抗ErbB2嵌合抗体chA21的大规模纯化工艺的建立及质量研究

发布时间：2018-08-10 21:47

【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。
[Abstract]:ErbB2 is a transmembrane glycoprotein with tyrosine kinase activity, which belongs to epidermal growth factor receptor family and is highly expressed in many cancer cells, especially breast cancer. The anti-ErbB2 chimeric antibody chA21 developed by our laboratory can inhibit the growth of breast cancer cells with high expression of ErbB2. In this paper, the mass purification process, quality research and process improvement of chA21 were studied. 1. The supernatant of CHO engineering cell line was cultured in packed bed bioreactor as raw material, and affinity chromatography was used. Gel filtration desalination, cation exchange chromatography and molecular sieve were used to isolate and purify chimeric antibody chA21, and a large-scale purification process was established. This process can effectively solve the problem of polymer formation in antibody purification process, remove endotoxin and DNA residues, and ensure the supernatant of each batch of purified 10L~20L culture. The total recovery rate of protein is more than 50 and the purity can reach 98.2. According to the quality requirements of biological products, the products of chA21 raw liquid produced on a large scale have been comprehensively identified and their quality control has been carried out, including content identification. Detection of purity and impurity pollutants, identification of physical and chemical properties, determination of biological activity, etc. The results showed that the product of chA21 was in accordance with the requirements of animal experiment and clinical trial. 3. Finally, the new strategy of purification process was explored, and the alternative purification scheme was discussed. The determination of mass purification process of chimeric antibody and preliminary exploration of quality study have laid a foundation for the application of drugs in the future.
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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