Preparation And Purification Of Insulin Aspart With Short C Peptide

Diabetes mellitus has become one of the most important disease that threats to human health,researchs of insulin which is most commonly used drug for treatment of diabetes drug has been the hot spot of the biomedical field.Insulin aspart is a newly developed insulin analogue,it can control the blood sugar level significantly,works faster and has less side effects compared to human insulin,these make it becomes the most commonly used short-acting insulin drug on the market.There are few reports about the downstream purification process of insulin aspart,in this paper,used insulin aspart precursor provided by ChongQing Fagen biological pharmaceutical co,LTD to establish a successful downstream purification process of insulin aspart.Firstly,used Ni Sepharose and SP Sepharose to extract and purify the insulin aspart precursor to remove most pigment in the precursor and increase the purity and concertration of the precursor.Based on it,used Lys-C enzyme to digest the insulin aspart precursor to remove the signal peptide and C peptide one-time and defined the process and condition of this reaction.Moreover,compared this reaction to the digestion of the precursor using trypsin.Secondly,after getting the intermediate DesB30 of the insulin aspart's downstream purification,the transpeptidation process was performed by using threonine methyl and threonine tert butyl ester respectively and then established the transpeptidation method using these two substrates for comparison.It is concluded that using threonine methyl as the transpeptidation's substrate is better than another.In addition,the effect of temperature on the reaction using threonine methyl was researched.After the transpeptidation process,RP-HPLC was used for purifing products in the reaction and the conditions and parameters of this process was studied to prepare insulin aspart ester whose purity over 97%.Then,in view of the insulin aspart tert-butyl ester,trifluoroacetic acid was used to remove protection group on the threonine tert butyl and then removed trifluoroacetic acid by rotary evaporation;for insulin aspart methyl ester,influence of pH on the deprotection process was studied and established the deprotection condition,at the same time,created a new deprotection method by using Q Sepharose.After transfer insulin ester into the mature insulin aspart,RP-HPLC was used to purify the extracted insulin aspart to make sure the purity of the insulin above 98%.Based on the RP-HPLC eluent,the influence of pH,zinc content,protein concentration and sodium chloride on the insulin crystallization was sudied and then established the crystallization system of insulin aspart which is 5 mg/ml insulin aspart,0.5 mol/L glycine,0.5 mol/L sodium chloride,0.2% phenol,pH6.5 and zinc to ASP mole ratio is2.5:1.Applied this system,crystal of insulin aspart which was uniform and met the demand was obtained.In this experiment,HPLC with mass spectrometer was used on every step of the process to confirm the downstream purification and preparation technology of insulin aspart is credible.