Method for purifying plasma functional proteins from Cohn fraction IV

A technology with four components and functions, applied in the field of protein preparation, can solve the problems of being unsuitable for green production, long production cycle, and numerous steps, and achieve the effect of easy industrial automatic production, high purity and activity, and short purification cycle

Inactive Publication Date: 2017-01-04
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In a complete production cycle, the chromatography method is used many times, the steps are numerous, the production cycle is long, and it is not suitable for industrial scale-up
In addition, more chromatography is used, which not only reduces the efficiency of the separation column, but also has a poor concentration effect on the sample, which increases the production cost and prolongs the production cycle.
And it does not selectively separate the medicinal value plasma protein in Cohn component 4, which is not suitable for the concept of "green production" in industrial production

Method used

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  • Method for purifying plasma functional proteins from Cohn fraction IV
  • Method for purifying plasma functional proteins from Cohn fraction IV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Dissolve 50 g of Cohn component four solids in 100 mL of Tris buffer (pH 7.5-8.0) and fully dissolve. Then, centrifuge at 10000 rpm for 20 min at 0° C. to remove the filter aid and obtain supernatant I. Adjust the pH value of the supernatant I to 2.0, separate the foreign protein to obtain the supernatant II, and use S-SOURCE 30 to pack the column with a column volume of 40 mL. First equilibrate the column with pH 2.0 maleic acid buffer, equilibrate for 10 column volumes, inject supernatant II, carry out gradient elution with pH 2.0 maleic acid buffer with a concentration of 0 to 10M, and collect each column on the map. peak.

Embodiment 2

[0026] Dissolve 50 g of Cohn component four solids in 150 mL of phosphate buffer (pH 7.0 to 7.5) and fully dissolve. Then, centrifuge at 8000 rpm for 25 min at 4°C to remove the filter aid and obtain supernatant I. Adjust the pH value of the supernatant I to 4.0, separate the foreign protein to obtain the supernatant II, and use SP-Sepharose HP to pack the column with a column volume of 40 mL. First equilibrate the column with pH 4.0 formic acid buffer, equilibrate for 10 column volumes, inject the supernatant II, perform gradient elution with pH 4.0 formic acid buffer with a concentration of 0 to 4M, and collect the peaks on the spectrum.

Embodiment 3

[0028] Dissolve 50 g of solid Cohn component four in 120 mL of pH 7.4 phosphate buffer, and dissolve fully. Then, centrifuge at 8000 rpm for 20 min at 4° C. to remove the filter aid and obtain supernatant I. Adjust the pH value of the supernatant I to 7.8, separate the miscellaneous proteins to obtain the supernatant II, and use Q Sepharose to pack the column with a volume of 50 mL. First equilibrate the column with pH 7.8 phosphate buffer, equilibrate for 10 column volumes, inject the supernatant II, perform gradient elution with pH 7.8 phosphate buffer with a concentration of 0 to 2M, and collect the peaks on the spectrum. Chromatogram see figure 2 .

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Abstract

The invention discloses a method for purifying plasma functional proteins from a Cohn fraction IV. The method comprises the following steps: carrying out pretreatment and removing a filter aid and other proteins in the raw materials; carrying out gradient separation by chromatography and collecting the plasma functional proteins in sequence; carrying out sterilization and freeze-drying. The method is high in protein yield and low in cost, comprises the purification process with the pipeline characteristic, has high degree of automation, is suitable for industrialization and achieves comprehensive utilization of the byproduct in plasma product production.

Description

technical field [0001] The invention relates to the field of protein preparation, in particular to a method for comprehensively utilizing Cohn component IV. Background technique [0002] Cohn Fraction IV (Cohn Fraction IV) is a by-product of the conventional industrial plasma protein production process (low temperature ethanol method), and is now usually treated as waste, which contains a variety of functional plasma proteins, such as albumin, apolipoprotein, transgenic ferritin etc. [0003] Apolipoprotein A-I (ApolipoproteinA-I, apoA-I) is an important component of human high-density lipoprotein (high density lipoprotein, HDL), maintains the normal structure of HDL, and has the effect of activating lecithin cholesterol transacylase in the human body. It can also directly act on the arterial wall to promote the outflow of cholesterol from the arterial wall and prevent the occurrence of arteriosclerosis. In addition, apoA-I also has anti-inflammatory and anti-endotoxin fun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/81C07K14/795C07K14/79C07K14/775C07K1/12C07K1/18C07K1/16A61K38/57A61K38/38A61K38/41A61K38/40A61K38/17A61P9/10A61P9/00A61P31/00A61P1/16A61P25/28A61P7/00
CPCC07K14/765A61K38/00C07K14/775C07K14/79C07K14/795C07K14/8128
Inventor 周建平丁杨赵紫强
Owner CHINA PHARM UNIV
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