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Method for extracting human antithrombin III from plasma

An antithrombin and plasma technology, applied in the field of biopharmaceuticals, can solve the problems of human antithrombin III remaining in laboratory development, failing to obtain new drug certificates and production approvals, and late production research, etc., and reduce chromatography. System and supporting facilities and site equipment, good virus filtering effect, high security effect

Pending Publication Date: 2022-04-26
广东双林生物制药有限公司
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  • Abstract
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  • Claims
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Problems solved by technology

[0009] There are also several domestic companies that have conducted in-depth research on the separation and purification of human antithrombin III, which can reach a purity of more than 85% at the laboratory level. Most of the research on human antithrombin Ⅲ is still in the stage of laboratory development, and so far there have been no reports of obtaining new drug certificates and production approval documents. The main reason is that the preparation process suitable for industrial scale has not been established. Therefore, how to transform laboratory research results into processes suitable for industrial production has become the key to industrial development

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  • Method for extracting human antithrombin III from plasma

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Embodiment 1

[0038] (1) Removal of cryoprecipitate from fresh frozen human plasma: take 500L of fresh frozen human plasma, melt and mix evenly, remove cryoprecipitate through a flow centrifuge, and obtain plasma supernatant after centrifugation;

[0039] (2) DEAE Sephadex A-50 gel adsorption: Add DEAE Sephadex A-50 gel to the plasma supernatant obtained in step (1), the amount of dry gel added is 500g, and the dry gel is pre-treated with a temperature above 70°C Swell with hot water for injection, then cool with cold water for injection below 25°C, and finally balance with equilibrium buffer, stir for 1.0 hour at an appropriate speed (to ensure that the gel is evenly mixed and distributed in the solution and not too fast), filter to obtain the filtrate, and condense The gel can be reused after being regenerated; the balance buffer formula is: 0.02M sodium citrate, 0.2M sodium chloride, pH 7.0

[0040] (3) Polyethylene glycol (PEG-4000) purification treatment: adjust the pH of the filtrate ...

Embodiment 2

[0050] (1) Removal of cryoprecipitate from fresh frozen human plasma: take 800L of fresh frozen human plasma, melt and mix evenly, remove cryoprecipitate through a flow centrifuge, and obtain plasma supernatant after centrifugation;

[0051] (2) DEAE Sephadex A-50 gel adsorption: Add DEAE Sephadex A-50 gel to the plasma supernatant obtained in step (1), the amount of dry gel added is 960g, and the dry gel is pre-heated with a temperature above 70°C Swell with hot water for injection, then cool with cold water for injection below 25°C, and finally balance with equilibrium buffer, stir for 1.5 hours at an appropriate speed (to ensure that the gel is evenly mixed and distributed in the solution and not too fast), filter to obtain the filtrate, and condense The gel can be reused after being regenerated; the equilibrium buffer formula is: 0.015M sodium citrate, 0.2M sodium chloride, pH 7.5

[0052] (3) Polyethylene glycol (PEG-4000) purification treatment: adjust the pH of the filt...

Embodiment 3

[0062] (1) Remove cryoprecipitate from fresh frozen human plasma: take 1000L fresh frozen human plasma, melt and mix evenly, remove cryoprecipitate through a flow centrifuge, and obtain plasma supernatant after centrifugation;

[0063] (2) DEAE Sephadex A-50 gel adsorption: Add DEAE Sephadex A-50 gel to the plasma supernatant obtained in step (1). Swell with hot water for injection, then cool with cold water for injection below 25°C, and finally balance with equilibrium buffer, stir for 2 hours at an appropriate speed (to ensure that the gel is evenly mixed and distributed in the solution and not too fast), filter to obtain the filtrate, and condense The gel can be reused after being regenerated; the equilibrium buffer formula is: 0.015M sodium citrate, 0.2M sodium chloride, pH 7.5

[0064] (3) Polyethylene glycol (PEG-4000) purification treatment: adjust the pH of the filtrate obtained in step (2) to 6.5, add 15% (w / v) polyethylene glycol (PEG-4000), and centrifuge to remove ...

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Abstract

The invention discloses a method for extracting human antithrombin III from plasma, and belongs to the field of biological pharmacy. The invention discloses a method for extracting human antithrombin III from cryoprecipitate-removed human plasma. The method comprises the following steps: removing cryoprecipitate from fresh frozen human plasma; carrying out DEAE (Diethylaminoethyl) SephadexA-50 gel adsorption; carrying out purification treatment on polyethylene glycol (PEG-4000); carrying out affinity chromatography; carrying out pasteurization virus inactivation; carrying out affinity chromatography; performing ultrafiltration concentration; sterilizing and filtering; virus removal and filtration with a nano-film; performing split charging; and freezing and drying. By improving the extraction process of the human antithrombin III, the prepared human antithrombin III is good in stability, high in product purity, safe and reliable, the specific activity of the human antithrombin III can reach 13IU / mg protein or above, the market requirements are met, and the utilization value of precious plasma resources is also improved.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to an extraction process of blood products, in particular to an extraction method of human antithrombin III. Background technique [0002] Human antithrombin III (Human Antithrombin III, AT-III) is an important factor that controls the physiological blood coagulation reaction process and plays an anticoagulant effect. It mainly exists in the human plasma system. The content of AT-III in normal human plasma is 180 -300mg / L, mainly synthesized in the liver, but also synthesized in vascular endothelial cells. AT-III is a potent natural anticoagulant and serine protease inhibitor, which can inactivate a variety of enzymes in the coagulation cascade, mainly through the inhibition of thrombin and FXa, and also through It acts through the inhibition of FIXa, FXIa, FXIIa and plasmin. AT-III can exert anticoagulant effect alone in the absence of heparin, but in the presence of heparin, AT-I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81C07K1/14C07K1/22C07K1/34C07K1/36
CPCC07K14/8128
Inventor 庾昌文殷如胡川杨波波陈相
Owner 广东双林生物制药有限公司
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