Extracting norfibroase from snake venom and preparation process for liquid drugs injection of norfibroase

A defibrase and snake venom technology, applied in hydrolytic enzymes, pharmaceutical formulas, anti-enzyme immunoglobulins, etc., can solve the problems of reducing the specific activity of products, increasing the possibility of polymer production, and low purity of products, so as to reduce Effect of specific activity, increased possibility, and high purity

Active Publication Date: 2005-03-23
BEIJING SAISHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional separation and purification of defibrase adopts multi-step column chromatography, including ion exchange chromatography and molecular sieve gel chromatography, and even adopts high-performance liquid phase preparation, so that it is feasible to obtain a trace single-component medicinal enzyme, but It is almost impossible to obtain single-component medicinal enzymes in large-scale production, and there is also the problem of low purity of the obtained products
However, the affinity chromatography using polysaccharides as ligands, the preparation process can reach more than ten milligrams of samples, but multi-step operations must be taken to obtain a single component
[0004] At present, foreign snake venom thrombin preparations are all aqueous injections. Due to the limitation of the preparation level, the prescriptions all contain preservative ingredients to maintain the stability of the preparation, such as Ancrod, Batroxobin, and Japan's Donglingxomysin; To ensure the stability of potency during long-term storage, all are made into freeze-dried powder, which reduces the specific activity of the product and increases the possibility of polymer production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The method for extracting defibrase from snake venom comprises the following steps:

[0041] 1). Preparation of monoclonal antibody: After the purified and qualitative defibrase (1g / L) extracted from snake venom was mixed and emulsified with an equal volume of Freund's adjuvant, the 7-week-old Balb / C mouse was subcutaneously Inject 0.2ml each time, once every 2 weeks, and immunize 3 times in total; inject 0.2ml intravenously 3 days before fusion; then use the Koher method to collect splenocytes from immunized mice, and add the splenocytes from immunized mice to 1640 culture medium , to obtain a cell suspension; the resulting cell suspension was mixed with mouse SP2 / 10 myeloma cells at a ratio of 10:1, and fused under the action of 50% polyethylene glycol to obtain a fusion body; the fusion body was selectively cultivated, first Screen out hybridoma cells that can secrete monoclonal antibodies that react with predetermined antigens, and then screen out hybridoma cells wi...

Embodiment 2

[0054] The method for extracting defibrase from snake venom comprises the following steps:

[0055] 1). Preparation of monoclonal antibody: After the purified and qualitative defibrase (1g / L) extracted from snake venom was mixed and emulsified with an equal volume of Freund's adjuvant, mice were immunized; For immunized splenocytes, add the taken splenocytes to 1640 culture medium to obtain a cell suspension; mix the obtained cell suspension with SP2 / 10 myeloma cells at a ratio of 8:1, and mix under the action of 80% polyethylene glycol The fusion is obtained by fusion; the fusion is selectively cultured, and the hybridoma cells that can secrete the monoclonal antibody that reacts with the predetermined antigen are first screened out, and then the hybridoma cells with predetermined specificity are screened out, and finally the hybridoma cells that can be used for practical use are selected. Apply uniform cell clones with stable growth and specific functions, obtain monoclonal ...

Embodiment 3

[0067] The method for extracting defibrase from snake venom comprises the following steps:

[0068] 1). Preparation of monoclonal antibody: After the purified and qualitative defibrase (1g / L) extracted from snake venom was mixed and emulsified with an equal volume of Freund's adjuvant, mice were immunized; Splenocytes of immunized mice were added to 1640 culture medium to obtain a cell suspension; the resulting cell suspension was mixed with mouse SP2 / 10 myeloma cells at a ratio of 15:1, The fusion is obtained under the action of 30% polyethylene glycol; the fusion is selectively cultured, and the hybridoma cells that can secrete the monoclonal antibody that reacts with the predetermined antigen are first screened out, and then the hybridoma cells with predetermined specificity are screened out. Tumor cells, and finally select the uniform cell clones that can be used in practice and have stable growth and specific functions, and obtain monoclonal antibodies by ascitic fluid me...

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PUM

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Abstract

The invention discloses a method of extracting defibrase from snake poison and preparing defibrase injection preparation, and the process of extracting defibrase includes the steps: preparing monoclonal antibody; preparing crude defibrase; preparing antibody affinity chromatography column; preparing defibrase; and the process of preparing the defibrase injection preparation includes the steps: taking and adding purified defibrase to a proper amount of injection water for dissolving so as to obtain a solution, adding 5-20g NaCl stabilizer to every 1000ml solution, making split charging and obtaining the defibrase injection preparation.

Description

technical field [0001] The invention relates to a method for extracting defibrase with thrombolytic effect from snake venom and a preparation method for defibrase aqueous injection preparation, in particular to a method for extracting high-purity biologically active enzymes by using monoclonal antibody affinity chromatography technology And the preparation process of high stability aqueous injection preparation. Background technique [0002] Defibrase is a proteolytic enzyme that can dissolve thrombus, inhibit thrombus formation, and improve microcirculation. Clinically used for acute cerebral infarction, including prevention of cerebral thrombosis, cerebral embolism, transient ischemic attack (TIA) and recurrence of cerebral infarction; prevention of myocardial infarction, unstable angina pectoris and recurrence of myocardial infarction; extremity blood vessels Diseases, including femoral artery embolism, thromboangiitis obliterans, Raynaud's disease; also applicable to bl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48C07K16/40C12N9/50
Inventor 王光马骉李莉
Owner BEIJING SAISHENG PHARMA
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