Round 1: DOE setup: 3 factors (seeding density, temperature, total feeding) & 2 levels. Factor B "temperature" revealed to be the only significant factor
Round 2: Exploring optimal temperature control strategies for shake flasks. Under optimal condition, the expression level reaches 11.21g/L, exceeding the desired target titer (10g/L)
Round 3: Assess reproducibility of the shake flask process. Scale-up & process confirmation in 3L bioreactors