High-cell-density Escherichia coli-based nanobody production: benchmarking of subcellular targeting, strain...

Nanobodies (NB) are compact single-domain antibody fragments with substantial diagnostic and therapeutic potential, but scalable microbial production under controlled bioreactor conditions remains insufficiently characterized. In this study a high-cell-density cultivation (HCDC) fed-batch process for an anti-human CD45 NB in Escherichia coli was established and evaluated how process parameters, host background, and subcellular targeting (cytoplasmic vs. periplasmic) affect yield and product quality including correct disulfide bond formation.

Using a two-stage design-of-experiments (DoE) strategy in 2 L stirred-tank bioreactors, OD_600nm at induction and post-induction temperature were identified as the main process variables controlling the post-purification nanobody concentration, whereas inducer concentration had no relevant effect within the tested range. Late induction at OD_600nm 125-150 combined with production temperatures around 21-22.5 °C defined a robust operating window and yielded more than 2.5 g L^-1 purified nanobody after Strep-tag affinity chromatography. Benchmarking of five host strain-plasmid combinations under standardized HCDC conditions further increased NB yield indicating that production performance was directly governed by the combined host background and expression architecture. While E. coli NEBExpress® with periplasmic SP_PelB targeting reached around 2.4 g L^-1 anti-hCD45 NB and the highest volumetric productivity (0.122 g L^-1 h^-1), E. coli SHuffle® T7 Express produced 2.2 g L^-1 anti-hCD45 NB intracellularly. Nanobody-quality analysis revealed a pronounced difference between production routes. NB preparations obtained from periplasmic targeting constructs formed thermally uniform species with single nano differential scanning fluorimetry (DSF) unfolding transitions at 73.27-74.05 °C, whereas cytoplasmic derived preparations showed biphasic unfolding profiles with lower first transitions at 51.62-55.31 °C and second transitions at 68.30-71.53 °C indicating a higher thermal stability and also folding uniformity for secreted anti-hCD45 NB. Finally, flow cytometry confirmed functional binding of all tested anti-hCD45 NB to CD45-positive Jurkat cells.

High-cell-density fed-batch cultivations of E. coli can reach > 2.5 g L^-1 production of functional anti-CD45 nanobodies when induction timing, production temperature, host strain genetics, and subcellular location of the final product are aligned. The highest yield, most uniform, and thermally stable anti-hCD45 NB preparation was obtained with signal-peptide-mediated periplasmic targeting.