Optimization of toxins and bacteria inactivation process in industrial-scale production of Clostridium perfringens bacterin-toxoid vaccine

Production of anaerobic bacterin-toxoid vaccines relies on the complete inactivation of toxins and bacteria cells. This study focuses on optimizing the inactivation process of Clostridium perfringens type D at an industrial scale. A combined modeling, simulation, and experimental method was used to examine how operational parameters affect the inactivation efficiency. Initially, using the constant mixing time method, scale-down experiments in a 20-L bioreactor simulated industrial conditions, and then various mixing speeds (0, 150, 300 rpm), formalin concentrations (0.3 %, 0.5 %, 0.7 %), and temperatures (room temperature and 37 °C) were tested. Based on scale-up relationships, optimal operational conditions were identified for a 1500-L bioreactor. Experiments in the 1500-L bioreactor were carried out under the following conditions: (I) 80 rpm, 0.5 % formalin, and 37 °C; (II) 40 rpm, 0.5 % formalin, and 37 °C; and (III) 20 rpm, 0.7 % formalin, and 37 °C. Throughout these experiments, the levels of free formaldehyde, pH, post-inactivation viability test, and residual toxicity of the products were regularly assessed. Results indicated that the optimal inactivation conditions in 20-L scale were 0.5 % formalin with a 300 rpm mixing rate for 6 days at 37 °C. Using the scale-up relationships, the optimal mixing speed in the 1500-L bioreactor was determined to be 80 rpm. Computational fluid dynamics (CFD) simulations of the 1500-L bioreactor confirmed that 80 rpm provided effective distribution of the culture medium (including toxins), bacteria, and formaldehyde. Post-inactivation viability test and toxicity assessments showed that the fast inactivation process in 1500-L scale can occur under condition (I) in 7 days, yielding a stable, irreversible product maintained for two months at 4 °C. This work provides critical insights for the industrial-scale inactivation of C. perfringens type D toxins and cells, with implications that optimized inactivation strategies can be adapted to a wide range of microbial vaccines and bioprocesses to improve throughput, consistency, and product quality. • Industrial inactivation process for bacterin-toxoid vaccine production was studied. • 0.5 % formalin, 300 rpm, and 37 °C for 6 days were effective in a 20-L bioreactor. • Corresponding optimal mixing speed for a 1500-L bioreactor was 80 rpm. • CFD confirmed effective distribution of formalin at 80 rpm in 1500-L bioreactor. • Inactivation in 1500-L scale occurred in 7 days, leading to an irreversible product.