Purification,Modification Process And Biological Assay Of Insulin Degludec

As a representative of a new generation of long-acting basal insulin,Insulin Degludec has the advantages of stable blood concentration,flexible administration time and site,and is expected to be the drug of choice for the majority of diabetic patients.Insulin Degludec was developed by Danish Novo Nordisk and approved by the FDA in 2015.At present,many domestic enterprises have obtained clinical approvals,but there are no listed products.In this paper,we will develop the technical difficulties in the purification process of Insulin Degludec and the quantitative analysis of biological activity,in order to obtain cheap and good Insulin Degludec.The main research contents of this paper include:1.The inclusion body solution obtained by fermentation of recombinant engineering bacteria HS631/BL21,was denatured and renatured to obtain a soluble protein solution.Then,it is separated and purified by membrane ultrafiltration using concentrated displacement and anion chromatography to obtain Pro-precursor of Insulin Degludec.In this process,by determining the amount of ?-mercaptoethanol and the renaturation time of the protein,the renaturation efficiency of the protein in the renaturation process is improved;the membrane pore size selection and TMP control conditions in the membrane ultrafiltration process are determined to ensure super The filtration yield is>95%;the optimization conditions of the anion chromatography process are determined,the chromatographic yield is>90%,and the chromatographic elution sample purity is>95%.2.On the basis of obtaining the pro-precursor,the recombinant trypsin is used to digest the proglutinin precursor protein,the guide peptide and C-peptide fragment are excised,and further purified by cation chromatography.The precursor protein of insulin Degludec is obtained.In this process,by optimizing the conditions of protease digestion,the yield of digestion reaction is improved,the formation of by-products is reduced,and a major by-product(DTI-IP01)is identified;Optimize to determine the appropriate buffer,loading capacity,elution conditions,so that the final chromatographic yield is above 90%,and the purity of the eluted sample is over 95%.3.On the basis of obtaining the precursor protein of Insulin Degludec,methylhexadecandioyl-Glu(OMe)-Osu is attached,and insulin Degludec is obtained by C8 reverse phase chromatography.In this process,by studying the reaction temperature,pH value and feed ratio in the connection process,the connection efficiency is improved and the by-product production is inhibited.After further purification by C8,the purity of Degu insulin is over 99%,and four main impurities(Ideg-IP01,Ideg-IP02,Ideg-IP03,Ideg-IP04)remaining in the process are identified.Through the above research,the purification route of Insulin Degludec is established as:inclusion body-protein renaturation-ultrafiltration concentration-anion chromatography(obtaining Pro-precursor protein of Insulin Degludec)-protease-cation chromatography(obtaining precursor protein of Insulin Degludec)-linked to hexadecandioic acid-C8 reverse phase chromatography purification(obtaining Insulin Degludec).The process determined by the above experiment is further enlarged to the production scale(500L),and the data of three batches of production show that the purification production process of Insulin Degludec is stable and the product quality is good.The biological activity of Insulin Degludec is analyzed by using mouse blood glucose method and fat cell glucose uptake method,and is compared with the original drug Tresiba.The results show that there is no significant difference between 3 batches of Insulin Degludec and 2 batches of Tresiba in reducing the glycemic activity of mice,and there is also no significant difference in the glucose uptake activity of fat cells,showing basically the same performance.