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基于中国仓鼠卵巢细胞表达的Fc融合蛋白的制备及表征
赵永强
Thesis Advisor苏志国
2017-01
Degree Grantor中国科学院研究生院
Degree Name硕士
Degree Discipline化学工程
KeywordFc融合蛋白,培养基优化,补料策略优化,Protein A亲和层析,阴离子交换层析
Abstract

Fc融合蛋白是指将目标生物活性的蛋白质或多肽分子,利用基因工程等技术手段,与免疫球蛋白G(Immunoglobulin , IgG)结构的Fc片段进行融合,产生新的由目标物与IgG融合组成的基因重组蛋白。解决Fc融合蛋白药物的制备工艺中一些关键工艺步骤的问题,对于此类药物的开发应用具有十分重要的意义。本文以本实验室构建的一株表达Fc融合蛋白的CHO细胞为对象,探索上游细胞培养工艺中的培养基优化和补料策略优化,以及下游纯化工艺中的Protein A亲和层析和阴离子交换层析步骤的优化,具体研究结果如下:1. 通过摇瓶培养的方式,进行培养基的混合配方试验。用于混合筛选的培养基为商品化的适宜CHO细胞生长的培养基。在考察细胞生长与表达的同时,兼顾降低成本。结果为:以种子培养基(Mab-Works medium C):基础培养基(CD OptiCHO)=1:1的比例配方,比单一使用种子培养基培养的细胞密度提高了161.8%,蛋白表达量提高了100%;为考虑降低培养基成本而进一步设计的试验,则获得了种子培养基(Mab-Works medium C):基础培养基(CD OptiCHO):基础培养基(CDM4Mab)=2:1:1的比例配方,相比于种子培养基:基础培养基(CD OptiCHO)=1:1比例的混合配方进行细胞培养,细胞密度高出13.3%,蛋白表达量高出18.1%,还可以降低成本25%,并能维持较好的活率; 2. 针对抗体类生物药(包括Fc融合蛋白)通用的Fed-Batch培养方式,进行补料方案的研究。发现:流加策略中流加方案二(流加培养基量为终体积的30%,平均至第3、6、9天流加)优于流加方案一(流加培养基量为终体积的30%,平均至第2、4、6、8天流加),细胞密度高出30.9%,蛋白表达量高出13.2%; 3. 在5L生物反应器规模,充分利用反应器的精准参数控制功能,对摇瓶试验的结果成功进行了放大;4. 对细胞收获液的亲和层析工艺进行探索,得出结果:pH值为3.3的洗脱条件适合本实验的Fc融合蛋白Protein A亲和层析步骤,此条件下获得了最高的纯度98.64%,同时收率较好达到94.7%;针对离子交换步骤的条件优化繁琐问题,从目标蛋白等电点测定到工艺中的缓冲液pH值、电导率对纯化效果的影响进行研究,结果发现:在电导率2 mS/cm的较低水平下,本试验蛋白在pH 值5.0~5.5之间时阴离子纯化效果最好。;Fc fusion protein means fusing the biological activity protein or peptide to the IgG’s Fc fragment by using genetic engineering technology, with the aim to produce a new recombinant protein. With the wide application of antibodies’ biopharmaceutical, it’s of great significance to solve the key downstream process issues about Fc fusion protein preparation. This thesis focuses on investigation and optimization of the cell culture medium and feeding strategy, the purification through the Protein A affinity chromatography, as well as the anion exchange chromatography. The study involves:1. Carry out medium mixing experiment through shake flask culture to optimize the medium component. The selected mediums are commercialized and suitable for CHO cell growth. The aime of this step is to inspect the influences of different mediums on the cell growth and protein expression, at the same time to reduce the cost. Compare with single seed medium, the formula of seed medium (Mab - Works medium C) : basic medium (CD OptiCHO) = 1:1 ratio increases cell density up to 161.8% and increase protein expression level up to 100%. Further experiment designed for saving cost indicated that the formula of seed medium (Mab - Works medium C): basic medium (CD OptiCHO) : basic medium (CDM4Mab) = 2:1:1 is better than that of seed medium(Mab-Works medium C): basal medium (CD OptiCHO) = 1:1 formula for cell culture. The cell density, protein expression level and the cost for the former is respectively 13.3% higher, 18.1% higher, and 25% lower than those for the later. Besides, cells can maintain good viability for the 2:1:1 formula.2. Optimize the fed-batch strategy for cell culture. It is found that: strategy 2 (the fed medium volume is 30% of the final volume, averagely fed at 3, 6, 9 days) is better than strategy 1 (fed medium volume is 30% of the final volume and averagely fed at 2, 4, 6, 8 days), with 30.9% increase of the cell density and 13.2% inctrase of protein expression.3. Succeed in scaling up cell culture in 5L bioreactor. The cell density and the taget protein expression is comparable and repeatable with the shake flask results. 4. Determine the optimal operation for the Protein A affinity chromatography process. After loading the supernante of culture both, the Fc-fused protein was eluted by decreasing the buffer pH value to 3.3. The purity of the eluted protein is about 98.64% and the recovery is about 94.7%;5. Anion exchange chromatography is used to further remove other impurities. Based the determination of the PI of the Fc-fused protein, a passthrough chromatographic model was adopted at pH 5.0 ~ 5.5 and the buffer conductivity is below 2 mS/cm. 

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/26838
Collection中国科学院过程工程研究所
研究所(批量导入)
Recommended Citation
GB/T 7714
赵永强. 基于中国仓鼠卵巢细胞表达的Fc融合蛋白的制备及表征[D]. 中国科学院研究生院,2017.
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