Optimization Of Production Technology Of Recombinant Human Granulocyte Colony-stimulating Factor(rhG-CSF)

The thesis consists of six chapters. High-density fermentation and its scaling-up for producing recombinant human granulocyte colony-stimulating factor (rhG-CSF) were systematically studied, which includes the studies on the potimigations of the culture media and culture conditions in both shake flask and 5L fermenter methods, respectively. The highest yield of rhG-CSF was 439mg/L. Furthermore, the conditions for the purification of rhG-CSF were also investigated. With only one step of high performance hydrophobic interaction chromatography (HPHIC), the purity of rhG-CSF was from 53% to 97.8% and the mass recovery was 24.0%.1. The optimization of the culture media of recombinant E.coli (DH5cc/pBV220)The culture media of fermentation for producing recombinant E.coli DH5a/pBV220 was investigated with shake culture method. The M9 mediums elected from several kinds of basic culture media was finally used. With single factor experiment, the substances providing carbon and nitrogen sources were screened. In addition, the optimized culture medium marked as M9III was obtained by orthogonal method.2. The fermentation of recombinant bacteria in shake cultureThe process conditions, such as incubating temperature, initial pH of culturing broth, inducing time, inoculum age, inoculum volume and so on were optimized by shake flask method. The experimental results showed that the optimum conditions are : temperature 30癈, initial pH 7.0 -7.2, approximate rotating speed 220r/min, inoculum age was 1.5 (ODeoo)- It was suitable for inducing cultivation when the growth of bacteria was in the initial logarithm growth phase and expressing 4 hours. Under these culture conditions, the expression and weight of dry cell yield of the rhG-CSF by theshake flask method were 36.5% and 4.45g/L, respectively, when the M9III mediumwas used.3.The batch culture of recombinant E.coli (DH5a/pBV220) in 5L fermenterBased on the optimum results of shake culture method, the effects of dissolve oxygen, inducing time, inoculum volume etc. on the batch culture and fed-batch culture of engineering bacteria in 5L fermenter were studied.Firstly, the processing conditions of batch culture of recombinant bacteria in 5L fermenter, including whether adding trace element and vitamin to culture media and so on were optimized. The yield of 6.61g/L of dry cell weight (DCW) could be obtained, and the expression of recombinant rhG-CSF was 38.1% under these optimal conditions.Secondly, the stable fermentation parameters of fed-batch culture of rhG-CSF in 5L fermenter were obtained by studied from initial pH, dissolve oxygen and other factors. Under the optimized fed-batch culture conditions, the yield of dry cell weight (DCW) and the expression of recombinant rhG-CSF were 12.29g/L and 41.9%, respectively. 4. Purification of rhG-CSF by one stepThe chromatographic separation conditions of elution gradient and flow rate were optimized with high performance hydrophobic interaction chromato graphy (HPHIC). With only one chromatographic run, the purification of rhG-CSF was accomplished. The purity of the obtained rhG-CSF was 97.8% and its mass recovery was 24.0%.