摘要
优化了离子交换色谱法分析抗表皮生长因子(Epidermal growth factor,EGF)单克隆抗体表面电荷不均一性的条件,分析糖基化对单克隆抗体表面电荷不均一性分布的影响。应用凝胶等电聚焦电泳方法测定单克隆抗体电荷不均一性组分等电点分布范围,再优化离子交换色谱法分析抗EGF单克隆抗体表面电荷不均一的条件,包括色谱柱、流动相pH、梯度及柱温,选择最佳的分析条件分析糖基化对单克隆抗体的电荷不均一性分布的影响。凝胶等电聚焦电泳方法分析抗EGF单克隆抗体电荷不均一性组分等电点分布范围在8.0～8.6范围内,离子交换色谱法分析表面电荷不均一性的最佳条件:TSK-gel CM-STAT离子交换色谱柱、流动相pH 6.0、合适的起始梯度及梯度陡度有利于电荷不均一性各组分的分离,柱温为40℃;去除糖基化的单克隆抗体分布在酸性区的表面电荷不均一组分相对含量增加。合适的分析条件能够使单克隆抗体表面电荷不均一性各组分在离子交换色谱柱上得到最佳分离及获得较高灵敏度,单克隆抗体的糖基化修饰使表面电荷不均一性向碱性区发生偏移。
Optimal analytical condition for ion exchange chro- matography analyzing surface charge heterogeneity of anti- EGF monoclonal antibody,and analysis of the effect of glyco- sylation on surface charge heterogeneity of anti-EGF mono- clonal antibody. The isoelectric point （pl） range of charge heterogeneous components of anti-EGF monoclonal antibodyhas been analyzed by gel isoelectric focusing electrophoresis, and analytical condition for ion exchange chromatography ana- lyzing charge heterogeneity of monoclonal antibody has been optimized,which included column type, mobile phase pH, gra- dient,and column temperature. The effect of glycosylation on surface charge heterogeneity of anti-EGF monoclonal antibodyhas also been analyzed in the optimum condition. The range of pl for charge heterogeneous components was from 8.0 to 8.6, and the optimum analytical condition was that TSK-gel CM- STAT column,mobile phase at pH 6.0, and column tempera- ture at 40 ℃ were selected for analysis of surface charge het- erogeneity using ion exchange chromatography, and correct combination initial gradient and gradient step could be effec- tive for separation of surface charge heterogeneity of anti-EGF monoclonal antibody;relative content of components distribu- ted in the acid region increased for anti-EGF monoclonal anti- body with removed glycan, optimal analytical condition for ion exchange chromatography was effective to separate charge het- erogeneous components and increase sensitivity,and glycan of anti-EGF monoclonal antibody had the tendency to render charge distribution to migrate for basic region.
出处
《化学试剂》
CAS
CSCD
北大核心
2012年第5期437-440,472,共5页
Chemical Reagents
基金
河南省教育厅自然科学研究指导项目(2011B-150041)
关键词
抗EGF单克隆抗体
凝胶等电聚焦电泳
离子交换色谱法
表面电荷不均一性
糖基化
anti-EGF monoclonal antibody
gel isoelectric fo-cusing electrophoresis
ion exchange chromatography
surfacecharge heterogeneity
glycosylation
