Method for preparing fibrinogen by expanded bed adsorption (EBA) technique

A technology of fibrinogen and fibrin, which is applied in the direction of preparation methods of fibrinogen and peptides, chemical instruments and methods, etc., can solve problems such as no change, achieve reduction of chemical substances, improvement of process continuity and automation, The effect of improving safety and quality

Inactive Publication Date: 2015-07-01
SHENZHEN POLYTECHNIC
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Problems solved by technology

The preparation method of fibrinogen disclosed in Chinese patent application CN201110188019.X is also mainly based on traditional low-temperature ethanol technology as the core, and the innovation is to add auxiliary agents, such as anticoagulants: trisodium citrate or potassium oxalate; Agents: tributyl phosphate and Tween 80; such as dissolving agents: disodium hydrogen phosphate buffer system, carbonic acid buffer system, citric acid buffer system, this process does not use column chromatography as a purification step
Chinese patent application CN201410524351.2 discloses a preparation process for extracting human fibrinogen from the waste of coagulation factor VIII extracted from cryoprecipitate. -III inactivation of thrombin, EDTA in addition to Ca 2+ and nanofiltration membrane filtration to remove viruses. Although the above measures help to reduce the activation of fibrinogen to fibrin and avoid the use of chemical reagents, and improve the safety and activity of the product, cryoprecipitate is still used as the initial isolate. And the core technology of secondary low-temperature ethanol precipitation has not changed

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  • Method for preparing fibrinogen by expanded bed adsorption (EBA) technique
  • Method for preparing fibrinogen by expanded bed adsorption (EBA) technique
  • Method for preparing fibrinogen by expanded bed adsorption (EBA) technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation of ion exchange chromatography medium for expanded bed (EBA)

[0049] 1. Preparation of cross-linked agarose-tungsten carbide composite beads

[0050] Take 2 g of drained agarose gel and 0.5 g of NaCl to make 4% (W / W) agarose-water suspension. According to the traditional method of thermal regeneration of reverse phase suspension, 50g of 4% agarose-water suspension was transferred to the flask, stirred and mixed at room temperature for 15 minutes, and a series of different amounts of tungsten carbide (xlwc100, Chaozhou Xianglu Tungsten Co., Ltd., China, average particle size 9-11μm), add tungsten carbide and agarose gel at mass ratios of 0.2, 0.4, 0.6, 0.8, 1 and 2, stir the mixture at 600rpm for 15min, heat to 93°C for 60min, as the water phase spare. Then, 200mL of paraffin and petroleum ether (10:1, V / V), 4% emulsifier (Span80:Tween80=10:4) mixture was ultrasonicated for 5-10min, and preheated in a 93°C water bath as the oil phase for later us...

Embodiment 2

[0080] The liquid preparation of embodiment 2 EBA chromatography, the treatment of medium and packing column

[0081] Chromatographic medium: Agarose (tungsten carbide)-DEAE was synthesized according to the method in Example 1; XP 2*60 chromatography column, STREAMLINE 100, 10*100 were purchased from GE Company.

[0082] Chromatography buffer: A balance solution (40mM sodium citrate pH5.0), B eluent (10mM sodium citrate pH5.0), B1 eluent 1 (B+5g / L sodium octanoate / HCl pH6. 0), B2 eluent 2 (1M sodium citrate pH8.0+0.3M sodium chloride pH8.0), B3 eluent 3 (20mM sodium citrate+0.1M sodium chloride pH8.0), C regeneration Solution (1M NaOH) was prepared according to routine.

[0083] Rinse the agarose (tungsten carbide)-DEAE medium with 1:1 (V / V) double distilled water. When the medium is completely precipitated and separated from water, pour the supernatant and repeat three times. Evacuate the air bubbles through the bottom of the column, add water to soak the bottom surface, an...

Embodiment 3

[0084] Example 3 Expanded bed action column chromatography (EBA) separation of raw plasma (column bed 25cm high)

[0085] Column equilibration (A balance solution 2.5CV) → plasma preparation (dilution × 3, adjusted to pH 5.0 with HCl) → sample loading (injection from the lower end of the column) (1.5L plasma / L adsorbent amount) → non-adsorbing Plasma fraction and eluate (2.5CV)→elution fraction 1 (1CV)→elution fraction 2 (1CV)→elution fraction 3 (1CV)→CIP (1CV) / equilibrium / first elution (Ultraviolet spectrophotometer adjusted to 280nm)

[0086] XP 2*60 column was used in the experiment (column bed 25cm high), one chromatography was performed with a linear velocity of 5cm / min, and one chromatography was performed with a linear velocity of 10cm / min. Dilute the raw plasma (liquid state) three times and load it directly. According to the amount of 1.5L plasma / L adsorbent (diluted and adjusted pH) / sample, the experimental results of the two chromatography are shown in Table 3.

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Abstract

The invention provides a method for preparing fibrinogen, which comprises the following steps: 1) by using an agarose tungsten carbide-DEAE (diethylaminoethanol) ion exchange medium as a column chromatography filler, preparing an expanded bed action chromatographic column; 2) passing plasma through the chromatographic column in the step 1) to perform primary purification, and collecting a fibrinogen initial product; 3) carrying out acid precipitation on the fibrinogen initial product obtained in the step 2) to remove impurities; 4) removing viruses from the impurity-removed fibrinogen obtained in the step 3); and 5) further purifying the virus-removed fibrinogen in the step 4) by using MacroCap Q column chromatography to obtain the target fibrinogen solution. The method has the advantages of higher technical continuity and higher degree of automation, greatly reduces the residual chemical substances, and enhances the safety and quality of the product.

Description

technical field [0001] The invention relates to a method for preparing protein, in particular to a method for preparing fibrinogen. Background technique [0002] Blood products (boold products) refer to the plasma protein components and the formed components of blood cells that are separated, purified or made by recombinant DNA technology from healthy human plasma or specifically immunized human plasma. At present, according to the different functions of blood products, they can be divided into five categories: human albumin, immunoglobulins, coagulation factors, special proteins and trace proteins, and fibrin adhesives. In addition to market supply and demand factors, another important factor that contributes to the different market share of the above products is the separation technology. [0003] During World War II, the low-temperature ethanol method developed by Cohn officially pioneered the separation and purification of blood products, and had a profound impact on th...

Claims

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Application Information

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IPC IPC(8): C07K14/75C07K1/18
CPCC07K14/75
Inventor 王妍林孝发喇文军张英
Owner SHENZHEN POLYTECHNIC
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