Screening method of cell strain of GS expression system

An expression system and screening method technology, applied in the field of high-throughput screening of GS expression system cell lines, can solve the problems of heavy workload, long experiment cycle, cumbersome operation, etc., and achieve high clone formation rate, shortened screening cycle, and stable sex enhancing effect

Inactive Publication Date: 2016-08-10
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The most common method for monoclonal screening is limiting dilution cloning (LDC), which is low-cost and easy to operate, that is, the cell density is 95% Cell pool, the highest cloning efficiency is only about 7%, and the average level is 3% to 4%, and the operation is cumbersome, the workload is heavy, the experiment period is long, the experiment efficiency is low, and high-throughput screening cannot be achieved

Method used

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  • Screening method of cell strain of GS expression system
  • Screening method of cell strain of GS expression system
  • Screening method of cell strain of GS expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of cell pool A and monoclonal screening of cell pool A for stable cell lines with high expression

[0032] 1. Construction of expression constructs

[0033] Referring to WO 2007 / 014162 A2 patent and GS Xceed TM Gene Expression System (purchased from Lonza Company) manual independently prepared Anti-VEGF humanized antibody GS double gene expression vector.

[0034] 2. Transfection of Mammalian Cells

[0035] use HiPure Plasmid DNA Purification Kit (Invitrogen, K2100-04) was used to extract the double-gene expression vector of Anti-VEGF humanized antibody, linearized and purified by PvuI (TaKaRa, 1242A), and the total amount of 40 μg DNA / electroporation system was used CHOK1SV GS-KO cells (Lonza, GS Xceed TM GeneExpression System), resuspend the cells after three electric shocks in a 125mL conical culture flask (corning, 431143-50EA) containing 30mL CD CHO Medium (Invitrogen, 12490-25), 140rmp, 37°C, 8.0% CO 2 Shaking culture under conditions. ...

Embodiment 2

[0073] Example 2 Preparation of Cell Pool B and Cell Pool B Monoclonal Screening for High Expression Stable Cell Lines

[0074] 1. Construction of expression constructs

[0075] Referring to WO 2007 / 014162A2 patent and GS Xceed TM The Gene Expression System (purchased from Lonza Company) was used to independently prepare the Anti-VEGF humanized antibody GS double gene expression vector.

[0076] 2. Transfection of Mammalian Cells

[0077] use HiPure Plasmid DNA Purification Kit (Invitrogen, K2100-04) was used to extract the double-gene expression vector of Anti-VEGF humanized antibody, linearized and purified by PvuI (TaKaRa, 1242A), and the total amount of 40 μg DNA / electroporation system was used CHOK1SV GS-KO cells (Lonza, GS Xceed TM GeneExpression System), resuspend the cells after three electric shocks in a 125mL conical culture flask (corning, 431143-50EA) containing 30mL CD CHO Medium (Invitrogen, 12490-25), 140rmp, 37°C, 8.0% CO 2 Shaking culture under condition...

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Abstract

The invention relates to a screening method of a cell strain of a GS expression system. The method includes the steps of: pressurizing methionine sulphoximine, selecting a cell pool being 25-35% in cell activity for performing monoclonal screening, and adding the cell pool to a semi-solid culture medium containing 10% of a conditional culture medium to form mono-clone (wherein final cell concentration is 150 cells/ml), and then performing screening and amplified cultivation to obtain a high-expression cell strain through a high-throughput cell screening system, and performing stability evaluation to the screened high-expression cell strain. The method can be used for obtaining the cell strain being more than 90% in stability.

Description

technical field [0001] The invention relates to the field of cell engineering, in particular to a high-throughput screening method for GS expression system cell lines. technical background [0002] Lonza's glutamate synthetase (glutamine sythetase, GS) expression system came out in 1992 and has been improved. This system inserts foreign genes into the downstream of the GS gene expression vector, and host cells can choose endogenous non- Recombinant mouse myeloma cells NS0 expressing GS gene and Chinese hamster ovary cell CHOK1SV GS-KO of GS gene knockout type were added with GS inhibitor aminosulfomethionine (methioninesulfoximine, MSX) during cell culture to make GS gene and The connected target gene can be amplified together to achieve the purpose of increasing the expression level of the target gene. So far, Lonza's GS expression system has been widely used in the biopharmaceutical industry, including the marketed (Roche) and (Medimmune). [0003] The purpose of cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65
CPCC12N15/65C12N15/85C12N2800/107
Inventor 刘静张彩霞李扬杨彬孙文正倪健
Owner SUNSHINE LAKE PHARM CO LTD
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