Department of Biology, Taiyuan Normal University, Shanxi, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Oct 1;906:63-8. doi: 10.1016/j.jchromb.2012.08.022. Epub 2012 Aug 23.
This work aimed to develop a rapid capillary zone electrophoresis (CZE) method to provide abundant purity and identity information of monoclonal antibodies. The CZE running buffer system was optimized to be 20mM acetate-acetic acid (pH 6.0) together with the co-addition of 0.3% polyethylene oxide (PEO) and 2mM triethylenetetramine (TETA), which was further tested with advantages on the peak resolution improvements. The conditioning period was scheduled to 1 min for both 0.1M HCl and CZE running buffer to reduce total separation time. Additionally, the applied voltage and effective separation length were optimized at 30 kV and 20 cm separately. Compared with the method reported by Yan [1], this newly developed method showed a higher resolution in separating the two unknown basic peaks by testing monoclonal antibody sample (mAb1). The further validation results showed that for all five of charge isoform peaks of test mAb1, repeatability, intraday and interday precision had a RSD less than 0.58% for migration time and less than 3.18% for corrected area percent. The correlation coefficients of more than 0.98 for all peaks also demonstrated the good linearity for the method. In addition to the application of distinguishing intact antibody from C-terminal Lys variants, the method also has advantage in separating the Fab, Fc and intact antibody-relevant substances quickly, which facilitated the rough evaluation of papain induced digestion.
本工作旨在开发一种快速毛细管区带电泳（CZE）方法，以提供单克隆抗体的丰富纯度和身份信息。优化了 CZE 运行缓冲液系统，为 20mM 乙酸-乙酸（pH6.0），并添加 0.3% 聚氧化乙烯（PEO）和 2mM 三乙烯四胺（TETA），进一步测试了该系统在提高峰分辨率方面的优势。条件期设定为 0.1M HCl 和 CZE 运行缓冲液各 1 分钟，以减少总分离时间。此外，优化了施加电压和有效分离长度，分别为 30kV 和 20cm。与 Yan [1] 报道的方法相比，该新开发的方法在测试单克隆抗体样品（mAb1）时，通过分离两个未知碱性峰显示出更高的分辨率。进一步的验证结果表明，对于测试 mAb1 的所有五个电荷异构体峰，重复性、日内和日间精密度的迁移时间 RSD 小于 0.58%，校正面积百分比的 RSD 小于 3.18%。所有峰的相关系数均大于 0.98，表明该方法具有良好的线性。除了应用于区分完整抗体和 C 末端赖氨酸变体外，该方法还具有快速分离 Fab、Fc 和完整抗体相关物质的优势，有利于木瓜蛋白酶诱导消化的粗略评估。
