Sosic Zoran, Houde Damian, Blum Andy, Carlage Tyler, Lyubarskaya Yelena
Analytical Development, Biogen Idec, Cambridge, MA 02142, USA.
Electrophoresis. 2008 Nov;29(21):4368-76. doi: 10.1002/elps.200800157.
In this work several aspects of imaging capillary IEF (icIEF) application for charge heterogeneity analysis of recombinant proteins and monoclonal antibodies have been discussed. Advantages of the method as compared with traditional approaches for determination of biomolecule charge heterogeneity, such as gel and IEC, have been demonstrated. Correlation of icIEF-detected protein isoforms with the charge heterogeneity determined by IEC has been shown for a representative recombinant monoclonal antibody. Identification of charged variants collected from IEC has been performed by ESI-MS. Qualification of an icIEF method for use in quality control environment for quantitative analysis of recombinant protein charge heterogeneity and monitoring protein stability has also been discussed. The intermediate precision for determination of pI of main or main acidic species was </=0.2% RSD. Relative % peak areas for acidic, main and basic species were reproducible within 1.9, 0.9 and 16.6% RSD, respectively. Based on the assay performance evaluation, icIEF assay has been shown to allow for fast method development, short analysis time and high sample throughput. Some aspects of the method specificity for use as an identity test in biopharmaceutical development have been discussed.
在本工作中，已讨论了成像毛细管等电聚焦（icIEF）在重组蛋白和单克隆抗体电荷异质性分析中的几个应用方面。已证明该方法与用于确定生物分子电荷异质性的传统方法（如凝胶和离子交换色谱法）相比的优势。对于一种代表性的重组单克隆抗体，已显示icIEF检测到的蛋白异构体与通过离子交换色谱法确定的电荷异质性之间的相关性。已通过电喷雾电离质谱法（ESI-MS）对从离子交换色谱法收集的带电变体进行了鉴定。还讨论了一种icIEF方法在质量控制环境中用于重组蛋白电荷异质性定量分析和监测蛋白稳定性的验证。主要或主要酸性物种的等电点测定的中间精密度为相对标准偏差（RSD）≤0.2%。酸性、主要和碱性物种的相对峰面积百分比分别在RSD为1.9%、0.9%和16.6%范围内可重现。基于分析性能评估，已表明icIEF分析能够实现快速方法开发、短分析时间和高样品通量。还讨论了该方法在生物制药开发中用作鉴别试验的特异性的一些方面。
