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Method for separating and purifying alpha 1-antitrypsin from human blood plasma component FIV precipitation

An anti-trypsin, separation and purification technology, applied in the field of separation and purification of α1-antitrypsin, achieves the effects of high yield, less equipment occupancy and low energy consumption

Active Publication Date: 2008-10-01
广东双林生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no production of this product in our country, and the market demand is mainly met by imported products.

Method used

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  • Method for separating and purifying alpha 1-antitrypsin from human blood plasma component FIV precipitation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (1) FIV precipitation pretreatment

[0036] Weigh 3kg of Cohn’s method component precipitation FIV, add 50L of 15mM, pH8.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, stir overnight at 2-8°C, add 3kg of diatomaceous earth, and stir at 2-8°C 1 hour, high-speed centrifugation, the temperature is controlled at 4°C, add 1.5kg of perlite to the filtrate, stir at 2-8°C for 3h, adjust the pH of the solution to 6.2 with 1M HCl, high-speed centrifugation, the temperature is controlled at 4°C, take the supernatant The liquid is the upper column liquid of the chromatography.

[0037] (2) DEAE-Sepharose Fast Flow Anion Exchange Chromatography

[0038]Take out the DEAF-Sepharose Fast Flow gel and place it at room temperature to achieve temperature equilibrium. Take 6L of the gel and pack it into the column, and wash the column with 15mM sodium phosphate buffer, pH 6.0. The volume of the balance solution is 8 times the column volume. Load 10L of the upp...

Embodiment 2

[0053] (1) FIV precipitation pretreatment

[0054] Weigh 3kg of Cohn’s method component precipitated FIV, add 50L of 30mM sodium acetate buffer solution, stir overnight at 2-8°C, add 3kg of diatomaceous earth, stir at 2-8°C for 1 hour, and filter under pressure on the sample plate frame filter. Control the temperature at 4°C, add 1.5kg of perlite to the filtrate, stir at 2-8°C for 3 hours, adjust the pH of the solution to 6.2 with 1M HCl, pressurize and filter the sample plate frame filter, control the temperature at 4°C, and take the supernatant as a layer Analyze the upper column liquid.

[0055] (2) DEAE-Sepharose Fast Flow Anion Exchange Chromatography

[0056] Take out the DEAF-Sepharose Fast Flow gel and place it at room temperature to achieve temperature equilibrium. Take 6L of the gel and pack it into the column, and wash the column with 30mM sodium acetate buffer solution, pH 6.0. The volume of the balance solution is 12 times the column volume. Load 10L of the uppe...

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Abstract

The invention discloses a method for separating and purifying alpha1-antitryptase from the FIV component precipitation of human plasma, which is characterized in that the method comprises the following steps of (A) pretreatment of plasma precipitation; (B) anion gel chromatography; (C) gel-filtration chromatography; (D) ultrafiltration desalting concentration; (E) Baculovirus inactivation; (F) lyophilization and subpackaging; (G) dry heat sterilization. The method takes the FIV component precipitation which is waste material produced from human plasma during the production process of human serum albumin as raw material and adopts chromatography technique to separate and purify a protease inhibitor, namely, alpha 1- antitryptase (alpha 1-AT) and builds the production technique of alpha 1-AT concentrate. Products prepared by the method has good pureness, high yield and simple and easy operation as well as few occupied equipment, little labor intensity, low energy consumption and low production cost due to taking the waste material generated by albumin as raw material.

Description

technical field [0001] The invention relates to a preparation method of α1-antitrypsin, in particular to a method for separating and purifying α1-antitrypsin from human plasma component precipitation. Background technique [0002] α1-AT is mainly a glycoprotein synthesized by liver cells, with a molecular weight of 54KD and a half-life of 6 days in vivo. α1-AT is the most abundant serine protease inhibitor in human plasma, which can inhibit a variety of serine endopeptidases, such as inhibiting the activity of elastase, trypsin, plasmin, collagenase, thrombin and Hageman factor, etc. . α1-AT is the most important antitrypsin in the blood, accounting for more than 90% of serum trypsin inhibitory activity. α1-AT has strong vascular permeability, high concentration in lung tissue, and is more specific to elastase activity. Its main physiological function is to inhibit the activity of lung elastase and protect the lungs from elastase. enzymatic damage. [0003] The diseases ...

Claims

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Application Information

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IPC IPC(8): C07K1/18C07K1/34C07K1/14C07K14/81
Inventor 朱光祖吕应年
Owner 广东双林生物制药有限公司
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