Production Technology Study Of Recombinant Gly-Thymosin Beta 4

Myocardial infarction has become a serious disease, which is deleterious to human health. Although many drugs and methods were developed for the treatment of this disease, there still exist considerable problems, especially, quite few drugs or methods could improve myocardial resistance to ischemic myocardium and myocardial cell repairing capacity. It had been demonstrated that thymosinβ4 (referred to Tβ4) could help to prevent and repair of ischemic heart damage after a heart attack, which can be utilized for the treatment of myocardial ischemic injury. It is crucial to explore the production process and quality standards for the development of high efficient Tβ4 drug. In this study, the production process, quanlity standards and pharmacodynamic of recombinant Glycine-Thymosinβ4 (referred to Gly-Tβ4) were studied. The main contents are summarized as follows:Firstly, the conditions of engineering bacteria growth and protein expression in flask were studied, and the optimum fermentation conditions were determined. Additionally, the conditions of engineering bacteria growth and protein expression in 30 liter fermenter were also studied, and the optimum fermentation conditions were established. Under the optimum conditions, the target fusion protein was highly expressed.Secondly, the bench-scale purification process of Gly-Tβ4 was investigated, including the use of nickel-ion metal chelate chromatography to purify target fusion protein, the use of gel filtration chromatography to remove imdozole, the use of thrombin to digest of fusion protein and release target protein, the use of anion exchange chromatography for initial purification of target protein, the use of reverse phase chromatography to acquire pure target protein and to remove endotoxin, and the use of anion-exchange chromatography to remove organic solvents and to concentrate target protein. Then, the process parameters for bench-scale purification were futher optimized, including feeding concentrations of imdozole in nickel metal ion chelating chromatography, the conditions of thrombin digestion and reverse phase chromatography elution buffer. Then, the stability of the small scale purification process was proved by several batches of purification. Thirdly, the pilot-scale purification process of Gly-Tβ4 was studied, mainly by linear amplification of bench-scale purification process. The principle of amplification is to amplify column diameter and to retain the same media height at the same linear flow rate. Then, the stability of the pilot scale purification process was proved by several batches of purification.Fourthly, the quality standards of recombinant Gly-Tβ4 were determined. The method of reverse phase HPLC for determination of the purity of Gly-Tβ4, and the method of E-rosette for evaluation of bioactivity of Gly-Tβ4, were studied and optimized. Thus, the quality standards of stock solution and target product of Gly-Tβ4 were established.Finally, the pharmacodynamic of Gly-Tβ4 was studied in the rat model. It was shown that Gly-Tβ4 has a therapeutic effect for treatment of myocardial infarction caused by coronary artery ligation.