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Preparation method for recombinant human antibody fusion protein

A fusion protein and human antibody technology, applied in the field of biomedicine, can solve the problems of cumbersome operation, low expression amount, and many processing opportunities.

Active Publication Date: 2021-01-12
SHANGHAI JINGZE PHARMA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The prokaryotic expression system is characterized by a short time course and low cost. The disadvantage is that the expression of eukaryotic proteins has not been accurately modified; a large number of proteins are often precipitated into insoluble inclusion body polymers, which require complex denaturation and renaturation processes; the secretion of a large number of proteins is relatively slow. difficulty
The eukaryotic expression system is characterized by many opportunities for protein post-translational processing, and the protein produced is closer to the natural protein, but its expression is low, the operation is cumbersome, and the product purification is complicated.
Biological activity and immunogenicity of recombinant proteins produced sometimes vary due to post-translational processing that is not identical

Method used

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  • Preparation method for recombinant human antibody fusion protein
  • Preparation method for recombinant human antibody fusion protein
  • Preparation method for recombinant human antibody fusion protein

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preparation example Construction

[0099] 2. The preparation method of the present invention can express a high-purity recombinant human antibody fusion protein with a suitable glycosylation pattern.

[0100] 3. Compared with other eukaryotic expression systems, the DG44 expression system can efficiently express foreign genes with a high expression level;

[0101] 4. During the purification process, use affinity chromatography-anion-cation to obtain antibody protein with a purity greater than 99.8%, which is a simple, convenient, low-cost, and high-yield antibody fragment purification and preparation method;

[0102] 5. Since trace process-related impurities such as host cell protein (HCP) and host cell DNA (HCD) residues in biopharmaceuticals may have a certain impact on the safety and efficacy of biopharmaceuticals, precise HCP and HCD control is of great importance to The quality and safety of recombinant human antibody fusion proteins are of particular importance.

[0103] 6. The recombinant human antibody...

Embodiment 1

[0106] Example 1 Engineering Cell Line Construction of Recombinant Human Antibody Fusion Protein Gene

[0107] After the pCHO 1.0 vector was digested with AvrII (CCTAGG) and PacI (TTAATTAA), it was ligated with the recombinant human antibody fusion protein gene fragment, and the ligated product was transformed into DH5α E. coli competent cells, and spread on LB agar culture plates containing ampicillin resistance On the plate, through resistance screening, a single colony transformant was obtained on the plate. The recombinant plasmids were extracted after single-clonal colonies were selected and cultured, and the extracted recombinant plasmids were digested with AvrII / PacI double enzymes for DNA sequencing. The sequencing results of the recombinant plasmids were consistent with the expected sequence comparison.

[0108] The correct recombinant plasmid Pvu I of clone is digested and transformed into the CHO-DG44 host cell (purchased from Invitrogen Company) in the logarithmic ...

Embodiment 2

[0109] Example 2 Purification and Identification of Recombinant Human Antibody Fusion Protein

[0110] The engineered cell strain of Example 1 was induced by IPTG, and 1000 ml of culture supernatant was taken.

[0111] 1. Affinity chromatography

[0112] Get the filtrate and load it on the Protein ADiamond affinity chromatography column (filler comes from Bogelong Shanghai Biotechnology Co., Ltd.) equilibrated with "20mmol / L PB, 150mmol / L NaCl, pH 7 ± 0.5" for chromatography, use "50mmol / L CB, pH5±1” eluting, “50mmol / L CB, pH4.5±0.5” elution, detected by ELISA Book Example Recombinant Human Antibody Fusion Protein Peak, Collection of Flow Through.

[0113] 2. Low pH incubation and depth filtration

[0114] 2.1 Low pH incubation

[0115] Selection of buffer: the affinity chromatography flow-through in step 1 is directly adjusted to pH 5.5 with 1mol / L Tris, and placed at 2-8°C for testing; the same batch of affinity chromatography flow-through in step 1 is used Adjust the...

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Abstract

The invention provides a purification method for a recombinant human antibody fusion protein. The method comprises the following steps: (a) providing a transfection vector for expressing a gene of therecombinant human antibody fusion protein; (b) transfecting the transfection vector into a mammalian cell and performing culturing to obtain a raw material solution containing the recombinant human antibody fusion protein and impurities; (c) carrying out affinity chromatography on the raw material solution to obtain an affinity chromatography solution; and (d) carrying out low-pH incubation and deep filtration on the affinity chromatography solution to prepare a first purified solution, namely the purified recombinant human antibody fusion protein, wherein the pH value of the low-pH incubation is 3-4, and citric acid or salt thereof is utilized to adjust the pH value. The method disclosed by the invention not only can remove the impurities efficiently, but also keeps the activity of the target protein-the recombinant human antibody fusion protein, so that the high-purity and high-concentration recombinant human antibody fusion protein is prepared so as to be used for treating ocular neovascularization diseases of mammals.

Description

technical field [0001] The invention relates to the field of biomedicine. Specifically, the present invention relates to a method for preparing high-purity recombinant human antibody fusion protein. Background technique [0002] Vascular endothelial growth factor (VEGF) is a heparin-binding growth factor capable of inducing blood vessel formation (angiogenesis) in vivo and expressed specifically in vascular endothelial cells. The human VEGF protein was identified in 1989, and in addition, its gene has been cloned and its gene sequence sequenced. [0003] Antibody fusion protein refers to the product obtained by using genetic engineering technology to fuse antibody fragments with other biologically active proteins. It can be expressed in prokaryotic cells (such as Escherichia coli) and also in eukaryotic cells. The prokaryotic expression system is characterized by a short time course and low cost. The disadvantage is that the expression of eukaryotic proteins has not been ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C07K19/00C07K1/22C07K1/36C07K1/34C07K1/18A61K38/17A61P27/02
CPCC12N15/85C07K14/71C12N9/12C12Y207/10001A61P27/02C07K2319/30C12N2800/107A61K38/00
Inventor 彭红卫韩为跃张磊王冲万云雷俞亚波
Owner SHANGHAI JINGZE PHARMA CO LTD
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