產工藝、提高細胞密度、提高單位產量、降低生產成本是保證大規(guī)模生產的關鍵。通過細胞擴培實現(xiàn)懸浮的無血清培養(yǎng),通過細胞鑒定確證細胞的安全性,通過大規(guī)模培養(yǎng)工藝的研究實現(xiàn)真正的放大生產。 本課題通過細胞生物反應器大規(guī)模培養(yǎng)工藝從小試到中試放大的研究,確定可實現(xiàn)放大生產的CHO細胞培養(yǎng)表達目的蛋白rhFSH的工藝,并通過一系列質量研究證明所表達目的蛋白具有與同類已上市銷售產品相同的質量特性。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329
本文編號：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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329
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