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Optimization Of Production Technology Of Recombinant Human Interferon-γ And Studies On Renaturation Of α-Chymotrypsin

Posted on:2002-10-17Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y H ShenFull Text:PDF
GTID:1101360062485411Subject:Analytical Chemistry
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The thesis includes two parts. First, the high-density fermentation and its scaling-up for producing recombinant human interferon-y (rhlFN-y) were systematically studied. The highest yield was 7.13g/L, being 9-folds of that reported in literature. Second, the investigation of the renaturation of a-Chymotrypsin (a-Chy), one of proteins being difficult to be renatured by high performance hydrophobic interaction chromatography (HPHIC) was presented. On the basis, the conditions for the renaturation with simultaneous purification of rhIFN-y was also investigated. With only one chromatographic run, the purity of rhIFN-y was more than 95% and the highest specific activity 1.8X108IU/mg being 11-folds of that requested by the domestic drug standard.Part one High-density fermentation and its scaling-up forproducing rhIFN-y (recombinant E.coli DH5 II medium forproducing the recombinant E.coli DH5ot/pBV220 in shake flasks was analyzed. The results showed that carbon and nitrogen sourcs were rich in Mg II medium, but the dissolved oxygen was insufficient and the pH was acidic, which may be the unfavorable factors in the whole fermentation process.3. Batch of fermentation in 5L fermentorThe optimal conditions of the batch of fermentation for producing recombinant E.coli DH5a/pBV220, such as rotating speed, inhalant volume, expression time and so on were investigated in 5L fermentor. The experimental results showed the same result as that in shake flask pointed above. In addition, besides bacteria harvest and expression yield, the results of renaturation with simultaneous purification of rhIFN-y were firstly considered as a criterion to optimize different fermentation conditions. The experimental results indicated that the optimal process conditions were decided by the bacteria yield, rhIFN-y expression, the efficiency of the renaturation, and purity of the purified rhlFN-y.4. High-density fed-batch culture in 5L fermentor and optimization by model builtThe optimal conditions of high-density fed-batch fermentation for producing recombinant E.coli DH5oc/pBV220 were selected and the production yield of rhIFN-y was 7.13g/L, being 9.1 folds of that reported in literature. From the parameters of fed-batch fermentation, carbon source, nitrogen source, and the dissolve oxygen were sufficient. Because pH in the culture medium was adjusted to be greater than 7.0, the unfavorable factor for the whole production process could be removed. Based on the obtained experiment result by single factor optimization and the model built by gradual regression technology, a new experimental design method for optimizing the prescriptions of culture media was established. According to the new method, some main factors were selected to obtain the best prescription of culture medium by analyzing the experimental data with a computer. The experimental design method could reduce experimental time and some local optimizations.5. Scaling-up in SOL fermentorWith a suitable enlarged principle, the process conditions for a batch offermentation and fed-batch of fermentation were successfully enlarged and, thus, used in the fermentation in a SOL fermentor. For the latter, the yield of rhIFN-y reached 2.33g/L, while by using fed-batch fermentation, the OD6oo value...
Keywords/Search Tags:culture media, experimental design method, high-density fermentation, fed-batch cultivation, recombinant E.coli, experimental scaling-up, recombinant human interferon-γ, renaturation, purification, α-chymotrypsin, high performance liquid chromatography
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