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Method for purifying recombinant human proinsulin

A technology of recombinant human insulin and restoration, which is applied in the field of biomedicine, can solve problems such as poor process connection, complicated operation process, and unfavorable enzyme digestion steps, and achieve the effects of ensuring process connection, simple process operation, and reducing production costs

Active Publication Date: 2012-07-04
LUNAN PHARMA GROUP CORPORATION
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese invention patent application CN101717442A discloses the technical scheme of PEGylated recombinant human DesB30 insulin and its preparation method and application. In the technical scheme, the host cell of the recombinant expression vector is Pichia pastoris, and the purification method of proinsulin is fermentation broth After filtration, it is adsorbed by a macroporous resin and eluted with 60% ethanol, and then further purified by strong cation exchange chromatography, and then purified by zinc chloride precipitation. The proinsulin precipitate is collected and redissolved for enzyme digestion to obtain insulin. The production process for the preparation of proinsulin by this scheme is cumbersome, and proinsulin exists in the form of precipitation, which is not conducive to the direct implementation of the enzyme digestion step.
[0005] Chinese Invention Patent Specification CN88102311.6 discloses a human crystalline proinsulin and its production method. In this technical scheme, a cationic salt is first added to the solution containing human proinsulin, and then the pH is adjusted to 5.4-6.5 to form crystals, and then the crystals are recovered. Human proinsulin, although this method simplifies the production process, but the product yield is low, and proinsulin still exists in the form of precipitation
Chinese invention patent specification ZL98812757.1 discloses the preparation method of human proinsulin. The technical scheme is: the inclusion body is firstly dissolved and sulfonated, then treated with cyanogen bromide and purified by ion exchange chromatography, and then refolded. Human proinsulin is subjected to adsorption chromatography and zinc chloride precipitation to obtain human proinsulin in the form of precipitation, which can obtain a yield of 90%. The production process of this method is complicated, and the product still exists in the form of precipitation
[0006] In the method disclosed above, the operation process is complicated, and the precursor of insulin, proinsulin, finally exists in the form of precipitation, which is not conducive to the direct operation of the subsequent enzymatic digestion step, and the process connection is poor, and the yield and purity of proinsulin cannot be taken into account at the same time.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Preparation of recombinant human proinsulin refolding solution:

[0037] The engineered Escherichia coli bacteria containing the recombinant human proinsulin gene are fermented by high-density cells and centrifuged to obtain about 17.5 kg of bacteria. The bacteria are homogenized and centrifuged to obtain recombinant human proinsulin in the form of inclusion bodies. After washing the inclusion bodies Dissolve under denaturing conditions and then refold to obtain about 500L of refolding liquid.

Embodiment 2

[0038] Example 2. Macroporous adsorption resin chromatography purification:

[0039] Pack D-101 macroporous adsorption resin on a glass chromatography column (diameter-to-height ratio of 1:15), deionized water (adjusted to pH 3.5 with acetic acid) to equilibrate the chromatographic medium, and the refolding solution (adjusted with hydrochloric acid) obtained in Example 1 (pH=3.5) A total of about 500L was loaded with a peristaltic pump, and the sample flow rate was maintained at 300cm / h. After the sample was loaded, 5-10 column volumes were washed with deionized water (pH adjusted to 3.5 by acetic acid), and the cleaning flow rate was 300cm / h, followed by elution with 95% ethanol (adjusted to pH 3.5 by acetic acid), the elution flow rate was maintained at 100 cm / h, and about 11.8 L of eluate was collected.

Embodiment 3

[0040] Example 3. Chromatographic purification of macroporous adsorption resin:

[0041] Pack AB-8 macroporous adsorption resin on a glass chromatography column (diameter-to-height ratio of 1:20), deionized water (adjusted to pH 5.0 by acetic acid) to equilibrate the chromatography medium, and refolding solution (adjusted by hydrochloric acid) obtained in Example 1 (pH=5.0), a total of about 500L is loaded with a peristaltic pump, and the sample flow rate is maintained at 200cm / h. After the sample is loaded, 5-10 column volumes are cleaned with deionized water (pH 5.0 adjusted by acetic acid), and the cleaning flow rate is 200cm / h, followed by elution with 65% ethanol (adjusted to pH 5.0 by acetic acid), the elution flow rate was maintained at 50 cm / h, and about 12.5 L of eluate was collected.

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PUM

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Abstract

The invention belongs to the field of biomedicine, and particularly relates to a method for purifying recombinant human proinsulin, in particular to a method for purifying recombinant human proinsulin by using macroporous absorbent resin and cation exchange resin. In the method, a recombinant human proinsuli plural liquid is adsorbed and concentrated through the macroporous absorbent resin, and is loaded onto the cation exchange resin for further purifying, so that the collected proinsulin has high yield and high purity, and can be directly applied to enzyme digestion to obtain insulin. The method is easy and convenient to operate, has high process cohesion, and is suitable for industrial production.

Description

Technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a method for purifying recombinant human proinsulin, in particular to a method for purifying recombinant human proinsulin by using a macroporous adsorption resin and a cation exchange resin. Background technique [0002] Insulin is a special medicine for diabetes that cannot be replaced so far. There are more than 120 million diabetic patients worldwide, and this number is increasing every year. It is estimated to be 300 million by 2025. Each diabetic patient needs to use 1.5 to 2.0 mg of insulin every day. , The world consumes about 5000 kg of insulin every year. Therefore, it is imperative to develop low-cost and large-scale insulin preparation methods. [0003] Insulin is a protein hormone secreted by pancreatic β-cells stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc. It is the only hormone in the body that lowers blood...

Claims

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Application Information

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IPC IPC(8): C07K1/36
Inventor 赵志全熊继元
Owner LUNAN PHARMA GROUP CORPORATION
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