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Method for purifying alpha1-antitrypsin from Cohn component IV precipitate

An anti-trypsin and albumin technology, which is applied in the field of protein purification, can solve the problems of unsuitable process production and cumbersome preparation methods, and achieve the effects of simplifying the production process, reducing ultrafiltration and concentration steps, and ensuring the safety of viruses

Active Publication Date: 2017-01-25
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the introduction of affinity chromatography into the above-mentioned AAT purification method can effectively remove albumin, the preparation method is cumbersome and not suitable for process production

Method used

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  • Method for purifying alpha1-antitrypsin from Cohn component IV precipitate
  • Method for purifying alpha1-antitrypsin from Cohn component IV precipitate
  • Method for purifying alpha1-antitrypsin from Cohn component IV precipitate

Examples

Experimental program
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Effect test

Embodiment 1

[0058] a) Dissolving and concentrating Cohn component IV precipitate: 200g Cohn component IV precipitate was dissolved in 2000ml water, stirred at 4°C for 3h, adjusted to pH 7.5, and centrifuged to remove diatomaceous earth to obtain Cohn component IV solution (electrophoretic diagram See figure 2 ). The solution of Cohn component IV was concentrated by ultrafiltration to 4 times the volume to 500ml to obtain the concentrated solution of Cohn component IV with a protein content of 20-22mg / ml.

[0059] b), PEG precipitation: adjust the pH value of the Cohn component IV concentrate to 5.0, add 50 g of PEG4000 while stirring at 4°C to a final concentration of 10% (w / v, g / 100ml), and continue stirring for 30 -45min to fully dissolve PEG4000; stand at 4°C for 30-45min, centrifuge at 6000g for 20min, discard the precipitate, and take the supernatant; the precipitate includes macromolecular (molecular weight > 180kDa) miscellaneous proteins, transferrin, albumin and vitamin D bind...

Embodiment 2

[0073] a) Dissolving and concentrating Cohn component Ⅳ precipitation: 1.2kg Cohn component Ⅳ precipitation was dissolved in 10.8L water, stirred at 0°C for 4h, adjusted to pH 7.8 with 0.5M NaOH, and used a filter press for solid-liquid separation to remove silicon Celite to obtain Cohn component IV solution. The solution of Cohn component IV was concentrated by ultrafiltration to 7 times volume to 2L to obtain the concentrated solution of Cohn component IV with a protein content of 29-31 mg / ml.

[0074] b), PEG precipitation: Cohn component IV concentrate was adjusted to pH 5.2 with 0.5M HCl, and 300g PEG 4000 was added while stirring at 0°C to a final concentration of 15% (w / v, g / 100ml). Continue to stir for 30-45min to fully dissolve PEG4000 after completion; stand at 0°C for 30-45min, centrifuge at 4000g for 30min, discard the precipitate, and take the supernatant.

[0075] c), one-time virus inactivation: S / D method virus inactivation, the supernatant is adjusted to pH 7.0...

Embodiment 3

[0087] a) Dissolving and concentrating Cohn component Ⅳ precipitate: 4kg Cohn component Ⅳ precipitate was dissolved in 32L water, stirred at 10°C for 2h, adjusted to pH 8.0 with 0.5M NaOH, and removed diatomaceous earth by solid-liquid separation using a filter press , to obtain Cohn component IV solution. The solution of Cohn fraction IV was concentrated by ultrafiltration to 10 times volume to 3.6 L to obtain the concentrated solution of Cohn fraction IV with a protein content of 38-40 mg / ml.

[0088] b), PEG precipitation: the Cohn component IV concentrate was adjusted to pH 5.5 with 0.5M HCl, and 432g PEG 4000 was added while stirring at 10°C to a final concentration of 12% (w / v, g / 100ml). Continue to stir for 3-4 hours to fully dissolve PEG 4000; stand overnight at 10°C, centrifuge at 4000g for 60 minutes, discard the precipitate, and take the supernatant.

[0089] c), one-time virus inactivation: S / D method virus inactivation, the supernatant is adjusted to pH 7.5 with ...

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Abstract

The invention discloses a method for purifying alpha1-antitrypsin from a Cohn component IV precipitate. The method comprises the following steps of sequentially carrying out dissolution and concentration, PEG precipitation, primary viral inactivation, primary ultrafiltration and concentration, thrice chromatography refining, freeze-drying, and secondary viral inactivation on the Cohn component IV precipitate, thus finally obtaining the alpha1-antitrypsin. According to the method provided by the invention, the Cohn component IV precipitate which is a waste of albumin produced by a low-temperature alcohol method is adopted as a starting material for preparing AAT, so that the comprehensive utilization ratio of raw blood plasma is favorably improved. During the whole process of the method, PEG precipitation is combined with anion exchange chromatography and twice blue dyestuff affinity chromatography, so that the impurity protein can be effectively removed, an AAT preparation with the purity being larger than 95 percent is finally obtained, and the specific activity is larger than 3,900U / mg.

Description

technical field [0001] The invention relates to the technical field of protein purification, in particular to a method for purifying α1-antitrypsin from Cohn component IV precipitation. Background technique [0002] α1-antitrypsin (alpha 1-antitrypsin, AAT) is a single-chain glycoprotein composed of 394 amino acids and 3 oligosaccharide side chains, with a molecular weight of 52kDa, an isoelectric point of 4.8, and an active site located at positions 358-359 Met-Ser region. 70-80% of AAT is synthesized and secreted by liver cells. In addition, monocytes, macrophages, and alveolar cells can also produce a small amount of AAT. [0003] The main physiological function of AAT is to maintain the balance of protease-anti-protease system and protect normal tissues from enzymatic damage. The concentration of AAT in normal human plasma is 1.0-2.0mg / ml. When it is lower than 0.5mg / ml, it is difficult to resist the release of neutrophil elastase (neutrophil elastase, NE), which can d...

Claims

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Application Information

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IPC IPC(8): C07K14/81C07K1/36C07K1/34C07K1/22C07K1/18
CPCC07K14/8125
Inventor 章金刚张晋超赵雄吕茂民马玉媛皇甫超济贾俊婷
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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