A defined medium and fed-batch feeding process for the production of a yeast biocatalyst, developed at the 23-L scale, was scaled up to the 600-L pilot scale. Presterilized 100-L-vol plastic bags were implemented for the pilot-scale nutrient feeding. Medium of increased concentration Oqs implemented at the pilot scale, and equivalent dry cell weights were reached with a medium 80% more concentrated than that used at the laboratory scale. The higher medium concentration was believed to be necessary at the pilot scale owing to the additional heat stresses on key components (e.g., complexing of magnesium sulfate with phosphate), increased dilution during sterilization, lower evaporation rate owing to the lower vessel volume per minute air flow rate, and increased dilution owing to nutrient feeding or shot additions. Peak cell density was found to be somewhat insensitive to variations in residual glucose levels. These results suggest that defined medium developed at the laboratory scale may need to be further optimized at the pilot scale for equivalent performance.