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Process development and optimization studies were performed in order to improve the purification process of (rhIFN-gamma). 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- gamma, 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-gamma. 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-gamma. 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.

To reduce the degradation pathway of rhIFN-gamma (recombinant human interferon-gamma), the susceptibility against oxidative stress during fermentation in Escherichia coli cells and in purification process were investigated. Fermentations of recombinant E. coli were performed at 5, 30 and 60% DO (dissolved oxygen) concentrations. The expressed rhIFN-gamma is purified in three-step chromatography processes with 99.9% purity under reducing and non-reducing conditions. Recombinant IFN-gamma production, carbonyl and dimeric contents and antiviral-specific activity of purified protein were monitored. The increase in carbonyl content was taken to be an indicator of protein oxidation. DO concentration did not show any noticeable effect on rhIFN-gamma production. During fermentation, the carbonyl-group content showed no significant increase at 5 and 30% DO but a 10-fold increase at 60% DO concentration was observed. The antiviral-specific activity of purified rhIFN-gamma was decreased 35 and 69% at 30 and 60% DO concentrations respectively. After the purification process, under non-reducing conditions, the activity of purified protein decreased to 30% of its original value. The degree of dimeric forms of protein was found to depend on the O2 concentration during fermentation and purification.