| Thymopentin(TP5) is a short-chain peptide. It is composed of five amino acids and its molecular structure is like protein. Thymopentin is synthesized by chemical method. It is the sequence from the 32th to 36th amino acids of the active center in thymopoietin. Thymopentin have all the functions of thymopoietin. Its main physiological function is inducing the transformation, multiplication and maturation of T cell. It is mostly used in curing cancer, herpes simplex, rheumatoid arthritis, AIDS, severity immunodeficiency syndrome, chronic hepatitis II on clinic.In this paper, the separation methods of thymopentin were studied. The main method is ion-exchange chromatography and reversed phase chromatography, and we scaled up the method. The main content is following:1) Analyzed the physical and chemical characters of thymopentin. Firstly, we analyzed the isoelectric point, curve of titration, hydrophoby. Secondly, we did some experiments about thymopentin and we measured the hydrophobic constant of thymopentin that is -1.4. It showed that it is hydrophilic. At the same time, we studied the stability of thymopentin, and the result showed that it is stable in water for a short time. We summarized the analytic results and found that ion-exchange method and reverse phase chromatography method are fit for separating thymopentin.2) Studied on the separation conditions of thymopentin. We got the result by selecting the resin, selecting the buffer solution, compared the effects of pH value on separation, choosing of elution gradient, comparing the chromatography and analyzing the HPLC chromatography. We found that the effect would be better when using acetic buffer solution3) Then we studied the adsorption of SP Sepharose FF on TP5, including static and dynamic adsorption. We measured the value of k which is 0.0396 s-1 and fitted the curve. We found it was a single layer adsorption and fitted toLangmuir model. The Langmuir formula is q =(128.44·c)/(2.38+c) and themaximum adsorption capacity is 128.44 mg/g.resin. The result shows it isfavorable at the pH value 4.4. And then, we studied dynamic adsorption of SP Sepharose FF on TP5. We mainly studied the effects of feed sample speed on adsorption.4) We studied the conditions of separation TP5 by RPLC. We compared the effects of separation with different columns, concentration of ion-couple solvent and elution gradient. The result shows that the effects of separation will be better when column is MKF and ion-couple solvent is 0.1% TFA at a gradient from 5% to 30% of B phase in 20 minutes.5) Finally, we scaled up the method, which combined the ion-exchange chromatography and RPLC. We got the TP5 with purity of 96.55% and the recycle ratio was 82.4% by ion-exchange chromatography. The effects were good when we desalted of the sample with RPLC. The purity is 98.23% and recycle ratio is 95.8%.6) We freeze-dried the sample and analyzed it with HPLC and MS. The purity is more than 98% and the total yield is 78.9%. This method can decrease the costing compared with traditional method that separated peptide with RPLC singly.
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