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Preparation method of recombinant anti-TNF-alpha completely humanized monoclonal antibody

A monoclonal antibody, fully human technology, applied in the direction of recombinant DNA technology, anti-animal/human immunoglobulin, chemical instruments and methods, etc., can solve the problem of antibody expression, stability, titer difference, technical means level Unevenness, the quality of life of patients cannot be effectively guaranteed, etc.

Inactive Publication Date: 2018-06-15
安徽未名生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the face of foreign technological monopoly on adalimumab, the technical means of the domestic generic drug industry are uneven, resulting in significant differences in the results of antibody expression, stability, and potency. The level of domestic antibody drugs has always lagged behind foreign companies. The quality of life of domestic patients cannot be effectively guaranteed

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  • Preparation method of recombinant anti-TNF-alpha completely humanized monoclonal antibody
  • Preparation method of recombinant anti-TNF-alpha completely humanized monoclonal antibody
  • Preparation method of recombinant anti-TNF-alpha completely humanized monoclonal antibody

Examples

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Effect test

Embodiment 1

[0027] Example 1 Obtainment and Synthesis of Light Chain and Heavy Chain Sequences

[0028] The present invention determines the IgG1 light chain constant region kappa sequence (IGK, Gene ID: 50802) and heavy chain constant region gamma sequence (IGHG1, Gene ID: 3500) of IgG1 according to human IgG1 related literature and GeneBank data, and according to the original drug Humira Publicly available data identified the amino acid and DNA sequences of the light and heavy chain variable regions of adalimumab. After sequence comparison and confirmation, the codons preferred by Chinese hamsters were selected to artificially synthesize the full-length light chain L, heavy chain variable region VH and heavy chain constant region CH sequences, and the required enzyme cutting sites and protection sites were added.

[0029] Wherein the nucleotide sequence of the light chain has the sequence of SEQ ID NO.1, the amino acid sequence of the light chain has the sequence of SEQ ID NO.3; the nuc...

Embodiment 2

[0030] Embodiment 2pHL101-GS / Neo vector construction

[0031] (1) The synthetic light chain full-length sequence L, heavy chain variable region VH and heavy chain constant region CH sequences are placed in the amplification plasmid pEASY, and amplified in large quantities to obtain sufficient target fragments.

[0032] (2) First select heavy chain constant region CH double enzyme digestion (HindⅢ and NheI) and connect it into a mammalian expression vector such as pcDNA3.1(+) to obtain the connection vector of heavy chain constant region CH, which is convenient for integration with other heavy chain The V regions are connected. A double BbsI structure restriction site (gtcttcgagaagac) is added to the N-terminus of the CH sequence, which is connected to the heavy chain V region. The restriction endonuclease characteristics of BbsI are different from those of traditional restriction endonucleases. The base at the position forms a gap with a difference of 4 bases. In the case of...

Embodiment 3

[0035] Embodiment 3 transfection host cell CHO-GS -Screening for high expression clones

[0036] CHO cells can be grown in high density in bioreactors, easy to perform genetic manipulation, N-glycosylation type is similar to human beings, and have low risk of virus transmission, so they are widely used in the field of biopharmaceuticals. The present invention uses the GS-deficient cell CHO-GS independently transformed and identified by our commercialized CHO-K1 cell line - , to further reduce the expression of endogenous GS, using the GS-Neo dual screening system.

[0037] The linearized pHL101-GS / Neo plasmid was transfected by electroporation, and the GS-Neo dual screening system was used for gradient pressurization screening to obtain stable cell clones that highly express recombinant anti-TNF-α fully human monoclonal antibody. After multiple rounds of transfection and screening, after identification and detection, cell clones with expression levels greater than 35pg / cell·...

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Abstract

The invention discloses a preparation method of a recombinant anti-TNF-alpha completely humanized monoclonal antibody. The method comprises: synthesizing a nucleic acid sequence of a recombinant anti-TNF-alpha completely humanized monoclonal antibody, using the obtained nucleic acid sequence of the recombinant anti-TNF-alpha completely humanized monoclonal antibody to construct a carrier, transfecting a host cell, and screening high-expression cell clone to establish a cell bank; and using the obtained cell strain for culture, and performing separation and purification to obtain the recombinant anti-TNF-alpha completely humanized monoclonal antibody. Light chains and heavy chains of the recombinant anti-TNF-alpha completely humanized monoclonal antibody is designed and synthesized according to preferred codons of Chinese hamsters, an eukaryotic expression carrier is established, a host cell CHO-GS- is transfected, serum-free culture is adopted, and separation and purification are adopted to obtain the high-purity recombinant anti-TNF-alpha completely humanized monoclonal antibody. The preparation method is simple to operate, short in production period, and suitable for industrial production.

Description

technical field [0001] The invention relates to the technical field of monoclonal antibodies, in particular to a method for preparing recombinant anti-TNF-α fully human monoclonal antibodies. Background technique [0002] Rheumatoid arthritis (Rheumatoid Rrthritis, RA) is a chronic, erosive, symmetrical, small joint, and multi-articular systemic autoimmune disease mainly characterized by inflammation of the joint synovium, which seriously affects the patient's Quality of Life. [0003] TNF-α is a common cytokine that promotes synovial inflammation and occupies an important position in the network of pathogenesis. TNF-α is a pleiotropic pro-inflammatory factor, one of the earliest and most important inflammatory mediators in the process of inflammatory response: it activates mononuclear macrophages in an autocrine and paracrine manner to produce a large amount of IL-1 , IL-6, IL-8 and other pro-inflammatory factors can cause a cascade reaction and amplify signal transductio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24C12N15/85C12N5/10
CPCC07K16/241C07K2317/21C07K2317/56
Inventor 王强
Owner 安徽未名生物医药有限公司
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