Imaged capillary isoelectric focusing (icIEF) tandem high resolution mass spectrometry for charged heterogeneity of protein drugs in biopharmaceutical discovery

Abstract

Since the first commercial imaged capillary isoelectric focusing (icIEF) instrument was developed twenty years ago, the technology has become the gold standard of quality and manufacturing process control in the biopharmaceutical industry. This is owing to its high-resolution and high-throughput characterization of protein charge heterogeneity. In addition to a charge variant profiling, mass spectrometry (MS) analyses are also desirable to obtain an in-tact molecular weight (MW) and further identification of these charged species. While offline fractionation technologies including isoelectric focusing (IEF) and free flow electrophoresis (FFE) followed by liquid chromatography (LC)-mass spectrometry (MS) coupling have been employed for this purpose, there have been much fewer reported applications of icIEF-based MS connection and fraction collection. Factors that have impeded the development of these icIEF applications include difficulties with a direct connection to the MS interface as well as high background signal of carrier ampholytes and incompatible coated capillary cartridges. In this work, we developed a robust and flexible icIEF-MS platform which overcomes these challenges to achieve both the rapid icIEF separation and high-resolution MS (HRMS) identification of protein charged variants simultaneously. We demonstrate how this methodology proves highly-sensitive and highly reliable for the characterization of commercial monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs). The whole workflow of icIEF-MS for protein heterogeneity is straight forward and accurate and can be performed within 45 min. Furthermore, the developed icIEF-MS configuration can flexibly switch to icIEF-based fraction collection model allowing the user to perform additional in-depth characterization such as peptide mapping by high performance liquid chromatography (HPLC) tandem mass spectrometry (LC-MS/MS).