大腸菌における品質設計アプローチを用いたサブユニットロタウイルスワクチン抗原発現のための堅牢な培養プロセスの迅速な開発

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 OD₆₀₀ for induction (ODI), induction temperature (ITmp), the final concentration of IPTG (Con), and speed of feed addition (SOFA) were explored based on a 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 vertexes 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 antigen expression process in Escherichia coli.