重组抗HER2人源化单克隆抗体大规模制备及活性鉴定方法学建立  

A method for expression and activity determination of Anti-Her2 humanized monoclonal antibody

作　　者：陈观平[1] 汪一帆[1] 应栩华[1] CHEN Guan-ping;WANG Yi-fan;YING Xu-hua(Cancer Institute of Integrative Medicine,Tongde Hospital of Zhejiang Province,Hangzhou 310012,China)

机构地区：[1]浙江省立同德医院中西医结合肿瘤研究所,浙江杭州310012

出　　处：《中国药理学通报》2021年第6期884-888,共5页Chinese Pharmacological Bulletin

基　　金：浙江省基础公益技术应用研究计划(No LGF18H290003)。

摘　　要：目的建立大规模表达重组人源化HER2单克隆抗体(Anti-her2)并测定重组抗体活性的方法。方法利用Anti-her2重链、轻链真核表达载体质粒共同转化CHO细胞,筛选稳定表达株,然后放大培养,并利用Protein A亲和层析柱纯化融合蛋白,SDS-PAGE、HPLC检测产物纯度,Western blot检测产物特异性,同时比较重组抗体的活性。结果筛选得到1株稳定表达的细胞株,收液后蛋白BCA定量为135.9 mg·L-1。纯化后获得的目的蛋白相对分子质量约为185 kD,蛋白纯度在99%以上,与商品化的“赫赛汀”一致;活性检测显示其活性与“赫赛汀”相当,细胞实验揭示其能显著抑制BT-474细胞增殖及胞内HER2的表达。结论该方法筛选获得的重组蛋白,与商品化药物性质基本一致,这为重组抗体药物的大规模悬浮培养放大和工业化生产奠定了基础。Aim To establish a method for the stable expression of recombinant humanized Anti-her2 monoclonal antibody and to measure its bioactivity.Methods Anti-her2 heavy chain and light chain eukaryotic expression vectors were used to transfect CHO cells,and then scaled-up after screening the stably expressed cell line.Protein A affinity chromatography was utilized to purify the fusion protein.Western blot was used to identify the properties of the product,SDS-PAGE and HPLC were employed to detect its specificity,and CCK8 assay was used to detect its effect on cell viability.Results One cell line with stable expression of recombinant antibody was selected for scale-up.The supernatant was gained for the purification with protein concentration of 135.9 mg·L-1.The molecular weight of purified protein was about 185 kD,and its purity was higher than 99%.Its bioactivity was assayed in vitro test.The results indicated that it showed strong bioactivity consistent with Herceptin.And it could significantly inhibit cell proliferation and HER2 expression.Conclusions The antibody protein can be successfully obtained by purification after scaling-up by suspension cultivation.Its bioactivity is basically consistent with the commercialized drug.This method has laid a foundation for large-scale fermentation of this recombinant protein.

关 键 词：重组 HER2 人源化 单克隆抗体 中国仓鼠卵巢细胞 纯化 

分 类 号：R-332[医药卫生] R322.65