Advancing protein heterogeneity analysis: Nano-flow pressure mobilization for precise icIEF fractionation and online MS detection.

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.