Sodium dodecyl sulfate-capillary gel electrophoresis with native fluorescence detection for analysis of therapeutic...

A 280 nm light-emitting diode (LED) was used as the excitation source for native fluorescence detection (NFD) of proteins in capillary electrophoresis. The NFD scheme was evaluated in sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) for monoclonal antibody (mAb) characterization. Utilizing a technique by which we filtered the LED emission through a 280 nm bandpass filter, we were able to increase overall concentration sensitivity of SDS-CGE-NFD ~2.3-fold. Under the optimized conditions, the assay linear dynamic range was > 4 orders of magnitude with a correlation coefficient (r²) of 0.9999, and the limit of detection was 8.3 ng/mL. The SDS-CGE-NFD assay was applied to quantitation and purity analysis of Etanercept, a therapeutic protein. Over the range of 50 - 150% of the target concentration, 200 µg/mL, recoveries were in the range of 97.02 - 101.6%. The SDS-CGE-NFD assay allowed for simultaneous quantitation of high- and low-molecular-weight species in Etanercept.