毛细管电泳法测定重组人源化IgG2抗体的纯度Determination of purity of recombinant humanized IgG2 antibody by capillary electrophoresis
黄碧君,黄艳,胡红琴,郭占云
HUANG Bi-jun,HUANG Yan,HU Hong-qin,GUO Zhan-yun
摘要(Abstract)：
目的建立利用毛细管电泳法测定重组人源化免疫球蛋白G(immunoglobulin G,IgG)纯度的方法,并进行优化和验证。方法采用单因素法,对稀释液、碘乙酰胺浓度(非还原条件)、加热方式、进样电压、进样时间、分离电压、样品盘和卡盒温度进行多水平优化,并利用优化的方法测定IgG2的纯度。以峰面积对样品加样量进行线性回归,确定该方法的线性范围,并对该方法进行精密度、准确度及溶液稳定性验证。结果最佳电泳条件为:选用超纯水作为稀释液,250 mmol/L碘乙酰胺(非还原条件),70℃干热10 min,进样电压为5 kV,进样时间为30 s,分离电压为15 kV,样品盘和卡盒温度为20℃。在优化的色谱条件下,在还原条件下,IgG2纯度检测的线性范围为50~150μg,相关系数为0.998,重复性的相对标准偏差(relative standard deviation,RSD)为0.14%,中间精密度的RSD为0.24%,且20℃放置13 h稳定;在非还原条件下,IgG2纯度检测的线性范围为50~150μg,相关系数为0.999,重复性的RSD为0.36%,中间精密度的RSD为0.16%,且20℃放置13 h稳定。结论该方法线性关系良好,且精密度、准确度、灵敏度较高,测定结果稳定可靠,适用于重组人源化IgG2的纯度分析。
Objective To develop,optimize and verify a capillary electrophoresis(CE) for determination of purity of humanized immunoglobulin G(IgG). Methods A series of experimental conditions,including diluent,iodoacetamide concentration for non-reduced sample,heating method,inject-voltage and duration,separate-voltage and temperatures of sample storage and cartridge were optimized by single factor experiment. The developed method was used for determination of IgG2 purity,determined for linear range by linear regression of peak area against sample amount,and verified for precision,accuracy and stability. Results The condition for CE was optimized as follows:samples were prepared with ultrapurified water containing 250 mmol/L iodoacetamide in non-reduced forms by using dry bath thermostat at 70 ℃ for 10 min. The temperatures of sample storage and cartiadge were maintained at 20 ℃. Samples were injected using CE 30 s at 5 kV. The time for sample loading was 30 s. Separation was performed at 15 kV. By the optimized method under reduced condition,the linear range for determination of IgG2 purity was 50 ~ 150 μg with a correlation coefficient of 0. 998. The RSD in reproducibility test was 0. 14%,while that in intermediate precision test was0. 24%. The IgG2 protein was stable after storage at 20 ℃ for 13 h. However,under non-reduced condition,the linear range for determination of IgG2 purity was 50 ~ 150 μg,with a correlation coefficient of 0. 999,while the RSD in reproducibility test was 0. 36% and that in intermediate precision test was 0. 16%,and IgG2 protein was stable after storage at 20 ℃ for 13 h. Conclusion The developed method showed good linearity,precision,accuracy and sensitivity,and the test result was stable and reliable,which was suitable for the analysis of purity of recombinant humanized IgG2.
关键词(KeyWords)：
免疫球蛋白G;纯度;毛细管电泳法
Immunoglobulin G(IgG2);Purity;Capillary electrophoresis(CE)
基金项目(Foundation):
作者(Author):
黄碧君,黄艳,胡红琴,郭占云
HUANG Bi-jun,HUANG Yan,HU Hong-qin,GUO Zhan-yun
DOI: 10.13200/j.cnki.cjb.002123
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