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Culture technology of mammalian cells capable of efficiently expressing recombinant cat interferon [omega]2

A mammalian, high-efficiency expression technology, applied in the field of biopharmaceuticals, can solve the problems of low-efficiency and high cost of mammalian cell expression

Inactive Publication Date: 2021-09-07
成都彤琦恩生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a mammalian cell culture process for efficiently expressing rFeINF-ω2, so as to overcome the problems of low efficiency and high cost of expression in mammalian cells

Method used

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  • Culture technology of mammalian cells capable of efficiently expressing recombinant cat interferon [omega]2
  • Culture technology of mammalian cells capable of efficiently expressing recombinant cat interferon [omega]2
  • Culture technology of mammalian cells capable of efficiently expressing recombinant cat interferon [omega]2

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1 Recombinant cat interferon-ω2 expression cell expansion culture

[0028] Resuscitate mammalian cells expressing recombinant feline interferon-ω2 at 5 x 10 5 The cells / ml density was inoculated into cell shaker flasks and placed on a shaker for shaking culture. The shaker parameters were set as: 36.5°C, 5% CO 2 , 140rpm / min; after continuous culture for about 3 days, the cell density reached 2-3×10 6 cells / ml, with 5×10 5 The cells / ml density is subcultured until the number of cells is sufficient to inoculate the bioreactor, and the seed cell liquid is obtained.

Embodiment 2

[0029] Embodiment 2 Recombinant cat interferon-ω2 expression cell bioreactor culture

[0030] Seed cell liquid at 5×10 5 Cells / ml density was inoculated into a bioreactor (working volume: 3L), cultured with ActiPro medium, and the process parameters were set as follows: temperature 36.5°C, dissolved oxygen 40%, pH 6.8±0.5, stirring speed 130 rpm On the 6th day of culture, the temperature was lowered to 33°C; on the 3rd, 6th, 8th, 10th, and 12th day of culture, 5%, 5%, 5%, 5%, and 3% Cell Boost 7a / Cell Boost 7b (10: 1 ratio) feed feeding, supplemented with 1% 200mM glutamine on the 3rd day of cultivation; the cell supernatant was taken every day for biochemical index determination, and when the glucose concentration was lower than 2g / L, 1% 400g / L glucose solution was added, Keep the glucose concentration above 2g / L. The cells were continuously cultured for 12 days, and the viability was still maintained at a constant level, slightly decreased until the 13th day, and the cell ...

Embodiment 3

[0031] Example 3 Yield Evaluation of Recombinant Feline Interferon-ω2

[0032] The supernatant samples of the 10th, 12th, and 16th days of culture in the bioreactor were taken for SDS-PAGE electrophoresis and Coomassie brilliant blue staining for identification. There was an obvious rFeIFN-ω2 protein band at about 20KDa, and with the extension of culture time, rFeIFN-ω2 expression levels are increasing ( image 3 ). At the same time, affinity chromatography was used to purify the supernatant sample, and then BCA was used to quantify the purified rFeIFN-ω2. The results also showed that the protein expression level increased with the increase of culture time, reaching as high as 2.11g / L( Figure 4 ). In addition, the evaluation of the anti-VSV virus specific activity of the purified product showed that the biological specific activity of rFeIFN-ω2 of the present invention against VSV>2×10 8 / mg, significantly higher than the specific activity of rFeIFN-ω2 expressed in Esche...

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Abstract

The invention provides a suspension culture technology of mammal cells capable of efficiently expressing recombinant cat interferon-[omega]2 (rFeIFN-[omega]2). The suspension culture technology is carried out in a bioreactor and comprises the following steps of: (1) setting the initial culture temperature to be 36.5 DEG C, the dissolved oxygen to be 40% and the pH to be 6.8 + / -0.5, and lowering the temperature to 33 DEG C on the sixth day of culture; and (2) supplementing 5%, 5%, 5%, 5% and 3% serum-free feed-batch culture medium on the third, sixth, eighth, tenth and twelfth days of culturing, maintaining the dissolved oxygen to be 40% and the pH to be 6.8 + / -0.5, and finishing culturing on the sixteenth day. The suspension culture technology has the advantages of a simple culture technological process, and long motility keeping time of cells under a condition of high-density growth, target protein expression can be 2g / L or above, antiviral activity is good, production cost is low, and the suspension culture technology is suitable for large-scale industrial application.

Description

[0001] manual technical field [0002] The invention relates to a mammalian cell culture process for highly expressing rFeIFN-ω2, belonging to the technical field of biopharmaceuticals. [0003] Background of the invention [0004] Feline interferon-ω (FeiIFN-ω) belongs to type I interferon and has similar antiviral activity to feline interferon-α, but it has been developed into an antiviral agent so far, and only rFeiIFN-ω has been approved for marketing , the preparation is called Virbagen Omega, which has been widely used in Japan and European and American countries. Virbagen Omega is approved for the treatment of feline immunodeficiency virus and feline leukemia virus infectious diseases and canine parvovirus infectious diseases, but for feline viral rhinotracheitis, feline calicivirus, canine distemper virus, infectious hepatitis virus, For the treatment of rabies virus, pseudorabies virus, infectious gastroenteritis virus, canine coronavirus, canine herpes virus and ot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12P21/02C07K14/555
CPCC12N5/0682C12P21/02C07K14/555C12N2510/02
Inventor 代燕平王雯茜高小平罗弟祥
Owner 成都彤琦恩生物科技有限公司
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