Preparation Of A Novel Long Acting Human Insulin Analog

As a drug for treating diabetes, especially IDDM (insulin dependent diabetes mellitus), insulin is still absolutely necessarily. To reconstruct the basal insulin secretion for diabetes patients with little number of injecting times is the ideal curative effect of long acting insulin. Insulin Glargine and Insulin Detemir that had been used in clinic in home and abroad recently are both true basal insulins. Though they metabolised with essentially no peaks or valleys, the duration of the pharmacodynamic action are no more than 24 hours. Diabetes patients incurred inconvenience and suffering by injecting insulin frequently, so it is significant to develop insulins with longer effect to protract the interval of insulins injection. Based on the clinical requirements and the achievements of the structure of nature insulin, a new long acting insulin analog, which can remain active for 48 hours with essentially no peaks or valleys, and not necessary to adjusting the dosage by monitering the blood glouse of the patients could be produced by genetic engineering and chemical modification.Objectives: To construct the P. pastoris expression system of Human Mini-proinsulin DesB30 (the C peptide is AAK), choose the high level expression strain of GS115, and obtain the recombination protein of HMPIDesB30. To establish the preparation, purification and renaturation (to obtain Human Insulin DesB30, HIDesB30) pilot process of HMPIDesB30. Chemical modify HIDesB30 by myristic acid, MPS (3-maleimidopropionic acid N-hydroxysuccinimide ester) and mPEG-SPA (methyl polyethylene glycol succinimidyl propionate). To study the hypoglycemic activity of each chemical modified human insulin analog, and screen a new long acting insulin analog, which have the duration of more than 48 hours and the metabolic activity was constant over the study period.Methods:①The high level expression of HMPIDesB30 in P. pastoris: 1) vector pPIC9K and pPICZalphA were used to choose the high level expression strain of GS115. HMPIDesB30/pPIC9K: pPIC9 was used to construct the recombinant plasmid. Firstly, the fragment from pPIC9 digested by XhoⅠand NotⅠw as connected to HMPIDesB30 to construct plasmid HMPIDesB30/pPIC9. Then the fragment from HMPIDesB30/pPIC9 digested by SalⅠand SacⅠwas subcloned into the pPIC9K which had been digested by the same enzyme to construct the plasmid HMPIDesB30/pPIC9K; HMPIDesB30/pPICZalphA: HMPIDesB30 was inserted into pPICZalphA between site of XhoI and NotI, to establish the recombinate plasmid of HMPIDesB30/pPICZalphaA. 2) The plasmid of HMPIDesB30/pPIC9K and HMPIDesB30/pPICZalphaA had been linearized and integrated into the P. pastoris strain of GS115's genome by electrotransformation. 3) The multiple clones were obtained by YPD plates which contains increasing concentrations of G418 or Zeocin. 4) Expression of HMPIDesB30 and choose the high level expression strain of GS115.5) Optimize the fermentation condition and ferment with high density.②The purification and renaturation of HMPIDesB30: The protein was purified by macroporous resin (SIPI-40), SP ion-exchange chromatography and precipitated with 10-20 mmol/L Zn2+, then the products were dissolved in 25 mmol/L Tris-HCl (pH9.0) containing 10-20 mmol/L EDTA. The purified HMPIDesB30 was digested by trypsin at 4℃overnight with the quality ratio of trypase/HMPIDesB30=1/500.③Chemical modification of HIDesB30: HIDesB30 was modified by myristic acid, MPS and mPEG-SPA, and the productions were purified.④Study the hypoglycemic activity of modified HIDesB30: Chemical modified HIDesB30 were injected to STZ (streptozotocin)-induced diabetic mice to study the duration of the pharmacodynamic action and the relation between pharmacodynamic action and dosage. Then do research on the metabolic characteristics of the modification products that offer longer duration of action than Detemir.Results:①The expression level of HMPIDesB30 reached 1.0 g/L, the purified protein was characterized by 16.5% Tricine SDS-PAGE, HPLC, and mass spectrometry, the molecular mass of the expressed products are consistent with the theory value.②The recovery of HMPIDesB30's purification are about 60%, the purity of HMPIDesB30 are about 95%, and the recovery of HIDesB30 are about 90%.③The yield of myristic acid modified HIDesB30 are about 50%, the molecular mass and hypoglycemic activity of myristic acid modified HIDesB30 are consistent with Detemir. The yield of MPS modified HIDesB30 are about 80%, the HIDesB30-MPS had hypoglycemic activity. The yield of mPEG-SPA modified HIDesB30 (HIDesB30-PEG1, HIDesB30-PEG2) in different pH are 60% and 95% respectively, they both have hypoglycemic activity.④The duration of pharmacodynamic action of chemical modified HIDesB30 are dependent on the dosage. The order of the duration of action are as follows: HIDesB30-PEG1>Detemir> HIDesB30-PEG2>MPS. ⑤HIDesB30-PEG1 has the duration of more than 48 hours and the metabolic activity was constant over the study period.Clusions:HMPIDesB30 had been secretorily expressed in P. pastrois successfully, and the expression level reached 1 g/L.The recombinant protein of HMPIDesB30 had satisfactory biological activity, and it neither had glycosylation modification nor formed dimer or multimer. The optimal conditions of the fermentation were established. To over come the defect that the ultrafiltration could not remove the pigmentosus, macroporous had been used in purificationan to boost the recovery rate. The methods of removing the C peptid by singular trypsin were established. Though the molecular mass and the hypoglycemic activity of myristic acid modified HIDesB30 are both consistent with Detemir, the expression system, purification artwork and the methods of modification were different from Detemir. The yields of the modification products were higher than report one. The elected PEGylated human insulin analog: HIDesB30-PEG1 has the duration of 48 hours that was longer than Detemir, and it metabolised with essentially no peaks or valleys. HIDesB30-PEG1 could be a potential long-acting insulin analog for the treatment of diabetes.