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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 ~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. [ABSTRACT FROM AUTHOR]

Abstract: Process development and optimization studies were performed in order to improve the purification process of (rhIFN-γ). The objective was to generate material with higher purity and quantity. An in-process control screening was developed to obtain the optimal condition for column chromatographic purification by measuring LPS, nucleic acids, rhIFN- γ, monomer and its covalent dimers. A new resin screening method was applied to select optimal resin for each of the chromatographic columns. The resulting process used Butyl and Q-Sepharose, refolding and SP-Sepharose for purification of IFN-γ. Effects of different process conditions such as cell lysis, removal of impurity and oxygen concentration were evaluated. Removal of impurities was evaluated by washing of inclusion bodies with 1% Triton X-100 and 3M urea and different chromatography steps. The results reveal that Triton removed about 43% of the LPS but urea had no effect on removal of nucleic acids and LPS. Further analysis show that removal of impurities by column chromatography decreases aggregation and increases the process yield. Oxygen concentration was identified as parameter that could have a significant impact on covalent dimers formation, as an unacceptable pharmaceutical form of rhIFN-γ. On the basis of small-scale studies, optimum operating conditions were chosen and the purification process was successfully scaled-up to a pilot scale process with step yield and product quality that were better than previous reports. [Copyright &y& Elsevier]