Biotherapeutics are a complex and heterogeneous class of drug molecules that require comprehensive and rapid characterization throughout the development process. Charge heterogeneity of monoclonal antibodies (mAbs) can result from cellular processes, chemical degradation and production conditions during manufacturing, which may impact the efficacy, safety and potency of therapeutic mAbs. Imaged capillary isoelectric focusing (iCIEF) is an established platform technique for separating and quantifying charge variants based on changes to the molecule’s isoelectric point that result from post translational modifications (PTMs). However, identifying those PTMs that result in changes to the iCIEF profile often requires fractionation, LC method development, offline MS characterization and reanalysis by iCIEF, which is a lengthy and laborious process.
To overcome these bottlenecks, we describe advances in microfluidic chip technology development that have enabled the direct coupling of iCIEF with MS to provide charge variant separation, UV quantitation and MS characterization. These advancements provide unprecedented insights into critical PTMs, including glycan analysis, charge variant peak ID and intact mass analysis of biotherapeutic mAbs. Several examples of charge variant characterization demonstrating high-resolution iCIEF separation coupled with sensitive detection and mass identification of major and minor proteoforms will be highlighted.
This technology holds promise to reduce the timeline, investment and risk associated with the development of biotherapeutics.