Preparation Process Of High-puritv Thymosin α1

Thymosin alpha1(Tα1) which composed of28amino acids is an immunomodulator with high-active. It belongs to relatively strong acid which has an N-terminal acetylation. There is an alpha-helix and a beta-turn in secondary structure. These properties determine the difficulty in the preparation of high-purity and high-quality thymosin alpha1.This paper is mainly composed of two aspects. The best synthesis process was screened to reduce the racemic impurities and deletion impurity in synthesis step. Optimization of purification process was studied to remove the difficulty impurity. Finally the high-purity thymosin alpha1was successfully prepared.Thymosin alpha1was synthesized in SPPS with Wang resin as the solid phase carrier. Aiming at the possible synthesis of racemic peptide impurities and deletion peptide impurity, corresponding measures were taken to avoid or reduce these impurities.The reagent with high efficiency and little side reaction was used as condensation reagents. For the purpose of complete reaction secondary reaction was carried out in the amino acid sites where the alpha-helix and beta-turn formed. Synthesis condition including condensation reagent system, amino acid feed ratio in the experimental process, reaction time and reaction temperature were optimized. The TBTU/HOBt/DIEA reaction system was adopted as the main condensation reagent, which has a high efficience and little side reaction, to reduce the the probability of racemic impurities and shorten the synthesis cycle. In the5-8,17-24amino acid sites HOBt/DIC condensation reaction system was used for the secondary reaction to reduce the generation of deletion impurities. In the study on the feed ratio screening experiment molar ratio of5:1was choused. The main reaction time was80-100min, and reaction temperature was30±2℃. The secondary reaction time was2-3h, reaction temperature was30～35℃. Amino acid Wang resin with alternative range0.25～0.35mmol·g-1was selected as the most appropriate substitute in order to reduce the coupling difficulty which brought by steric effect and promote the complete reaction of amino acid coupling. The yield by this synthesis process increased by28.6%compared with the control group.The crude peptide was separated and purified by reversed-phase ion-pair liquid chromatography. High-purity of thymosin alpha1was successfully prepared. Reversed-phase C18packing was used as the stationary phase and ion-pair reagent with a certain concentration as the elution reagent of A phase, with the acetonitrile as the elution reagent of B phase. The high-purity thymosin alpha1was finally prepared by multi-step purification. This paper combined the two elution reagent of trifluoroacetic acid (TFA)/acetonitrile system and tetramethylammonium hydroxide (TMAOH)/acetonitrile system. Tetramethylammonium hydroxide elution reagent with different concentration and pH was used as elution A phase, and finally high-purity thymosin alpha1was successfully prepared. The purity of the refined solution was improved after removal of most salt by RPLC with methanol water solution of certain concentration as the elution. The using of reversed-phase ion-pair liquid chromatography solved the problem of preparation for the high-purity long peptide product.The organic salts in the thymosin alpha1refined solution were removed by ion-exchange-reversed-phase chromatography. Ion-exchange resin was used as the stationary phase with the thymosin alpha1hanging on it. The charged state of thymosin alpha1was changed and ion exclusion formed between the objective molecular and tetramethylammonium hydroxide. A certain concentration ethanol ammonia solution was used to separate the thymosin alpha1and ion-pair reagent, and the organic salt was removed. The inorganic salt introduced in previous step was removed by reverse phase liquid chromatography. Finally thymosin alpha1lyophilized powder was prepared by vacuum freeze drying. A full preparation process of thymosin alpha1was established in this paper. It provides a good reference for the preparation of other long-chain polypeptide.