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Method for preparing antithrombin

An antithrombin and heparin technology, applied in the field of plasma protein, achieves the effects of small operation volume, low cost, and improved effectiveness and safety

Active Publication Date: 2013-03-20
CHENGDU RONGSHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, Heparin-Sepharose in-situ virus inactivation and in-place cleaning (CIP&SIP) have certain problems, so there are limitations in industrial applications

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1 Preparation of antithrombin of the present invention

[0056] a. Precipitate dissolution: 0.3kg Kistler and Nitschmann method component IV precipitate was dissolved in 2.4L 0.005M sodium citrate + 0.01M disodium hydrogen phosphate pH 8.1 buffer solution, stirred for 1 hour, then heated at 40°C for 1 hour , stirred for 4 hours. The whole dissolution process takes 6 hours. The pellet was discarded and 2% Celite was added to the supernatant. Concentrate to 0.4L by ultrafiltration after pressure filtration. Concentrate protein concentration 5.3%;

[0057] b. PEG precipitation: Cool the FIV precipitation solution to 5°C and add PEG4000 to 6% (g / ml). After stirring for 1 hour, adjust the pH to 5.95 with 1N HCl and filter, and adjust the pH of the supernatant to pH 7 with 0.5N NaOH .35;

[0058] c. S / D virus inactivation: use 1% Tween80 / 0.3% TNBP at 24±1°C for 6 hours. After the end, it was diluted with an equal volume of buffer solution. In this example, 0....

Embodiment 2

[0068] Embodiment 2 Preparation of antithrombin of the present invention

[0069] a. Precipitate dissolution: 0.3kg Kistler and Nitschmann method component IV precipitate was dissolved in 3.6L 0.005M sodium citrate + 0.01M disodium hydrogen phosphate pH 9.0 buffer solution, stirred for 1 hour, then heated at 40°C for 1 hour , stirred at room temperature 16-25°C for 14 hours. Discard the precipitate, add 2% Celite to the supernatant; press filter and concentrate to 0.5L by ultrafiltration, the protein concentration of the concentrate is 5.8%;

[0070] b. PEG precipitation: Cool the FIV precipitation solution to 5°C and add PEG4000 to 10% (g / ml). After stirring for 1 hour, adjust the pH to 6.05 with 1N HCl and filter, and adjust the pH of the supernatant to pH 7 with 0.5N NaOH .45;

[0071] c. S / D virus inactivation: use 1% Tween80 / 0.3% TNBP at 24±1°C for 6 hours. Dilute the suspension with an equal volume of buffer after completion. In this example, 0.0025M sodium citrate +...

Embodiment 3

[0081] Embodiment 3 Preparation of antithrombin of the present invention

[0082] a. Precipitate dissolution: 0.3kg Kistler and Nitschmann method component IV precipitate was dissolved in 3.0L 0.005M sodium citrate + 0.01M disodium hydrogen phosphate pH 9.0 buffer solution, stirred for 1 hour, then heated at 40°C for 1 hour , stirred at room temperature 16-25°C for 14 hours. The whole dissolution process is 16 hours;

[0083] b. PEG precipitation: Cool the FIV precipitation solution to 5°C and add PEG4000 to 10% (g / ml). After stirring for 1 hour, adjust the pH to 6.05 with 1N HCl and filter, and adjust the pH of the supernatant to pH 7 with 0.5N NaOH .45;

[0084] c. S / D virus inactivation: use 1% Tween80 / 0.3% TNBP at 24±1°C for 6 hours. Dilute with an equal volume of buffer to stop. In this example, 0.0025M sodium citrate + 0.005M disodium hydrogen phosphate pH 7.45, about 3L was used to terminate the dilution;

[0085] d. Adjust the pH, the conductance is consistent wit...

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Abstract

The invention provides a method for preparing antithrombin, which comprises the following steps of: (a) precipitation and dissolution; (b) PEG precipitation; (c) virus inactivation; (e) demodulation; (h) adsorption of residual heparin; (i) virus inactivation; and (j) bacterium removal sub-packing and freeze-drying. Through the invention, the hard silica gel matrix (HeparinHyperD) with higher chemical stability is subjected to heparin affinity chromatography; the matrix has better ability against alkali resistance, and is more suitable for needs of industrial production; and the inventor improves the purification technology and the virus inactivation removal and conducts organic combination to expect deep comprehensive utilization of blood plasma and improve the effectiveness and safety of the AT-III preparation, thereby providing a new option to clinical treatment.

Description

technical field [0001] The invention relates to a method for preparing human antithrombin by precipitation of component IV of the improved K-N method, belonging to the field of plasma proteins. Background technique [0002] Human antithrombin (Human Antithrombin-Ⅲ, AT-Ⅲ) is an important anticoagulant substance in the body. It is a single-chain α2 glycoprotein with a molecular weight of 58KD. It consists of 432 amino acids and contains 3 disulfide bonds and 4 oligosaccharide side chains, polysaccharide content of about 15%. Distributed in the kidneys, lungs, especially liver vascular endothelial cells in the body, the normal plasma level of AT-Ⅲ is 80-300mg / L, and its activity is 70%-130%. The active site of AT-Ⅲ and serine protease is located at Arg393-Ser394. The protease attacks the bond to cleave it and cause AT-Ⅲ allosteric, thereby forming a 1:1 complex between AT-Ⅲ and the enzyme. This covalent binding Is irreversible and can be greatly enhanced by heparin or heparan...

Claims

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Application Information

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IPC IPC(8): C07K14/81C07K1/36C07K1/30C07K1/22
CPCC07K1/30C07K1/36C07K1/34C07K1/18C07K14/81
Inventor 吴强蔡骏廖颖初毅波胡晓东
Owner CHENGDU RONGSHENG PHARMA
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