A fed-batch fermentation process was developed to culture recombinant Pichia pastoris at high cell density and with high protein expression. High levels of the thrombomodulin fragment were obtained from an SMD1168 strain (pep4(-)) that had a methanol utilization slow (mut(s)) phenotype. Dissolved oxygen concentration (DO) was controlled by cascading DO with agitation and pure oxygen supplementation, thereby avoiding oxygen limitation during the entire process. Wet cell density reached 420 g/litre and the concentration of protein, an 86 amino acid thrombomodulin fragment, was 360 mg/litre.