BACKGROUND The effects of alternative co-substrate feeding strategies on recombinant human erythropoietin (rHuEPO) production by Pichia pastoris-Mut(+) strain were investigated. RESULTS Five different production strategies were designed and performed in the production phase of the bioprocesses with the use of either mannitol or sorbitol as the co-substrate. The highest rHuEPO production was achieved as C-rHuEPO= 0.65 g L-1 at t=9 h, with the cell concentration C-x=55 g L-1 in the production phase; wherein methanol was fed to the bioreactor with a predetermined dynamic feeding rate calculated for constant mu(M0)=0.03 h(-1); and three consecutive pulses of the co-substrate mannitol were introduced at t=0, 6, and 12 h, so that C-Man=50 g L-1. The overall cell and product yields on the substrates methanol and mannitol were found, respectively, as Y-X/St=0.22 g g(-1) and Y-rHuEPO/St=3.74 mg g(-1). CONCLUSIONS The use of mannitol as the co-substrate enhanced rHuEPO production and furthermore shortened the bioprocess time. The design of semi-batch feeding strategy, based on the selection of the substrates and the co-substrates, and their dynamic feeding into the bioreactor, is important to increase the production and productivity in r-protein production by Mut(+) strains of P. pastoris, and the developed strategy would be especially important for therapeutic glycoprotein productions by P. pastoris. (c) 2013 Society of Chemical Industry