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

A high-density, magnetic bead technology, applied in peptide preparation methods, chemical instruments and methods, immunoglobulins, etc., can solve the problems of long chromatographic operation, gap in antibody binding efficiency, and expensive protein A affinity purification packing. , to achieve the effect of saving production time and equipment requirements, improving antibody binding efficiency, and fast antibody capture speed

Inactive Publication Date: 2012-12-26
BEAVERNANO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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-viscosit

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Using SL-PA magnetic beads to extract human IgG from human serum, including the following steps:

[0044] For the preparation method of SL-PA magnetic beads, see Example 1 of the Chinese patent application "A method for directional immobilization of functional proteins using SLP" (patent application number: 201210107107.7).

[0045] (1) Take 100mg of SL-PA magnetic beads, add them to a 5ml centrifuge tube, then add 5ml of washing buffer (pH 7.5, containing 50mM phosphate buffer and 150mM NaCl; the same below), oscillate to resuspend, and place in a magnetic separator After 2 minutes, the supernatant was sucked off after the solid-liquid separation. Repeat this step once;

[0046] (2) Mix the washed magnetic beads with 3ml of human serum (purchased from Guangzhou Ruite Biotechnology Co., Ltd.) (that is, human serum S before purification), and incubate at room temperature for 10 minutes, shaking from time to time during the period to maintain the balance between the magn...

Embodiment 2

[0063] Extracting human IgG1 monoclonal antibody from CHO cell expression supernatant, including the following steps:

[0064] The recombinant CHO cell expression line containing human IgG1 gene (purchased from Guangzhou Yuansheng Pharmaceutical Technology Co., Ltd.) was taken, and after serum-free suspension culture, the recombinant CHO cell line expressed human IgG1 and secreted it into the culture supernatant, and the expression level was 1.15mg / ml. Take 20ml of the unfiltered culture solution and mix it with 100mg of SL-PA magnetic beads. The purification steps are the same as steps 1-7 of Example 1.

[0065] Results: 21.62 mg of human IgG1 was successfully extracted from the culture supernatant of recombinant CHO cells, the purification yield was 96%, the product purity was 94%, and the purification operation took 37 minutes.

[0066] The same volume and the same sample were purified using antibody affinity chromatography (chromatographic system: AKTA purifer 10, GE life...

Embodiment 3

[0069] Extract Rituximab monoclonal antibody from CHO cell mixture, including the following steps:

[0070] Take 20 mg of rituximab (purchased from Roche Pharmaceuticals, USA) and mix it with 20 ml of CHO cell culture medium to obtain a CHO cell mixture containing rituximab. Use 100 mg SL-PA magnetic beads to extract rituximab from this mixture, and the steps are the same as steps 1-7 in Example 1. As a result, 19.49 mg of rituximab was successfully extracted from the mixture, with a purification yield of 97.45% and a purity of 95%.

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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...

Claims

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Application Information

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IPC IPC(8): C07K16/00C07K1/14
Inventor 任辉张曙光韩蓝青乌韦・斯莱泰安德烈亚斯・布韦特莱瑟刘蔼珊
Owner BEAVERNANO TECH
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