A kind of preparation method of insulin desb30

Abstract

The invention discloses a method for preparing insulin or an insulin derivative precursor. The method comprises the following steps that S1, protease is added to fermentation broth of insulin precursor protein; S2, the pH value and temperature of the solution treated in S1 are adjusted, so that protease is subjected to digestion, and a solution containing the insulin or insulin derivative precursor is obtained after digestion is carried out. The method for preparing the insulin or insulin derivative precursor can further comprises the step that S3, the solution containing the insulin or insulin derivative precursor obtained after digestion is carried out is subjected to isoelectric precipitation recycling operation, and the higher-purity insulin or insulin derivative precursor is obtained.Compared with the prior art, according to the scheme, the method for preparing the insulin or insulin derivative precursor is easy and convenient to operate, low in equipment investment and high in technological process yield and has good industrial application prospects.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to an insulin DesB 30 method of preparation. Background technique [0002] Insulin is one of the important drugs in the treatment of diabetes. At present, the insulin produced in the market mainly adopts recombinant technology. The production process of recombinant insulin usually includes the following steps: S1. Expressing proinsulin by microorganisms such as E. coli or yeast; S2, remove most of the impurities in the fermentation broth after purification; S3, obtain the insulin (DesB) that removes the 30th amino acid residue of the B chain by trypsin or lysyl endonuclease digestion 30 Insulin) solid powder; S4, DesB obtained by the above operation 30 Insulin can be modified differently to obtain recombinant insulin derivatives with different efficacy. For example, rapid-acting insulin aspart can be obtained by transpeptide reaction and other processes; or long-acting insulin...

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Problems solved by technology

Chinese invention patent document CN105111304B discloses a method for the purification and enzyme switching of recombinant human insulin precursor, specifically disclosing the purification of proinsulin fermentation broth by ion exchange, followed by enzymatic digestion by adding organic solvent acetonitrile, followed by rotary evaporation DesB was obtained by organic solvent and isoelectric precipitation 30 Insulin, although this method can obtain relatively high-purity insulin precursors, the overall process is very cumbersome, and the overall molar yield is only about 80%

Chinese invention patent document CN1699412B discloses a new method for the preparation of insulin and insulin analogues through genetic engineering, specifically disclosing the application of XAD-7 hydrophobic adsorption packing for separation and purification-rotary evaporation to remove organic reagents-gel filtration to remove pigments and some impurities , and then use the cation exchange column SP-Fast flow to remove impurities such as lipids and collect the insulin precursor. This method can also improve the purity of the product. However, the process steps used are long, the consumption of organic reagents is large, and the hydrophobic adsorption layer Low analytical exchange capacity, low yield in the desalination step, not suitable for large-scale production

Chinese invention patent application document CN105153294A discloses a purification method for recombinant insulin and insulin analogue precursors, specifically disclosing the use of more advanced ion-exchange fillers that can tolerate high conductivity for the recovery of insulin precursors, such as Capto MMC can Purification is carried out directly without diluting the fermentation broth, and the yield is about 85%. However, the price of similar packing materials is often more than 5 times that of commonly used ion exchange packing materials, resulting in a greatly increased production cost, and is still not suitable for large-scale industrialization. Production

[0004] In summary, in the prior art, insulin precursors prepared by recombinant expression methods are used in the preparation of DesB 30 In the insulin process, there are many problems such as the inability of the conventional chromatography packing precursor purification process to tolerate high-salt loading, ultrafiltration and desalination, large-volume dilution, and difficulty in connecting with the subsequent enzyme digestion process after high-salt elution. Hydrophobic fillers have problems such as large amount of organic reagents, high cost of fillers, and serious pollution in chromatography.

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