Method for purifying antibody by using high-density protein A coated magnetic beads

Abstract

The invention discloses a method for purifying an antibody by using high-density protein A coated magnetic beads. The method comprises the following steps: adding magnetic beads and an expression product containing the antibody into a reaction vessel, applying a magnetic field to the outside or inside of the reaction vessel, and then, transferring out a supernatant; adding a washing buffer solution into the reaction vessel, and removing the magnetic field; repeatedly washing the magnetic beads for 2-4 times; adding an eluting buffer solution, removing the magnetic field, and incubating for 5-15 minutes at room temperature; enabling the magnetic beads to be attached to the wall through applying the magnetic field, transferring out the solution, and adjusting the pH value of the solution to be neutral, thereby obtaining a purified antibody. According to the method, antibody purification is carried out by using a magnetic bead affinity separation technology, so that the antibody capture speed is higher compared with that of the traditional antibody affinity chromatography; in addition, the purifying operation has no need of complicated chromatography systems, and samples have no need of clarifying treatment, so that the samples with relatively high viscosity can be directly subjected to purifying operation; moreover, the unique protein A coated magnetic beads are used, so that the antibody binding efficiency is increased greatly, and the shedding rate of a protein A ligand reaches a very low level.

Description

technical field [0001] The invention relates to a method for purifying antibodies, in particular to a method for purifying antibodies by using protein A high-density coated magnetic beads. Background technique [0002] Monoclonal antibodies are produced by hybridoma cells formed by the hybridization of B lymphocytes and myeloma cells, and the domains formed by their heavy and light chains can recognize and bind specific antigens. In 1997, the FDA approved the first chimeric monoclonal antibody drug Rituxan Anti-CD20 antibody for the treatment of lymphoma. By 2007, the FDA had approved more than 20 therapeutic monoclonal antibody drugs, about half of which were used to treat cancer. It has become a "bomb drug" with annual sales exceeding one billion US dollars. The global market for monoclonal antibody drugs is growing rapidly. About 200 kinds of monoclonal antibodies are in the clinical trial stage, accounting for more than one-third of the total number of clinical biotechn...

AI Technical Summary

Problems solved by technology

Large-scale industrial clarification operations usually require flow centrifuges or ultrafiltration systems, which are time-consuming, expensive and complicated to maintain

[0006] 2. Chromatographic operation is time-consuming and expensive

Large-scale chromatographic equipment and protein A affinity purification media are very expensive, and basically rely on imports, which is the bottleneck of the antibody purification industry

[0007] 3. The chromatography system cannot handle high viscosity samples

Due to the limitation of the pressure that the filler can withstand, high-viscosity samples (such as serum or high-protein concentration samples) need to be diluted and filtered before they can be applied to chromatography operations, resulting in the amplification of sample volume and the dilution of target products.

[0008] 4. Protein A affinity chromatography media is prone to protein A ligand falling off during repeated washing

However, because the antibody binding efficiency of protein A-coated magnetic beads currently on the market is still far behind that of traditional agarose fillers, it has not yet been widely used in the antibody purification industry

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