The Missing Link: Connecting Cultivation Conditions and Refolding Performance via Inclusion Body Biophysical Properties.

Inclusion bodies (IBs) frequently form during the expression of heterologous proteins in Escherichia coli and are therefore important in pharmaceutical production. While it is accepted that cultivation conditions affect cultivation performance, the link to refolding performance remains unexplored. This study proposes that this interplay relies, at least partially, on the inherited biophysical properties of the IBs. Using a design of experiments approach, this study systematically explored how cultivation conditions-postinduction temperature, pH, and feed rate-affect the production of IBs containing anti-desipramine single-chain variable fragment antibodies as a model therapeutic protein. Various biophysical properties of the IBs-including hydrophobicity, secondary structure, and particle size-were characterized, and their relationship to cultivation parameters and refolding performance was analyzed. Key findings revealed that higher feed rates and temperatures increased the product titer and IB size. Larger IBs facilitated refolding, while a higher content of amyloid structures, occurring locally without a strong link to the cultivation parameters, hampered protein solubilization and refolding efficiency. Higher protein content in the IBs adversely affected the refolding yield due to a hidden coupling between cultivation and refolding. This study establishes IB biophysical properties as critical factors for linking upstream and refolding process performance, offering actionable insights to enhance bioprocess robustness and efficiency.