Quantifying Critical Quality Attributes of Protein Therapeutics by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis With Native Fluorescence Detection.

In the biopharmaceutical industry, the sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) assay is often used to assess therapeutic critical quality attributes (CQAs). Traditional detection methods for SDS-CGE methods, such as ultraviolet (UV) absorbance and laser-induced fluorescence (LIF), are widely used but come with limitations. A native fluorescence detection (NFD) scheme was previously developed to enhance sensitivity and reduce gel matrix interference without requiring sample derivatization, and the SDS-CGE-NFD assay exhibited high precision and accuracy for absolute quantification of monoclonal antibodies (mAbs). In this work, we assessed the suitability of SDS-CGE-NFD to quantification of CQAs in protein therapeutics, which is generally relative rather than absolute. NFD was compared with UV absorbance and LIF detection for quantifying CQAs of protein therapeutics in SDS-CGE. Three lots of NIST monoclonal antibody (NISTmAb) were assayed by SDS-CGE with NFD, UV, and LIF detection, and the relative abundance of total fragments was compared and found similar. Analysis with NFD measured abundances at a range of 1.77%-2.00%, compared to the range of 1.53%-1.78% measured with UV absorbance and 1.63%-1.86% measured with LIF. Aggregates were not recognized with UV absorbance but were apparent with measured relative abundance of 0.38%-0.40% using NFD and 0.35%-0.40% using LIF. Under the reducing conditions, glycosylation site occupancy on the heavy chain was measured in the range of 99.30%-99.33% with all three detection approaches. The comparable results measured with three detection modes suggested that SDS-CGE-NFD was suitable to quantify CQAs of protein therapeutics. The SDS-CGE-NFD workflow was successfully applied to analyze two commercial protein therapeutics, a bispecific mAb of ∼146 kDa (Emicizumab) and an Fc-fusion protein of ∼63 kDa (Dulaglutide).