Tailoring high-cell-density fed-batch fermentation strategies to produce Russell's viper venom-specific single-domain...

Attributes of camelid-derived single-domain antibodies (sdAbs), such as enhanced antigen specificity, improved solubility and stability, efficient tissue penetration, and superior biodistribution, make them preferable therapeutics for diverse applications, including the treatment of cancers, neurological disorders, infectious diseases, and intoxication. Limited availability and high cost of antivenoms necessitate the discovery of alternative neutralizers. This investigation presents a proof of concept for developing a cost-effective Escherichia coli-based platform to produce high yields of an sdAb specific to Russell's viper venom, denoted as 'FRV'. The gene encoding the FRV protein was cloned into the pET28a vector, and expression was evaluated in shake flasks using both complex and chemically defined media at various temperatures, achieving a maximum concentration of ~ 190 mg/L and a specific product yield (Y_P/X) of 38 mg/g DCW. Solubility assessments of FRV sdAb indicated the formation of inclusion bodies (IBs) under all tested conditions. Multiple strategies were employed to simplify and enhance IB solubilization, refolding, and Ni-NTA purification, yielding ~ 85% recovery and ~ 90% purity. Additionally, FRV immunoreactivity and neutralization activity were further validated by immunoblotting, ELISA, and in vitro phospholipase inhibition assay. FRV sdAb expression was subsequently scaled up via fed-batch fermentation in 1 L and 2.5 L cultures using different optimization strategies. Systematic evaluation of cultivation parameters, including induction OD₆₀₀, post-induction temperature, feed composition, and antibiotic levels, yielded a maximum FRV concentration of ~ 3.8 g/L and a Y_P/X of 59 mg/g DCW. Among the fermentation strategies evaluated, induction temperature was identified as a key factor influencing product yields and plasmid stability.