Capillary Zone Electrophoresis with Light-Emitting Diode-Induced Fluorescence Detection for the Analysis of Monoclonal Antibodies: Detector Optimization through Design of Experiments and Comparison to UV Detection

Abstract

Capillary zone electrophoresis (CZE) is an important technique for the analysis of monoclonal antibodies (mAbs). A recently released light-emitting diode (LED)-induced fluorescence (LEDIF) detector equipped with a 275 nm LED for the detection of proteins through their native fluorescence was used in this study and compared to results obtained using the predominant detection mode, the measurement of the absorption of ultraviolet light (UV detection). This was accomplished using an established CZE method for the analysis of three mAbs: NISTmAb, matuzumab, and Intact Mass Check Standard (Waters). For this purpose, the detector’s settings were first optimized using a design of experiments approach. Three factors, rise time, photomultiplier high voltage supply, and acquisition frequency, were optimized by means of a D-optimal design. The optimal settings were then used for the investigation of signal-to-noise ratios (S/Ns), linearity, and precision. LEDIF detection offered a similar separation quality, up to 12 times higher S/Ns, and lower limits of detection compared to UV detection. Repeatability was excellent, with relative standard deviations (RSDs) of approximately 1% for percentage areas. For intermediate precision, RSDs of <2% (n = 3 × 10) were typically achieved. Overall, LEDIF detection was found to be an excellent and easily optimizable alternative to UV detection.

Capillary Electrophoresis: A Three-Year Literature Review

“…The high-throughput detection allows accurate location of each channel and LODs lower than 0.1 μg/mL. Zagst et al introduced a light-emitting diode-induced fluorescence detector for native fluorescence detection of proteins . The detector was compared to conventional UV detection in the analysis of three mAbs, exhibiting up to 12 times higher signal-to-noise (S/N) ratios.…”

“…Zagst et al introduced a light-emitting diode-induced fluorescence detector for native fluorescence detection of proteins. 111 The detector was compared to conventional UV detection in the analysis of three mAbs, exhibiting up to 12 times higher signal-to-noise (S/N) ratios. Petersen et al developed a highly sensitive fluorescence detector for CE analysis using silicon photomultipliers, which allows for a dynamic range spanning 5 orders of magnitude.…”