The Research Of Recombinant Human Thymosin Tα1 Manufacturing Technique

Thymosin al is a kind of thermal stability acidic peptide which composed of 28 amino acids,amino acid sequence:NH2-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-COOH.Recombinant human thymosin Tal has a good effect to the treatment of immune dysfunction, hepatitis B, hepatitis C, cancer, enhance the body's immune system and so on.There are about 300 million hepatitis B patients all over the world. There are about 100 million patients only in china,and 30 million hepatitis B patients increasing every year.This figure is on the rise currently;the incidence and mortality rates of cancer have been on the rise,cancer hazard is getting worse.Based on the good therapeutic effect of thymosin on hepatitis and cancer diseases,which has a broad market prospect.Following the outbreak of Influenza A H1N1 after the SARS,we urgent need a kind of drugs for increasing human resistance rapidly,in order to resist the spread of various infectious diseases.Domestic thymosin is mainly remain at extraction products of animal thymosin thymus, and the majority are peptides material extracted from the pigs,cattle thymus. The fresh or frozen thymus of pig (or calf) are removed fat parts,minced, added saline,placed in broken machine to made of homogenized,high-speed centrifugation,get extract. Impurity extraction, salt precipitation,ultrafiltration,drying,to be thymosin fraction (Hanxiong gonadotropin al,α5,α7,β3,andβ4,etc.).Some preparation contain the active ingredient less than 1%,while the main component is the not purified impurities.It has more side effects and its clinical efficacy has also been greatly affected.Thymosin widely used internationally are chemically synthesized.Synthetic thymosin Tal commonly used solid-phase peptide synthesis technology,but because of its high cost of synthesis,it is very expensive.This topic used the monomer secretion expression vector PSTⅡ-Tα1 to transform host cells(E.coli) and fermentation expression of human thymosin al monomer.Since secretory expression technology,only destruction of cellwall to collect the recombinant protein in the production process,while the cell membrane remain intact,ensuring the end product rarely contaminate by bacterial protein,so that it easier for purification.This topic study the process of fermentation and purification process parameters,including medium preparation, fermentation temperature,pH,P02,carbon,nitrogen and the ratio of other nutrients supply, purification buffer preparation,equilibrium time,the buffer flow rate and other process parameters to be confirmed;intermediate products and finished products inspection, including the purity and impurity analysis,physical and chemical indicators,such as bacterial endotoxin,and sterile to confirm whether the established manufacturing process to achieve the requirements of the appropriate quality standards and,based on test results options a suitable production process for large-scale production.A suitable production process is choosed by comparison of the results of product testing, the entire production process meet the regulatory requirements of GMP, the purity of product is over 96% and appropriate to improve the yield of the product.