Time-Resolved Native Fluorescence Imaging of the Migration and Band Broadening of Monoclonal Antibody During Capillary Gel Electrophoresis With Sodium Dodecyl Sulfate.

We developed an imaging-based approach to directly visualize and monitor molecular migration during capillary electrophoresis and applied it to the separation of monoclonal antibodies (mAbs) by capillary gel electrophoresis with sodium dodecyl sulfate (CGE-SDS). Proteins were detected through their intrinsic fluorescence at 350 nm following excitation at 280 nm, enabling real-time observation of migration behavior without additional labeling. Time-resolved imaging allowed direct visualization of both migration and band broadening of mAbs under reducing and non-reducing conditions throughout the separation process. Analysis of electrophoretic mobilities suggests that mAb separation in the gel matrix is largely governed by the Ogston sieving mechanism. Quantification of band broadening revealed that peak dispersion increased with applied electric field strength, as evidenced by the marked reduction in broadening when the separation voltage was removed. Qualitative evaluation of the dispersion behavior suggests that mAb band broadening arises from the combined effects of electromigration dispersion (EMD) and gel-sieving-induced dispersion, with glycosylation-related heterogeneity providing an additional contribution for glycosylated species such as the heavy chain (HC) and intact mAb. These results demonstrate the utility of real-time imaging for elucidating separation mechanisms and dispersion phenomena in CGE-SDS and provide new insights into factors governing the electrophoretic behavior of therapeutic antibodies.