Insulin precursor precipitation recovery method for removing amino acid residue at 30th of chain B

Abstract

The invention provides an insulin precursor precipitation recovery method for removing amino acid residue at 30th of a chain B. The method comprises carrying out digestion on an insulin precursor through trypsin at 30 DEG C for 1-2h, adding divalent metal ions into a reaction system subjected to digestion, adjusting pH of the reaction system to close to the isoelectric point of the reaction product, and collecting the precipitates, wherein the divalent metal ions comprise water-soluble divalent metal ions of Ni<2+>, Ca<2+>, Mg<2+>, Zn<2+>, Cu<2+>, Fe<2+> and Mn<2+>, a mole ratio of the divalent metal ions to the insulin precursor is in a range of 0.5:1 to 4:1, pH is in a range of 4.0 to 6.0, the precipitation temperature is 0-30 DEG C and the precipitation time is 0-12h. The method is freeof treatment on a digestion reaction system, realizes direct isoelectric point precipitation and has simple processes. Compared with the prior art, the method reduces the residual rate of the productin the supernatant by 10 times or more. The insulin precursor precipitation recovery method has obvious advantages.

Description

Technical field [0001] The invention belongs to the field of preparation of recombinant human insulin, and specifically relates to a method for recovering insulin precursor precipitation. Background technique [0002] Insulin and its analogues are the most direct and effective drugs for the treatment of diabetes, especially for patients with type I and type II advanced diabetes. There are still no alternative drugs. The development of safe, effective and convenient insulin and new analogs has always been a hot spot in the development of biological drugs. With the development of biotechnology, insulin produced by recombinant technology has gradually replaced the original animal-derived insulin. The production process of recombinant insulin is generally to express proinsulin through microorganisms such as E. coli or yeast, and then digest by trypsin or lysyl endonuclease to obtain an insulin precursor (DesB30 insulin) with the amino acid residue at position 30 of the B chain remov...

AI Technical Summary

Problems solved by technology

CN105111304A discloses a method for purification and enzymatic conversion of recombinant human insulin precursor. In this invention, the fermentation supernatant of the secreted and expressed recombinant human insulin precursor is purified with a cationic chromatography column, and successively use Washing with two different washing liquids can elute the target protein more quickly, shorten the elution time, reduce the precipitation of the target protein, and reduce the volume of the eluted sample at the same time. elution, the eluted product does not need to be inverted, and trypsin is directly added for enzyme conversion, and the enzyme conversion efficiency is above 95%, which simplifies the production process and saves costs. If there are too many enzymes and fermentation impurities, etc., the enzymatic digestion product DesB30 is generally recovered from the reaction system after enzymatic digestion by using the principle of isoelectric precipitation recovery. Due to the different purification methods of proinsulin from various manufacturers, the enzymatic digestion system Substances are also very different. The use of isoelectric point precipitation cannot completely precipitate DesB30, and some DesB30 will still exist in the supernatant, resulting in a large loss of process yield.

The digestion rates of these three steps are very different. The first step is very fast, the second step is relatively slow, and the third step is not completely reacted. Even after overnight digestion, there are still nearly 20% of the double-chain insulin precursor. Unable to convert to Des B30 insulin

The spatial structure of the aggregate formed by the insulin precursor is the reason why the yield of DesB30 cannot be further improved. In the paper, the enzyme digestion was carried out in the presence of the organic solvent acetonitrile, and the isoelectric point precipitation was directly used after enzyme digestion. It was difficult to precipitate DesB30. Using high-temperature rotary evaporation to remove acetonitrile and then isoelectric point precipitation, 6.3% of the product cannot be recovered, and rotary evaporation at high temperature will have obvious degradation products that will affect the subsequent process steps

[0005] In summary, there is still a lack of effective methods to increase the yield of DesB30 insulin

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