Purifying method for recombinant humanized anti-human tumor necrosis factor monoclonal antibody

A technology of tumor necrosis factor and monoclonal antibody, applied in the direction of microorganism-based methods, biochemical equipment and methods, anti-cytokine/lymphokine/interferon immunoglobulin, etc., can solve the problem that cannot meet the actual production requirements, antibody General effect of protein acidic components, etc.

Inactive Publication Date: 2014-10-15
SHANGHAI UNION BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of cationic chromatography to separate and remove acidic components in WB

Method used

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  • Purifying method for recombinant humanized anti-human tumor necrosis factor monoclonal antibody

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Embodiment 1

[0048] Example 1 Purification of recombinant human anti-human tumor necrosis factor monoclonal antibody using the method provided by the present invention

[0049] Use POROS XS to fill the cation exchange chromatography column;

[0050] The column is equilibrated with a phosphate buffer with a phosphate ion concentration of 50mmol / L and a pH value of 6.2;

[0051] The recombinant human anti-human tumor necrosis factor monoclonal antibody with a mass volume concentration of 27.3 mg / mL is loaded onto the equilibrated cation exchange chromatography column;

[0052] The concentration of phosphate ions is 50mmol / L, and the phosphate buffer solution with a pH value of 6.2 is used to wash the cation exchange chromatography column after loading the sample;

[0053] Use 12 times column volume of eluent to elute the washed cation exchange chromatography column, in the eluent:

[0054] Liquid A is a phosphate buffer with a concentration of phosphate ions of 50mmol / L, a pH value of 6.2,...

Embodiment 2

[0059] Example 2 Purification of recombinant human anti-human tumor necrosis factor monoclonal antibody using the method provided by the present invention

[0060] Use POROS XS to fill the cation exchange chromatography column;

[0061] The column is equilibrated with a phosphate buffer solution with a concentration of phosphate ions of 20mmol / L and a pH value of 5.8;

[0062] The recombinant human anti-human tumor necrosis factor monoclonal antibody with a mass volume concentration of 27.3 mg / mL is loaded onto the equilibrated cation exchange chromatography column;

[0063] The concentration of phosphate ions is 20mmol / L, and the phosphate buffer solution with a pH value of 5.8 is used to wash the cation exchange chromatography column after sample loading;

[0064] Use 12 times column volume of eluent to elute the washed cation exchange chromatography column, in the eluent:

[0065] Liquid A is a phosphate buffer solution with a concentration of phosphate ions of 20mmol / L, ...

Embodiment 3

[0070] Example 3 Purification of recombinant human anti-human tumor necrosis factor monoclonal antibody using the method provided by the present invention

[0071] Use POROS XS to fill the cation exchange chromatography column;

[0072] The column is equilibrated with a phosphate buffer solution with a concentration of phosphate ions of 80mmol / L and a pH value of 6.6;

[0073] The recombinant human anti-human tumor necrosis factor monoclonal antibody with a mass volume concentration of 27.3 mg / mL is loaded onto the equilibrated cation exchange chromatography column;

[0074] The concentration of phosphate ions is 80mmol / L, and the phosphate buffer solution with a pH value of 6.6 is used to wash the cation exchange chromatography column after loading the sample;

[0075] Use 12 times column volume of eluent to elute the washed cation exchange chromatography column, in the eluent:

[0076] Liquid A is a phosphate buffer solution with a concentration of phosphate ions of 80mmol...

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Abstract

The invention relates to the technical field of biological pharmacy, and especially relates to a purifying method for a recombinant humanized anti-human tumor necrosis factor monoclonal antibody. The method comprises: sampling a to-be purified recombinant humanized anti-human tumor necrosis factor monoclonal antibody sample to a balanced cation exchange chromatographic column, and performing washing, leaching, rebalancing and elution, so as to obtain a purified recombinant humanized anti-human tumor necrosis factor monoclonal antibody solution. A preparation method for the to-be purified recombinant humanized anti-human tumor necrosis factor monoclonal antibody sample comprises: getting Chinese hamster ovary cells integrated with humanized anti-human tumor necrosis factor monoclonal antibody gene, culturing, getting a cell culturing supernatant, and employing protein A to perform affinity chromatography, so as to obtain the to-be purified recombinant humanized anti-human tumor necrosis factor monoclonal antibody sample. Leaching in the method means linear gradient elution. A buffer for rebalancing in the method is a citric acid buffer, an acetic acid buffer or a phosphoric acid buffer. Through the leaching step, the purity of the recombinant humanized anti-human tumor necrosis factor monoclonal antibody is successively improved.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a method for purifying recombinant human anti-human tumor necrosis factor monoclonal antibody. Background technique [0002] WBP204 (also known as UBP1211) is a recombinant human anti-human tumor necrosis factor (hTNF-α) monoclonal antibody. It is a protein molecule with a molecular weight of about 150kDa, which can effectively antagonize hTNF-α in the human body. A variety of immune diseases caused by abnormal expression of α. At present, its preparation is usually obtained by purification after expression in eukaryotic cells. However, in the process of expression in eukaryotic cells, DNA is translated and expressed into proteins, such as glycosylation, oxidation, deamination, phosphorylation, Modifications such as N-terminal pyroglutamination and C-terminal lysine excision reaction, these modification processes will cause the surface charge heterogeneity of the mono...

Claims

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Application Information

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IPC IPC(8): C07K16/24C07K1/18C07K1/22C12P21/08C12R1/91
Inventor 沈克强陈智胜鲁钱达朱建王威
Owner SHANGHAI UNION BIOPHARM
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