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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-gamma(hIFN-gamma). The feeding rate was adjusted to achieve an appropriate specific growth rate. 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 rate feeding 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-gamma in the variable specific growth rate strategy were 0.35 g rHu-IFN-gamma g(-1) dry cell wt and 0.9 g rHu-IFN-gamma l(-1) h(-1), respectively. A new chromatographic purification procedure involving anion exchange and cation exchange chromatographies was developed for purification of rHu-IFN-gamma from inclusion bodies. The established purification process is reproducible and the total recovery of rHu-IFN-gamma was ~30% (100 mg rHu-IFN-gamma g(-1) dry cell wt). The purity of the rHu-IFN-gamma 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 final purified rHu-IFN-gamma has a specific antiviral activity of ~2 x 10(7) IU/mg protein, as determined by viral cytopathic effect assay. These results certify the product for clinical purposes.

Human interferon-gamma (hIFN-gamma) was expressed in Escherichia coli BL21(DE3) under the control of the T7 promoter. Glucose was used as the sole source of carbon and energy with simple exponential feeding rate in fed-batch process. Cell density of recombinant E. coli was reached to 100 g dry wt l(-1) under both constant (0.12 h(-1)) and variable (0.12-0.52 h(-1)) specific growth rates. In the variable specific growth rate fed-batch process, plasmid stability and specific yield of rhIFN-gamma were greater than constant specific growth rate fed-batch process. The final specific yield and overall productivity of rhIFN-gamma were 0.35 +/- 0.02 g rhIFN-gamma g(-1) dry cell wt and 0.9 +/- 0.05 g rhIFN-gamma l(-1) h(-1) in the variable specific growth rate fed-batch process, respectively.

Different feeding strategies for the production of human interferon-gamma using an isopropyl beta-D-thiogalactoside-inducible expression system in recombinant Escherichia coli BL21(DE3) (plasmid pET3a-ifngamma) were studied. Four fed-batch modes were designed to compare the effect of mu (specific growth rate) on recombinant-protein production, substrate consumption, by-product formation and plasmid stability during pre- and post-chemical induction in high-cell-density cultures of E. coli. It was found that Y(p/s), the product/substrate yield of interferon-gamma was significantly affected by mu throughout the process, but product/biomass yield (Y(p/x)) was influenced by mu at the pre-induction stage. By applying an efficient feeding strategy, in which the mu was maintained at the maximum attainable level, recombinant protein was accumulated up to a level of 60% of the total cell protein and its productivity was increased significantly. In this case, the overall productivities of biomass and recombinant protein were 6.36 g l(-1) h(-1) and 2.1 g l(-1) h(-1) respectively, in comparison with 1.91 g l(-1) h(-1) and 0.16 g l(-1) h(-1) during exponential feeding, in which the specific growth rate was kept constant throughout the entire process.

Development of inexpensive and simple culture media and appropriate induction conditions are always favorable for industry. In this research, chemical composition and stoichiometric data for gamma-interferon production and recombinant Escherichia coli growth were used in order to achieve a simple medium and favorable induction conditions. To achieve this goal, the effects of medium composition and induction conditions on the production of gamma-interferon were investigated in batch culture of E. coli BL21 (DE3) [pET3a-ifngamma]. These conditions were considered as suitable conditions for the production of gamma-interferon: 2.5x M9 medium, supplemented with a mixture of amino acids (milligram per liter), including glutamic acid 215, aspartic acid 250, lysine 160, and phenylalanine 90, and induction at late-log phase (OD(600) = 4.5). Under these conditions, dry cell weight of 6 +/- 0.2 g/l and gamma-interferon concentration of 2.15 +/- 0.1 g/l were obtained. Later, without changing the concentration ratio of amino acids and glucose, the effect of increase in the primary glucose concentration on productivity of gamma-interferon was investigated. It was found that 25 g/l glucose will result in maximum attainable biomass and recombinant human gamma-interferon. At improved conditions, a dry cell weight of 14 +/- 0.2 g/l, concentration and overall productivity of gamma-interferon 4.2 +/- 0.1 g/l and 420 +/- 10 mg/l h, respectively, were obtained.

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The fed-batch process using glucose as the sole source of carbon and energy with exponential feeding rate was carried out for high cell density cultivation of recombinant Escherichia coli BL21 (DE3) expressing human granulocyte-colony stimulating factor (hG-CSF). IPTG was used to induce the expression of hG-CSF at 48 g dry cell wt l(-1) during high cell density culture of recombinant E. coli BL21 (DE3) [pET23a-g-csf]. The final cell density, specific yield and overall productivity of hG-CSF were obtained as approximately 64 g dry cell wt l(-1), 223 mg hG-CSF g(-1) dry cell wt and 775 mg hG-CSF l(-1) h(-1), respectively. The resulting purification process used cell lysis, inclusion body (IB) preparation, refolding, DEAE and Butyl-Sepharose. Effects of different process conditions such as cell lysis and washing of IB were evaluated. The results reveal that the cells lyzed at 1,200 bar, 99.9% and Triton removed about 64% of the LPS but sarcosyl had no effect on removal of nucleic acids and LPS. Further analysis show that DEAE column removes DNA about 84%. Cupper concentration was identified as parameter that could have a significant impact on aggregation, as an unacceptable pharmaceutical form that decrease process yields. The purity of purified hG-CSF was more than 99%. Also the comparison of activity between purified hG-CSF and commercial form do not show valuable decrease in activity in purified form.

In order to increase the productivity of human IL-2 (interleukin-2), a stoichiometric model has been used to determine the most essential amino acids and precise values of their amounts to be added to the culture during expression of human IL-2 (as a model protein) by recombinant Escherichia coli BL21 (pET21a-hil2). Experiments were performed to investigate the effect of chosen amino acids and their interactions on expression of human IL-2. Glutamine, a mixture of leucine, aspartic acid and glycine, and a mixture of leucine, glutamine and aspartic acid, were the most effective for the expression of IL-2. The most promising amino acids were then chosen for further experiments at three different levels to determine whether altering their stoichiometry can lead to better expression levels. The optimized value of glutamine in the flask was 0.316 g/l; a mixture of leucine, glutamine and aspartic acid at concentrations of 0.124, 0.316 and 0.212 g/l respectively and of leucine, aspartic acid and glycine in concentrations of 0.124, 0.212, 0.111 g/l respectively were chosen to be added to the flask. The effect of glutamine, as one of the amino acids most influencing the expression of IL-2 in batch and fed-batch high-cell-density cultures, was studied. The results revealed that the amount of expressed IL-2 compared with the control culture increased from 81 to 195 mg/l in the shake flask, 403 to 594 mg/l in the fermentor and 5.15 to 10.01 g/l in the fermentor under fed-batch cultivation.