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Medicinal kininogenase and its preparation method and application

A technology of kininogenase and drugs, which is applied in the field of protein drugs, can solve the problems of difficult separation and purification of porcine pancreas-derived kininogenase, and the inability to industrialize it, and achieve the effects of high biological activity, high efficacy and low side effects

Active Publication Date: 2020-03-31
ZONHON BIOPHARMA INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Technical problem to be solved: the present invention mainly solves the problem of how to further improve the product quality of porcine pancreas-derived kininogenase, and provides a kind of kininogenase with higher activity and better stability; The source kininogenase is difficult to separate and purify, and cannot be industrialized. A single-component method for separating and purifying KLK1b, KLK1a and KLK1c that can be applied to large-scale production is provided.

Method used

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  • Medicinal kininogenase and its preparation method and application
  • Medicinal kininogenase and its preparation method and application
  • Medicinal kininogenase and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Example 1 Comparison of Purified KLK1 Single Component in Different Media

[0058] In order to purify the single-component sample in KLK1, the purification conditions were explored and a large number of media were screened. The specific results are shown in Table 1.

[0059] Table 1 Screening of different media

[0060] Media Name Purification yields the purity of single-component KLK1b Total protein yield CaptoQ ImpRes(GE) 80％ 10％ CaptoQ adhere(GE) 84％ 10％ DEAE Sepharose FF(GE) 50％ 20％ HP Q(GE) 98％ 30％ Q FF(GE) 90％ 20％ SOURCE Q(GE) 88％ 10％ Capto DEAE (GE) 60％ 25％ Super Q650(TOSH) 86％ 18％ Q-600AR(TOSH) 80％ 15％

[0061] Through a large number of media screening results, it can be concluded that HP Q media (purchased from GE) has the best separation effect, the purity of single-component KLK1b can reach 98%, and the yield can reach 85%.

Embodiment 2

[0062] Example 2 Separation and purification of porcine pancreatic kininogenase single components KLK1b, KLK1a and KLK1c

preparation example 1

[0064] Porcine pancreatic kininogenase (from Changzhou Qianhong Biochemical Pharmaceutical Co., Ltd.) was diluted to 6 mg / mL with liquid A, and purified by ion-exchange chromatography. Purified chromatography conditions: ion exchange medium (QFF), A solution: 50mM Tris-HCl (pH9.0), B solution: 50mM Tris-HCl (pH9.0) containing 1M NaCl; flow rate 10mL / min, The detection wavelength is 280nm.

[0065] Loading: the above dilution of porcine pancreatic kininogenase was bound to a QFF ion exchange column.

[0066] Equilibration: Flush 5 column volumes with solution A.

[0067] Elution: The mobile phase ratio is 12% B, 88% A, after elution of 10 column volumes, gradient elution is performed. Gradient conditions were 12% B to 30% B, 30 column volumes.

[0068] Collection: collect part of the eluted product before peak 1 as KLK1b, such as Figure 1a Shown; Partial elution product after peak 2 was collected as KLK1a, as Figure 2a As shown, the partially eluted product after peak 3 w...

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Abstract

The invention relates to medicinal kallikrein (KLK1) and a preparing method and application thereof. The kallikrein does not contain KLK1c. Tissue kallikrein from porcine pancreas is composed of three components with different glycosylation degrees. A protein electrophoresis analysis result is three stripes, named KLK1b, KLK1a and KLK1c respectively in a molecular weight descending order, wherein KLK1c has the lowest glycosylation degree and the lowest specific activity. Compared with existing tissue kallikrein from porcine pancreas available in the market, the tissue kallikrein with KLK1c removed has the advantages of being higher in purity and stability, higher in biological activity, good in pharmacodynamic effect and smaller in side effect. The invention also provides a method for purifying porcine tissue kallikrein. The problem that KLK1b, KLK1a and KLK1c are hard to separate and industrialization can not be achieved is solved.

Description

technical field [0001] The present invention relates to protein drugs, especially medicinal kininogenase, its preparation method and its application in drug preparation. Background technique [0002] Kininogenase, also known as kallikrein (kininogenase) or kallidinogenase (kallidinogenase), it is a kind of serine protease, exists in various tissues and biological fluids, and catalyzes macromolecular precursors (kinin Original, Kininogen) releases biologically active peptides (kinin, Kinin). The physiological effects of kininogenase include capillary and arterial vasodilation and increased permeability, increasing the blood supply of coronary arteries, brain, and retina. , vasospasm, thromboangiitis obliterans, frostbite and trauma embolism also have therapeutic effect. At the same time, kininogenase can also be used as an activating factor to activate plasminogen into plasmin, hydrolyze insoluble fibrin into soluble small peptides, and thus play a therapeutic role in cereb...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64A61K38/48A61P13/12A61P9/10
CPCA61K38/482C12N9/6424
Inventor 马永王俊
Owner ZONHON BIOPHARMA INST
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