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

A technology of blood plasma component IV and buffer solution, which is applied in the production process field of preparing high-purity apolipoprotein ApoA-I, can solve the problems of loss of ApoA-I, unfavorable safety production, small preparation amount, etc., and achieve safe and convenient operation Effect

Active Publication Date: 2013-09-04
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] Example 1: ApoA-I sample preparation

[0036] (1) Four precipitation dissolution and pretreatment of plasma components

[0037] Weigh 5 kg (wet weight) of component 4, dissolve it in 45 kg of sodium acetate buffer (control temperature is 0~8℃), adjust the pH to about 9.00, stir well to dissolve, and then centrifuge with Beckman The machine (7000rpm) removes diatomaceous earth and insoluble materials, and collects the centrifugal supernatant.

[0038] (2) Centrifugation to obtain ApoA-I precipitation

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

[0040] (3) Reconstituted ApoA-I precipitation

[0041] 500g of ApoA-I precipitate was fully dissolved in 5kg of 2wt% sodium chloride solution at about 5°C, and then...

Embodiment 2

[0051] ApoA-I sample preparation

[0052] (1) Four precipitation dissolution and pretreatment of plasma components

[0053] Weigh 3 kg (wet weight) of component four, dissolve it in sodium acetate buffer (control the temperature at 0~8℃), adjust the pH to 8.00, stir well to dissolve it, and then centrifuge at 7000rpm to remove the diatomaceous earth and Insoluble matter, collect the centrifuged supernatant.

[0054] (2) Centrifugation to obtain ApoA-I precipitation

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

[0056] (3) Reconstituted ApoA-I precipitation

[0057] 300 g of ApoA-I precipitate was fully dissolved in a 1 wt% sodium chloride solution at 0°C, and then filtered with a 0.45 μm filter membrane.

[0058] (4) ...

Embodiment 3

[0062] ApoA-I sample preparation

[0063] (1) Four precipitation dissolution and pretreatment of plasma components

[0064] Weigh 6 kg (wet weight) of component four, dissolve it in sodium acetate buffer (control temperature is 0~8℃), adjust the pH to 10.00, stir well to dissolve, and then centrifuge at 7000rpm to remove diatomaceous earth And insoluble matter, collect the centrifuge supernatant.

[0065] (2) Centrifugation to obtain ApoA-I precipitation

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

[0067] (3) Reconstituted ApoA-I precipitation

[0068] Dissolve 600 g of ApoA-I precipitate in a 3 wt% sodium chloride solution at about 10° C., and then filter with a 0.45 μm filter membrane.

[0069] (4) Column chromato...

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Abstract

The present invention provides a method for preparing high purity ApoA-I from the precipitate of plasma fraction IV, which including the following steps: dissolving the precipitate of plasma fraction IV, removing the diatomite and impurities by centrifugation, and collecting the supernatant; adding sodium chloride to said supernatant to precipitate ApoA-I protein, and obtaining ApoA-I precipitate by centrifugation; redissolving ApoA-I precipitate and filtrating it; separating the filtrate from the above procedure first by anion column chromatography then by hydrophobic column chromatography to obtain high purity ApoA-I solution.

Description

Technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to a production process for preparing high-purity apolipoprotein ApoA-I from four precipitations of plasma components. Background technique [0002] Apolipoprotein A-I (ApoA-I) is the main apolipoprotein of High Density Lipoprotein (HDL). It is a single polypeptide chain composed of 243 amino acid residues and has a molecular weight of 28.3kD. The main function of HDL is to participate in the reverse cholesterol transport (Reverse Cholesterol Transport, RCT), remove the cholesterol in the peripheral tissue cells and transport it to the liver for transformation and clearance, so it has an important role in the occurrence and development of atherosclerosis (AS) ApoA-I is the main bearer of the anti-AS function of HDL. At the same time, ApoA-I also has anti-inflammatory and anti-endotoxin functions, so it is one of the focuses of lipid metabolism research. In additio...

Claims

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Application Information

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Patent Type & Authority Patents(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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