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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 of humanized anti-CD20 monoclonal antibody and to screen its crystallization conditions. Methods The purity of the antibody was detected by reduction and non-reducing SDS-PAGE and SEC-HPLC. The relative molecular weight of the heavy and light chain of antibody was determined by reduction of SDSPAGE. The ion exchange chromatography (IEC-HPLC) method was used to detect the content of antibody charge carriers. The suspension was initially used to screen the antibody crystallization conditions. Results The purity of non-reducing SDS-PAGE of humanized anti-CD20 monoclonal antibody was 100% and the purity of reduced SDS-PAGE (light chain and heavy chain) was 100%. The relative molecular weights of heavy chain and light chain were 58 000 and 27 000, the SFC-HPLC purity was 99.2%; the isoelectric point was 9.2; the surface charge distribution of the antibody was more uniform; the crystal protein obtained was twins with a resolution of about 8 angstroms. Conclusion The purity of this antibody reaches the crystallization requirement. Based on its physicochemical properties, the primary screening of the crystallization conditions has laid the foundation for the study of its structure and function.