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Low-immunogenicity lumbrukinase, preparation method and application thereof

An immunogenicity and lumbrokinase technology, which is applied in the field of preparation of various dosage forms of pharmaceutical preparations, lumbrokinase, and preparation of low immunogenicity lumbrokinase. Response and other issues

Active Publication Date: 2014-01-08
中科优术(黑龙江)科技产业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the shortcomings of lumbrokinase with many components, difficult quality control, easy to produce allergic reactions, and low utilization, and provide a method for preparing molecularly modified lumbrokinase with low immunogenicity and the lumbrokinase prepared by the method. Lumbrokinase with low immune activity, the characteristic of this method is that the specific activity of the prepared lumbrokinase is ≥30000IU / mg, the molecular weight is 25500±2000 Daltons, the immunogenicity is smaller than that of natural enzymes, and less than that of natural enzyme immunogens 12% of sex

Method used

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  • Low-immunogenicity lumbrukinase, preparation method and application thereof
  • Low-immunogenicity lumbrukinase, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] The preparation of embodiment 1 lumbrokinase (F-III-2)

[0064] 1. Refining of lumbrokinase component (F-III-2):

[0065] 1. Preparation of crude lumbrokinase

[0066] Take 50g of fresh worms, wash them with water for several times, and soak them in clean water to remove the dirt in the viscera. Then add 50ml of water, and add 0.02 per thousand sodium benzoate for anticorrosion, heat and autolyze in a water bath at 45°C for 9 hours, centrifuge the cloudy solution at 4000rpm for 10 minutes, discard the precipitate, and filter with suction. Filtrate plus solid (NH 4 ) 2 SO 4 Make (NH 4 ) 2 SO 4 The final concentration reached 30.0%, stored at 4°C for 4h, centrifuged at 15,000r / min for 10min in a centrifuge, obtained the supernatant, added (NH4) 2 SO 4 , so that (NH4) 2 SO 4 The final concentration reached 75.0%, centrifuged at 15,000r / min for 10min, collected the precipitate, and dissolved it with deionized water. Dialyze with a dialysis bag for 48 hours, desa...

Embodiment 2

[0086] With low immunogenic lumbrokinase (calculated as 20000IU / mg lumbrokinase) as raw material, in a 100,000-class clean area, 50 g of low immunogenic lumbrokinase and 15 kg of starch are placed in a wet mixing granulator, and mixed After 2-2.5 minutes, add starch slurry (total amount: 5%) in batches during the stirring process, and stir to make a soft material, and quickly granulate for 4-5 minutes. Take out the wet granules and dry them in a drying room at 60-70°C, add magnesium stearate and mix them to obtain dry granules. Compress in a tablet press. During the tablet compression process, pay attention to the appearance of the tablet at any time, and check the weight difference, hardness and disintegration of the tablet regularly according to the regulations.

Embodiment 3

[0088] With low immunogenic lumbrokinase (calculated as 20000IU / mg lumbrokinase) as raw material, in a 100,000-class clean area, 50 g of low immunogenic lumbrokinase and 15 kg of starch are placed in a wet mixing granulator, and mixed After 2-2.5 minutes, add starch slurry (total amount: 5%) in batches during the stirring process, and stir to make a soft material, and quickly granulate for 4-5 minutes. Take out the wet granules and dry them in an oven at 60-70°C. Fill 0# capsules after whole grains.

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Abstract

The invention discloses low-immunogenicity lumbrukinase, a preparation method and application thereof, and belongs to the field of biological pharmacy. The method provided by the invention comprises the following steps of: first, extracting to obtain a lumbrukinase crude product from an earthworm, and then, passing the crude product through an ion exchange column to elute, purifying, colleting a component with fibrinolytic activity, freezing, drying, concentrating, separating a concentrated lumbrukinase solution by using a gel filtration chromatographic column, detecting the fibrinolytic activity by using an agarose-hemaleucin flat band method, collecting a second eluting peak with the fibrinolytic activity, wherein detected by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electropheresis), the molecular weight of the second eluting peak is 23500-27500 Da; and after the eluting peak is dialyzed, desalted and subjected to low-temperature freeze drying, carrying out molecular modification on the eluting peak by utilizing activated monomethoxy polyethyleneglycol and dextran to obtain the low-immunogenicity lumbrukinase. The lumbrukinase prepared by the invention has the characteristics of low immunogenicity, high enzyme activity and the like, and can be used for the research and the preparation of an antithrombotic drug and a drug for treating or preventing cardiovascular and cerebrovascular diseases.

Description

technical field [0001] The invention relates to a method for preparing lumbrokinase, in particular to a method for preparing lumbrokinase with low immunogenicity and pharmaceutical preparations prepared therefrom which can be used in various dosage forms for oral administration and injection. It belongs to the field of biopharmaceuticals. Background technique [0002] Thrombosis and cardiovascular and cerebrovascular diseases are major diseases that seriously threaten human health, especially the common diseases of middle-aged and elderly people over the age of 50. The number of people who die from thrombosis and cardiovascular and cerebrovascular diseases is as high as 25 million every year in the world, ranking first in each country. The number one cause of death. Thrombosis and cardiovascular and cerebrovascular diseases have become the number one killer of human death. Thrombosis and cardiovascular and cerebrovascular diseases have the characteristics of "four highs an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64A61K38/48A61P7/02A61P9/00
Inventor 陈喜君王辉
Owner 中科优术(黑龙江)科技产业有限公司
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