Faculty of Chemistry, Aalen University, Aalen, Germany.
Electrophoresis. 2018 May;39(9-10):1151-1154. doi: 10.1002/elps.201800013. Epub 2018 Mar 12.
Imaged capillary isoelectric focusing (iCIEF) is a powerful separation technique applied frequently for the analysis of biotherapeutics. However, direct mass spectrometric characterization is usually not possible. Here, focused peaks from an iCIEF system are transferred to a four-port nanoliter valve where peaks of interest are cut and transferred directly (flow injection) or via capillary zone electrophoresis to electrospray mass spectrometry. At first, flow injection coupling was tested with a mixture of peptides showing intraday precision (RSD) of 20.0 and 3.4% in area and 15.2 and 6.3% in intensity for angiotensin I and leucine-enkephalin, respectively. For the analysis of charge variants in an intact mAb, flow injection and CZE as second dimension were compared, demonstrating the usability of flow injection even for large proteins. However, improved spectra quality was achieved when CZE-MS was applied. In this way, accurate masses were obtained not only for the main isoform, but also for the main acidic and basic variants. These results demonstrate the power of iCIEF-CZE-MS for the analysis of biotherapeutics.
成像毛细管等电聚焦（iCIEF）是一种强大的分离技术，常用于生物治疗剂的分析。然而，通常无法直接进行质谱表征。在这里，iCIEF 系统中的聚焦峰被转移到四端口纳升阀，其中感兴趣的峰被直接切割和转移（流动注射）或通过毛细管区带电泳转移到电喷雾质谱。首先，用混合肽测试了流动注射偶联，结果表明血管紧张素 I 和亮氨酸脑啡肽的面积和强度的日内精密度（RSD）分别为 20.0%和 3.4%、15.2%和 6.3%。为了分析完整 mAb 中的电荷变异体，比较了流动注射和 CZE 作为二维，证明了即使对于大蛋白，流动注射也是可行的。然而，当应用 CZE-MS 时，获得了更好的光谱质量。通过这种方式，不仅可以获得主要异构体的准确质量，还可以获得主要的酸性和碱性变异体的准确质量。这些结果证明了 iCIEF-CZE-MS 用于生物治疗剂分析的强大功能。
