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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。
The condition of surface charge heterogeneity of anti-epidermal growth factor (EGF) monoclonal antibody was optimized by ion exchange chromatography, and the effect of glycosylation on the surface charge heterogeneity distribution of monoclonal antibody was analyzed. Gel isoelectric focusing electrophoresis was used to determine the isoelectric point distribution range of the non-homogeneous charge components of monoclonal antibodies. Ion exchange chromatography was used to analyze the conditions of non-uniform surface charge of anti-EGF monoclonal antibodies, including chromatographic column, mobile phase pH , Gradient and column temperature, the optimal analytical conditions were selected to analyze the effect of glycosylation on the charge heterogeneity distribution of the monoclonal antibodies. Gel isoelectric focusing electrophoresis analysis of anti-EGF monoclonal antibody charge heterogeneous components isoelectric point distribution in the range of 8.0 to 8.6, ion exchange chromatography analysis of surface charge heterogeneity of the best conditions: TSK-gel CM -STAT ion exchange column, the mobile phase pH 6.0, the appropriate initial gradient and gradient steepness conducive to the separation of the charge heterogeneity of the components, the column temperature was 40 ℃; removal of glycosylated monoclonal antibodies in acidic The relative content of non-uniform surface charge increases. Appropriate analytical conditions can make the surface of the monoclonal antibody non-uniformity of each component in the ion exchange chromatography column to obtain the best separation and obtain higher sensitivity, monoclonal antibody glycosylation modification of the surface charge heterogeneity to the basic region Offset.