Method for screening high-output stable cell strain by HTRF

A cell line, high-yield technology, applied in the field of cell line screening, to achieve the effects of short time-consuming, stable results, and saving manpower and material costs

Inactive Publication Date: 2013-09-25
无锡药明生物技术股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] However, there is currently no method for scree

Method used

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  • Method for screening high-output stable cell strain by HTRF
  • Method for screening high-output stable cell strain by HTRF
  • Method for screening high-output stable cell strain by HTRF

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0031] Example 1

[0032] 1) In a 96-well plate, culture the cell line monoclonal (DG44) expressing the target antibody (Avastin) according to conventional methods;

[0033] 2) After cell culture for 10 days, perform 3 / 4 change of medium. After one day of medium change, draw 10μL of supernatant and add it to a 384-well screening microplate, and add 5μL of Human-IGg-XL665 (Cisbio) and 5μL of Anti to the well -Human-IGg-Fc-Cryptate (Cisbio), and use 10μL of the Avastin target antibody to be detected as a standard for control, react at room temperature for 2.5h or overnight, according to the principle of HTRF method, detect the absorbance ratio of A665nm / A615nm This ratio is used to judge the expression yield of the cell line clone. When the ratio is lower, the target antibody yield is higher. The result is figure 2 .

Example Embodiment

[0034] Example 2

[0035] For a single clone of the cell line (DG44) expressing the target antibody (Rituxin), the medium was changed 3 / 4 after 8 days of culture, and the supernatant was aspirated one day after changing the medium. The test was carried out according to the method of Example 1. The results are shown in image 3 .

Example Embodiment

[0036] Example 3

[0037] For a single clone of a cell line (CHO-M) expressing the target antibody (Humira), the medium was changed 3 / 4 after 7 days of culture, and the supernatant was aspirated one day after the medium was changed. The test was performed according to the method of Example 1. The results are shown in Figure 4 .

[0038] by Figure 2-4 It can be seen that the OD ratio of different cell lines varies, and the lower the ratio, the higher the antibody yield. Among them, for Example 3, the first 26 clones with higher yields were selected for expansion culture and process optimization, and finally a cell line with an expression level of up to 2.5g / l and a number of cells with a yield higher than 1.5g / l were selected Experiments prove that the method of the present invention is simple and feasible for cell strain screening.

[0039] To sum up, the current HTRF technology is mainly used for compound screening in drug research and new drug screening, while the present inven...

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Abstract

The invention discloses a method for screening high-output stable cell strain by HTRF, comprising the following steps of: (1) in a microwell plate, culturing cell strain monoclone for expressing an object antibody or a fusion protein; (2) absorbing cultured cell supernatant, and added the cell supernatant into the microwell plate for screening, adding Human-IGg-XL665 and Anti-Human-IGg-Fc-Cryptate into micropores, contrasting by using the object antibody or the fusion protein to be detected as a standard substance, reacting under room temperature for 2.5h or overnight, detecting an absorbance ratio of A665nm to A615nm based on a HTRF method principle, and judging expression output of the cell strain clone based on the ratio. The method has advantages of short time consumption, high flux, simple operation and stable result, can effectively screen high-expressed cell strain clone in a short time, and accelerates a process in a bio-pharmaceutical industry.

Description

technical field [0001] The invention relates to a method for screening cell strains, in particular to a method for screening high-yield stable cell strains using HTRF (homogeneous time-resolved fluorescence technique). Background technique [0002] At present, domestic and foreign biopharmaceutical industries use mammalian engineering cell lines to express and produce monoclonal antibodies or fusion proteins. When screening high-yielding cell lines, ELISA is mainly used. As a routine protein expression identification method, ELISA has some insurmountable shortcomings in detecting the expression of monoclonal antibody or FC fusion protein in mammalian engineered cell lines, such as: 1) The experimental steps are time-consuming; 2) The experiment needs to be coated , plate washing, blocking, primary antibody binding, plate washing, secondary antibody binding, plate washing, color development and other operations, the steps are more complicated and take one day; 3) Due to many ...

Claims

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Application Information

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IPC IPC(8): G01N21/64C12Q1/04
Inventor 蔡洁行徐淑荣徐亚平李鹃李浩强
Owner 无锡药明生物技术股份有限公司
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