In previous studies for shake flask cultures, the commonly used protocol recommended by Invitrogen [10] for methanol feeding has been modified individually depending on the product. For the expression of proteins in shake flask cultures it is proposed that methanol is added twice per day [11, 12] . However, due to the small window in which the methanol concentration must be kept for an optimal process, pulse-based methanol addition for optimization of processes in shake flasks seems not to be the best way to provide data for the development of a fermentation process.