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Method for purifying adalimumab by aid of cation exchange chromatography

A cation exchange and adalimumab technology, which is applied in the field of cation exchange chromatography purification of adalimumab, can solve the problems of inconvenient removal of polymers, denaturation and inactivation of antibodies, and high concentration of additives

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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  • Method for purifying adalimumab by aid of cation exchange chromatography
  • Method for purifying adalimumab by aid of cation exchange chromatography
  • Method for purifying adalimumab by aid of cation exchange chromatography

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specific Embodiment approach

[0026] The specific embodiment of the present invention comprises the following steps:

[0027] 1) After fermenting and expressing CHO cells, separate the bacterial liquid;

[0028] 2) Dilute the fermentation broth with water, and adjust the pH to 6.00±0.10;

[0029] 3) Pass through a cation exchange column, use a buffer solution with a pH of 5.5-6.5 containing 0.1-0.5 mol / L urea for the first elution, and then use a beet with a pH of 5.5-6.5 and a mass fraction of 0.1%-1.0% The buffer solution of the alkaline surfactant is eluted for the second time, and the eluted components are collected;

[0030] 4) The collected components are eluted by hydrophobic interaction chromatography, and the eluted components are collected.

[0031] In some embodiments, the betaine surfactant is selected from dodecyl dihydroxyethyl betaine, octadecyl dihydroxyethyl betaine or cocamidopropyl betaine.

[0032] In some embodiments, the separation in step 1) is to centrifuge the fermentation broth...

Embodiment 1

[0044] 1) Fermentation broth acquisition

[0045] 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.

[0046] 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.

[0047] 2) Cation exchange column (CEX)

[0048] 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;

[0049] 2) Equilibration: AKTA purifer, a chromatography system of GE Company, is used, and an XK16 / ...

Embodiment 2

[0070] 1) Fermentation broth acquisition

[0071] 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.

[0072] 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.

[0073] 2) Cation exchange column (CEX)

[0074] 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;

[0075] 2) Equilibration: AKTA purifer, a chromatography system of GE Company, is used, and an XK16 / ...

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Abstract

The invention discloses a method for purifying adalimumab by the aid of cation exchange chromatography. The method has the advantages that pollutants in mixtures with antibodies and the pollutants are removed by urea and betaine surfactants which are added in CEX (cation exchange) multi-step elution procedures, polymers are separated from the antibodies, then hydrophobic interaction chromatography is carried out, and accordingly good pollutant separation effects can be realized; samples are easy to regulate in chromatography procedures, drastic change of pH (potential of hydrogen) values can be prevented, and the contents of polymers in ultimately purified products can be obviously lowered.

Description

technical field [0001] The invention relates to the preparation of monoclonal antibodies, in particular to a cation exchange chromatographic purification method of adalimumab. Background technique [0002] Adalimumab is the world's first approved fully human monoclonal antibody against tumor necrosis factor α (TNFα). 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 separating and purifying antibodies through an affinity chromatography capture step. Protein A columns are often preferred for antibody capture due to their high selectivity and strong ability to remove pollutants, but they are expensive and washable. The low pH can easily lead to the formation of high molecular polymers...

Claims

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Application Information

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IPC IPC(8): C07K16/24C07K1/20C07K1/18
CPCC07K16/241
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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