Optimization of a robust and reliable FITC labeling process for CE-LIF analysis of pharmaceutical compounds using design of experiments strategy

Emonts, Paul; Avohou, Hermane T.; Hubert, Philippe; Ziémons, Eric; Fillet, Marianne; Dispas, Amandine

doi:10.1016/j.jpba.2021.114304

Cited by 7 publications

(1 citation statement)

References 13 publications

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“…DoE approaches are important pieces in (analytical) quality-by-design concepts and frequently used in (analytical) method development [30,31]. Examples of DoE-guided CE-based separation method developments include labelling reactions in CE-LIF [32], CZE for adenovirus particles [33], chiral separations [34,35], as well as CE-SDS [36] and CZE [37] methods for mAb analysis. Within this manuscript, the separation method remained unmodified; the focus was on the LEDIF detector optimization.…”

Section: Introductionmentioning

confidence: 99%

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

Zagst,

Hartung,

Menges

et al. 2023

Separations

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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.

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