| Epothilone B is a kind of sixteen membered macrolides secondary metabolites produced by Myxobacteria sorangium cellulosum. Its anti-tumor mechanism is similar to paclitaxel. At present, several epothilone compounds are on the stage of clinical trails and achieve good results which are expected to be approved in recent years and become one of the most promising new antitumor drugs. However, the production cost of epothilone is still restricting factor to the market. The separation and purification technology of laboratory scale for epothilone B was studied in this paper. First, resin collected in fermentation liquid were studied on conditions of adsorption. Then three processes of separation and purification for epothilone B were studied, which laied the foundation for process of low cost separation and industrial scale.In this thesis, the main research results were as follows:The best solution on adsorption conditions of XAD-16 macroporous adsorption resin for epothilone B were studied. The results showed that the optimal conditions were that the concentration of methanol solution 80%, desorption p H 6.5, desorption time 135 min, desorption times 3, shaking speed 200 r/min. Desorption quantity Q2 and desorption rate D of XAD-16 on epothilone B achieved the optimal solution respectively, namely(17.42±0.11)mg/g and(86.32±0.98)%.Sephadex LH-20 with Preparative high performance liquid chromatography(PHPLC) combined studied effects of various factors on epothilones separation and purification process. The optimal process conditions of LH-20 column chromatography: purity and recovery rate of Epo B were(91.6±1.3)% and(85.9±1.0)% respectively when acetone as mobile phase, flow rate 1.0m L/min, sample concentration 3.0 mg/m L and the column bed height diameter ratio 8:1. Epo B samples were purified and separated further by PHPLC. The best separation condition of PHPLC were that acetonitrile:0.2% glacial acetic acid aqueous solution(70:30) as mobile phase, flow rate 15 m L/min, sample volume 800 μL. Under the conditions, the purity and recovery rate of Epo B were up to(99.1±1.4)%,(88.3±0.9)% respectively. In a word, the purity and the recovery rate of Epo B were(99.1±1.4)%,(75.5±0.7)% when Epo B crude extract was purified in accordance with two optimized conditions.Atmospheric pressure reversed phase C18 column chromatography process route. Impacts of multiple chromatographic parameters on the recovery rate and purity were studied by adding packing C18 into the Chromatography column. When the sample volume of Epo B was below 2.0 mg/m L, the purity of Epo B was(95.2±1.2)% and the recovery rate was(88.2±0.7)% with using 55% acetonitrile water solution as mobile phase who’s flow rate was 1.5 m L/min.In the third process routes, epothilone B was isolated and purified by molecular imprinting technique. Firstly, Synthesis process of epothilone B Molecularly Imprinted Polymers(MIPs) was studied. Epothilone B MIPs were obtained by hybrid template method, with using the adsorption amount as main index of optimize synthesis conditions. Finally, the optimal synthesis conditions were that Epo B and Epo D(1:1) as template molecular, MAA as the functional monomer, EGDMA as cross-linking agent, whose molar ratio was 1:6:20, and acetic acid acetate/acetone(8:2) as pore forming agent. The adsorption effect of polymer was best and unit adsorption capacity could reach 24.316 mg/g under this condition. Secondly, the adsorption and selectivity of the polymers were studied, the polymers was characterized by SEM, and characteristics of polymers was evaluated by Scatchard model. The results showed that there were two classes of adsorption epothilone B binding sites in MIPs. Thirdly, using Epothilone B MIPs as filler packing column, the extraction conditions were optimized: 60% methanol as sample solution, deionized water as eluent, 80%methanol/PBS as eluent solution. Under the condition, the purity and the recovery rate of Epo B were(95.8±1.4)% and(82.5±1.1)% respectively.Compare three kinds of process route, Sephadex LH-20 with Preparative high performance liquid chromatography(PHPLC) combined could reach a high purity which could reach more than 99%. But its purity was(75.5±0.7)%, and the filler was 11400 yuan/500 g. The separation and purification operation had two steps which was complex relatively; And the filler of atmospheric reversed-phase C18 column chromatography was 3000 yuan/500 g which was cheap relatively. The purity was about 95%, the recovery rate was high, and the equipment was simple; The purity and recovery of molecularly imprinted polymer had little difference with atmospheric reversed-phase C18 column chromatography. But its cost was lower, and operation process was convenient. It had strong affinity for emplate molecule, which almost had specific recognition properties. Therefore, the the optimum technology separation and purification of epothilone B was molecular imprinting chromatography. |
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