A method for extracting antithrombin III and various functional proteins from the precipitation of component IV of plasma cohn method

A technology of protein and plasma, which is applied in the direction of serum albumin and peptide preparation methods, chemical instruments and methods, etc., can solve the problems of lack of data support and non-disclosure for scale-up to large-scale production, and achieve the improvement of comprehensive utilization efficiency of plasma, high-efficiency Effects of capture, huge economic and social benefits

Active Publication Date: 2019-07-26
LANZHOU INST OF BIOLOGICAL PROD
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Problems solved by technology

The above two comprehensive utilization methods of Cohn method component IV precipitation are only laboratory-scale experiments, which do not involve the virus inactivation and activity detection of each functional protein, and some key quality indicators related to yield and specific activity are not disclosed. , from the perspective of scaling up to large-scale production, there is still a lack of sufficient data support, and there is an urgent need in this field for a comprehensive utilization method for the Cohn method component IV precipitation that can be used for large-scale production

Method used

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  • A method for extracting antithrombin III and various functional proteins from the precipitation of component IV of plasma cohn method
  • A method for extracting antithrombin III and various functional proteins from the precipitation of component IV of plasma cohn method
  • A method for extracting antithrombin III and various functional proteins from the precipitation of component IV of plasma cohn method

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Experimental program
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Embodiment 1

[0081] Embodiment 1, small-scale preparation process

[0082] 1. Precipitation dissolution: 1kg of component IV precipitate was dissolved in 5L pH8.0, 20mmol / L PB buffer solution, stirred at 4°C for 60min, and after pressure filtration, the filtrate was filtered and clarified with a 0.22μm hollow fiber column to obtain the filtered over liquid.

[0083] 2. S / D inactivation: use final concentration of 1% Tween-80 / 0.3% TNBP (tributyl phosphate) for virus inactivation, stir and incubate at 24°C for 6 hours, adjust to pH 6.0 with 0.1mo / L HCl. 20.

[0084] 3. Anion exchange chromatography: use DEAE Sepharose FF chromatography packing material, the chromatography column is XK 50 / 20, the column height is 10cm, equilibrate the chromatography column with 20mmol / L PB buffer solution of pH 6.20 for 4 column volumes and load the sample 500mL, wash the column with the same buffer for 2 column volumes, and then use 50mmol / L Tris-HCl buffer containing 30mmol / L NaCl, pH7.4 for elution.

[...

Embodiment 2

[0107] Embodiment 2, large-scale preparation technology

[0108] 1. Precipitate dissolution: 5kg of component IV precipitate was dissolved in 50L pH8.0, 20mmol / L PB buffer solution, stirred at 8°C for 30min, and after pressure filtration, the filtrate was filtered and clarified with a 0.22μm hollow fiber column to obtain over liquid.

[0109] 2. S / D inactivation: virus inactivation was carried out with a final concentration of 1% Tween-80 / 0.3% TNBP, stirred and incubated at 24°C for 6 hours, and adjusted to pH 6.80 with 0.1mo / L HCl.

[0110] 3. Anion exchange chromatography: use DEAE Sepharose FF chromatography filler, the chromatography column is BPG 140 / 50, the column height is 20cm, equilibrate the chromatography column with 20mmol / L PB buffer solution of pH 6.8 for 4 column volumes and load the sample 50L, wash the column for 2.5 column volumes, and then elute with 50mmol / L Tris-HCl buffer containing 20mmol / L NaCl, pH7.4.

[0111] 4. Tandem affinity chromatography: TOYOP...

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Abstract

The invention provides a method for extracting antithrombase III from Cohn method component IV sediment. According to the method, AT-III with the purity approximate to 100% can be extracted, and compared with an existing method, the specific activity and yield are remarkably increased. In addition, the invention provides a method for comprehensively extracting and utilizing alpha1-antitrypsin, antithrombase III and albumin from the Cohn method component IV sediment. Separation and extraction of the three products are achieved on the same process line at the same time, the production cost of alpha1-antitrypsin and antithrombase III can be effectively reduced, and large-scale industrial production can be achieved. According to the method, the comprehensive utilization efficiency of plasma can be remarkably improved, and high application value is achieved.

Description

technical field [0001] The invention belongs to the field of blood products and relates to the comprehensive utilization of blood plasma, in particular to a method for recovering and preparing antithrombin III and various functional proteins from the waste of preparing human serum albumin. Background technique [0002] Blood protein products are plasma protein components or blood cell component products with clear clinical application significance, such as human albumin, human immunoglobulin, and human coagulation factor, which are separated and purified from healthy human blood, plasma or specific immune human plasma (natural or recombinant), red blood cell concentrates, etc., are used in diagnosis, treatment or passive immune prevention, and play an important role in clinical practice. [0003] Separation and purification of plasma proteins, separating various plasma proteins according to their physical and chemical properties or biological characteristics. The methods cu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/81C07K14/765C07K1/22C07K1/18C07K1/16
CPCC07K14/765C07K14/8125C07K14/8128
Inventor 梁凌宇蒋琳郭玉婷杨军
Owner LANZHOU INST OF BIOLOGICAL PROD
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