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Method for preparing alpha 1-antitrypsin

An anti-trypsin and low-temperature ethanol method technology is applied in the preparation methods of peptides, protease inhibitors, chemical instruments and methods, etc., which can solve the problems of insufficient virus inactivation, long operation time, and high equipment requirements, and save equipment. The effect of investment, simplified process and less dosage

Active Publication Date: 2013-03-27
CHENGDU RONGSHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are some manufacturers and researchers in the world, such as Burnouf et al: "Biochemical and Biological Properties of anα1-antitrypsin Concentrate" VoxSang 52:291-297 (1987) using Kistler and Nitschmann method of component A or Component A+I supernatant is separated and purified by chromatographic column for α1-antitrypsin. This method has high requirements for equipment, large-scale chromatographic system and supporting facilities, long operation time and insufficient virus inactivation. and other defects

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 The preparation method of anti-α1-antitrypsin of the present invention

[0042] a. Precipitation dissolution: 3kg Kistler and Nitschmann method component IV precipitate was dissolved in 24L 0.02MPBS pH 8.1 buffer solution, stirred for 1 hour, heated and stirred at 40°C for 1 hour, then stirred at room temperature for 14 hours. The whole dissolution process was 16 hours, the precipitate was discarded, and 2% Celite was added to the supernatant. After pressure filtration, it was concentrated to 4L by ultrafiltration, and the protein concentration was 5.1%.

[0043] b. PEG precipitation: Cool the FIV precipitation solution to 5°C, add PEG4000 to 6% (g / ml), stir for 1 hour, adjust the pH to 5.05 with 1N HCl, add 1.5% of 2% Celite, and use pressure filtration , was filtered, and the pH of the filtrate was adjusted to pH7.05 with 0.5N NaOH.

[0044] c. S / D virus inactivation: use 1% Tween80 / 6%TNBP, 24±1°C, 6 hours, 24±1°C, 6 hours. After the S / D virus is inact...

Embodiment 2

[0053] Embodiment 2 The preparation method of anti-α1-antitrypsin of the present invention

[0054] a. Precipitation dissolution: 3kg Kistler and Nitschmann method component IV precipitation was dissolved in 36L 0.03MPBS pH 9.0 buffer solution, stirred for 1 hour, heated at 40°C for 1 hour, then stirred at room temperature for 4 hours. The whole dissolution process takes 6 hours. The pellet was discarded and 3% Celite was added to the supernatant. After pressure filtration, it was concentrated to 5L by ultrafiltration, and the protein concentration was 6.0%.

[0055] 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 5.35 with 1N HCl, and add 1.5-2% of the filter aid. Filtration was performed, and the pH of the filtrate was adjusted to pH 7.01 with 0.5N NaOH.

[0056] c. S / D virus inactivation: use 1% Tween80 / 0.3% TNBP at 24±1°C for 6 hours. After the S / D virus is inactivated, it is di...

Embodiment 3

[0065] Embodiment 3 The preparation method of α1-antitrypsin of the present invention

[0066] a. Precipitation dissolution: Dissolve 3kg of Kistler and Nitschmann method component IV precipitation in 30L 0.03MPBS pH 8.5 buffer solution, stir, disperse and suspend for 1 hour, then heat at 40°C for 1 hour, then continue at room temperature for 16-25 °C for 4 hours.

[0067] b. PEG precipitation: Cool the FIV precipitation solution to 5°C, add PEG4000 to 9% (g / ml), stir for 1 hour, adjust the pH to 5.10 with 1N HCl, add 2% filter aid, and use pressure filtration , was filtered, and the pH of the filtrate was adjusted to pH 7.00 with 0.5N NaOH.

[0068] c. S / D virus inactivation: use 1% Tween80 / 0.3% TNBP at 24±1°C for 6 hours. After the S / D virus is inactivated, it is terminated by dilution with an equal volume of PBS buffer, in this example, about 30 L of 0.015M PBS pH6.5 is used for termination.

[0069] d. Adjust the pH, the conductance is consistent with the balanced DEAE-...

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Abstract

The invention provides a method for preparing alpha 1-antitrypsin. The method for preparing alpha 1-antitrypsin comprises the following steps of: a. precipitating and dissolving; b. precipitating PEG (polyethylene glycol); c. inactivating viruses; d. purifying; e. ultra-filtering; f. purifying; g. ultra-filtering and dialyzing; and i. degerming and subpackaging, and freezing to be dry. By the method provided by the invention, alpha 1-antitrypsin is prepared from waste component precipitated FIV (feline immunodeficiency virus) by adopting an improved Kistler and Nitschmann method technology, not only is the long-time puzzled difficulty of alpha 1-antitrypsin preparation by a K-N technology or the improved technology thereof solved, the comprehensive utilization of blood plasma resources is realized, and the method has no interference influence to the blood plasma protein main body separation technology; the PEG4000 using amount is less; the operation volume is small; a centrifugal device is not used; the technology is simplified; device input is saved; and the method provided by the invention has a favorable application prospect.

Description

technical field [0001] The invention relates to a method for preparing human α1-antitrypsin by using improved K-N method component IV precipitation, which belongs to the field of plasma protein. Background technique [0002] Alpha-1-antitrypsin (Alpha-1 antitrypsin AAT). Molecule is composed of 394 amino acids, glycoprotein with a molecular weight of 52000, exists in two forms of natural activity and denatured inactivity, produced by the liver, the main function is to inhibit the activity of trypsin and elastase of neutrophils, and can also inhibit Chymotrypsin, plasmin, etc., are one of the most powerful enzyme inhibitors in the human body. Congenital deficiency of AAT such as gene mutation or acquired factors such as long-term smoking or bacterial infection will cause the inactivation of AAT active center, which will lead to functional deficiency of AAT and then cause the imbalance of protease and anti-protease system in the body, and the ability of lung tissue to resist ...

Claims

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Application Information

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IPC IPC(8): C07K14/81C07K1/36C07K1/34C07K1/30C07K1/18
CPCC07K1/30C07K1/36C07K1/34C07K14/8125C07K1/18C07K14/81
Inventor 蔡骏李泽林刘波初毅波邓红吴强
Owner CHENGDU RONGSHENG PHARMA