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Preparation method for human antithrombin III

An antithrombin, gel technology, applied in the direction of protease inhibitors, animal/human peptides, peptide sources, etc., can solve the problem of low AT-III production qualification rate, lack of targeted treatment measures, lack of removal measures, etc. problems, to achieve the effect of improving the safety of clinical use, reducing the probability of protein activation, and reducing the load

Inactive Publication Date: 2016-02-10
上海洲跃生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is not much research on AT-III in China, and there is no AT-III product available at present, and there is a lack of targeted treatment measures for various diseases caused by AT-III deficiency; in recent years, there have been sporadic research reports, such as patent CN103059129A
The conventional preparation process has a low yield, and due to the lack of measures to remove vitamin K-dependent coagulation factors, it is easy to cause protein activation during the preparation process, resulting in a low pass rate of AT-III production

Method used

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  • Preparation method for human antithrombin III
  • Preparation method for human antithrombin III

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. Put 3kg of component IV precipitate into 30kg buffer solution, the buffer solution composition is 0.02M HCL-TRIS, 0.15M sodium chloride, pH7.50-7.60, the temperature is controlled at 10°C, and stirred for 6 hours to fully dissolve ;

[0044] 2. Add polyethylene glycol to the above suspension to 6%, stir for 0.5 hours, then filter with a Supradur50P filter plate produced by Pall in series with a 0.45 μm filter element, collect the clarified filtrate, and pre-wash the filter plate with dissolution buffer before filtering and filter elements;

[0045] 3. Add pre-swelled and balanced DEAESephadexA-50 gel to the above filtrate, add 33g according to the dry gel, the buffer solution is composed of 0.02MTRIS-HCL, 0.15M sodium chloride, pH6.50-6.50; slowly stir for 1.5 hours, Stand still for 5 minutes; Then filter and collect the filtrate;

[0046] 4. Add Tween80 to 1.0% (wt%) and TNBP (tributyl phosphate) to 0.3% (wt%) to the above filtrate, stir well, heat up to 24-26°C, ke...

Embodiment 2

[0055] 1. Put 3kg of component IV precipitate into 60kg of buffer solution, the buffer solution is composed of 0.01M HCL-TRIS, 0.15M sodium chloride, pH8.50-8.60, the temperature is controlled at 20°C, and stirred for 4 hours to fully dissolve it ;

[0056] 2. Add polyethylene glycol to the above suspension to 3%, stir for 1.5 hours, then filter with a Supradur50P filter plate produced by Pall in series with a 0.45 μm filter element, collect the clarified filtrate, and pre-wash the filter plate with dissolution buffer before filtering and filter elements;

[0057] 3. Add pre-swelled and balanced DEAESephadexA-50 gel to the above filtrate, add 33g according to the dry gel, the buffer solution is composed of 0.01MTRIS-HCL, 0.15M sodium chloride, pH6.90-7.10; slowly stir for 1 hour, After standing for 10 minutes; then filter and collect the filtrate;

[0058] 4, with embodiment one;

[0059] 5. Put the above filtrate on the HeparinSepharose6FF chromatography column. The column...

Embodiment 3

[0064] 1. Put 3kg of component IV precipitate into 45kg buffer solution, the buffer solution composition is 0.02M HCL-TRIS, 0.1M sodium chloride, pH9.40-9.50, the temperature is controlled at 25°C, and stirred for 2.5 hours to fully dissolve it ;

[0065] 2. Add polyethylene glycol to the above suspension to 4.5%, stir for 1 hour, then filter with a Supradur50P filter plate produced by Pall in series with a 0.45 μm filter element, collect the clarified filtrate, and pre-wash the filter plate with dissolution buffer before filtering and filter elements;

[0066] 3. Add pre-swelled and balanced DEAESephadexA-50 gel to the above filtrate, add 36g according to the dry gel, the buffer solution is composed of 0.02MTRIS-HCL, 0.1M sodium chloride, pH7.40-7.50; slowly stir for 0.5 hours, Stand still for 15 minutes; Then filter and collect the filtrate;

[0067] 4, with embodiment one;

[0068] 5. Put the above filtrate on the HeparinSepharose CL-6B chromatography column. The column ...

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Abstract

The invention discloses a preparation method for human antithrombin III (AT-III). The preparation method comprises the following steps: (1) precipitation and dissolution of a blood plasma fraction IV; (2) polyethylene glycol (PEG) precipitation and impure protein removing; (3) DEAE Sephadex A-50 gel adsorption; (4) S / D virus inactivation; (5) heparin affinity column chromatography; (6) ultrafiltration and concentration; (7) addition of a stabilizer and regulation; (8) nanofilm virus-removing filtration; (9) sterilizing filtration; (10) freeze-drying; (11) dry and fever virus inactivation. According to the preparation method, blood coagulation factors, which remain in the raw materials and are depended by vitamin K, are removed, so as to greatly lower the possibility of protein activation in the production process, and improve the product qualification ratio; PEG is adopted for the impure protein removing, so as to reduce the load of a chromatographic column; a liquid obtained from lyophilized powder re-dissolution is clear, transparent, and free of protein precipitation and opalescence; through adoption of a three-step virus inactivation method, the safety of human AT-III for clinical use can be greatly improved.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a preparation method of blood products, in particular to a preparation method of human antithrombin III. Background technique [0002] Antithrombin III (antithrombin III, AT-III) is a vitamin K-dependent single-chain glycoprotein, which is synthesized in the liver, vascular endothelial cells and megakaryocytes. Serine protease inhibitor, molecular mass 58-64KD, isoelectric point 4.8; half-life about 70 hours, plasma content 260-320mg / L; contains 4 N-glycosylated oligosaccharide chains and 3 disulfide bonds , composed of 9 α-helical structures, 3 β-sheets and 1 reaction center loop; AT-Ⅲ forms a complex with serine such as thrombin, FXa, FXIIa, XIa, IXa, plasmin, etc. at a ratio of 1:1 Heparin can accelerate this reaction by more than a thousand times. After heparin combines with the lysine contained in AT-Ⅲ, it will cause the conformation change of AT-Ⅲ, so that the arginine cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81C07K1/36C07K1/34C07K1/30
CPCC07K14/8128
Inventor 李春洲
Owner 上海洲跃生物科技有限公司
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