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Method for fermentative production of recombinant human interleukin-12

A technology of human interleukin and seed cells, applied in biochemical equipment and methods, fermentation, microorganisms, etc., can solve the problem of low expression of recombinant human interleukin-12, and achieve the cost saving of culture medium, high survival rate and time saving. Effect

Active Publication Date: 2018-07-06
KANGLITAI BIOMEDICAL (QINGDAO) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] In order to solve the above problems, the present invention provides a method for fermentatively producing recombinant human interleukin-12, which uses CHO cells containing recombinant human interleukin-12 gene, and overcomes the limitations of existing culture methods by optimizing serum-free culture conditions. The problem of low expression of recombinant human interleukin-12

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  • Method for fermentative production of recombinant human interleukin-12
  • Method for fermentative production of recombinant human interleukin-12
  • Method for fermentative production of recombinant human interleukin-12

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1、5

[0029] Embodiment 1, 5L scale cell culture process development

[0030] According to the previous experiments, CD OptiCHO medium was selected as the basic medium, CHO CD EfficientFeed B was used as the feed medium, and a 5L (tank volume, working volume 3L) Sartorius fermenter fed-batch fermentation culture was carried out to establish a fermenter The technological process of cultivation, and the influence of different rotation speeds of the stirring paddles in the 5L fermenter on the growth, production and metabolic parameters of cells were compared and analyzed, and the parameter values ​​with better performance were selected.

[0031] The feed addition scheme is to add feed FeedB once every 2-3 days, and control the total amount of FeedB added to be 40% of the final volume of culture; according to the detection of Nova Bioprofile400 biochemical analyzer, add glutamine (Gln) and glucose (Gluc ), control the Gluc concentration above 2g / L, and the Gln content between 1.5-5mM....

Embodiment 2、7

[0062] Example 2, 7L pilot scale cell culture process optimization

[0063] Based on the previous 5L Sartorius fermenter fed-batch fermentation process, the 7L (10L tank volume, 7L actual culture volume) scale process was explored according to the 5L fermenter process, and the 7L pilot scale fermentation process was determined. Production process such as figure 1 shown.

[0064] With a preheated CD OptiCHO TM AGT + 0.18% F68 + 8mM L-glutamine medium for recovery of rhIL-12 production cells, the density of recovered cultured cells is 4.0-5.0×10 5 individual / mL. The culture conditions are: T-125 shake flask, rotation speed 110-120rpm, temperature 37°C, 5% CO 2 incubator. Cells were subcultured every 2 days and adapted to 2-3 passages until the cell doubling time returned to normal. After the cells grow normally, the cell expansion is performed, and the inoculation is performed when the cells expand to the required volume. Inoculate the cell density at 5.0×10 5 Individu...

Embodiment 3

[0071] Example 3. 7L three batches of continuous production

[0072]The Sartorius fermenter (BIOSTAT B plus 10L) used in the three batches of 7L continuous production tests in this embodiment. Rinse the fermenter with tap water until there is no visible foreign matter, and then soak in 0.2M sodium hydroxide for 6-24 hours to soften or dissolve the attachment; scrub the soaked fermenter with tap water at least 3 times, and rinse until it is invisible to the naked eye. Remove any residue; rinse 3 times with pure water; finally wash 3 times with Mill-Q ultrapure water. After the tank body is assembled, prepare 1 / 3 tank volume of PBS solution and add it to the fermenter tank. After completing the electrode calibration, connect it to the tank body. Sterilize at 121°C for 35 minutes according to the tank body manual and sterilizer verification parameters.

[0073] To seed CHO cells, use pre-warmed CD OptiCHO TM Culture medium + 0.18% F68 + 8mM L-glutamine medium for resuscitat...

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Abstract

The invention provides a method for fermentative production of recombinant human interleukin-12, and belongs to the field of biological product production processes. The method comprises: 1) resuscitating and activating Chinese hamster ovary cells (CHO) containing a recombinant human interleukin-12 expression gene by using a serum-free culture medium, and carrying out enlarging culture to obtain seed cells; 2) after the seed cells normally grow, carrying out adaptive enlarging culture on the seed cells; 3) inoculating the obtained seed cells into a bioreactor, and culturing; 4) periodically adding a serum-free supplemented culture medium and glutamine during the culturing, and monitoring cell growth; and 5) terminating the culturing and harvesting the cells when the cell viability is lessthan 80%. With the method of the present invention, a large amount of the recombinant human interleukin-12 can be expressed, and the downstream purification is easily performed.

Description

technical field [0001] The invention belongs to the field of biological product production technology, in particular to a method for fermenting and producing recombinant human interleukin-12 by cultivating eukaryotic cells Chinese hamster ovary cells (CHO) in a serum-free medium. Background technique [0002] Interleukin-12 (IL-12) was discovered by Trinchieri and Gately in 1989 and 1990. It was originally named NK cell stimulating factor (NKSF) and cytotoxic lymphocyte maturation factor (CLMF). In 1991, it was named interleukin- 12. [0003] Interleukin-12 is a 70 kDa heterodimer consisting of two covalently linked subunits, one of 35 kDa (P35 subunit) and the other of 40 kDa (P40 subunit). The P35 subunit is produced by a variety of cells, including T, B lymphocytes, NK cells, and large monocytes, while the P40 chain is mainly produced by activated monocytes and B cells, and neither P35 nor P40 has any biological active. [0004] IL-12 has the activity of regulating and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12P21/02
CPCC07K14/5434C12N5/0682C12N2500/32C12N2500/90C12N2510/02
Inventor 郑彬彬卢世香黄冰姜会春赵毅
Owner KANGLITAI BIOMEDICAL (QINGDAO) CO LTD
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