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A high-throughput screening method for cho-dhfr expression system cell lines

An expression system and screening method technology, applied in the field of cell engineering, can solve problems such as low cloning efficiency, and achieve the effects of high clone formation rate, shortened screening period and high expression amount

Active Publication Date: 2019-06-25
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Specifically, this method overcomes the problem of extremely low cloning efficiency by limiting dilution method by selecting cell pools with cell viability of 40% to 50%, performing monoclonalization on semi-solid medium, and using a high-throughput cell screening system. , so that it is easier to screen high-quality cell lines with stable and high expression

Method used

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  • A high-throughput screening method for cho-dhfr expression system cell lines
  • A high-throughput screening method for cho-dhfr expression system cell lines
  • A high-throughput screening method for cho-dhfr expression system cell lines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of cell pool

[0033] 1. Construction of Expression Constructs

[0034] Refer to OptiCHO TM Antibody Express Kit (purchased from Invitrogen), pOptiVEC TM - TA Cloning Kit (purchased from Invitrogen) and pcDNA TM 3.3- TA Cloning Kit (purchased from Invitrogen Company) instruction manual, independently prepared the expression vectors expressing the heavy chain and light chain of anti-TNFα fully human antibody (represented by item number A- before the vector name), they are: A- pOptiVEC-VL (containing Dhfr-VL) and A-pcDNA3.3-VH (containing Neo-VH).

[0035] 2. Transfection into Host Cells

[0036] use A-pOptiVEC-VL and A-pcDNA3.3-VH of Anti-TNFα fully human antibody were extracted with HiPure Plasmid DNA Purification Kit (purchased from Invitrogen Company), and were linearized and purified by PvuI (purchased from TaKaRa Company). Equivalent confluence, take a total amount of 40 μg DNA / electroporation system, use an electroporator (Bio-Rad ...

Embodiment 2

[0041] Example 2 The effect of conditioned medium on colony formation

[0042] 1. Preparation of conditioned medium and semi-solid medium

[0043] Preparation method of conditioned medium: take the stable cell line that has been transfected with pOptiVEC (the vector contains Dhfr gene, and does not contain other exogenous protein genes) for batch culture in 300 mL of OptiCHO Medium of 8 mM glutamine, and the cells are cultured to density at 0.9×10 7 ~1.1×10 7 Between cells / mL and cell viability >95%, centrifuge to separate the supernatant and cells, take the supernatant and filter it with a 0.22 μm membrane, and divide it into 10mL / tube, 25mL / tube, 50mL / tube, -80 Store at ℃ until use. Estimate the dosage when using it. Choose an appropriate volume of conditioned culture based on 37°C, thaw in a water bath, and discard the rest.

[0044] Preparation of semi-solid medium: The ingredients in the semi-solid medium formula in Table 1 were mixed vigorously at room temperature ac...

Embodiment 3

[0062] Example 3 Screening of high-expression stable cell lines by monoclonalization

[0063] 1. Monoclonalization of cell pools A, B, and C

[0064] According to the preparation system 2 in Table 1, prepare 3 parts of semi-solid medium, and centrifuge appropriate amount of cells in cell pool A (viability 41.58%), cell pool B (viability 49.66%) and cell pool C (viability 95.86%), and resuspend them respectively. In 1.5mL of 2.5×OptiCHO Medium, three cell suspensions with a cell density of 3000cells / mL were obtained. Three cell suspensions were mixed with three semi-solid medium, and after gentle mixing, 2 mL / well was added to two 6-well culture plates, named AK(5)~(10), and cultured for 10 minutes. ~12 days, 37°C, 5.0% CO 2 Under the conditions, wait for the cell mass to slowly form.

[0065] 2. ClonePix2 imaging and picking clones

[0066] After the cells were cultured in 6-well plates AK(5)~(10) for 12 days, the cells were evenly distributed and moderate in size (0.3~0.6...

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Abstract

The invention relates to the field of cell engineering, in particular to a high-throughput screening method for CHO-Dhfr expression system cell strains. The method comprises the following steps: pressurizing by adopting methotrexate for three turns; selecting a cell pool with the cell activity of 40 to 50 percent; performing monoclonal treatment in a specific semisolid medium. According to the method, the colony forming efficiency is remarkably improved; more stable strains with high expression quantity can be screened within a relatively short time.

Description

technical field [0001] The invention relates to the field of cell engineering, in particular to a high-throughput screening method for cell lines of CHO-Dhfr expression system. technical background [0002] The "engineered cell" lines used for the production of monoclonal antibodies or recombinant protein drugs with larger molecular weight should have the following characteristics: good growth characteristics, high serum-free suspension culture density (1-2 × 10 7 cell / ml); strong heterologous protein expression ability (20-70 pg / cell / day, pcd), with correct post-translational modification ability; host and recombinant cell lines with clear genetic background and stable phenotype, in line with relevant regulatory requirements. [0003] Currently, one of the most commonly used screening systems for cell line construction in the industry is the CHO-Dhfr system. CHO cells are Chinese Hamster Ovary cells (Chinese Hamster Ovary). DHFR refers to dihydrofolate reductase, which cat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10C12Q1/02
CPCC12N15/85C12N2800/107G01N33/5005
Inventor 刘静杨彬张彩霞陈华林陈莉孙文正李文佳
Owner SUNSHINE LAKE PHARM CO LTD
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