全人源化抗人TNF-α单克隆抗体毛细管区带电泳鉴别方法的建立及验证  

Development and validation of capillary zone electrophoresis method for identification of fully humanized anti TNF-α monoclonal antibody

作　　者：杨兰兰[1] 桂芳[2] 张银川[2] 张雅婷[2] 潘勇兵[2] 闭兰[2] 吴小丽[2] 

机构地区：[1]湖北医药学院附属襄阳医院超声科,湖北襄阳441000 [2]武汉生物制品研究所有限责任公司抗体研究室,湖北武汉430207

出　　处：《微生物学免疫学进展》2016年第6期23-30,共8页Progress In Microbiology and Immunology

基　　金：国家科技重大专项课题资助项目(2011ZX09506-005)

摘　　要：目的建立及验证用于全人源化抗人TNF-α(Tumor necrosis factor-α)单克隆抗体(简称抗人TNF-α单抗)鉴别的毛细管区带电泳(Capillary zone electrophoresis,CZE)方法。方法采用DB-1毛细管,以样品检测分离度、迁移时间、电流及峰形为判断标准,筛选CZE过程中的各种参数(缓冲液p H、ε-氨基乙酸浓度、乙酸钠浓度、羟丙基纤维素浓度、样品稀释剂、进样时间及运行电压等),建立CZE方法,并对方法的专属性、精密度、中间精密度进行验证。结果建立的CZE方法参数条件为:缓冲液p H为5.0,EACA浓度为300 mmol/L,乙酸钠浓度为20 mmol/L,HPMC质量分数为0.05%,用50 mmol/L Tris稀释样品,进样时间为15 s,运行电压为20 k V。样品制备重复性主峰迁移时间RSD为0.459%,中间精密度RSD为1.145%。结论 CZE方法分离度高、专属性强、重复性和精密度好、简单快速,可作为全人源化抗人TNF-α单抗的鉴别方法。Objective Development and validation of capillary zone electrophoresis（ CZE） method for the identification of fully humanized anti TNF-α monoclonal antibody. Methods The CZE method was developed by using DB-1-coated capillary tube,the judgement based on resolution,migration time,current and peak shape for the tested sample,through optimazation of the key parameters in the CZE test,such as,the p H of the buffer; the concentration for ε-aminocaproic acid（EACA）,sodium acetate,and hydroxypropylmethyl cellulose（HPMC）; the type of sample dilution; the time of sample injection and the operating voltage,etc,the methd of CZE was developed and its validation of specificity and precision was conducted. Results The key parameters of CZE was selected as follows： the buffer p H was 5. 0,concentration as 300 mmol / L EACA,20 mmol / L sodium acetate,0.05% HPMC,respectively; the sample dilution was in 50 mmol / L Tris; the sample injection time was 15 s and the operating voltage was 20 k V. The result of the validation for CZE showed that the main peak＇s relative standard derivations（ RSDs） of migration time of sample preparation was 0.459%,and the main peak＇s RSD of migration time of intermediate precision was 1.145%. Conclusion The developed CZE showed a good resolution,repeatability,specificity,and with an easy and fast in operation. It could be applied in identification for fully humanized anti TNF-α monoclonal antibody.

关 键 词：人肿瘤坏死因子Α 单克隆抗体 毛细管区带电泳 鉴别 

分 类 号：R392[医药卫生—免疫学]