摘要
目的建立IgG2型单抗毛细管区带电泳(CZE)电荷异质性分析方法。方法通过对三乙基四胺(TETA)和6-氨基己酸(EACA)浓度,以及电泳缓冲液pH和毛细管分离温度等参数进行优化,建立能够有效分离和定量IgG2单抗电荷异构体的CZE分析方法,并对该方法进行方法学验证和应用评价。结果 TETA和EACA浓度,以及电泳缓冲液pH和毛细管分离温度的变化,会影响IgG2单抗的电荷异构体分离效果,通过一系列参数的优化,最终确定了方法参数(400 mmol·L^(-1)EACA/0.05%TETA,pH 6.2,分离电压30 kV,分离温度35℃)。建立的CZE分析方法可以有效地对IgG2单抗电荷异构体进行分离,并具有良好的精密度,峰面积的RSD值小于3.0%。该方法同样适用于IgG1和IgG4型单抗的电荷异质性分析,同时,根据电泳图谱和迁移时间,能够对不同亚型和靶点的单抗进行有效的区分鉴别。结论建立的IgG2型单抗毛细管区带电泳方法,可有效分离电荷异构体,并具有良好的精密度,可作为平台方法应用于IgG2型单抗的电荷异质性分析和鉴别。
OBJECTIVE To establish a capillary zone electrophoresis（CZE）-based method for charge heterogeneity analysis of IgG2 monoclonal antibody.METHODS The concentrations of TETA and EACA,pH of separation buffer and temperature of capillary were optimized,to obtain a method which can efficiently separate the charge variants of IgG2 mabs.The reproducibility and repeatability of the optimized CZE method were validated and a battery of mabs were evaluated.RESULTS The separation effect was significantly influenced by TETA/EACA concentration,pH and separation temperature.Through a series of optimization,a CZE method which showed good resolution and precision（RSD of area percentage was below 3.0% was established and the method parameters were as below：400 mmol·L-1 EACA/0.05% TETA,pH 6.2,separation under 30 kV voltage at 35 ℃.This method was also suitable for IgG1 and IgG4 mabscharge heterogeneity analysis.Based on the specific electrophorogram and migration time,it could also be used as an identification method for monoclonal antibodies.CONCLUSION The optimized CZE method can efficiently separate the charge variants of IgG2 mabs and shows good precision and specificity,which can be used to analyze the charge heterogeneity and identification of monoclonal antibodies.
作者
王文波
于传飞
张峰
武刚
高凯
王兰
WANG Wen-bo;YU Chuan-fei;ZHANG Feng;WU Gang;GAO Kai;WANG Lan(Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech products, National Institutes for Food and Drug Control, Beijing 100050, China)
出处
《中国药学杂志》
CAS
CSCD
北大核心
2018年第8期627-631,共5页
Chinese Pharmaceutical Journal
基金
国家"重点研发计划"生物安全关键技术研发专项资助(2016YFC1200904)
国家"重大新药创制"军队特需生物防治药质量控制研究项目资助(2016ZX09J16101)
关键词
IgG2单抗
电荷异质性
毛细管区带电泳
方法优化
质量控制
IgG2 monoclonal antibody charge heterogeneity capillary zone electrophoresis method optimization quality control
