Kwok Teresa, Chan She Lin, Xu Niusheng, Huang Tiemin, Bo Tao
Advanced Electrophoresis Solutions Ltd., Cambridge, Canada.
Advanced Electrophoresis Solutions Ltd., Cambridge, Canada.
Anal Biochem. 2025 Jun;701:115825. doi: 10.1016/j.ab.2025.115825. Epub 2025 Mar 2.
This study addresses the challenges of high-resolution protein charge variant fractionation and efficient online mass spectrometry (MS) detection in imaged capillary isoelectric focusing (icIEF)-based workflows. icIEF often faces limitations in efficiency, peak integrity, and detection sensitivity due to diffusion and uncontrolled mobilization. To overcome these, we developed a novel icIEF fractionation framework that integrates nano-flow pressure mobilization with the capillary diameter transformation technique (CDTT). Using a model system with a 320 μm ID separation channel and a 50 μm ID transfer capillary, we investigated the electrophoretic and nano-flow transport mechanisms influencing fractionation efficiency. The impact of these innovations on peak area, height, and width for charge proteoforms was assessed, showing improvements in precision. These insights were applied to a 200 μm ID separation channel system, resulting in enhanced separation efficiency and icIEF-MS sensitivity. This study offers a scalable, high-precision solution for charge heterogeneity analysis in biopharmaceutical development and regulatory applications.
本研究探讨了基于成像毛细管等电聚焦（icIEF）的工作流程中高分辨率蛋白质电荷变体分离和高效在线质谱（MS）检测所面临的挑战。由于扩散和不受控制的迁移，icIEF在效率、峰完整性和检测灵敏度方面常常面临限制。为了克服这些问题，我们开发了一种新型的icIEF分离框架，该框架将纳流压力迁移与毛细管直径转换技术（CDTT）相结合。使用内径为320μm的分离通道和内径为50μm的转移毛细管的模型系统，我们研究了影响分离效率的电泳和纳流传输机制。评估了这些创新对电荷蛋白变体的峰面积、高度和宽度的影响，结果显示精度有所提高。这些见解被应用于内径为200μm的分离通道系统，从而提高了分离效率和icIEF-MS灵敏度。本研究为生物制药开发和监管应用中的电荷异质性分析提供了一种可扩展的高精度解决方案。
