Scale-up problems of processes have been known in all producing sectors and technologies. However biotechnology with the extraordinary complexity of working with living organisms puts itself in an exceptionally challenging position. Scale-up issues can become very time consuming and costly since it delays progress to full-scale production and can even lead to a stop of development. This thesis focused on scaling-up a P. pastoris fermentation from a 5L lab scale fermenter to a 60L pilot plant fermenter. Prior to this, CFD simulations of the 60L fermenter with different fermenter setups were performed. The result was an identification of the most suitable setting of agitator and feed position as well as agitation to ensure homogeneity in the reactors liquid phase. These calculations were verified by several E. coli fermentations with the output that the simulated highest homogeneity results in the highest substrate to biomass yield. A scale-up of a P. pastoris fermentation in lab scale reactor, whose liquid phase is considered to be highly homogeneous, was chosen to consolidate the findings for parameters by the CFD simulations. The process started with a batch phase and continued with a fed batch phase, both with glycerol as the C-source to gain biomass. A pulse with methanol was conducted afterwards so the organism adapted to the new C-source methanol and changed its metabolic state, in which P. pastoris metabolized methanol to biomass as well as the product horseradish peroxidase (HRP), an extensively used enzyme in biotechnological and medical applications. After the complete consumption of methanol, a fed batch phase followed with a step-wise increase of cell specific substrate uptake rate. An intended accumulation of methanol identified the maximum cell specific uptake rate of methanol of 0.12 gS/(gX*h). The fermentation process was successfully executed in both fermenters. Metabolic rates and quotients were calculated to quantify the comparability of the fermentation in different scales. Calculated values for all other phases were equivalent and do not differ significantly. For example, for the batch phase, the cell specific growth rate was found to be 0.23 h-1 in 5L bioreactor and 0.21 h-1 in the 60L fermenter, substrate to biomass yield 0.72 gS/gX (5L) and 0.63 gS/gX (60L). Those values resulted in a cell specific substrate uptake rate of 0.32 gS/(gX*h) in the small scale and 0.33 gS/(gX*h) in the pilot plant scale.