Native fluorescence detection with a laser driven light source for protein analysis in capillary electrophoresis

Abstract

While ultraviolet light (UV) absorbance detection is the most widely used detection mode in capillary electrophoresis (CE), it can yield poor concentration sensitivity and has tendencies to exhibit baseline fluctuations. In order to overcome these challenges, alternative detection strategies, including the use of dedicated wavelength lasers, have been applied, resulting in enhancements of concentration sensitivity as well as decreased baseline disturbance. In this work, using a laser driven light source for excitation, we reported a native fluorescence detection (NFD) scheme for use in a commercial CE platform, PA 800 Plus Pharmaceutical Analysis System, for protein analysis. The CE-NFD system was characterized using tryptophan and a reduced IgG. We compared NFD with UV absorbance detection as applied to sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and capillary isoelectric focusing (cIEF). In SDS-CGE, with the reported NFD a non-reduced IgG standard sample yielded a signal-to-noise ratio which was 14.6 times higher than with UV absorbance detection at 214 nm. In cIEF analysis of NISTmAb, Humanized IgG1k, with NFD ∼170 times less sample mass was needed to obtain similar profile quality to that with UV absorbance detection at 280 nm. NFD also eliminated baseline anomalies observed with UV absorbance detection and showed less interference by other absorbing species. These results suggest that CE-NFD is a practical and powerful tool for protein characterization in the biopharmaceutical industry.