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Cell strain constructing method for preparing anti-EGFR completely-humanized monoclonal antibody

A technique of monoclonal antibody and construction method, which is applied in the field of cell line construction for the preparation of fully humanized anti-EGFR monoclonal antibody, which can solve the problems of limiting the research and development and industrialization of biosimilar drugs, and the impact of final product expression or activity. Achieve the effects of mild reaction conditions, easy industrialization, and cost reduction

Pending Publication Date: 2019-07-09
JINING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Minor changes in each production step will affect the expression level or activity of the final product; development of high-expression cell lines and cell culture processes, protein purification process and process control, quality standards for antibody drugs, etc., all limit biosimilars. R&D and Industrialization

Method used

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  • Cell strain constructing method for preparing anti-EGFR completely-humanized monoclonal antibody
  • Cell strain constructing method for preparing anti-EGFR completely-humanized monoclonal antibody
  • Cell strain constructing method for preparing anti-EGFR completely-humanized monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Transfection experiment:

[0050] Experimental Materials:

[0051] Host cell: DG44 (Chinese hamster ovary cell dihydrofolate reductase-deficient strain CHO / DHFR-, ATCC)

[0052] Transfection reagent: Freestyle Max Reagent (Invitrogen 16447-100)

[0053] OPTI PRO SFM (GIBCO 12309-019)

[0054] Linearized plasmid: pCHO1.0invitrogen

[0055] DG44 complete medium: CD DG44 (Invitrogen 12610-010) contains

[0056] 4mM GlutaMAX-I (Invitrogen 35050-061)

[0057] 18ml / L PLURONIC F68(PF68)(Invitrogen 24040-032)

[0058] Consumables and related instruments

[0059] CORNING 125ml Erlenmeyer Flask, Item No. 431143;

[0060] Shaker INFORS HT Model: Multitron II

[0061] Parameter setting: 110rpm, 36.5℃, 6%CO 2 , humidity 75%;

[0062] Cell Counter BECKMAN COULTER Model: VI-CELL XR

[0063] Biological Safety Cabinet Shanghai Shangjing Model: BSC-ⅡA2

[0064] Transfection experimental steps:

[0065] 1. 24h before transfection, the DG44 cells for standby transfection were ...

Embodiment 2

[0136] Equipment and Consumables

[0137] Constant temperature water bath Julabo Model: TW20

[0138] Biological Safety Cabinet Shanghai Net Model: BSC-Ⅱ A2

[0139] CO2 static incubator THERMO SCIENTIFIC model: HERA cell240i

[0140] Cell Counter BECKMAN COULTER Model: VI-CELL XR

[0141] Centrifuge EPPENDORF Model: Centrifuge 5810R

[0142] ClonePix GENETIX Model: Clonepix FL

[0143] 96-well plate (greiner bio-one CELLSTAR, Cat. No. 655185)

[0144] 24-well plate (greiner bio-one CELLSTAR, Cat. No. 662102)

[0145] 6-well plate (greiner bio-one CELLSTAR, Cat. No. 657185)

[0146] Shaker KUHNER Model: Climo-Shaker ISF1-XC

[0147] Parameter setting: 225rpm, 36.5°C, 6% CO2, 85% humidity.

[0148] Shaker TPP TUBESPIN BIOREACTOR 50, Item No.: 87050

[0149] The culture medium and reagents are shown in Table 5:

[0150] table 5

[0151] Medium name brand article number SFM4 ADCF Hyclone SH3A2156.01 Glutamine Sigma G8540 MTX Sigma ...

Embodiment 3

[0207] The invention selects the stable cell line JK-I with high expression of Vectibix through POOL construction, batch feeding experiment screening and plating, clone picking, HTRF, clone amplification, and clone batch feeding experiment. Through process optimization, the expression amount reached 2.05g / L, and the product SEC, pI-cIEF, deglycosylated molecular weight, and cell-based antibody activity were significantly improved. Example 3 Cell Line Stability Test

[0208] The genetic stability of cell lines is the basis for subsequent stable production processes and product quality. The genetic stability of the cells in the PCB (Primary Cell Bank), MCB (Master Cell Bank), and WCB (Work Cell Bank) three cell banks in the project was studied by molecular biology methods, including heavy chain (HC) and light Stranded (LC) DNA sequence stability and copy number stability. The project plans to carry out cell line stability research for no less than 12 weeks, including cell grow...

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Abstract

The invention discloses a cell strain constructing method for preparing an anti-EGFR completely-humanized monoclonal antibody. The method comprises the following steps: in a POOL screening step aftercells of Chinese hamster ovary cell dihydrofolate reductase deficient strains CHO / DHFR- complete a transfection step, screening cell strains integrated with a heavy chain gene by using an SFM4CHO culture medium without containing HT in the first stage, and screening out the cell strains integrated with a light chain gene by using Blasticidin in the culture medium at the same time; and in the second stage, adding MTX to increase copy numbers of the light and heavy chain genes integrated into cell strain genomes, as to improve an expression quantity. An efficient stable cell strain, of which theexpression quantity reaches 2.0-3.0 g / L, suitable for a large trial production process is firstly cloned and screened out, product SEC, pI-cIEF, a de-sugaring molecular weight, and antibody activitybased on the cells are remarkably improved, so possibility is provided for realizing industrialization of recombining the anti-EGFR completely-humanized monoclonal antibody in China.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for constructing a cell line for preparing an anti-EGFR fully humanized monoclonal antibody. Background technique [0002] Monoclonal antibody (mAb) is an antibody produced by a single B cell clone with a high degree of uniformity and targeting only a specific antigenic epitope. It has specific targeting and is mainly indicated for tumors, autoimmune diseases, Infectious diseases, etc. Since the world's first therapeutic monoclonal antibody Rituxin was approved by the FDA in 1997, monoclonal antibodies have developed rapidly in the global biomedical field in just 20 years, becoming one of the research hotspots in recent years. [0003] In recent years, a number of companies and research institutions have emerged in China to study therapeutic monoclonal antibody drugs, among which therapeutic antibodies targeting epidermal growth factor receptor (EGFR) are of particu...

Claims

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Application Information

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IPC IPC(8): C12N5/20
CPCC07K16/2863C12N2510/02
Inventor 李明丽李明轩尹春光魏衍刚
Owner JINING UNIV
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