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Unraveling what makes a monoclonal antibody difficult‐to‐express: From intracellular accumulation to incomplete folding and degradation via ERAD
Author(s) -
Mathias Sven,
Wippermann Anna,
Raab Nadja,
Zeh Nikolas,
Handrick René,
Gorr Ingo,
Schulz Patrick,
Fischer Simon,
Gamer Martin,
Otte Kerstin
Publication year - 2020
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27196
Subject(s) - endoplasmic reticulum , protein disulfide isomerase , chinese hamster ovary cell , endoplasmic reticulum associated protein degradation , monoclonal antibody , microbiology and biotechnology , intracellular , chemistry , antibody , biochemistry , foldase , mhc class i , proteasome , biology , major histocompatibility complex , unfolded protein response , immunology , gene , receptor , escherichia coli , groel
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.