BACKGROUND: The influence of methanol feed rate on recombinant human growth hormone (rhGH) production by Pichia pastoris hGH-Mut(+) in medium containing sorbitol was investigated at three different specific growth rates (mu), namely, 0.02 (MS-0.02), 0.03 (MS-0.03), and 0.04 (MS-0.04). RESULTS: Increasing methanol feed rate above MS-0.03 did not affect sorbitol consumption, showing that mu = 0.03 h(-1) is a threshold limiting value, above which sorbitol utilization became independent of methanol feed rate. Moreover, when sorbitol was consumed, no further cell growth was observed. Increase in methanol feed rate triggered cell synthesis and the highest cell concentration was obtained at MS-0.04 as 48 g L-1 (t = 18 h); whereas, the highest rhGH production, 270 mg L-1, was obtained at MS-0.03 as a consequence of lower extracellular protease production and higher AOX activity (41 U g(-1) CDW). Oxygen uptake rate increased with increasing mu, having the maximum value, 76.6 mmol m(-3) s(-1), at MS-0.04. K(L)a had a tendency to increase with mu, having a maximum value of 0.15 s(-1) at MS-0.04 (t = 15 h). CONCLUSION: By considering rhGH concentration and oxygen transfer characteristics, the bioprocess can be improved by a two-stage feeding strategy, operating at MS-0.04 at the beginning of fermentation, and thereafter shifting to MS-0.03. (c) 2009 Society of Chemical Industry