人源化抗CD20单克隆抗体的理化性质及结晶条件的筛选  

Physicochemical properties and screening of condition for crystallization of humanized anti-CD20 monoclonal antibody

作　　者：何睦[1] 谭小钉[2] 卢日峰[1] 刘大涛[2] 王志武[1] 

机构地区：[1]吉林大学公共卫生学院,吉林长春130021 [2]上海医药集团股份有限公司中央研究院,上海201203

出　　处：《中国生物制品学杂志》2014年第5期657-660,共4页Chinese Journal of Biologicals

摘　　要：目的分析人源化抗CD20单克隆抗体的理化性质,并初步筛选其结晶条件。方法分别采用还原、非还原SDS-PAGE及高效液相体积排阻色谱(size exclusive chromatography-HPLC,SEC-HPLC)法检测抗体纯度;还原SDSPAGE测定抗体轻重链相对分子质量;毛细管电泳测定抗体等电点;高效液相阳离子交换色谱(ion exchange chromatographyHPLC,IEC-HPLC)法检测抗体电荷异质体含量;悬滴气相扩散法初步筛选抗体的结晶条件。结果人源化抗CD20单克隆抗体的非还原SDS-PAGE纯度为100%,还原SDS-PAGE纯度(轻链+重链)为100%,重链、轻链相对分子质量分别为58 000和27 000,SFC-HPLC纯度为99.2%;等电点为9.2;抗体表面电荷分布较均一;得到的蛋白晶体为孪晶,分辨率约为8埃。结论该抗体的纯度达到结晶要求,以其理化性质为基础,初步筛选出了结晶条件,为其结构和功能的研究奠定了基础。Objective To analyze the physicochemical properties and preliminarily screen the condition for crystallization of humanize anti-CD20 monoclonal antibody. Methods The antibody was analyzed for purity by size exclusive chromatography-HPLC（SEC-HPLC）as well as reduced and non-reduced SDS-PAGE,for the relative molecular masses of light and heavy chains by reduced SDS-PAGE,for isoelectric point by capillary electrophoresis,and for electric charge heterogeneity content by ion-exchange chromatography. The condition for crystallization of antibody was preliminarily screened by hanging-drop vapor-diffusion method. Results The purity of antibody was 99. 2% by SEC-HPLC,while were 100% by both non-reduced and reduced SDS-PAGE. The relative molecular masses of heavy and light chains were 58 000 and 27 000 respectively,while the isoelectric point was 9. 2. The electric charges were distributed uniformly on the surface of antibody. Twin crystal of protein was obtained,with a resolving power of 8 魡. Conclusion The purity of antibody met the requirement for crystallization. The condition for crystallization was preliminarily screened based on the physicochemical properties,which laid a foundation of study on the structure and function of the antibody.

关 键 词：人源化抗体 单克隆抗体 CD20 纯度 理化性质 结晶 

分 类 号：R392-33[医药卫生—免疫学] O782[医药卫生—基础医学]