Repair Polypeptide Pilot Production Process And Preliminary Animal Studies

Aims:Repair polypeptide (RP) is a micro molecule peptide composed of 15 amino acid residues, and has the ability of cytoprotection. In this paper, we groped the technique for synthesis and purification of repair polypeptide in laboratory and the optimized technique laid a foundation for immunization research and application as new drugs.Methods:Repair peptide was synthesized in samll-scale by Fmoc-solid phase peptide synthesis method and the crude products were purified and identified by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). The production and purification procedure of RP was then scaled up and three batches of peptides were manufactured. In order to identify the bioactivity of RP, skin-scald model was built in rats and the protevtive effects of RP were investigated.Results:Experimental conditions of peptide synthesis was established and take Wang resins as the solid-phase carrier, catalyst using DMAP/HOBT, solvents and cleaner using DMF, cutting agents using TFA/H2O. The purity of that repair polypeptide crude products was about 78%. HPLC was used for purifying and identifying of RP and water-acetonitrile containing 0.1% trifluoroacetic acid (TFA) was used as mobile phase. Chromatography conditions for purification were as follow: C18 reverse-phase chromatography column,20* 250 mm, was equilibrated with 0.1% TFA aqueous solution, samples were eluted by 0.1% TFA acetonitrile solution; flow rate is 10 ml/min and peptides was detected with 215 nm ultraviolet wavelength. The element composition of the products is C62H98N18O22, with the molecular weight about 1419.6Dr. and has the maximum absorption at 275 nm and 207 nm ultraviolet, which fits the characteristics of general peptide absorption peaks. Results showed that the amino acid sequence and composition of purified RP was in accordance with the theoretical data. The chromatographic retension value of RP in HPLC is stable and RP can be 1.22±0.03-fold purified with purity and yield 98% and 71.83%±0.21%, respectively. RP can promote epidemis restoration and wegener maturation in skin-scard model of rats.Conclusion:Established a techique for synthesis and purification of repair polypeptides in large-scale and verified the cytoprotective effects of the peptide, which showed potential for therapeutic application.