全柱成像毛细管等电聚焦(iCIEF)技术分析重组人IgG VEGFR:Fc-融合蛋白电荷异质性  

Charge heterogeneity analysis of human IgG VEGFR:Fc-fusion protein with imaging capillary isoelectric focusing electrophoresis(iCIEF)

作　　者：刘振东 韩国华 杨勇 王庆民 LIU Zhen-dong, HAN Guo-hua, YANG Yong, WANG Qing-min(The Research and Development Department, Qilu Pharmaceutical Co., Ltd., Jinan 250100, China)

机构地区：[1]齐鲁制药有限公司药物研究院,济南250100

出　　处：《药物分析杂志》2018年第3期539-544,共6页Chinese Journal of Pharmaceutical Analysis

摘　　要：目的:建立全柱成像毛细管等电聚焦法(iCIEF)分析重组人IgG VEGFR:Fc-融合蛋白(血管内皮生长因子受体-抗体Fc融合蛋白)的电荷异质性。方法:首先对全柱成像毛细管等电聚焦方法在蛋白浓度、助溶剂、两性电解质、聚焦时间等方面进行了条件优化。并应用优化的方法进行Fc-融合蛋白的电荷异质性及等电点检测及重复性验证,并将结果与毛细管等电聚焦(cIEF)及平板胶等电聚焦(IEF)结果进行对比分析。结果:在优化的实验条件下,分析Fc-融合蛋白的电荷异质性及等电点具有良好的分离度和重复性,分离效果明显好于cIEF和IEF,且主成分的pI重复测定RSD均小于0.5%。结论:iCIEF作为一种新的技术手段,用于重组蛋白类药物电荷异质性及等电点分析,方法快速、准确,重复性好,能够较好地检测高度糖基化的重组VEGFR:Fc-融合蛋白产品的电荷异质性,为保障Fc-融合蛋白类产品生产工艺的稳定性及质量控制提供了一种快速、可靠的分析方法。Objective： To develop an imaged capillary isoelectric focusing electrophoresis method for charge heterogeneity analysis of recombinant human IgG VEGFR ： Fc-fusion protein. Methods： This study first optimized the imaged capillary isoelectric focusing on the electrophoresis method including working concentration of protein, additives, pH range of ampholytes, focusing time, and then analyzed the charge heterogeneity and isoelectric points of Fc-fusion protein using the optimized method. The results were compared with the cIEF and IEF. Results： Under the optimal conditions, the charge heterogeneity and isoelectric points of Fc-fusion protein were very accurate with a perfect resolution and repeatability. The iCIEF separation is significantly better than cIEF and tEF, and the RSD of pI was less than 0. 5% with repeat assay. Conclusion： iCIEF as a new technique can be used to determine the charge heterogeneity and isoelectric points of recombinant proteins, which is rapid, accurate and repeatable. The charge heterogeneity of VEGFR： Fc-fusion protein, which was highly glycosylated detected by iCIEF excellently. ICIEF provides a rapid and reliable assay method for charge heterogeneity research and quality control of Fc-fusion proteins.

关 键 词：血管内皮生长因子受体-抗体Fc融合蛋白 电荷异质性 条件优化 全柱成像毛细管等电聚焦 

分 类 号：R917[医药卫生—药物分析学]