ORIGINAL PAPER Theppanya Charoenrat Æ Mariena Ketudat-Cairns Helle Stendahl-Andersen Æ Mehmedalija Jahic Sven-Olof Enfors Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes Received: 8 March 2005 / Accepted: 20 May 2005 / Published online: 4 August 2005 Ó Springer-Verlag 2005 Abstract An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood b-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q O /q O,max ), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance de- mand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90–95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing. Keywords Oxygen-limited fed batch (OLFB) Æ Methanol-limited fed batch (MLFB) Æ Pichia pastoris Æ b-glucosidase List of symbols AOX Enzyme alcohol oxidase AOX1 Alcohol oxidase gene 1 CPR Carbon dioxide production rate (mol h 1 ) DOT Dissolved oxygen tension (%) MLFB Methanol limited fed-batch OLFB Oxygen limited fed-batch OUR Oxygen uptake rate (mol h 1 ) PI Propidium iodide q o Specific oxygen uptake rate (mol g cell 1 h 1 ) q o,max Maximum specific oxygen uptake rate (mol g cell 1 h 1 ) q p Specific b-glucosidase productivity (U g cell 1 h 1 ) Q i Inlet air flow rate (L h 1 ) Q o Outlet air flow rate (L h 1 ) RRR Relative rate of respiration V Medium volume (L) V m Molar volume of gas (L mol 1 ) X Biomass concentration from dry weight (g L 1 ) Y C CO2/S Carbon yield coefficient of carbon dioxide from methanol (mol mol 1 ) Y C X/S Carbon yield coefficient of biomass from methanol (mol mol 1 ) Introduction Pichia pastoris is a methylotrophic yeast that is often genetically engineered to express proteins [1]. It is suited for foreign protein expression for three main reasons: it can be easily manipulated at the molecular genetic level; it can express and secrete proteins at high levels; and it can perform many of the ‘higher eukaryotic’ protein modifications such as glycosylation, disulfide-bond for- mation and proteolytic processing [2]. Pichia pastoris can be grown to very high cell densi- ties (more than 130 g dry cell weight L 1 )[3, 4]. It also contains a tightly methanol-controlled alcohol oxidase (AOX1) promoter that is induced by methanol and can be used to drive expression of foreign genes [5]. The strong promoter, coupled with the high cell-density fermentation, has allowed production of recombinant product at very high levels. Product concentrations can T. Charoenrat Æ H. Stendahl-Andersen Æ M. Jahic S.-O. Enfors (&) School of Biotechnology, Royal Institute of Technology, AlbaNova University Centre, Roslagstullsbacken 21, 106 91, Stockholm, Sweden E-mail: enfors@biotech.kth.se Tel.: +46-8-55378302 Fax: +46-8-55378323 M. Ketudat-Cairns Æ T. Charoenrat School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand Bioprocess Biosyst Eng (2005) 27: 399–406 DOI 10.1007/s00449-005-0005-4