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Method for purifying vitamin k-dependent proteins such as coagulation factor ix

A vitamin-dependent technology, applied in coagulation/fibrinolytic factors, peptide preparation methods, chemical instruments and methods, etc., can solve expensive problems, achieve the effect of inhibiting aggregates and maintaining natural molecular structure

Inactive Publication Date: 2016-06-01
OCTAPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of commonly used immunoaffinity chromatography is that it is relatively expensive and uses monoclonal antibodies of animal origin as affinity ligands

Method used

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  • Method for purifying vitamin k-dependent proteins such as coagulation factor ix
  • Method for purifying vitamin k-dependent proteins such as coagulation factor ix
  • Method for purifying vitamin k-dependent proteins such as coagulation factor ix

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] raw material

[0117] Using Bloodborne FIX (pdFIX, ). Lyophilized Nanotiv was dissolved and diluted in equilibration buffer prior to loading onto CaptoMMC columns.

[0118] Chromatography Resins and Columns

[0119] GE Healthcare's mixed-mode resin CaptoMMC (cat No. 17-5317) was used as the capture step for the FIX molecules. CaptoMMC is a weak cationic resin with hydrophobic and thiophilic interactions and hydrogen bonding. A Tricorn 5 / 150 column was packed with CaptoMMC resin to a bed height of 15.7 cm. The column volume (CV) was 3.1 ml.

[0120] buffer

[0121] Equilibration buffer: 20mM sodium citrate, 0.1M NaCl, 0.02% Polysorbate80, pH 7.0

[0122] Low Salt Wash Buffer: 20mM Sodium Citrate, 0.2M NaCl, 0.02% Polysorbate80, pH 6.5

[0123] High Salt Wash Buffer: 20mM Sodium Citrate, 0.7M NaCl, 0.02% Polysorbate80, pH 6.5

[0124] Elution buffer: 20mM sodium citrate, 0.2M NaCl, 0.5M arginine hydrochloride, 0.02% Polysorbate80, pH 6.5

[0125] experiment setti...

Embodiment 2

[0133] raw material

[0134] Recombinant human FIX (rhFIX) was produced in HEK293 cells. Cells are removed and the cell-free supernatant is the starting material for loading onto the CaptoMMC column.

[0135] Chromatography Resins and Columns

[0136] GE Healthcare's mixed-mode resin CaptoMMC (cat No. 17-5317) was used as the capture step for the FIX molecules. CaptoMMC is a weak cationic resin with hydrophobic and thiophilic interactions and hydrogen bonding. The XK26 column was packed with CaptoMMC resin to a bed height of 15 cm. The column volume (CV) was 80ml.

[0137] buffer

[0138] Equilibration buffer: 20mM sodium citrate, 0.1M NaCl, 0.02% Polysorbate80, pH 7.0

[0139] High Salt Wash Buffer: 20mM Sodium Citrate, 0.7M NaCl, 0.02% Polysorbate80, pH 6.5

[0140] Elution buffer: 20mM sodium citrate, 0.2M NaCl, 0.5M arginine hydrochloride, 0.02% Polysorbate80, pH 6.5

[0141] experiment settings

[0142] The column was equilibrated with equilibration buffer and th...

Embodiment 3

[0150] raw material

[0151] Recombinant human FIX (rhFIX) was produced in HEK293 cells. Cells are removed and the cell-free supernatant is the starting material for loading onto the CaptoMMC column.

[0152] Chromatography Resins and Columns

[0153] GE Healthcare's mixed-mode resin CaptoMMC (cat No. 17-5317) was used as the capture step for the FIX molecules. CaptoMMC is a weak cationic resin with hydrophobic and thiophilic interactions and hydrogen bonding. The XK26 column was packed with CaptoMMC resin to a bed height of 15 cm. The column volume (CV) was 80ml.

[0154] buffer

[0155] Equilibration buffer: 20mM sodium citrate, 0.1M NaCl, pH 7.0

[0156] High Salt Wash Buffer: 20mM Sodium Citrate, 0.7M NaCl, pH 6.5

[0157] Elution buffer: 20mM sodium citrate, 0.2M NaCl, 0.8M arginine hydrochloride, pH 6.5 Experimental setup and results

[0158] The column was equilibrated with equilibration buffer and then loaded with material at a flow rate of 26 ml / min. rhFIX bo...

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Abstract

A method for the preparation of prion-free vitamin K-dependent proteins in a purification sequence using chromatography, characterized in that: - at least one chromatographic step is performed using a multimodal resin; - vitamin K-containing dependent proteins are provided as an aqueous solution fraction of protein; - contacting fraction containing vitamin K-dependent protein with multimodal resin at pH 6-9; - optional washing with aqueous wash buffer prior to elution of vitamin K-dependent protein Multimodal resin for vitamin K-dependent proteins to wash away contaminants and retain vitamin K-dependent proteins; -Elute vitamin K-dependent from multimodal resins at pH 6-9 in arginine-containing buffers protein; and - optionally collecting the vitamin K-dependent protein-containing fraction in purified or enriched form.

Description

technical field [0001] The present invention relates to a method for purifying a vitamin K-dependent protein such as blood coagulation factor IX (abbreviated as FIX) and a fraction comprising a vitamin K-dependent protein such as FIX obtainable by the method of the present invention. Background technique [0002] Hemophilia is a genetic disorder that impairs the body's ability to control blood clotting, or blood clotting. In one of its forms, hemophilia B, a deficiency of blood clotting factor IX, or FIX for short, occurs in about 1 in 25,000 males. Factor IX (or Christmas factor) is a serine protease of the coagulation system. It is a vitamin K-dependent plasma protein, through the Ca 2+ Participates in coagulation by converting factor X to its active form in the presence of ions, phospholipids and factor VIIIa. FIX proteins are essential factors of blood coagulation with multifunctional properties. [0003] Factor IX (FIX) is a single-chain glycoprotein comprising 461 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64
CPCC07K14/745C12N9/6424C07K1/22C07K1/16C12N9/64B01D15/08C12N9/644
Inventor 古斯塔夫·吉尔加姆斯特凡·温厄
Owner OCTAPHARMA
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