Refining proteoform characterization in biopharmaceuticals: A paradigm for the impact of pI markers and carrier ampholytes in imaged capillary isoelectric focusing tandem mass spectrometry.

Proteoforms, structurally distinct yet closely related protein isoforms, play a pivotal role in biopharmaceutical development, directly influencing therapeutic efficacy, safety, and stability. These molecular variants arise from genetic polymorphisms, alternative splicing, and post-translational modifications, necessitating advanced analytical techniques for precise characterization. Imaged capillary isoelectric focusing (icIEF) coupled with mass spectrometry (MS) has become a powerful high-resolution tool for resolving and identifying proteoforms in complex biopharmaceutical samples. This study used a monoclonal antibody (mAb) as a paradigm to comprehensively evaluate the chemical properties of pI markers and carrier ampholytes in icIEF-MS. By investigating their effects on method accuracy, sensitivity, MS compatibility, and repeatability, we demonstrated how reagent selection can impact overall assay performance. The MS characterization of these reagents provided deeper insights into their influence on icIEF separation and proteoform identification, offering a critical case study for optimizing diverse icIEF reagent strategies. These findings contribute to the advancement of icIEF-MS methodologies, ensuring robust and reproducible proteoform characterization for biopharmaceutical research, process development, and quality control.