Rapid development of a robust bioprocess for subunit rotavirus vaccine production in Escherichia coli with the Quality by Design approach

Rotavirus are leading cause of severe diarrhea and mortality in children less than 5 years old. Vaccination is considered to be the most effective strategy for preventing rotavirus infection. Two live attenuated oral vaccines are licensed in many countries worldwide, with significant reductions in rotavirus-associated mortality and hospitalizations. However, the effectiveness of those vaccines is lower in low- and middle-income countries partly due to the high cost of current vaccines. The spike protein VP4 of rotavirus is the main antigen for inducing neutralizing antibodies and emerges as a promising candidate for cost-effective subunit vaccine against rotaviruses. In this study, we developed a robust bioprocess for VP4 protein production in Escherichia coli with yield higher than 620 mg/L and compliant with the Quality by Design approach. First, the process parameters with potential significant effect on VP4 protein yield were identify based on our experience in virus-like particle vaccine production and screened with a Fractional Factorial Design approach in 1-L parallel bioreactor system. Then, the robust setpoint and design space of the time of induction (TOI), induction temperature (ITmp), the final concentration of IPTG (Con), and speed of feed addition (SOFA) were explored based on a the Central Composite Design approach and criteria of VP4 protein yield > 500 mg/L and probability of failure < 3%. With process parameters set at the robust setpoint, the VP4 protein yield of 685 mg/L was obtained in 1-L bioreactor. Furthermore, the VP4 protein yields with process parameters at the robust setpoint and design space vertexs were higher than 620 mg/L and within the interval of model prediction. This study may serve as a reference for development of a robust and cost-effective subunit rotavirus vaccine production process in Escherichia coli .