Study On A Compound Of New Fast-acting Of Insulin Analog Gland Insulin Glargine

Insulin is the most effective medicine for insulin-dependent diabetics,and insulin analogs mainly include quick-acting,intermediate-acting,and long-acting insulin.Based on the injection of basal insulin one time per day,diabetics also need to receive the injection of fast-acting insulin to control the instant hyperglycemia during the meal.Frequent injections cause inconvenience and pain to diabetics.Therefore,the development of insulin with less injection is the research hotspot of biopharmaceutical enterprises.The goal of this project is to prepare a new fast-acting insulin analog GL,then prepare insulin GL and insulin glargine into a double insulin preparation,so as to meet the requirements of clinical diabetics requiring less injection,quick onset,and stable blood sugar lowering.This thesis consists of three parts.The first part starts with the renaturation of the inclusion body and establishes the purified preparation process of insulin GL.The second part develops the process of insulin GL and insulin GL + insulin glargine compound preparation and conducts a stability test.The third part conducts a preliminary efficacy test on insulin GL and compound preparation.Insulin glargine is one of the long-acting basic insulins in the market,which has the advantages of long onset time and low risk of nocturnal hypoglycemia.Insulin glargine can only remain stable at low pH(pH 3.5-4.5).The existing quick-acting insulin,insulin aspart and insulin lispro are all stable at neutral pH(pH 7.0-8.0),so basal insulin,insulin glargine and quick-acting insulin such as lispro and aspart can't be prepared into a compound preparation.Insulin GL is a new variety optimized by recombinant DNA technology.On the basis of the structure of the quick-acting insulin,insulin lispro,the aspartic acid amide of the 21st position of the chain A is replaced with glycine which is more stable under acidic conditions,so as to avoid the deamination reaction,making it suitable for preparing preparations which are stable under acidic pH(pH 3.5-4.5).This project develops a purified preparation process for insulin GL,which includes two-step ion-exchange chromatography,reversed-phase purification,crystallization and lyophilization,and trypsin digestion.More than 10 g of finished product can be obtained per kg of inclusion body.The reversed-phase purification process successfully converts the acetonitrile mobile phase into an ethanol mobile phase,which saves the cost,reduces environmental pressure,and achieves the same purification effect.The preparation of the pilot sample of the raw material is completed(the reversed-phase purity is over 99.5%,and the aggregate purity is over 99.3%).The molecular weight and amino acid sequence are inspected by mass spectra for insulin GL,and the theoretical values are identical.This project completes the process development of insulin GL injection and insulin GL + insulin glargine(50/50)injection preparations and the preparation of three batches of pilot samples,which completes 18-month long-term stability and 6-month accelerated stability test.The stability is good and has the performance of preparing finished medicine.A preliminary efficacy test is performed,and insulin GL injection and insulin GL+insulin glargine(50/50)injection preparations all have the function of lowering blood sugar.