RSM-based optimization and scale-up production of hIL-2 in Escherichia coli

Abstract

Human interleukin 2 (hIL-2) has become a pivotal molecule widely used in the treatment of various diseases. The development of a robust and highly productive high cell density cultivation process for producing hIL-2 from inclusion bodies represents a promising and attractive strategy for its commercial production. In this study, a 12-bioreactor 250 mL DASbox mini bioreactor system was employed to develop a model using Response Surface Methodology (RSM) aimed at understanding the impacts of cultivation parameters on cell growth and product yiled as well as maximizing hIL-2 production. The optimized process was further validated in a 5-L stirred tank bioreactor: an induction temperature of 31.3°C, an isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration of 1.0 mM, a post-induction feeding rate of 103.80 mL/h, and an induction OD₆₀₀ of 150.0. The maximum hIL-2 titer achieved 4.90 g/L, which correlated well with the predicted range. Subsequent solubilization and in vitro refolding process resulted in hIL-2 purity exceeding 99 %, and a the bioactivity of 0.88 × 10⁷ U/mL, comparable to that of commercial products. This study demonstrates the effectiveness of optimizing cultivation parameters to enhance hIL-2 yield and highlights the value of employing statistical methods as a powerful tool to guide manufacturing operations in recombinant protein production.