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Method for preparing insulin by separation and purification through ion exchange chromatography

An ion exchange chromatography, separation and purification technology, which is applied in the preparation of insulin by separation and purification of ion exchange chromatography, and the purification and separation of recombinant insulin or insulin analogs by chromatography technology, can solve the problem of high cost, low yield, poor purity, etc. problem, to achieve the effect of low cost, high yield, efficient separation, purification and preparation

Inactive Publication Date: 2021-02-09
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a method for the purification and preparation of insulin and its analogues. Specifically, the present invention proposes a new method for the separation and preparation of ion-exchange chromatography that improves the purity and yield of insulin. The problems of low yield, high cost and poor purity in the purification preparation process

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  • Method for preparing insulin by separation and purification through ion exchange chromatography
  • Method for preparing insulin by separation and purification through ion exchange chromatography
  • Method for preparing insulin by separation and purification through ion exchange chromatography

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Weigh 1.8g crude insulin and dissolve it in the organic salt corresponding to mobile phase A or the mixed buffer solution of organic salt and organic solvent (concentration 30mg / mL), add acetic acid to adjust the pH to about 3.5 to completely dissolve the solid until transparent, use 0.45 Filter through a μm filter. The high-performance liquid phase analysis method was used to analyze the polymer and related protein impurities in the original solution; the analysis results are shown in figure 1 and 2 . Mobile phase A was 40% 50mM sodium acetate / water+60% ethanol, pH 4; mobile phase B was 40% 50mM sodium acetate+1M NaCl / water+60% ethanol, pH 4.0.

[0039] Use mobile phase A to balance the cation-exchange chromatographic column (WorkBeads 40S, flow rate 80cm / h, column volume 60ml) with 3-4 column volumes, and load the processed sample to the balanced cation-exchange chromatographic column through the medium and low pressure chromatographic system, The amount of sample ...

Embodiment 2

[0042] Weigh 2g of crude insulin and dissolve it in the organic salt corresponding to mobile phase A or the mixed buffer solution of organic salt and organic solvent (concentration 20mg / mL), add acetic acid to adjust the pH to about 3.5 to completely dissolve the solid until transparent, use 0.45μm Membrane filtration. The high-performance liquid phase analysis method was used to analyze the polymer and related protein impurities in the original solution. Mobile phase A was 50% 50mM ammonium acetate / water+50% ethanol, pH 4.5; mobile phase B was 50% 50mM ammonium acetate+1M NaCl / water+50% ethanol, pH 4.5.

[0043] Use mobile phase A to balance the cation-exchange chromatographic column (WorkBeads 40S, flow rate 60cm / h, column volume 60ml) with 3-4 column volumes, and load the processed sample to the balanced cation-exchange chromatographic column through the medium and low pressure chromatographic system, The amount of sample loaded is 1.8g (33g / L filler). After the sample is ...

Embodiment 3

[0046] Weigh 1.8g crude insulin and dissolve it in the organic salt corresponding to mobile phase A or the mixed buffer solution of organic salt and organic solvent (concentration 18mg / mL), add acetic acid to adjust the pH to about 3.5 to completely dissolve the solid until transparent, use 0.45 Filter through a μm filter. The high-performance liquid phase analysis method was used to analyze the polymer and related protein impurities in the original solution. Mobile phase A was 45% 20mM sodium acetate / water+55% ethanol, pH 4.0; mobile phase B was 45% 20mM ammonium acetate+1M NaCl / water+55% ethanol, pH 4.0.

[0047] Use mobile phase A to balance the cation-exchange chromatography column (SP Sepharose FF, flow rate 100cm / h, column volume 60ml) for 3-4 column volumes, and load the processed sample to the balanced cation-exchange chromatography column through the medium and low pressure chromatography system , the loading amount is 1.8g (30g / L filler), after loading the sample, r...

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Abstract

The invention discloses a method for preparing insulin by separation and purification through an ion exchange chromatography. The method is used for separating and purifying insulin and analogues thereof by adopting the ion exchange chromatography technology, the preparation method adopts anion-cation ion exchange chromatographic packing, by adjusting condition parameters such as mobile phase buffer salt and organic phase types, pH, flow velocity and mobile phase elution gradient, the insulin has good peak shape control and proper retention behaviors during chromatographic preparation and purification, optimized purification conditions are finally determined through peak broadening comparison under different conditions, high-efficiency separation, purification and preparation of the insulin are realized, and an insulin sample with relatively high purity is obtained. According to the method, compared with an existing chromatographic purification method, the method has the advantages ofbeing large in sample loading amount, high in purity and yield, short in consumed time, small in environmental influence, easy to operate, good in stability, low in solvent usage amount and fraction volume and the like, the production cost of existing insulin and insulin analogue products can be reduced on a large scale, and the method is suitable for industrial production.

Description

technical field [0001] The invention relates to the technical field of biomedicine, and relates to a preparation method for purifying and separating recombinant insulin or insulin analogues by chromatographic technology, in particular to a method for preparing insulin by separation and purification by ion exchange chromatography. [0002] technical background [0003] Diabetes is a chronic metabolic disease with clinical symptoms of polydipsia, polyphagia, polyuria, and weight loss, especially cardiovascular, cerebrovascular, and nervous system complications, which seriously threaten the life and health of patients. In recent years, the incidence of diabetes in the world is showing an "explosive" growth, and the prevalence rate has reached more than 10%. There are many types of diabetes treatment drugs, and insulin is still the first choice of hypoglycemic drugs with the best treatment effect. China is the largest diabetes country in the world, and the market prospect of ins...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62C07K1/36C07K1/34C07K1/18
CPCC07K14/62
Inventor 梁鑫淼叶贤龙郭志谋于伟
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI