Unraveling what makes a monoclonal antibody difficult‐to‐express: From intracellular accumulation to incomplete folding and degradation via ERAD

Citations Over TimeTop 10% of 2019 papers

Abstract

Although most therapeutic monoclonal antibodies (mAbs) can routinely be produced in the multigram per litre range, some mAb candidates turn out to be difficult-to-express (DTE). In addition, the class of more complex biological formats is permanently increasing and mammalian expression systems like Chinese hamster ovary (CHO) cell lines can show low performance. Hence, there is an urgent need to identify any rate limiting processing step during cellular synthesis. Therefore, we assessed the intracellular location of the DTE antibody mAb2 by fluorescence and electron microscopy (EM) and revealed an accumulation of the antibody, which led to an aberrant morphology of the endoplasmic reticulum (ER). Analysis of underlying cellular mechanisms revealed that neither aggregation nor antibody assembly, but folding represented the reason for hampered secretion. We identified that the disulfide bridge formation within the antibody light chain (LC) was impaired due to less recognition by protein disulfide isomerase (PDI). As a consequence, the DTE molecule was degraded intracellularly by the ubiquitin proteasome system via ER-associated degradation (ERAD). This study revealed that with the continuous emergence of DTE therapeutic protein candidates, special attention needs to be drawn to optimization processes to ensure manufacturability.

Related Papers

• → Advances in the study of protein folding and endoplasmic reticulum-associated degradation in mammal cells(2024)7 cited
• → Entry of Protein Toxins into Mammalian Cells by Crossing the Endoplasmic Reticulum Membrane: Co-opting Basic Mechanisms of Endoplasmic Reticulum-Associated Degradation(2006)72 cited
• → Folding in vitro of bovine pancreatic trypsin inhibitor in the presence of proteins of the endoplasmic reticulum(1992)65 cited
• → Endoplasmic Reticulum-Associated Protein Degradation(2013)1 cited