【摘要】： ErbB2是具有酪氨酸激酶活性的跨膜糖蛋白,屬于表皮生長因子受體家族,在多種癌細(xì)胞特別乳腺癌中有高表達(dá),針對其的單克隆抗體具有潛在的治療效果。本實驗室自行研制的抗ErbB2嵌合抗體chA21具有抑制高表達(dá)ErbB2乳腺癌細(xì)胞生長的作用。本論文從chA21的大規(guī)模純化生產(chǎn)工藝、質(zhì)量研究以及工藝改進(jìn)進(jìn)行了研究: 1、以填充床生物反應(yīng)器大規(guī)模培養(yǎng)CHO工程細(xì)胞株表達(dá)的上清為原料,采用親和層析、凝膠過濾除鹽、陽離子交換層析、分子篩等步驟,分離純化嵌合抗體chA21,建立大規(guī)模純化工藝。該工藝能有效解決抗體純化過程形成的多聚體問題,去除內(nèi)毒素和DNA殘留;可以確保每批純化10L～20L培養(yǎng)上清,蛋白總回收率大于50%,純度可達(dá)98%。 2、按照國家對生物制品的質(zhì)量要求,對大規(guī)模生產(chǎn)的chA21原液產(chǎn)品進(jìn)行了全面鑒定和質(zhì)量控制,主要包括含量鑒定、純度和雜質(zhì)污染物的檢測、理化性質(zhì)鑒定、生物學(xué)活性測定等。鑒定結(jié)果表明制備的chA21產(chǎn)品符合動物實驗和臨床試驗的要求。 3、最后進(jìn)一步對純化工藝新策略摸索,探討可以替代的純化方案。 嵌合抗體大規(guī)模純化工藝的確定和質(zhì)量研究的初步摸索,為今后的藥物申請奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

【共引文獻(xiàn)】

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