Spectroscopy-Assisted Bayesian Optimization for Efficient Refolding of Inclusion Body Proteins

Abstract

The production of recombinant proteins in Escherichia coli often yields insoluble inclusion bodies, which require denaturation and refolding to obtain the native product. The pro- tein refolding step usually represents a major bottleneck. Conventional development and optimization typically rely on sequential Design of Experiments with high-performance liquid chromatography readouts. This approach is slow, labor-intensive, and requires an established chromatographic method as well as purified protein standards. At the beginning of process development, these prerequisites may not be met - especially for proteins that can only be expressed as inclusion bodies. We introduce a more efficient, data-driven workflow that pairs Bayesian optimization with a rapid, in-line readout from intrinsic tryptophan fluorescence. Using a disulfide-bonded single-chain variable fragment, we explored a five-dimensional design space of refolding buffer composition (dithiothreitol, oxidized glutathione, dilution factor, pH, and final urea concen- tration) guided by two spectroscopy-derived objectives. We showed that the spectral shift correlates with chromatographic yields, supporting its use as a fit-for-purpose sensor to guide process development. With 25 experiments, Bayesian optimization identified conditions that delivered a refolded protein concentration of 1.29 ± 0.06 g L⁻¹ at 58.7 ± 1.3 % refolding yield with a dilution factor of 3.14, whereas a three-stage Design of Experiments with more than 60 experiments concluded at 0.37 ± 0.02 g L⁻¹ and 61.4 ± 3.1 % with a dilution factor of 11.39. Thus, the presented workflow achieved roughly 3.5-fold higher product concentration at comparable yield, while operating at substantially higher protein concentrations. Therefore, spectroscopy- assisted Bayesian optimization was found to be a practical, sample-efficient tool for refolding optimization that is especially valuable in early development stages.