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Adsorbent, solid phase carrier and purification method

A purification method and solid-phase carrier technology, which can be used in peptide preparation methods, chemical instruments and methods, carrier binding/immobilization of peptides, etc., can solve problems such as ligand deterioration, and achieve prevention of modification, safe purification, high affinity The effect of harmony and resistance to deterioration

Active Publication Date: 2013-06-05
JNC CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although affinity chromatography using protein as a ligand can achieve highly selective and efficient purification, there are problems of ligand degradation due to the following reasons: modification or decomposition of the ligand due to alkali washing, Decomposition of ligands by proteolytic enzymes, or fragility of ligands themselves
However, it has not been known that a polypeptide fragment having a repeating structure of the above sequence binds to fibronectin, von Willebrand factor, etc. with high affinity

Method used

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  • Adsorbent, solid phase carrier and purification method
  • Adsorbent, solid phase carrier and purification method
  • Adsorbent, solid phase carrier and purification method

Examples

Experimental program
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Effect test

Embodiment 1

[0115] Cellufine Formyl (manufactured by JNC Co., Ltd.) was washed with 50 mM sodium carbonate and subjected to suction filtration, and 4.3 g of the obtained washed gel was placed in a 200 mL Erlenmeyer flask, and 16 g of an aqueous polypeptide solution containing 0.5% by weight of polyPHG was added. , 6.4 mL of 50 mM sodium carbonate, and 9.6 mL of water were thoroughly mixed, and shaken at 40° C. for 2 hours. 0.7 mL of an aqueous solution of 50 mg / mL of sodium borohydride was added, followed by further shaking at 40° C. for 2 hours. After the reaction, the reaction slurry was sucked and filtered to recover the gel. In order to remove unreacted substances remaining in the gel, the gel was washed with 50 mM sodium carbonate, aqueous hydrochloric acid (pH 3), and pure water, followed by suction filtration to obtain a polyPHG-immobilized gel.

[0116] The amount of polyPHG-containing polypeptide remaining in the washing liquid was quantified, and the amount of immobilized polyP...

Embodiment 2

[0122] Weigh 2.7 g of Shodex BIOACT gel EPO series (manufactured by Showa Denko Co., Ltd.) by dry weight, add to 30 mL of 0.25% by weight polyPHG-containing polypeptide, 50 mM sodium carbonate solution, and shake at 40° C. overnight reaction. After the reaction, the reaction slurry was sucked and filtered to recover the gel. In order to remove unreacted substances remaining in the gel, the gel was washed with 50 mM sodium carbonate, aqueous hydrochloric acid (pH 3), and pure water, followed by suction filtration to obtain a polyPHG-immobilized gel.

[0123] The amount of polyPHG-containing polypeptide remaining in the washing liquid was quantified, and the amount of immobilized polyPHG-containing polypeptide was calculated from the difference with the polyPHG-containing polypeptide used for the reaction. The amount of polyPHG-containing polypeptide in the washing solution was calculated by measuring the concentration with absorbance at 210 nm.

Embodiment 3

[0125] Except having changed reaction temperature into 4 degreeC from 40 degreeC, it carried out similarly to Example 2.

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Abstract

An adsorbent, a solid phase carrier and a purification method are provided. An objective of the invention is to provide an adsorbent allowing purification of a blood coagulation factor or a cell adhesion factor under mild conditions, according to simple procedures, and at a low cost and safely while having a high affinity and a high resistance to deterioration, and a method for purifying the blood coagulation factor or the cell adhesion factor; a solution is to apply a polypeptide having peptide fragments represented by formula (1) as the adsorbent for the blood coagulation factor or the cell adhesion factor, and to purify the blood coagulation factor or the cell adhesion factor using the adsorbent: -(Pro-Hyp-Gly)n- Wherein, in formula (1), n is an integer from 2 to 9,000.

Description

technical field [0001] The present invention relates to an adsorbent for blood coagulation factor or cell adhesion factor and a refining method. Background technique [0002] Chromatography is generally known as a method for purifying biomolecules such as proteins, and specific examples thereof include cation exchange chromatography, anion exchange chromatography, and gel filtration. Chromatography (Gel filtration Chromatography), Hydrophobic Interaction Chromatography (Hydrophobic Interaction Chromatography), Affinity Chromatography (Affinity Chromatography). Since the target protein can be purified to a high degree by combining these methods, chromatography is becoming important in the purification of erythropoietin and immunoglobulin, which are used as biopharmaceuticals, such as blood, plasma, or cell culture fluid. technology. In particular, affinity chromatography is very important in that it is highly selective for the target substance compared to other methods, so ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K7/08C07K14/00C07K17/00C07K14/745C07K14/78C07K14/705C07K14/47C07K1/14
CPCB01J20/3219C07K14/755B01J20/26C07K1/22C07K14/705B01J20/3274B01J20/24C07K14/78C07K14/745
Inventor 戸所正美岛谷明子
Owner JNC CORP
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