【作者】 蔡华；

【导师】 刘坐镇； 宁方红；

【作者基本信息】 华东理工大学 ， 生物化工， 2015， 硕士

【摘要】 免疫抑制剂他克莫司(FK506)发酵法生产时,会有与其结构极为类似的副产物子囊霉素(FK520)和双氢他克莫司(2H-FK506)产生,这两种类似物用色谱和结晶法都很难去除,FK506在含水环境中又易向其异构体Ⅰ/Ⅱ转化,这些因素导致了FK506分离纯化的难度大,收率低。本文旨在开发一种可以适用于工业化分离纯化FK506的新方法。通过静态吸附与解吸实验筛选了HZM-3型大孔吸附树脂作为色谱填料,优化中压制备色谱分离条件,使一次层析分离达到了与文献中报道用进口填料(XAD1180、CG161M)类似的效果,具有较好的经济性,但仍有2%左右的类似物未能去除。为了完全去除类似物FK520、2H-FK506,磺化HZM-3型大孔吸附树脂作为配位基体,动态载银,制得可以特异性吸附FK506的Ag+型配位树脂。考察配基密度对FK506吸附与解吸性能的影响,优选了具有合适配基密度和经济性能的Ag+型配位树脂；考察了Ag+型配位树脂分离纯化FK506的性能,中压制备色谱优化配位色谱的相关条件,当上样量/柱体积为8 mg/mL,流速为1 BV/h时,甲醇/乙酸乙酯(1/1)作为流动相,洗脱仅4.5 BV(柱体积),收集的FK506用HPLC检测纯度达到98.8%以上,收率为87%,未检测到类似物,乙醇结晶后FK506的纯度达到99.6%。通过Ag+型配位树脂制备色谱法分离纯化FK506粗品,FK506与其类似物可以达到基线分离,极大的提高了FK506的分离纯化效率以及收率。最后考察了不同种类和级别的流动相、储存条件对Ag+型配位树脂稳定性的影响；企业中试过程中Ag+型配位树脂的配基损耗情况以及获得FK506产品的质量分析,表明了Ag+型配位树脂具有较高的稳定性和较长的使用寿命,分离得到的FK506产品质量符合药典标准和企业生产要求。Ag+型配位树脂对FK506的分离性能和其自身的稳定性研究结果表明,本论文制得Ag+型配位树脂及其制备色谱分离工艺为快速、高效的分离纯化FK506提供了较好的方法,具有较高的工业应用价值。更多还原

【Abstract】 The fermentation production of immunosuppressant tacrolimus (FK506) has by-products which were ascomycin (FK520) and dihydro-tacrolimus (2H-FK506), and they have similar structures. It was difficult to remove analogues by chromatography and crystallization. In water-solvent solutions, FK506 was converted into its isomers of tautomer I and II easily. These factors made it difficult for isolation and separation of FK506, and the yield was low. This paper was aimed to develop a new method which can be applied to isolate and purificate FK506 in industrialization.This paper screened out the macroporous adsorption resin of HZM-3 as chromatographic packings by static adsorption and desorption, and optimized these conditions of MPLC. The effect of single chromatography separation was similar to import packings, like XAD1180 and CG161M, and it had good economic performance. But there ware still about 2% of analogues that was unable to remove. In order to completely remove analogues FK520 and 2H-FK506, this paper sulfonated the macroporous adsorption resin of HZM-3 as the ligand substrate, and produced ligand resin by dynamic loading Ag+, which could specifically adsorb FK506. Examining and optimizing ligands density that effected on the properties of adsorption and desorption of FK506, this paper achieved more suitable and economical ligands density of ligand resins with Ag+. Studying on purification performance of FK506 by ligand resins with Ag+, this paper optimized these conditions of MPLC. When sample amount/column volume was 8 mg/mL, and flow rate was 1 BV/h, and methanol/acetic acid ethyl ester was 1/1 as mobile phase, and elution was only 4.5 BV, the purity of FK506 could achieve to above 98.8% without analogues, and the yield was 87%. The final purity of FK506 was 99.6% after crystallization by ethanol. Using ligand resin with Ag+ as chromatography packings of MPLC for isolation and purification of FK506, FK506 and its analogues could be baseline separated, and it could greatly improve the efficiency on isolation and purification of FK506 and yield.Finally, this paper examined the stability of ligand resin with Ag+ in different types and levels of mobile phase and conditions of storage. Enlarging pilot production of FK506 in enterprise, the loss of Ag+ and the quality of FK506 showed that ligand resin with Ag+ had high stability and long service life.The quality of product FK506 accorded with pharmacopoeia standards and enterprise’s production requirements.Studying on the performance of isolation and purification of FK506 and the stability of ligand resin with Ag+, it showed that this paper developed ligand resin with Ag+ and separation technology could provide a good way for fast and efficient purification of FK506, and had higher value for industrial application.更多还原