Characterization workflow for fragments detected in capillary electrophoresis sodium dodecyl sulfate analysis of therapeutic monoclonal antibodies

Abstract Product‐related fragments in monoclonal antibodies (mAbs) can have a significant impact on the efficacy and safety of the product. Capillary electrophoresis sodium dodecyl sulfate (CE‐SDS) is a commonly used method for fragment quantification, but it has challenges in peak identification due to the inability to enrich components and the incompatibility of SDS with mass spectrometry (MS). This article presents a workflow for identifying peaks in CE‐SDS analysis. The workflow involves comparing the migration time of peaks with that of standards and utilizing MS analysis to identify fragments. By employing this innovative systematic workflow, we successfully identified the CE‐SDS impurity peaks of seven antibody products. Among them, four products exhibited characteristic fragments associated with disulfide bonds (light chain [LC], heavy–light [HL] chain, heavy–heavy [HH] chain, and HH–LC) and a glycosylation‐related fragment non‐glycosylated heavy chain. Additionally, one product showed a fragment formed by the connection of HC_C 130 and HC_C 130 , which is associated with a thioether bond. Furthermore, two other products displayed amino acid backbone breakage, with one product showing clipping at the HC region of A 233 –G 285 and the other product showing clipping at the HC regions of A 97 –S 158 and N 342 –T 366 . This workflow can be applied in early drug research, process development, or during the biologics license application stage to characterize fragments in therapeutic mAbs analyzed by CE‐SDS.