【作者】 傅璐；

【导师】 刘兢；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 ErbB2是具有酪氨酸激酶活性的跨膜糖蛋白,属于表皮生长因子受体家族,在多种癌细胞特别乳腺癌中有高表达,针对其的单克隆抗体具有潜在的治疗效果。本实验室自行研制的抗ErbB2嵌合抗体chA21具有抑制高表达ErbB2乳腺癌细胞生长的作用。本论文从chA21的大规模纯化生产工艺、质量研究以及工艺改进进行了研究:1、以填充床生物反应器大规模培养CHO工程细胞株表达的上清为原料,采用亲和层析、凝胶过滤除盐、阳离子交换层析、分子筛等步骤,分离纯化嵌合抗体chA21,建立大规模纯化工艺。该工艺能有效解决抗体纯化过程形成的多聚体问题,去除内毒素和DNA残留;可以确保每批纯化10L～20L培养上清,蛋白总回收率大于50%,纯度可达98%。2、按照国家对生物制品的质量要求,对大规模生产的chA21原液产品进行了全面鉴定和质量控制,主要包括含量鉴定、纯度和杂质污染物的检测、理化性质鉴定、生物学活性测定等。鉴定结果表明制备的chA21产品符合动物实验和临床试验的要求。3、最后进一步对纯化工艺新策略摸索,探讨可以替代的纯化方案。嵌合抗体大规模纯化工艺的确定和质量研究的初步摸索,为今后的药物申请奠定了基础。更多还原

【Abstract】 The ErbB2 is a transmembrane glycoprotein with intrinsic tyrosine-kinase activities and belongs to epidermal growth factor receptor family. It overexpressed in several malignant human tumors like breast cancer. So using monoclonal antibodies to target overexpressed ErbB2 cancer for clinical therapy became a new strategy and powerful tool. Our libratory constructed single-chain Chimeric Anti-ErbB2 Antibody chA21 by assembling a single-chain Fv antibody and a human IgG1 Fc fragment. In my dissertation, we developed the purification procedure of chA21, identified the chA21 product and researched the new purification strategy, mainly including three chapters:1、chA21 from engineered CHO cells were cultured in the packed bed bioreactor. To establish mass-scale purification technology, culture supernatant was collected and purified in a sequence of downstream processing steps, namely, affinity chromatography, desalting, ion exchange chromatography and molecular sieve chromatography. By the developed mass-scale procedure, formed aggregates could be removed effetely; endotoxins and residual DNA were also extracted. 10L~20L of culture supernatant could be treated in a single operation. The purify rate reached more than 98% and the total recovery rate was over 50%.2、chA21 product was all analyzed according to the national standard of biologic product. The items included content, purity, foreigh material, contaminant, physoci-chemical property and biology activity. The results indicated that chA21 product was in line with the standard of injection medicine. So the purify rate of the produce technics was high and quality inspection methods were stability and reliable, which could be fit for in large-scale production of chA21.3、Researched the new purification strategy through the other chromatograghy.The study on purification and stability of chA21 may be the foundation for drug application.更多还原