抗VEGF单克隆抗体生物学活性分析方法的建立及其应用  

Development,optimization,and application of a cell-based assay for bioactivity determination of anti-VEGF monoclonal antibody

作　　者：莫琳 周冬梅 徐军 孙文正 杨彬 

机构地区：[1]广东东阳光药业有限公司,东莞523867

出　　处：《中国医药生物技术》2017年第1期24-28,共5页Chinese Medicinal Biotechnology

基　　金：广东省引进创新科研团队计划(201101Y0104990178)

摘　　要：目的建立抗VEGF单克隆抗体生物学活性分析方法,并对实验条件进行优化及方法学验证。方法从新生儿脐带分离获取人脐静脉内皮细胞(HUVEC),使用第4~6代细胞进行活性检测。HUVEC接种96孔板于CO_2培养箱培养4 h后加入不同浓度的抗VEGF单克隆抗体——VEGF165混合物孵育48 h,加入CCK8显色4 h,酶标仪读取各孔OD450吸光值,采用四参数拟合绘制标准曲线,计算参比品和样品的IC50值,获取样品的相对活性。结果该方法专属性强,仅在抗VEGF单克隆抗体上呈现相应的剂量关系曲线,标准曲线拟合度r^2>0.99,精密度样品相对活性RSD<20%,方法在50%~200%活性范围内具有良好的准确性。用该方法对两个候选药分别进行6次重复性检测,候选药相对活性值分别为(95.80±3.29)%,(101.10±3.81)%。结论本研究建立的HUVEC增殖抑制法特异性高、重复性好,并具有良好的准确性及精密度,可用于抗VEGF单克隆抗体生物学活性的评估。Objective To development,optimize a cell-base assay for bioactivity determination of anti-VEGF monoclonal antibody（m Ab） and to utilize the mA bs to inhibit VEGF induced proliferation of human umbilical vein endothelial cells（HUVEC）.Methods Fresh HUVEC from human umbilical cords were cultured up to the fourth generation but no more than sixth generation should be used.HUVEC were seeded into each well of a 96-well plate.The plate were incubated for 4 h at 37 ℃ and 5% CO_2 and then HUVEC were treated with anti-VEGF antibodies containing solution for 48 h.CCK8 were added to each well for another 4 h at 37 ℃ and 5% CO_2.The cellular response was recorded at a 450 nm absorbance using the Spectra Max M2 e.The EC50 values of the control and samples were obtained by using four parameters curve and then the bioactivity of samples were calculated.Results This assay possessed excellent specificity.Only anti-VEGF antibody showed a typical dose-response curve with more than 0.99 of a standard curve fit of r^2 value and less than 20% of a RSD for the precision validation.This assay also presented a good accuracy within the activity range of 50%- 200%.This assay was used to conduct 6 measure each for two different samples containing active anti-VEGF mA bs using a commercial anti-VEGF m Ab as the control.A mean relative activity of（95.80 ± 3.29）% and（101.10 ± 3.81）% was obtained for these two samples respectively.Conclusion We have successfully developed,optimized,and applied the HUVEC proliferation inhibitory assay for bioactivity determination of anti-VEGF mA bs with excellent specificity,reproducibility and accuracy.

关 键 词：人脐静脉内皮细胞 血管内皮生长因子类 抗VEGF单克隆抗体 

分 类 号：R392-33[医药卫生—免疫学]