Production, purification, and chemical stability of recombinant human interferon-γ in low oxygen tension condition: a formulation approach.

The low stability of recombinant human interferon-γ (rhIFN-γ) therapeutic protein imposes some restrictions in its medical applications. In the current study, the effect of oxygen tension on the stability of purified rhIFN-γ was investigated. The rhIFN-γ was purified (>99%) by a two-step chromatographic process. Storage vials were filled by purified formulated product under normal atmospheric oxygen and low oxygen tension conditions. At different time intervals, the amounts of rhIFN-γ covalent dimers and deamidated forms were analyzed using analytical high-performance liquid chromatography (HPLC; size exclusion and cation exchange) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods. To determine the biological activity of purified rhIFN-γ, an antiviral activity assay against vesicular stomatitis virus (VSV) was performed. Upon rhIFN-γ long-term storage in a low oxygen tension condition, the amounts of rhIFN-γ covalent dimers and deamidated forms and also the biological activity of rhIFN-γ changed a little. In contrast, by 9 months of storage of rhIFN-γ preparations under normal atmospheric condition, the amount of covalent dimers and deamidated forms increased with time and reached to approximately 3.5% and 11.5% of the initial amount, respectively. The antiviral specific activity of 9-month-old rhIFN-γ preparations decreased to 41% of the initial amount at normal storage condition, while no significant reduction was seen at the low oxygen tension condition. In conclusion, oxygen tension during storage could have a significant impact on rhIFN-γ stability and finally on the quality of pharmaceutical rhIFN-γ product.

A novel amino acid supplementation strategy based on a stoichiometric model to enhance human IL-2 (interleukin-2) expression in high-cell-density Escherichia coli cultures.

A novel amino acid supplementation strategy was developed for enhancing the production of IL-2 (interleukin-2; as a model protein) by recombinant Escherichia coli BL21 (pET21a-hil2) in fed-batch high-cell-density cultures. The amino acids most needed and their amounts were determined using a stoichiometric model, and full factorial design experiments were conducted to determine the effects of single amino acids and amino acid mixtures on production. One of the most effective amino acid mixtures was found to be leucine, aspartic acid and glycine. This amino acid mixture was utilized for the production of IL-2 in batch and fed-batch fermentations. The amount of IL-2 produced increased from 403 to 722 mg/l and from 5.15 × 10³ to 8.08 × 10³ mg/l in batch and fed-batch cultures respectively. The results also revealed that the above amino acid mixture specifically increases IL-2 concentration in the cells.

Prevention of human granulocyte colony-stimulating factor protein aggregation in recombinant Pichia pastoris fed-batch fermentation using additives.

rhG-CSF (recombinant human granulocyte colony-stimulating factor) was expressed in the yeast Pichia pastoris under the control of the AOX1 (alcohol oxidase 1) promoter. The production of rhG-CSF was induced by switching from growth on glycerol to growth on methanol. In the induction phase, the methanol feed rate had a significant effect on the specific expression rate of rhG-CSF. A constant feed rate of 16 ml.h(-1).l(-1) was found to be optimal for a high specific expression rate of rhG-CSF (0.058 mg(-1).h(-1).g DCW(-1); DCW is dry cell weight). Under this condition, a maximum concentration of 300 mg/l of rhG-CSF and the expression yield of 0.6 mg of rhG-CSF/g of methanol were attained. However, the secreted rhG-CSF was shown to exist as aggregates in the culture broth, owing to hydrophobic interactions. To prevent undesirable protein aggregation, the presence of additional additives in the P. pastoris culture medium was investigated. Among seven additives tested, Tween 20, Tween 80 and betaine exhibited the best results in respect of preventing the formation of rhG-CSF protein aggregates.

A proposed feeding strategy for the overproduction of recombinant proteins in Escherichia coli.

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.

Efficient harvesting of marine Chlorella vulgaris microalgae utilizing cationic starch nanoparticles by response surface methodology.

Harvesting involves nearly thirty percent of total production cost of microalgae that needs to be done efficiently. Utilizing inexpensive and highly available biopolymer-based flocculants can be a solution for reducing the harvest costs. Herein, flocculation process of Chlorella vulgaris microalgae using cationic starch nanoparticles (CSNPs) was evaluated and optimized through the response surface methodology (RSM). pH, microalgae and CSNPs concentrations were considered as the main independent variables. Under the optimum conditions of microalgae concentration 0.75gdry weight/L, CSNPs concentration 7.1mgdry weight/L and pH 11.8, the maximum flocculation efficiency (90%) achieved. Twenty percent increase in flocculation efficiency observed with the use of CSNPs instead of the non-particulate starch which can be due to the more electrostatic interactions between the cationic nanoparticles and the microalgae. Therefore, the synthesized CSNPs can be employed as a convenient and economical flocculants for efficient harvest of Chlorella vulgaris microalgae at large scale.

Enhancement of human gamma-interferon production in recombinant E. coli using batch cultivation.

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.

Process development for production of human granulocyte-colony stimulating factor by high cell density cultivation of recombinant Escherichia coli.

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.

Effect of oxidative stress on the production of recombinant human interferon-gamma in Escherichia coli.

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.