Abstract:The production of biopharmaceutical in lab scale differs from in pilot plant-scale particularly in process control or conditions control aspects. For larger volume of production, there is a chance of having deviation on controlling the operating conditions that is likely to have effects on specifications of biopharmaceuticals. Thus, this work focused on the optimization and robustness study of growth conditions on microbial fermentation using design of experiment methodology (DoE) and statistical analysis approach. Three factors for designing experiment were temperature, pH and % dissolved oxygen (DO). A high-throughput MRT-24 microbioreactor and Pichia pastoris KM71H which expresses Japanese Encephalitis virus envelope protein were used as a model study and the measured output was OD600. After 17 hours of fermentation, the data was analyzed and the conditions where temperature was 28.0 °C, pH was 6.37 and DO was 30% were calculated to be the optimized value. Then the robustness study was carried out in which 5 % loss of the highest yield was set as a criterion. The results predicted that the robust range was temperature of 28:Cto 29.5°C and pH of 6.0 to 6.5.