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Production technology of high-pure Apoa-I from fourth deposit of human blood plasma component

A technology of component four, human plasma, applied in the field of production technology for the preparation of high-purity apolipoprotein Apoa-I, can solve the problems of low protein yield, loss of Apoa-I, small preparation amount, etc., and achieve high protein recovery rate , Safe and convenient operation, short production cycle

Active Publication Date: 2012-10-17
SHANGHAI RAAS BLOOD PRODUCTS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the commonly used preparation methods of Apoa-I include ultracentrifugation, organic solvent precipitation and high-performance liquid phase method, etc. Although high-purity Apoa-I can be obtained, it has some obvious disadvantages: 1. The preparation amount is small, and it is not suitable for industrial production. ; 2. The protein yield is low. After multi-step treatment such as ultracentrifugation, organic solvent precipitation, and column chromatography, most of Apoa-I is lost in the preparation process; 3. The cost is high and expensive instruments such as ultracentrifuges are needed; 4. Poor safety , ethanol, acetone, trichloroacetic acid and other organic solvents are not only physiologically toxic, but also flammable and explosive, which is not conducive to safe production

Method used

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  • Production technology of high-pure Apoa-I from fourth deposit of human blood plasma component
  • Production technology of high-pure Apoa-I from fourth deposit of human blood plasma component
  • Production technology of high-pure Apoa-I from fourth deposit of human blood plasma component

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1, Apoa-I small test sample preparation

[0050] (1) Precipitation, dissolution and pretreatment of component four

[0051] Weigh 50 g of the precipitate of component 4 (wet weight, containing diatomaceous earth), dissolve it in 450 g of acetate buffer solution (0-2°C), adjust the pH value to about 6.0, and stir well to dissolve it.

[0052] (2) centrifugation to obtain Apoa-I precipitate

[0053] Add sodium chloride to the suspension to make the concentration reach about 6%, then adjust the pH value to about 4.8, and cool down to about -3°C. Then use Eppendorf centrifuge 5804R high-speed centrifugation (9000rpm), collect about 40g of the precipitate rich in Apoa-I, and discard the supernatant.

[0054] (3) Redissolve Apoa-I precipitation

[0055] Fully dissolve 40 g of Apoa-I precipitate in 360 g of acetate buffer solution at about 30°C, and then filter with a 0.45 μm filter membrane.

[0056] (4) S / D processing

[0057] Add Tween 80 and TNBP to the filtr...

Embodiment 2

[0069] Embodiment 2, Apoa-I small test sample preparation

[0070] (1) Precipitation, dissolution and pretreatment of component four

[0071] Weigh 50 g of the precipitate of component 4 (wet weight, containing diatomaceous earth), dissolve it in 450 g of tris buffer solution (28-30° C.), adjust the pH to about 10.0, and stir well to dissolve it.

[0072] (2) centrifugation to obtain Apoa-I precipitate

[0073] Add sodium chloride to the suspension to make the concentration reach about 0.2%, then adjust the pH value to about 5.8, and cool down to about 20°C. Then use Eppendorf centrifuge 5804R high-speed centrifugation (9000rpm), collect about 40g of the precipitate rich in Apoa-I, and discard the supernatant.

[0074] (3) Redissolve Apoa-I precipitation

[0075] Fully dissolve 40 g of Apoa-I precipitate in 360 g of tris buffer at about 5°C, and then filter with a 0.45 μm filter membrane.

[0076] (4) S / D processing

[0077] Add Tween 80 and TNBP to the filtrate and incub...

Embodiment 3

[0088] Embodiment 3, Apoa-I small test sample preparation

[0089] (1) Precipitation, dissolution and pretreatment of component four

[0090] Weigh 50 g of the precipitate of component 4 (wet weight, containing diatomaceous earth), dissolve it in 450 g of phosphate buffer (14-16°C), adjust the pH to about 8.5, and stir well to dissolve it.

[0091] (2) centrifugation to obtain Apoa-I precipitate

[0092] Add sodium chloride to the suspension to make the concentration reach about 1%, then adjust the pH value to about 6.50, and cool down to about 0°C. Then use Eppendorf centrifuge 5804R high-speed centrifugation (9000rpm), collect about 40g of the precipitate rich in Apoa-I, and discard the supernatant.

[0093] (3) Redissolve Apoa-I precipitation

[0094] Fully dissolve 40 g of Apoa-I precipitate in 360 g of phosphate buffer at about 15°C, and then filter with a 0.45 μm filter membrane.

[0095] (4) S / D processing

[0096] Add Tween 80 and TNBP to the filtrate and incubate...

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Abstract

The invention discloses a production technology of high-pure apolipoprotein Apoa-I from fourth deposit of a blood plasma component. The production technology is to prepare high-pure Apoa-I from fourth deposit of the blood plasma component through one step of centrifugation and one step of ion column chromatography. The production technology provided by the invention has advantages of simple equipment, convenient operation, few steps, short production period and high protein recovery rate, and is suitable for industrial large-scale production.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to a production process for preparing high-purity apolipoprotein Apoa-I from plasma component IV precipitation. Background technique [0002] Apolipoprotein a-I (Apolipoprotein a-I, referred to as Apoa-I) is the main apolipoprotein of high-density lipoprotein (High Density Lipoprotein, HDL), a single polypeptide chain, composed of 243 amino acid residues, and a molecular weight of 28.3kD. The main function of HDL is to participate in reverse cholesterol transport (Reverse Cholesterol Transport, RCT), moving cholesterol in peripheral tissue cells and transporting it to the liver for transformation and elimination, so it plays an important role in combating the occurrence and development of atherosclerosis (AS). And Apoa-I is the main bearer of HDL's anti-AS function. At the same time, Apoa-I also has anti-inflammatory and anti-endotoxin functions, so it is one of the focuse...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K1/18C07K1/14
Inventor 黄凯何秋李春洲陆晖李军辉包骧飞
Owner SHANGHAI RAAS BLOOD PRODUCTS CO LTD
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