In this study, the fermentation of recombinant Pichia pastoris, which expresses heterogeneous human augmenter of liver regeneration (rhALR), has been optimized in a 5-L fermentor. The optimal methanol feeding method to optimize rhALR production was identified. Three methods of methanol feeding, methanol-stat, DO-stat and exponential feeding, were investigated to identify the method that would optimize rhALR production. It was determined that the methanol-stat method resulted in increased rhALR production, compared to the other two methods, which limit the amount of methanol available. Additionally, the results showed that rhALR expression levels could be enhanced by maintaining the pH at 6 and lowering the temperature from 30 to 25 degrees C. Supplementation with riboflavin during the induction phase also increased rhALR production. After these optimizations, rhALR was produced at a concentration of 424 mg/L, a three-fold improvement over original process. (c) 2010 Elsevier Inc. All rights reserved.