Medicina

J Ind Microbiol Biotechnol (2004) 31: 63–69 DOI 10.1007/s10295-004-0117-x

O R I GI N A L P A P E R

R. Khalilzadeh Æ S. A. Shojaosadati Æ N. Maghsoudi J. Mohammadian-Mosaabadi Æ M. R. Mohammadi A. Bahrami Æ N. Maleksabet Æ M. A. Nassiri-Khalilli M. Ebrahimi Æ H. Naderimanesh

Process development for production of recombinant human interferon-c expressed in Escherichia coli
Received: 12September 2003 / Accepted: 22 December 2003 / Published online: 19 February 2004 Ó Society for Industrial Microbiology 2004

Abstract A simple fed-batch process was carried out using constant and variable specific growth rates for high-cell-density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-c(hIFN-c). The feeding rate was adjusted to achieve an appropriate specific growthrate. The dissolved oxygen level was maintained at 20–30% of air saturation by control of airflow and stirrer speed and, where necessary, by enrichment of inlet air with pure oxygen. Glucose was the sole source of carbon and energy and was provided by following a simple exponential feeding rate. The final cell density in the fed-batch fermentation with constant and variable specific growth ratefeeding strategies was $100 g dry cell wt l)1 after 36 and 20 h, respectively. The final specific yield and overall productivity of recombinant hIFN-c in the variable specific growth rate strategy were 0.35 g rHu-IFN-c g)1 dry cell wt and 0.9 g rHu-IFN-c l)1 h)1, respectively. A new chromatographic purification procedure involving anion exchange and cation exchange chromatographies was developed forpurification of rHu-IFN-c from inclusion bodies. The established purification process is reproducible and the total recovery of rHu-IFN-c was $30% (100 mg rHu-

IFN-c g)1 dry cell wt). The purity of the rHu-IFN-c was determined using HPLC. Sterility, pyrogenicity, and DNA content tests were conducted to assure the absence of toxic materials and other components of E. coli in the final product. The finalpurified rHu-IFN-c has a specific antiviral activity of $2·107 IU/mg protein, as determined by viral cytopathic effect assay. These results certify the product for clinical purposes. Keywords Fed-batch fermentation Æ High-cell-density cultivation Æ Purification Æ Recombinant human interferon-c Æ Recombinant Escherichia coli

Introduction
Interferon (IFN) was discovered in 1957 as a biological agentinterfering with virus replication [7]. IFN-c is secreted by lymphocytes stimulated by mitogen and is involved in the differentiation, maturation, and proliferation of hematopoietic cells. It also enhances nonspecific immunity to tumors, as well as to microbial, viral, and parasitic organisms [12, 24]. Natural human interferon-c(hIFN-c) is composed of 143 amino acid residues with a total molecularmass of 20–25 kDa. It is glycosylated and does not contain cysteine residues [6, 20]. In 1986, hIFN-c cDNA was successfully cloned and expressed in Escherichia coli, which made possible the production of recombinant hIFN-c (rHu-IFN-c) in relatively large amounts [34]. rHu-IFN-c produced in E. coli is not glycosylated and has methionine as its N-terminal residue instead of pyroglutamic acid. Thetotal molecular mass of rHu-IFN-c is less (17 kDa) than that of hIFN-c, but nonetheless it is physiologically active. Clinical trials indicate that rHu-IFN-c has therapeutic efficacy on kidney cell carcinoma, colon cancer, and rheumatoid arthritis [34]. E. coli is the most commonly used host for heterologous protein production [10, 19, 33]. Using expression vectors in batch and fed-batch cultivations,R. Khalilzadeh Æ S. A. Shojaosadati (&) Æ N. Maghsoudi A. Bahrami Biotechnology Group, Department of Chemical Engineering, Tarbiat Modarres University, Faculty of Engineering, P.O. Box 14155-4838, Tehran, Iran E-mail: shoja_sa@modares.ac.ir Tel.: +9821-8005040 Fax: +9821-8006544 J. Mohammadian-Mosaabadi Æ M. A. Nassiri-Khalilli M. Ebrahimi Æ H. Naderimanesh Biochemistry Group, Tarbiat...