A method for activating and preparing human thrombin from human coagulation factor ⅸ chromatography waste liquid

A technology of human coagulation factor and human thrombin, which is applied in the field of activation and preparation of human thrombin, can solve the problems of unmanned thrombin, potential safety hazards, and inability to prepare at the same time, achieving simple process, reduced incidence, and clinical virus safety guarantee Effect

Active Publication Date: 2017-11-03
SHANDONG TAIBANG BIOLOGICAL PROD CO LTD
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  • Abstract
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Problems solved by technology

[0004]On the one hand, the traditional activation method is generally with thromboplastin (Thromboplastin, generally animal brain tissue or other tissue extracts or human brain powder, etc.) or Snake venom is used as an activator, even after subsequent purification, it is still unavoidable that the finished product contains nucleic acid or animal-derived protein and nucleic acid, etc., and there are still certain safety hazards
On the other hand, since human prothrombin is one of the components of human prothrombin complex (Prothrombin Concentration Complex, PCC), a special drug for the treatment of hemophilia B in China, the two products cannot be prepared at the same time, so there is basically no domestic clinical practice. The application of human thrombin
With the gradual improvement of domestic purification technology, domestic manufacturers are currently conducting research on the separation and purification of human coagulation factor IX from PCC to replace PCC in the treatment of hemophilia B. During this process, human prothrombin is chromatographically It is a pity that the waste liquid is discarded as impurity protein

Method used

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  • A method for activating and preparing human thrombin from human coagulation factor ⅸ chromatography waste liquid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] In this example, the chromatographic flow-through was used as the chromatographic waste liquid, and the crude PCC and the finished product of human coagulation factor VIII activated human thrombin.

[0040] Step 1: Adjust the pH of 2L of human plasma to 7.0, add 3g of DEAE Sephadex A-50 gel at a ratio of 1.5g / L, and absorb for 60 minutes; with 20mmol / L sodium citrate and 200mmol / L sodium chloride solution, Wash miscellaneous protein 3 times at pH 7.0, elute 3 times with 20mmol / L sodium citrate, 1mol / L sodium chloride solution, pH 7.0, desalt the eluate by ultrafiltration until the conductivity is 20mS / cm, volume 90ml , which is the crude product of PCC, and the detected prothrombin activity is 15.1IU / ml.

[0041] The second step: Add the crude PCC obtained in the first step to the pre-prepared Tween-80 / TnBP at a ratio of 1:9, incubate at 24±1°C for 360 minutes, and inactivate the lipid-enveloped virus, and the volume after inactivation is 100ml.

[0042] The third step: ...

Embodiment 2

[0049] In this example, the chromatographic washing liquid was used as the chromatographic waste liquid, and the crude product of PCC and the finished product of human coagulation factor VIII activated human thrombin.

[0050] The first step: the eluate is desalted by ultrafiltration to a conductivity of 10mS / cm.

[0051] The third step: Divide the PCC crude product obtained in the second step after virus inactivation according to 1:19 (V / V) into two parts (1 / 20 is liquid a: 5ml, 19 / 20 is liquid b: 95ml), of which Liquid b was adsorbed by Heparin Sepharose FF gel, first washed with 0.1mol / L sodium citrate, 0.15mol / L sodium chloride solution pH7.0, and then washed with 0.1mol / L sodium citrate, 0.5mol / L The human coagulation factor IX product was obtained by eluting with sodium chloride solution at pH 7.0, and the chromatographic washing liquid was chromatographic waste liquid.

[0052] All the other conditions are with embodiment 1.

[0053] The ratio of human thrombin activi...

Embodiment 3

[0055] In this example, the chromatographic flow-through was used as the chromatographic waste liquid, and the finished product of PCC and the finished product of human coagulation factor VIII were used to activate human thrombin.

[0056] The third step: after the virus inactivation obtained in the second step, the PCC crude product was adsorbed on Heparin Sepharose FF gel, and eluted with 0.1mol / L sodium citrate and 0.5mol / L sodium chloride solution pH7.0 to obtain human Coagulation factor IX products. The chromatographic flow-through is the chromatographic waste liquid.

[0057] Step 4: Add 5% PCC (PCC finished product, 17IU / ml, 4ml), 1% human coagulation factor VIII (FVIII finished product, 22IU / ml, 580μl), 25mmol / L to the chromatographic waste liquid obtained in the third step CaCl 2 , incubated at 20°C for 3 hours, and then activated. The ratio of human thrombin activity / human prothrombin activity before and after activation was 114 times.

[0058] All the other condi...

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Abstract

The invention discloses a method for preparing human thrombin by activating human coagulation factor IX chromatography waste liquor. Human thrombin is prepared from a raw material which is waste liquor produced when a human coagulation factor IX is prepared by a chromatographic technology. The method specifically comprises the following steps: (1) obtaining a PCC (Prothrombin Concentration Complex) crude product from human blood plasma; (2) performing S / D inactivation; (3) obtaining a human coagulation factor IX crude product and chromatography waste liquor by chromatography; (4) activating the chromatography waste liquor; (5) purifying thrombin; (6) performing freeze-drying and dry-heat inactivation. According to the method, the F IX chromatography waste liquor is used as the raw material, and influence on the purification of F IX is prevented; the addition of any animal-derived substance in a waste liquor activation process is avoided; the method comprises two virus inactivation steps, so that high clinical safety is achieved.

Description

technical field [0001] The invention belongs to the technical field of medicine, and specifically relates to a method for activating and preparing human thrombin from human coagulation factor IX chromatography waste liquid. Background technique [0002] Human thrombin is a rapid hemostatic drug activated by human prothrombin (human coagulation factor II). Thrombin itself is suitable for ligation of small blood vessels, capillaries and solid organ bleeding that are difficult to stop bleeding; Hemostasis for trauma, surgery, oral cavity, ENT, urology, burns, orthopedics, etc., can be used as a medicine alone, or can be combined with human fibrinogen to produce fibrin glue. [0003] The research on the production technology of human thrombin mainly contains: Philip K. etc.: A novel one-step purification of human α-thrombin after direct activation of crude prothrombin enriched from plasma, Biochem, J. (1991) 280,805-808, the author also with Human plasma is used as raw material...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/74
CPCC12N9/6429C12Y304/21005
Inventor 菅长永马山高建锋董雪杨西建朱孟沼席智赢陈晨杨虎虎
Owner SHANDONG TAIBANG BIOLOGICAL PROD CO LTD
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