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A kind of purification method of antibody

A purification method and technology of equilibration solution, which are applied in the preparation methods of peptides, chemical instruments and methods, organic chemistry, etc., can solve the problem that the virus removal ability has no substantial effect, and can shorten the single cycle time, improve the linear flow rate, high load effect

Active Publication Date: 2021-02-12
HJB HANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Protein A affinity chromatography has a certain ability to remove HCP and DNA, so the removal effect of protein A affinity chromatography on HCP and DNA has become one of the key factors for the ability of anion exchange chromatography to remove viruses. However, the conventional protein A affinity layer The virus removal ability of the analytical process is very robust, and changes in process parameters have no substantial impact on its virus removal ability

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] MabSelect SuRe LX, chromatography column Vantage 11 / 250 (Millipore), diameter 1.1cm, column height 20cm, column volume 19.0mL, operating flow rate 220cm / h. The source of the sample is the monoclonal antibody expressed by CHO cells (No. Mab1). Rinse the chromatographic column with 0.2mol / L sodium hydroxide solution of 3CV, then use 5CV of equilibrium liquid (50mM Tris-HCl+150mM NaCl, pH7.4) to equilibrate the chromatographic column; the cell culture harvest liquid after clarification ( HCCF) is uploaded to the chromatographic column, and the loading capacity of the sample is 50g / L; the column bed is washed with 3CV of the equilibrium solution, and the intermediate wash buffer (50mM

[0048] NaAc-HAc+1M NaCl, pH5.5, conductivity 88.5mS / cm) to wash the column bed, then wash the column bed with 3CV equilibrium solution; then wash with 3.5CV eluent (50mM NaAc-HAc, pH3.5) Remove and collect the eluate; finally wash the chromatography column with 3CV of 0.2mol / L sodium hydrox...

Embodiment 2

[0055] MabSelect SuRe, chromatography column Vantage 11 / 250 (Millipore), diameter 1.1 cm, column height 20 cm, column volume 19.0 ml, operating flow rate 200 cm / h. The source of the sample is the monoclonal antibody (Mab2) expressed by CHO cells. Rinse the chromatographic column with 0.1mol / L sodium hydroxide solution of 3CV, then use 5CV of equilibrium solution (20mM Tris-HCl+1M NaCl, pH7.4) to equilibrate the chromatographic column; the cell culture harvest liquid after clarification ( HCCF) is loaded into the chromatographic column, and the loading capacity of the sample is 30g / L; the column bed is washed with 3CV of the equilibrium solution, and the intermediate cleaning buffer (1M NaAc-HAc, pH5.8, conductivity 47.3mS / cm ) to wash the column bed, and then wash the column bed with 3CV equilibrium solution; then use 3.5CV of eluent (100mM HAc, pH3.0) to elute, collect the eluent; finally use 3CV of 0.1mol / L of hydroxide Rinse the chromatography column with sodium solution, ...

Embodiment 3

[0062] MabSelect SuRe LX, chromatography column Vantage 11 / 250 (Millipore), diameter 1.1 cm, column height 20 cm, column volume 19.0 ml, operating flow rate 220 cm / h. The source of the sample is the monoclonal antibody (Mab1) expressed by CHO cells. Rinse the chromatographic column with 0.2mol / L sodium hydroxide solution of 3CV, then use 5CV of equilibrium liquid (50mM Tris-HCl+150mM NaCl, pH7.4) to equilibrate the chromatographic column; the cell culture harvest liquid after clarification ( HCCF) is loaded onto the chromatographic column, and the loading capacity of the sample is 50g / L; the column bed is washed with 3CV of the equilibrium solution, and the intermediate cleaning buffer (50mM NaAc-HAc+1M NaCl, pH5.0, conductivity 91.3 mS / cm) to wash the column bed, then wash the column bed with 3CV equilibrium solution; then use 3.5CV of eluent (50mM NaAc-HAc, pH3.5) to elute and collect the eluate; finally use 3CV of 0.2mol / L of sodium hydroxide solution to wash the chromato...

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Abstract

The invention provides an antibody purification method. A method for purifying an antibody, comprising a protein A affinity chromatography step; the method of the protein A affinity chromatography is: washing with an intermediate washing buffer after loading the sample and before eluting the target antibody with an eluent Column bed; the intermediate cleaning buffer is: a buffer with a pH value of 5-6 and a conductivity of 20-100mS / cm. The invention improves the ability to remove HCP and DNA by adding an intermediate cleaning step in the process of protein A affinity chromatography, and further improves the ability to remove viruses by anion exchange chromatography.

Description

technical field [0001] The present invention relates to the field of antibody purification, in particular to an antibody purification method. Background technique [0002] In the pharmaceutical industry, there are many drugs derived from biotechnology, and there is a growing interest in biopharmaceuticals. The characteristics of low toxicity and side effects, high specific efficacy, long half-life and platform-based production technology make monoclonal antibody in the leading position in the field of therapeutic biological products, and it is the most successful type of biological products. So far, most monoclonal antibodies are expressed by mammalian cells. This is because mammalian cells can complete complex glycosylation modifications, and glycosylation modifications can increase protein stability and solubility to achieve longer The half-life of the monoclonal antibody; and the most commonly used monoclonal antibody expression system is the CHO cell line, and the glyco...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/00C07K1/22
CPCC07K1/22C07K16/00
Inventor 金雄华徐志豪汤炜梁泊宁
Owner HJB HANGZHOU CO LTD
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