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Cation exchange chromatographic purification method anti-TNF alpha-type monoclonal antibody

A cation exchange and monoclonal antibody technology, which is applied in the preparation method of peptides, chemical instruments and methods, anti-animal/human immunoglobulin, etc., can solve the inconvenience of polymer removal, antibody denaturation and inactivation, high concentration of additives, etc. question

Inactive Publication Date: 2016-07-20
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the cationic packing with improved loading capacity can be used to treat the feed liquid treated after fermentation, but after the feed liquid is loaded on a specific column, due to the high content of pollutants, the competition caused by adsorption to the chromatographic column will affect the polymerization in the chromatography. Removal of polymers, which brings inconvenience to the removal of polymers in subsequent purification
Chinese patent CN102119168A discloses a chromatographic step using non-ionic polymers to remove protein aggregates, followed by ion-exchange chromatography using additives that enhance dissolution, but the concentration of the additives used is high and used in the antibody purification process, which may lead to Risk of antibody denaturation and inactivation

Method used

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  • Cation exchange chromatographic purification method anti-TNF alpha-type monoclonal antibody
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  • Cation exchange chromatographic purification method anti-TNF alpha-type monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1) Fermentation broth acquisition

[0047] CHO cells (Chinese hamster ovary cells) expressing fully human adalimumab were cultured and expressed adalimumab, and the expressed antibody was centrifuged at 4000-8000g for 10-15min on a SIGMA6K15 centrifuge to separate cells and For the fermentation broth, the supernatant was centrifuged for the second time, centrifuged at 6000-8000g for 10-15min to remove cell debris, and the supernatant was transferred to the next step.

[0048] Filtration: The supernatant after twice centrifugation is filtered through a 0.45μm filter membrane, and the filtered feed liquid can be directly loaded on the cationic column after adjustment.

[0049] 2) Cation exchange column (CEX)

[0050] 1) Sample adjustment: Dilute the clarified feed solution by 1 time, adjust the pH to 6.0 with 0.5M citric acid, and the volume is about 210ml at this time;

[0051] 2) Equilibration: AKTA purifer, a chromatography system of GE Company, is used, and an XK16 / ...

Embodiment 2

[0062] 1) Fermentation broth acquisition

[0063] CHO cells (Chinese hamster ovary cells) expressing fully human adalimumab were cultured and expressed adalimumab, and the expressed antibody was centrifuged at 4000-8000g for 10-15min on a SIGMA6K15 centrifuge to separate cells and For the fermentation broth, the supernatant was centrifuged for the second time, centrifuged at 6000-8000g for 10-15min to remove cell debris, and the supernatant was transferred to the next step.

[0064]Filtration: The supernatant after twice centrifugation is filtered through a 0.45μm filter membrane, and the filtered feed liquid can be directly loaded on the cationic column after adjustment.

[0065] 2) Cation exchange column (CEX)

[0066] 1) Sample adjustment: Dilute the clarified feed solution by 1 time, adjust the pH to 6.0 with 0.5M citric acid, and the volume is about 400ml at this time;

[0067] 2) Equilibration: AKTA purifer, a chromatography system of GE Company, is used, and an XK16 / 2...

Embodiment 3

[0076] 1) Fermentation broth acquisition

[0077] CHO cells (Chinese hamster ovary cells) expressing fully human adalimumab were cultured and expressed adalimumab, and the expressed antibody was centrifuged at 4000-8000g for 10-15min on a SIGMA6K15 centrifuge to separate cells and For the fermentation broth, the supernatant was centrifuged for the second time, centrifuged at 6000-8000g for 10-15min to remove cell debris, and the supernatant was transferred to the next step.

[0078] Filtration: The supernatant after twice centrifugation is filtered through a 0.45μm filter membrane, and the filtered feed liquid can be directly loaded on the cationic column after adjustment.

[0079] 2) Cation exchange column (CEX)

[0080] 1) Sample adjustment: Dilute the clarified feed solution by 1 time, adjust the pH to 6.0 with 0.5M citric acid, and the volume is about 400ml at this time;

[0081] 2) Equilibration: AKTA purifer, a chromatography system of GE Company, is used, and an XK16 / ...

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Abstract

The invention discloses a cation exchange chromatographic purification method an anti-TNF alpha-type monoclonal antibody. The method includes: adding urea and a betaine surfactant in the process of CEX multistep elution to remove pollutants in a mixture containing the antibody and the pollutants to separate a polymer from the antibody. The method has good pollutant separation effect, the chromatographic process is simple in sample adjusting, drastic changes of pH value are avoided, and content of the polymer in a final purified product is lowered remarkably.

Description

technical field [0001] The invention relates to the preparation of monoclonal antibodies, in particular to a cation exchange chromatographic purification method for anti-TNFα monoclonal antibodies. Background technique [0002] Anti-tumor necrosis factor α (TNF-α) monoclonal antibody (McAb) has broad application prospects in the clinical treatment of septic shock, rheumatoid arthritis and other diseases, such as infliximab (infliximab), adalimumab (adalimumab) and Golimumab are both TNFα antibodies. Among them, adalimumab is the first approved anti-tumor necrosis factor alpha (TNFα) fully human monoclonal antibody in the world. Adalimumab can prevent TNFα from binding to its cell surface receptors, thereby blocking the biological activity of TNFα, and finally reducing the inflammatory response and osteoclast activation, so as to control and relieve symptoms and signs. [0003] Traditional purification methods, such as Chinese patent CN102257006A, disclose a method for sepa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24C07K1/18
CPCC07K1/18C07K16/241
Inventor 洪艳杨彬马旭通林小鹊李文佳孙文正
Owner SUNSHINE LAKE PHARM CO LTD
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