Analytical Applications of Immobilized Enzyme Reactors (IMERs) Coupled to LC–MS/MS for Bottom- and Middle-Up Characterization of Proteins

Abstract

Identification, monitoring, and, more importantly, linkage of critical quality attributes (CQAs) in processing parameters in a biopharmaceutical product is required to ensure the quality and manufacturing consistency of the product, but also its safety and efficacy during clinical and commercial development. Recently, bottom-up and middle-up liquid chromatography–mass spectrometry (LC–MS) characterization strategies using immobilized enzyme reactors (IMERs) in combination with multidimensional liquid chromatography coupled with high-resolution MS (MDLC–HRMS), as well as sophisticated software solutions, have been added to the analytical toolbox. These strategies not only allow faster characterization of post-translational modifications (PTMs) present in biotherapeutic proteins but also have the potential to provide a fully automated and unified bottom-up, middle-up, and intact LC–MS characterization approach.