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A process 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: 2013-01-02
OCTAPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The disadvantage of commonly used immunoaffinity chromatography is that it is relat

Method used

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  • A process for purifying vitamin k dependent proteins such as coagulation factor IX
  • A process for purifying vitamin k dependent proteins such as coagulation factor IX
  • A process 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 Capto MMC columns.

[0118] Chromatography Resins and Columns

[0119] The mixed-mode resin Capto MMC (cat number 17-5317) from GE Healthcare was used as the capture step for the FIX molecules. Capto MMC is a weak cationic resin with hydrophobic and thiophilic interactions and hydrogen bonding. A Tricorn 5 / 150 column was packed with Capto MMC 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% Polysorbate 80, pH 7.0

[0122] Low Salt Wash Buffer: 20mM Sodium Citrate, 0.2M NaCl, 0.02% Polysorbate 80, pH 6.5

[0123] High Salt Wash Buffer: 20mM Sodium Citrate, 0.7M NaCl, 0.02% Polysorbate 80, pH 6.5

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

[012...

Embodiment 2

[0133] raw material

[0134] Recombinant human FIX (rhFIX) was produced in HEK 293 cells. Cells are removed and the cell-free supernatant is the starting material for loading onto a Capto MMC column.

[0135] Chromatography Resins and Columns

[0136] The mixed-mode resin Capto MMC (cat number 17-5317) from GE Healthcare was used as the capture step for the FIX molecules. Capto MMC is a weak cationic resin with hydrophobic and thiophilic interactions and hydrogen bonding. The XK26 column was packed with Capto MMC 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% Polysorbate 80, pH 7.0

[0139] High Salt Wash Buffer: 20mM Sodium Citrate, 0.7M NaCl, 0.02% Polysorbate 80, 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 equilibratio...

Embodiment 3

[0150] raw material

[0151] Recombinant human FIX (rhFIX) was produced in HEK 293 cells. Cells are removed and the cell-free supernatant is the starting material for loading onto a Capto MMC column.

[0152] Chromatography Resins and Columns

[0153] The mixed-mode resin Capto MMC (cat number 17-5317) from GE Healthcare was used as the capture step for the FIX molecules. Capto MMC is a weak cationic resin with hydrophobic and thiophilic interactions and hydrogen bonding. The XK26 column was packed with Capto MMC 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 / m...

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Abstract

A process of manufacturing a prion-free Vitamin K dependent Protein in a purification sequence employing chromatography characterized in that - at least one chromatography step is performed using a multimodal resin - providing a fraction containing Vitamin K dependent Protein in an aqueous solution; - contacting the fraction containing the Vitamin K dependent Protein with a multimodal resin at a pH between 6-9; - optionally washing the multimodal resin having the Vitamin K dependent Protein adsorbed with an aqueous washing buffer to wash away contaminants and retain the Vitamin K dependent Protein, before the Vitamin K dependent Protein is eluted; - the Vitamin K dependent Protein is eluting from the multimodal resin at a pH between 6 to 9 in a buffer comprising arginine; and - optionally collecting Vitamin K dependent Protein containing fractions 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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IPC IPC(8): C12N9/64
CPCC12N9/6424C07K1/22C07K14/745C07K1/16C12N9/64B01D15/08C12N9/644
Inventor 古斯塔夫·吉尔加姆斯特凡·温厄
Owner OCTAPHARMA
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