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Production process for preparing high-purity ApoA-I (Apolipoprotein A-I) from precipitates of plasma fraction IV

A technology of plasma component four and pH value, which is applied in the field of production technology for preparing high-purity apolipoprotein ApoA-I, can solve the problems of loss of ApoA-I, unfavorable safe production, small preparation amount, etc., and achieves safe and convenient operation. Effect

Active Publication Date: 2011-07-20
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 chromatography, etc. Although high-purity ApoA-I can be obtained, it has some obvious shortcomings: 1. The preparation amount is small and not suitable for industry Production; ②The protein yield is low, after multi-step treatment such as ultracentrifugation, organic solvent precipitation, column chromatography, etc., most of the ApoA-I is lost in the preparation process; ③The cost is high, and expensive instruments such as ultracentrifuges are required; ④Safety Poor performance, ethanol, acetone, trichloroacetic acid, urea and other organic solvents are not only physiologically toxic, but also flammable and explosive, which is not conducive to safe production
At present, there is no relevant report on the preparation of ApoA-I from plasma fraction IV

Method used

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  • Production process for preparing high-purity ApoA-I (Apolipoprotein A-I) from precipitates of plasma fraction IV
  • Production process for preparing high-purity ApoA-I (Apolipoprotein A-I) from precipitates of plasma fraction IV
  • Production process for preparing high-purity ApoA-I (Apolipoprotein A-I) from precipitates of plasma fraction IV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: ApoA-I sample preparation

[0036] (1) Plasma component four precipitation dissolution and pretreatment

[0037] Weigh 5 kg (wet weight) of component 4 precipitate, dissolve in 45 kg of sodium acetate buffer solution (control temperature 0-8°C), adjust the pH value to about 9.00, stir well to dissolve it, and then centrifuge it with Beckmann machine (7000rpm) to remove diatomaceous earth and insoluble matter, and collect the centrifuged supernatant.

[0038] (2) centrifugation to obtain ApoA-I precipitate

[0039] Sodium chloride is added to the supernatant to make the concentration reach about 2wt%, and then the pH value is adjusted to 6.0-6.5, and the temperature is lowered to -1-1°C to allow ApoA-I to coagulate and precipitate. Centrifuge at high speed, collect about 500 g of ApoA-I precipitate, and discard the supernatant.

[0040] (3) Redissolve ApoA-I precipitation

[0041] Fully dissolve 500 g of ApoA-I precipitate in 5 kg of 2 wt % sodium chlor...

Embodiment 2

[0051] ApoA-I sample preparation

[0052] (1) Plasma component four precipitation dissolution and pretreatment

[0053] Weigh 3 kg (wet weight) of the precipitate of component four, dissolve it in sodium acetate buffer (control temperature: 0-8°C), adjust the pH value to 8.00, stir well to dissolve it, and then centrifuge at 7000rpm to remove diatomaceous earth and Insoluble matter was collected by centrifugation supernatant.

[0054] (2) centrifugation to obtain ApoA-I precipitate

[0055] Sodium chloride is added to the supernatant to make the concentration reach 1wt%, then the pH value is adjusted to 6.0-6.5, and the temperature is lowered to -1-1° C. to allow ApoA-I to coagulate and precipitate. Centrifuge at high speed, collect about 300 g of ApoA-I precipitate, and discard the supernatant.

[0056] (3) Redissolve ApoA-I precipitation

[0057] Fully dissolve 300 g of the ApoA-I precipitate in 1 wt% sodium chloride solution at 0° C., and then filter with a 0.45 μm filt...

Embodiment 3

[0062] ApoA-I sample preparation

[0063] (1) Plasma component four precipitation dissolution and pretreatment

[0064] Weigh 6 kg (wet weight) of the precipitate of component four, dissolve it in sodium acetate buffer solution (control temperature is 0-8°C), adjust the pH value to 10.00, stir well to dissolve it, and then remove diatomaceous earth by centrifugation at 7000rpm and insoluble matter, and the centrifuged supernatant was collected.

[0065] (2) centrifugation to obtain ApoA-I precipitate

[0066] Sodium chloride is added to the supernatant to make the concentration reach 3wt%, and then the pH value is adjusted to 6.0-6.5, and the temperature is lowered to -1-1° C. to allow ApoA-I to coagulate and precipitate. Centrifuge at high speed, collect about 600 g of ApoA-I precipitate, and discard the supernatant.

[0067] (3) Redissolve ApoA-I precipitation

[0068] Fully dissolve 600 g of ApoA-I precipitate in 3 wt% sodium chloride solution at about 10° C., and then ...

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Abstract

The invention discloses a method for preparing high-purity ApoA-I (Apolipoprotein A-I) from a plasma fraction IV through purification, comprising the following steps of: dissolving the participates of the plasma fraction IV, centrifuging to remove diatomite and impurities, and collecting a supernate; adding sodium chloride to the supernate so that ApoA-I protein is coagulated and separated out, and centrifuging to obtain an ApoA-I precipitate; redissolving the precipitate and filtrating; and enabling the filtrate to be sequentially subjected to anion column chromatography and hydrophobic column chromatography and separating to obtain a high-purity ApoA-I solution. The ApoA-I prepared by adopting the process disclosed by the invention has high purity capable of reaching more than 95 percent and the ApoA-I yield capable of reaching 70 percent and is safe and convenient to operate, easy to realize process amplification and very suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular 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, ApoA-I) is the main apolipoprotein of high-density lipoprotein (High Density Lipoprotein, HDL), which is a single polypeptide chain consisting 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 th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/775C07K1/36A61P9/10
CPCC07K14/775A61P9/10
Inventor 黄凯何秋许必雄李春洲李军辉沈积慧郭颀然
Owner SHANGHAI RAAS BLOOD PRODUCTS CO LTD
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