Process development for production of recombinant human interferon-? expressed in Escherichia coli

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-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.

Increased yield of high purity recombinant human interferon-γ utilizing reversed phase column chromatography

“…Purified product yields from the published procedures previously discussed ranged from 0.3-14 mg per gram cell mass. Interestingly, one study [20] reported a batch process yield of 100 mg per gram of dry cell mass. Since E. coli is from 70-80% water, this still converts to a value less than what we obtained per gram of wet cell mass (although an exact conversion cannot be calculated).…”

“…In the food and animal industries, IFN-γ is effective against a variety of economically important animal viral diseases [16,17]. Hence, the commercial interests in efficiently optimizing the production yields and specific activities of IFN-γ preparations are tremendous [18][19][20][21][22].…”

High Yield Production of Heterologous Proteins with Escherichia coli

“…It is desirable to make the feed-solution as simple as possible by including sufficient non-carbon and non-nitrogen nutrients in the starting medium. One of the essential requirements during fedbatch operation is to supply nutrients to promote cell growth 18,40 . To limit their toxicity to the growing cells nutrients such as glucose, ammonia, salt are fed approximating their requirement.…”

“…Fed-batch processes have most often been used to obtain high cell density [11][12][13][14][15] . Cell concentrations of about ~100 g dry cell weight per litre can be yielded by fed-batch culture of both non-recombinant and recombinant E. coli [16][17][18] . However, this technique has several drawbacks, including substrate inhibition, limited oxygen transfer capacity, the formation of growth inhibitory by-products, and limited heat dissipation.…”

“…Several feeding strategies have been used for increasing cell concentrations in recombinant cultures, such as constant feed, step-wise increase in feed, feeding based on a feedback control technique so as to keep the dissolved oxygen or pH constant and exponential feed 18,[19][20][21][22][23] . Exponential feeding is widely used so that the cells can be grown at the desired specific growth rate, preventing the formation of toxic byproducts like acetate [24][25][26][27] .…”