Label-free protein quantification in complex formulations using intrinsic fluorescence gel electrophoresis

This study establishes a gel electrophoresis-based intrinsic fluorescence imaging (GE-IFI) method for dye-free detection of protein samples in complex buffer systems. The method utilizes the intrinsic fluorescence of proteins, primarily from tryptophan residues, as the signal source, avoiding the system dependence of extrinsic colorimetric reactions. The method was tested under conditions simulating critical steps in bioprocessing-denaturation (high urea), protein purification (weak acid), and high-salt formulation-using Denosumab and BSA to challenge its robustness. It exhibited stable signals across diverse systems and showed comparable or improved analytical robustness compared with conventional colorimetric methods under the evaluated conditions. The quantitative accuracy of GE-IFI was further validated by SEC-HPLC under acidic formulation conditions, demonstrating excellent agreement between fluorescence-based and chromatographic quantification. Based on the detection of intrinsic protein fluorescence, GE-IFI enables quantitative protein detection with reduced susceptibility to matrix effects from complex chemical environments. The method maintains high linearity and reproducibility across different formulation systems, offering a potential analytical strategy for monitoring protein concentration changes and aggregation-related molecular states during biopharmaceutical development.