Study On Production Process Of Insulin Lispro

Insulin Lispro,Humalog,the first rapid-acting insulin analogue developed by Eli Lilly,was approved by the US Food and Drug Administration in January 1996.Because of good efficacy and huge market benefits,insulin lispro as the third generation of insulin drugs has aroused great interest of pharmaceutical companies.However,two-thirds of domestic insulin product market is monopolized by foreign pharmaceutical companies.Indeed,few investigations report production technology of insulin and its analogues.This paper mainly studied production technology of insulin lispro.Utilizing the strategy of inclusion body renaturation developed by Lilly,fermentation conditions of shake flask and 5L fermentor was optimized on the basis of E.coli BL21(DE3)/pET28a-proLispro which has been constructed in the experiment.In 5L fermentor,high quality of inclusion body was obtained under appropriate fermentation conditions,such as addition of inducers in batches and reduction of microelement in the medium.Through exploring the composition of washing liquid,washing times,concentration of denaturing solution DTT and denaturing pH,the majority of impure proteins were eliminated,and protein concentration of inclusion body was up to 100.0 mg/mL(dry weight)in the denaturing solution.In addition,the renaturation conditions were optimized by single factor,orthogonal and response surface,including the concentration of denaturing solution DTT,renaturation solution pH,and renaturation liquid additive.The optimal renaturation conditions were as follows:Tris-Base 100.0 mmol/L,Glycine 50.0 mmol/L,EDTA 1.0 mmol/L,cystine 12.0 mmol/L,cysteine 1.0 mmol/L,sorbitol 8.0%,Urea 2.0 mol/L,and pH 10.0.The volume of renaturation solution was 4 times less than before,while the renaturation yield was 8 times higher than that.The conditions of enzyme digestion were explored,including the amount of Trypsin and the ratio of Trypsin to Carboxypeptidase B,to makes the digestion rate of proinsulin up to 60% and reaction time at approximately 210 min.After cation exchange chromatography and RP-HPLC,the purity of lispro was up to 99.02% and the total yield was 50.20%.Which can define the concentration of insulin Lispro,with the external standard method.The final yield of insulin insulin was 320 mg / L(the quality of insulin lispro / the volume of the fermentation broth).Besides,the results obtained from mass spectrometry and circular dichroism analysis were consistent with the theoretical value.Thus,the present study verified the stability of production technology of insulin lispro,which laid a solid foundation for industrial production.