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Method for purifying monoclonal antibody resistant to IL-17RA

A technology of monoclonal antibody and purification method, applied in the field of purification of anti-IL-17RA monoclonal antibody, can solve the problems of complex antibody purification process, high process purification requirements, limited administration volume, etc., to improve purification efficiency and simplify operation. Process, shorten the effect of purification cycle

Active Publication Date: 2020-09-22
BEIJING DONGFANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] At present, many problems are often encountered in the process of research and development of anti-IL-17RA monoclonal antibodies. Monoclonal antibody products are usually used in high doses, about 200mg / time, but the volume of subcutaneous injection is limited, 1.0- 1.5ml is already a limit, which will indicate that the concentration of anti-IL-17RA monoclonal antibody will be as high as 100-150mg / ml, which requires higher purification of the process
Brodalumab is the first and only fully humanized monoclonal antibody that selectively targets IL-17 receptor A (IL-17RA). However, the purification process of brodalumab antibody is complicated. Afterwards, it is necessary to adapt to the pH condition of the next step by changing or debugging the buffer solution. In the mass production process, a lot of time is wasted, and impurities are likely to be introduced during the debugging process, resulting in many intermediate steps and low recovery. Simultaneously cost increases, for this reason, be badly in need of developing a kind of purification method that can be specially applicable to the anti-IL-17RA monoclonal antibody provided by the present invention

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  • Method for purifying monoclonal antibody resistant to IL-17RA
  • Method for purifying monoclonal antibody resistant to IL-17RA
  • Method for purifying monoclonal antibody resistant to IL-17RA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Screening of anti-IL-17RA monoclonal antibodies

[0065] Antibody purification is the most important step in process development. Before the antibody purification method provided by the present invention, anti-IL-17RA monoclonal antibody should be obtained first. The present invention utilizes a fully synthetic ScFv single-chain phage antibody library to screen and obtain a fully human monoclonal antibody specifically binding to IL-17RA. At present, fully human antibodies are the main direction for the development of therapeutic antibodies, and the emergence of antibody library technology provides a good technical platform for the preparation and screening of fully human antibodies. Antibody library technology bypasses the hybridoma process that was necessary in the development of monoclonal antibodies in the past, and can obtain various antibody genes and antibody molecular fragments without even going through the immunization process. Phage antibody library...

Embodiment 2

[0113] Example 2, Gradual dilution of phage Elisa to compare the affinity of anti-IL-17RA monoclonal antibodies

[0114] 3.1 Preparation of monoclonal antibody purified phage:

[0115] The 11 monoclonal antibodies (mAb-1, mAb-2, mAb-3, mAb-4, mAb-5, mAb-6, mAb-7, mAb-8, mAb-4, mAb-5, mAb-6, mAb-7, mAb-8, mAb -9, mAb-10, mAb-11) were transferred to 2YTAG liquid medium, shake cultured to the logarithmic growth phase, and then added M13K07 auxiliary phage infection, after centrifugation, the bacteria were resuspended in 2YTAKA, cultured at 28°C overnight and expanded The phages were amplified, and the phages were purified by sedimentation with PEG6000-NaCl the next day. The purified phage of the anti-IL-17RA monoclonal antibody (AMH14 / AML14) provided by the core patent US7833527B2 of the marketed product Brodalumab was used as a positive control.

[0116] 3.2 Affinity comparison at the phage level

[0117] IL-17RA-ECD-His was coated with 0.01M PBS buffer solution of pH 7.2, 10...

Embodiment 3

[0121] Example 3, Preparation of Anti-IL-17RA Whole Antibody

[0122] The heavy chain variable region gene and the light chain variable region gene of the 11 strains of monoclonal antibodies screened in Example 1 were respectively cloned into the vector pTSE containing the heavy chain constant region and the light chain constant region (such as Figure 4 shown), the heavy chain constant region is a human IgG1 constant region (see SEQ ID NO: 14 for the amino acid sequence), the light chain constant region is a κ chain constant region (see SEQ ID NO: 15 for the amino acid sequence), and the vector pTSE is a PTT vector Obtained by basic transformation, the preparation process refers to paragraph [0019] on page 3 of CN103525868A specification, the structure is as follows Figure 4 shown.

[0123] SEQ ID NO: 14 (heavy chain constant region sequence of human IgG1):

[0124] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT...

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Abstract

The invention relates to the technical field of antibody engineering, and specifically provides a method for purifying a monoclonal antibody resistant to IL-17RA. The method includes affinity chromatography, cation exchange chromatography and anion exchange chromatography. Firstly, the IL-17RA-resistant monoclonal antibody which is purified by the purification method has high affinity and good biological activity, can be combined with an IL-17RA antigen specifically, and is a potential therapeutic drug for autoimmune diseases; secondly, the monoclonal antibody resistant to IL-17RA can be purified in three steps, affinity chromatography is used for capturing target protein, peaks of acidic substances is eliminated through cation exchange chromatography, and aggregate is removed through anion exchange chromatography so as to the qualified monoclonal antibody resistant to IL-17RA. The method is suitable for pilot scale-up production, no adjustment steps of intermediate samples are needed,the process connection is smooth, and the process cycle is shortened.

Description

technical field [0001] The invention relates to the technical field of antibody engineering, in particular to a method for purifying an anti-IL-17RA monoclonal antibody. Background technique [0002] Autoimmune diseases refer to diseases caused by the body's immune response to self-antigens, resulting in damage to its own tissues. Many diseases have been listed as autoimmune diseases, such as psoriasis, rheumatoid arthritis, ankylosing spondylitis, scleroderma, etc. Among them, psoriasis (Psoriasis), also known as psoriasis, is characterized by erythematous and scaly plaques of different sizes and clear borders on the skin, covered with a large number of dry silvery white scales. The histological features of psoriatic skin are excessive proliferation of epidermal keratinocytes, vascular proliferation, and infiltration of dendritic cells, macrophages, neutrophils, and T cells. The pathogenesis of psoriasis involves complex inflammatory responses and immune system. The prev...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K1/22C07K1/18
CPCC07K16/2866C07K2317/52C07K2317/56C07K2317/76C07K2317/92
Inventor 白义刘晓航孟艳敏
Owner BEIJING DONGFANG BIOTECH
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