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Cell culture medium for producing anti-PD-1 monoclonal antibody and optimizing method thereof

A monoclonal antibody and culture medium technology, applied in biochemical equipment and methods, reproductive tract cells, chemical instruments and methods, etc., can solve problems such as medium optimization, reduce production costs, increase antibody expression, and reduce resources and the effect on energy consumption

Inactive Publication Date: 2017-03-08
深圳万乐药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In terms of the use of CHO cells for monoclonal antibody expression, there is no report on medium optimization combined with amino acid analysis

Method used

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  • Cell culture medium for producing anti-PD-1 monoclonal antibody and optimizing method thereof
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  • Cell culture medium for producing anti-PD-1 monoclonal antibody and optimizing method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The optimization of embodiment 1 basal medium

[0050] 20 kinds of basal medium (see Table 1) were used for 125mL shake flask batch culture of genetically recombined CHO cells expressing anti-PD-1 monoclonal antibody in the DHFR-deficient system (batch culture means that after the cells are inoculated into the basal medium, Only glucose was added to meet the demand of carbon source, no feed medium was added until the cells entered the dying phase), and the culture conditions were: inoculation density 5×10 5 cells / mL, the culture volume is 30mL, the shaker speed is 110rpm, the culture temperature is 37℃, CO 2 The concentration is 5%. From the 0th, 3rd, and 5th days of culture to the end of the culture (cell viability is lower than 70%), samples are taken to detect the viable cell density (VCD) and cell viability (VIA). The viable cell density of 20 kinds of basal medium See the appendix for the change curve of cell viability figure 1 And attached figure 2 .

[0051]...

Embodiment 2

[0060] The optimization of embodiment 2 feeding medium

[0061] Use the basal medium determined in Example 1, 10 kinds of feed medium (see Table 2) to carry out 125mL shake flask batch feed culture, culture condition: inoculation density 8 * 10 5 cells / mL, culture volume 30mL, culture rotation speed 110rpm, culture initial temperature 37°C, temperature dropped to 33°C on the 5th day of culture, CO 2 The concentration is 5%, and the feed medium of 6% of the initial culture volume is supplemented on the 3rd day (D3), 5th day (D5), 7th day (D7), 9th day (D9), 11th day (D11), respectively, The primary reference standard for screening feed medium is the expression level of anti-PD-1 monoclonal antibody, so samples are taken at the end of the culture to detect the expression level of the antibody (Titer). See Figure 7 .

[0062] Such as Figure 7 As shown, the top three antibody expressions in the 10 feed media are: FA04 (OPM CHOCDFeed1), FH02 (Cell Boost 6) and FM09 (Cellvento ...

Embodiment 3

[0077] In order to further verify that the optimized basal and feed media have the advantages of improving cell growth and antibody expression compared with unoptimized media, a comparative test of cell batch fed culture on a 3L bioreactor was carried out.

[0078] The test conditions are shown in Table 6. There were 4 batches of co-cultivation, 2 batches used optimized base and feed medium, and 2 batches used unoptimized medium. Except for different culture media, other culture conditions were the same. The test results are shown in Figure 13-15 .

[0079] Table 6 Comparative test conditions before and after medium optimization

[0080]

[0081] Depend on Figure 13 It can be seen that the growth status of the cells before and after the optimization of the base and feed medium, the optimized medium can promote cell growth, and facilitate the maintenance of cell viability. The average PVCD of the optimized medium is 3.84×10 7 cells / mL, the average PVCD before optimizing ...

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Abstract

The invention provides a cell culture medium for producing anti-PD-1 monoclonal antibody and an optimizing method thereof; by applying an amino acid analysis technology, key amino acid influencing on cell growth and antibody expression is determined; on this basis, the culture medium type is determined, amino acid type and addition of the culture medium are optimized, so that the cell growth, maintenance and antibody expression effect are optimized.

Description

technical field [0001] The invention relates to the field of monoclonal antibody cell culture technology, and specifically relates to a cell culture medium for producing anti-PD-1 monoclonal antibody and an optimization method thereof. Background technique [0002] Programmed Death-1 (Programmed Death-1), also known as PD-1 and CD279, was first cloned and named from the apoptotic mouse T-cell hybridoma 2B4.11 in 1992. PD-1 is a member of the immunoglobulin superfamily CD28 family, a type I transmembrane glycoprotein with a relative molecular weight of 50-55KD. PD-1 is a T cell immunosuppressive receptor, highly expressed in activated T cells and B cells, can limit the function of T cell effectors in tumor cells, and plays a key role in regulating tumor immune escape and autoimmune response role. [0003] As a receptor, PD-1 has two ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC). The combination of PD-1 and PD-L1 can activate TCR and BCR, promote the phosphorylation of Tyr in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C07K16/28
CPCC12N5/0682C07K16/2818C12N2500/32
Inventor 张慧娟高玲梅于玉根袁庆陈泠
Owner 深圳万乐药业有限公司
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