Available online at www.sciencedirect.com Enzyme and Microbial Technology 42 (2008) 173–180 High level production and purification of human interferon 2b in high cell density culture of Pichia pastoris Atef Ayed a , Imen Rabhi a,b , Koussay Dellagi b , H´ ela Kallel a,∗ a Unit´ e de Biofermentation, Institut Pasteur de Tunis, 13, place Pasteur BP 74, 1002 Belv´ ed` ere, Tunis, Tunisia b Laboratoire d’Immunopathologie, de Vaccinologie et de G´ en´ etique Mol´ eculaire, Institut Pasteur de Tunis, Tunis, Tunisia Received 16 May 2007; received in revised form 30 August 2007; accepted 5 September 2007 Abstract Human interferon 2b gene was cloned in the methylotrophic yeast Pichia pastoris under the control of the AOX1 methanol inducible promoter. To optimise the volumetric productivity, we performed different fed-batch studies in a 5-L bioreactor. We demonstrated that hIFN2b was highly sensitive to proteases activity during high cell density culture. The target protein was totally degraded 20h after the start of methanol feeding. Replacement of culture medium with fresh medium after glycerol fed-batch culture mode as well as medium enrichment with casamino acids at 0.1% and EDTA at 10 mM, had significantly improved hIFN2b expression and prevented its proteolysis. Moreover, to further improve hIFN2b production, three different methanol fed-batch strategies had been assayed in high cell density culture. The optimal strategy resulted in a production level of 600 mg/l while residual methanol level was maintained below 2 g/l. Clarification of culture supernatant through a 0.1 m hollow fiber cartridge showed that almost 95% of the target protein was retained within the retentate. Triton X-100 or NaCl addition to the culture harvest before microfiltration had improved the recovery yield of this step. rhIFN2b was further purified by cation exchange on Sepharose SP resin followed by gel permeation on Sephacryl S-100. The overall yield of the process was equal to 30% (180 mg/l). The biological activity of the purified protein based on the antiviral activity test was 1.5 × 10 8 IU/mg. The optimised process has a great potential for large scale production of fully functional hIFN2b. © 2007 Elsevier Inc. All rights reserved. Keywords: Human interferon 2b; Pichia pastoris; High cell density culture; Purification process; Antiviral activity 1. Introduction Interferons (IFNs) are the first line of defence components of vertebrates that act against infectious agents and tumour devel- opment and progression. Recombinant interferon  has been used for almost two decades, as a therapeutic agent in a variety of diseases including viral infections and cancer. Recombinant human interferons have been expressed in Escherichia coli [1–3], yeast [4,5], baculovirus infected insect cells [6,7] and animal cells [8]. The yield of recombinant INFs using E. coli, is by far higher than that obtained using other systems. However, IFN protein expressed in large amount in E. coli often precipitates into insoluble, misfolded inclu- sion bodies that require subsequent solubilization and refolding steps [9,10]. Moreover, refolding from inclusion bodies presents ∗ Corresponding author. Tel.: +216 71 783 022; fax: +216 71 791 833. E-mail address: hela.kallel@pasteur.rns.tn (H. Kallel). several drawbacks such as the requirement for optimising the refolding conditions for each target protein. In addition, the res- olubilization procedures could affect the integrity of refolded proteins [11]. To overcome the shortcomings of E. coli expres- sion system, expression of human interferon alpha in Pichia pastoris was recently described [12–14]. P. pastoris is a widely used host for the production of het- erologous proteins. Some proteins are produced at gram per liter levels [15–17]. Complex proteins such as human glycoproteins or human collagens, are also successfully processed and secreted by P. pastoris [18–20]. Furthermore, this expression system presents many advantages [17,21–22]. The organism grows on defined media to high cell density on either glycerol or methanol as the sole carbon source and heterologous protein production is under the control of a strong but tightly regulated alcohol oxidase (AOX) promoter induced by methanol. P. pastoris can be grown to the desired cell density on glycerol as the carbon source and then on methanol for high level heterologous production [23]. In Pichia cells fed with methanol at growth limiting rates in biore- 0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2007.09.006