Biphasic cultivation strategy to avoid Epo-Fc aggregation and optimize protein expression.

•DoE was used to optimize a biphasic bioprocess for an aggregation-prone Epo-Fc fusion protein.•A synergistic effect of cultivation temperature and pH on product aggregation was found.•Aggregation was reduced from 75% to levels below 1%.•Optimized conditions lowered cell metabolism, nutrient consumption and by-product accumulation.•Optimized conditions increased cell-specific production rates resulting in a more economic bioprocess.