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Anti-TNF-alpha monoclonal antibody chromatographic method

A monoclonal antibody and composite layer technology, which is applied in the field of monoclonal antibody preparation, can solve the problems of increased volume, increased cost, low purity and yield, etc.

Inactive Publication Date: 2016-08-10
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the patent CN103509116A of AbbVie, the original research company of Adam, adopts the "three-step chromatography" with cations + anions + hydrophobic fillers with cations as the first step. Its cationic filler load is lower than 35 mg / ml. However, the feed liquid needs to adjust the conductivity and pH, and the volume becomes larger, resulting in an increase in cost
European patent EP1651665 uses MEP to capture and purify non-antibody proteins and fragments similar to antibodies. It uses two-step elution. 35% propylene glycol is added to the first elution, and 50% propylene glycol is added to the second elution. The recovery rate is 85%, but it is not optimized for monoclonal antibodies, and the purity and yield are low after being applied to monoclonal antibodies

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] 1) Clarification of cell fluid

[0048] Using an eppendorf centrifuge, centrifuge the CHO cell culture supernatant twice at 10,000 g to remove cells and cell debris, and pass through a 0.2 μm filter membrane to further reduce the turbidity of the sample.

[0049] 2) Composite chromatography

[0050] Using AKTA purifier-100 (GE Healthcare) chromatographic system, 1.2ml MEP (PALL company) composite chromatographic medium was loaded in Tricorn10 / 20 (GE company) chromatographic column. Fully equilibrate the recombination chromatography column with equilibration buffer (50mM PBS pH7.0), and start loading the sample when the UV absorption at 280nm returns to the baseline, and the conductance and pH remain stable, and then wash with the first washing buffer (50mM PBS pH7.0) .0) Wash the unbound whole protein, wait until the UV absorption at 280nm returns to the baseline, and after the conductivity and pH remain stable, then wash with the second washing buffer (50mM PBS pH6.0)...

Embodiment 2

[0072] 1) Clarification of cell fluid

[0073] Using an eppendorf centrifuge, centrifuge the CHO cell culture supernatant twice at 10,000 g to remove cells and cell debris, and pass through a 0.2 μm filter membrane to further reduce the turbidity of the sample.

[0074] 2) Composite chromatography

[0075] Using AKTA purifier-100 (GE Healthcare) chromatographic system, 1.2ml MEP (PALL company) composite chromatographic medium was loaded in Tricorn10 / 20 (GE company) chromatographic column. Fully equilibrate the recombination chromatography column with equilibration buffer (50mM PBS pH7.0), wait for the UV absorption at 280nm to return to the baseline, and start loading the sample when the conductivity and pH remain stable, and then wash with the first washing buffer (50mM PBS pH7.0) to wash the unbound whole protein, wait until the UV absorption at 280nm returns to the baseline, and after the conductivity and pH remain stable, then wash with the second washing buffer (50mM PBS...

Embodiment 3

[0098] 1) Clarification of cell fluid

[0099] Using an eppendorf centrifuge, centrifuge the CHO cell culture supernatant twice at 10,000 g to remove cells and cell debris, and pass through a 0.2 μm filter membrane to further reduce the turbidity of the sample.

[0100] 2) Composite chromatography

[0101] Using AKTA purifier-100 (GE Healthcare) chromatographic system, 1.2ml MEP (PALL company) composite chromatographic medium was loaded in Tricorn10 / 20 (GE company) chromatographic column. Fully equilibrate the recombination chromatography column with equilibration buffer (50mM PBS pH7.0), and start loading the sample when the UV absorption at 280nm returns to the baseline, and the conductance and pH remain stable, and then wash with the first washing buffer (50mM PBS pH7.0) .0) Wash the unbound whole protein, wait until the UV absorption at 280nm returns to the baseline, and after the conductance and pH remain stable, then wash with the second washing buffer (50mM PBS pH6.0),...

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PUM

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Abstract

The invention relates to an anti-TNF-alpha monoclonal antibody chromatographic method, and belongs to the field of biotechnology. In particular, the method for removing host proteins and multimers during a process of purification of a TNF-alpha monoclonal antibody is disclosed. An ion exchange and hydrophobic composite chromatographic filler is used, a low-pH buffer solution is used for removing contaminants, and then a lower-pH elution buffer solution is used for eluting the target antibody. The method can remove a part of the multimers s and most of the host proteins, significantly improves the purity of the antibody, is cheaper in price, has no protein A falling off, is simple and convenient, has no need for adjusting the sample pH value and electric conductance, and is suitable for process amplification and industrial production.

Description

technical field [0001] The present invention relates to the preparation of monoclonal antibodies, in particular to a chromatography method for anti-TNF-alpha monoclonal antibodies. Background technique [0002] Anti-tumor necrosis factor α (TNF-α) monoclonal antibody (Mc Ab) has broad application prospects in the clinical treatment of septic shock, rheumatoid arthritis and other diseases, such as infliximab (infliximab), adalimumab Anti-(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. At present, the vast majority of pharmaceutical companies use CHO (Chinese Hamster Ovary) cells, which are approved b...

Claims

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Application Information

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IPC IPC(8): C07K16/24C07K1/18C07K1/20
CPCC07K1/165C07K1/18C07K1/20C07K16/241
Inventor 杨辉马旭通杨彬孙文正李文佳苏彦景
Owner SUNSHINE LAKE PHARM CO LTD
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