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Recombinant insulin and insulin analogue precursor purification method

A technology for insulin analogs and purification methods, which is applied in the field of purification of recombinant insulin and insulin analog precursors, to achieve the effects of reducing process time and equipment investment costs, good process continuity, and simple operation steps

Inactive Publication Date: 2015-12-16
JINAN KANGHE MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Aiming at the technical deficiencies in the purification and preparation process of the existing recombinant insulin precursor and recombinant insulin analog precursor, the present invention proposes a preparation process with simple operation and lower cost

Method used

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  • Recombinant insulin and insulin analogue precursor purification method
  • Recombinant insulin and insulin analogue precursor purification method
  • Recombinant insulin and insulin analogue precursor purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Separation of recombinant human DesB using the filler CaptoS in this application and the reference filler SP-Sepharose6FF 30 insulin precursor

[0037] (1) Pretreatment of fermentation broth: the fermentation broth is derived from Pichia pastoris fermentation broth, adjusted to pH 2.0 with hydrochloric acid, centrifuged for 15 minutes, 8000g, 4-10°C, collected the centrifuged supernatant, and measured the conductivity value to 55mS / cm; The purity of the supernatant detected by HPLC was 38%, and the content was 3.5 mg / ml;

[0038] In order to test the influence of different conductivity on the purification results, the above-mentioned fermentation with a conductivity of 55mS / cm was adjusted to 3 parts of 40, 20 and 10mS / cm with water, and experiments were carried out respectively.

[0039] (2) Equipment / filler / buffer solution

[0040] Equipment: AKTAPureM1 chromatography equipment (GE Company, USA), full-wavelength ultraviolet detector, flow rate range 0.01-...

Embodiment 2

[0056] Example 2: Purification and separation of recombinant human DesB by CaptoMMC 30 insulin precursor

[0057] (1) Sample source: the same as in Example 1.

[0058] (2) the equipment / filler / buffer solution conditions used in this embodiment are as follows:

[0059] Equipment: AKTAPureM1 chromatography equipment (GE Company, USA), full-wavelength ultraviolet detector, flow rate range 0.01-20ml / min.

[0060] Chromatography column: XK16 / 20 column, detection wavelength: 280nm.

[0061] Chromatography packing material: CaptoMMC, column bed height: 10cm; column volume 20ml (self-filling);

[0062] Injection volume: 70-80ml, linear flow rate: 300cm / h, volumetric flow rate: 10ml / min.

[0063] Equilibrium buffer: 100mM glycine-hydrochloric acid buffer, pH2.0;

[0064] Elution buffer: 200 mM Tris-HCl buffer, pH 8.5.

[0065] (3) Operation steps: with embodiment 1.

[0066] (4) Experimental results

[0067] Table 2 CaptoMMC purification and isolation of recombinant human DesB ...

Embodiment 3

[0069] Example 3: Purification and separation of recombinant human DesB by UniPMMS 30 insulin precursor

[0070] (1) Sample source: the same as in Example 1.

[0071] (2) the equipment / filler / buffer solution conditions used in this embodiment are as follows:

[0072] Equipment: AKTAPureM1 chromatography equipment (GE Company, USA), full-wavelength ultraviolet detector, flow rate range 0.01-20ml / min.

[0073] Chromatography column: XK16 / 20 column, detection wavelength: 280nm.

[0074] Chromatography packing material: UniPMMS, column bed height: 10cm; column volume 20ml (self-filling);

[0075] Injection volume: 70-80ml, linear flow rate: 300cm / h, volumetric flow rate: 10ml / min;

[0076] Equilibrium buffer: 80mM citric acid-sodium citrate buffer, pH4.0;

[0077] Elution buffer: 100mM glycine-sodium hydroxide buffer, pH8.5;

[0078] (3) Operation steps: with embodiment 1.

[0079] (4) The experimental results are as follows:

[0080] Table 3 UniPMMS separation and purific...

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Abstract

The invention relates to the field of insulin production methods and discloses a recombinant insulin and insulin analogue precursor purification method. The invention solves the problems that conventional chromatographic packing cannot tolerate high-salinity sample loading, the amount of used organic reagent is great, the cost is high and the product purity is not high in the purification process of recombinant expressed insulin precursors and insulin analogue precursors. The invention adopts the technical scheme that the method comprises the steps of performing pH regulation and centrifugation to centrifuged fermentation supernatant, then directly loading a sample and performing adsorption, separation, purification and elution through a chromatographic column prepared by using any packing of Capto S, Capto MMC, Uni PMM S and Uni MSP to finally obtain high-purity recombinant insulin and insulin analogue precursors. Compared with the existing purification method, the recombinant insulin and insulin analogue precursor purification method has the advantages that the operation is simple, the yield is high, the spent time is short, the environmental influence is small, and the product production cost of the existing insulin and insulin analogues can be greatly reduced.

Description

technical field [0001] The invention relates to the field of insulin production methods, in particular to a method for purifying recombinant insulin and insulin analog precursors. Background technique [0002] Diabetes is the third leading cause of death after cardiovascular and tumors. In 2014, there were 387 million people with diabetes in the world, and the number is increasing every year. Insulin is a specific drug for the treatment of diabetes, especially for advanced patients, and there is no other drug that can replace it. With the continuous expansion of the population of diabetic patients and the diversification of insulin administration methods, the dosage of insulin is increasing rapidly. During the development of insulin, animal insulin has gradually been phased out clinically due to immunogenicity and the spread of infectious diseases such as mad cow disease and animal foot-and-mouth disease; insulin glargine, insulin aspart, insulin lispro, insulin detemir and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62C07K1/18
CPCC07K14/62
Inventor 张颖孔凡楼姜寿俊刘骏郭凤霞徐丹
Owner JINAN KANGHE MEDICAL TECH
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