Rapid development of a robust cultivation process for subunit rotavirus vaccine antigen expression in Escherichia coli with the quality by design approach.

Rotaviruses are the leading cause of severe diarrhea and mortality in children less than 5 years old. Vaccination is considered to be the most effective strategy to prevent rotavirus infection. Two live attenuated oral vaccines have been licensed in many countries worldwide, resulting in significant reductions in rotavirus-associated mortality and hospitalizations. However, access to these vaccines remains limited in low- and middle-income countries, partly due to their high cost of current vaccines. The spike protein VP4 of rotavirus is the main antigen responsible for inducing neutralizing antibodies. Therefore, VP4 protein could provide the antigen part of a new rotavirus subunit vaccine candidate. In this study, we developed a robust cultivation process for VP4 protein expression 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 OD