Induction Temperature and Feeding Profile Shape the Partitioning Between Soluble Product and Inclusion Body Formation in Escherichia coli

Inclusion body (IB) processes yielding the recombinant product only as IBs are long‐established in the biopharmaceutical industry. Yet, for recombinant proteins that accumulate both IBs and soluble fraction depending on the selected upstream processing (USP) strategy, the influence of process parameters on the partitioning between both forms remains poorly characterized. In this study, l ‐lactate dehydrogenase (LDH) was selected as a model protein as it yields IBs and soluble protein concurrently. We investigated how induction temperature and specific substrate uptake rate shift the relative amount of soluble product. A response surface model revealed a sharp switch between exclusively soluble production at 25°C, exclusive IB formation at 35°C and a mixed production regime at 30°C. Further, we investigated whether these trends hold for the structurally related proteins xylose isomerase (Xiso) and pyranose‐2‐oxidase (P2O) using a reduced diagonal design and a projection into the LDH latent space by principal component analysis (PCA). Results revealed that the shared structural features were not sufficient to predict the expression form. Still, the LDH model served as a reference framework for identifying the extent to which other mixed producers follow established process‐response trends.