Method for purifying antibodies using high-density peridium magnetic beads of protein A

Abstract

The invention discloses a method for purifying antibodies using high-density peridium magnetic beads of protein A. The method comprises the steps as follows: adding magnetic beads and expression products containing antibodies into a reacting container, applying magnetic fields outside or inside the reacting container and then taking supernatants out; adding washing buffers and removing the magnetic fields; rewashing the magnetic beads for two to four times; then adding washing buffers, removing the magnetic fields and hatching for 5 to 15 min at the room temperature; applying magnetic fields to making the magnetic beads stuck on walls and removing magnetic fields; and adjusting the pH value of solvents to be neutral so as to prepare the purified antibodies. The method provided by the invention adopts a magnetic bead affinity separation technology to purify the antibodies, and compared with the traditional antibody affinity chromatography technology, the method provided by the invention has a high antibody capturing speed; additionally, purifying operation does not need a complex chromatography system, samples do not need to be clarified, and samples with large viscosity can be purified directly; and specific protein A peridium magnetic beads are used, so that the combining efficiency of the antibodies is greatly improved, and the expulsion rate of protein A ligands 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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