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Method for preparing anti-canine distemper virus monoclonal antibody by adopting bioreactor

A technology of bioreactor and canine distemper virus, which is applied in the field of monoclonal antibodies, can solve the problems of unstable product quality, large batch-to-batch variation, and high content of miscellaneous proteins, so as to achieve controllable process and quality, reduce production costs, and product The effect of high purity

Active Publication Date: 2014-05-14
WUHAN CHOPPER BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for preparing anti-canine distemper virus monoclonal antibody using a bioreactor. The method of the present invention overcomes the high content of miscellaneous protein in the traditional animal production process, unstable product quality, and large batch-to-batch variation It has the advantages of clear medium composition, controllable process and quality, high antibody purity, easy operation, high production efficiency, high product purity, and good stability.

Method used

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  • Method for preparing anti-canine distemper virus monoclonal antibody by adopting bioreactor
  • Method for preparing anti-canine distemper virus monoclonal antibody by adopting bioreactor
  • Method for preparing anti-canine distemper virus monoclonal antibody by adopting bioreactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Hybridoma cells adapted to culture in serum-free suspension

[0042] The hybridoma cells used in this example are mouse hybridoma cells HB-216 purchased from the American Standard Biological Collection (ATCC), the Hybridoma-SFM serum-free medium was purchased from Invitrogen, and the cell culture shaker was purchased from Shanghai Zhicheng Analysis Instrument company, DMEM / F12 medium was purchased from Beijing Tsingda Tianyi Company, newborn calf serum was purchased from Wuhan Sanli Biotechnology Co., Ltd.

[0043] 1) Recovery of hybridoma cells

[0044] Take out the HB-216 hybridoma cells from liquid nitrogen, put them in a water bath at 37°C, and let them melt rapidly, then transfer the HB-216 hybridoma cells into a centrifuge tube of 10ml fresh DMEM / F12 medium containing 10% serum, gently Pipette to make it evenly mixed, centrifuge at 800r / min for 10 minutes, and discard the supernatant. Add fresh medium to resuspend, transfer the resuspended HB-216 hybri...

Embodiment 2

[0047] Example 2 Two-stage cultivation of hybridoma cells in a 14L stirred bioreactor

[0048] The bioreactor used in this example is a stirred bioreactor (purchased from Applikon Company in the Netherlands), equipped with a microbubble aeration device and a rotating Spin-Filter cell retention device, and the Hybridoma-SFM serum-free medium was purchased from Invitrogen Company of the United States, VERO The cells were provided by Wuhan Institute of Biological Products. CDV virus mouse-derived positive serum and CDV virus mouse-derived negative serum were prepared by Wuhan Zhongbo Biology Co., Ltd. according to conventional methods. DMEM / F12 medium was purchased from Beijing Tsingda Tianyi Company. Goat antibody The mouse IgG+IgM fluorescent secondary antibody was purchased from Sigma Company, and the fluorescence microscope was purchased from LEICA Company in Germany.

[0049] 1) Preparation of seed cells: Inoculate the HB-216 hybridoma cells adapted to Hybridoma-SFM serum-fr...

Embodiment 3

[0059] Example 3 Stirred bioreactor 14L-75L-650L scale-up cultivation

[0060] The bioreactor used in this example is a stirred bioreactor (purchased from Applikon Company in the Netherlands), equipped with a microbubble aeration device and a rotating Spin-Filter cell retention device, and the Hybridoma-SFM serum-free medium was purchased from Invitrogen Company of the United States, VERO The cells were provided by Wuhan Institute of Biological Products. CDV virus mouse-derived positive serum and CDV virus mouse-derived negative serum were prepared by Wuhan Zhongbo Biology Co., Ltd. according to conventional methods. DMEM / F12 medium was purchased from Beijing Tsingda Tianyi Company. Goat antibody The mouse IgG+IgM fluorescent secondary antibody was purchased from Sigma Company, and the fluorescence microscope was purchased from LEICA Company in Germany.

[0061] 1) Preparation of seed cells: inoculate the HB-216 hybridoma cells adapted to serum-free suspension culture in Examp...

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Abstract

The present invention relates to a method for preparing anti-canine distemper virus monoclonal antibody by adopting a bioreactor. The method is characterized by comprising the following steps: 1) carrying out serum-free suspension adaptation culture on a hybridoma cell line secreting anti-canine distemper virus monoclonal antibody; 2) culturing the hybridoma cells in a bioreactor in two stages; 3) harvesting a culture supernatant; and 4) carrying out ultra-filtration and freeze-drying to obtain the finished product. According to the method for preparing the anti-canine distemper virus monoclonal antibody by adopting the bioreactor, defects of high hybrid protein content in products, unstable quality, large batch difference and the like of the method for preparing the anti-canine distemper virus monoclonal antibody in animal bodies in the prior art are overcome, and advantages of definite culture medium components, controllable method and process, high antibody purity, convenient operation, high production efficiency and the like are provided.

Description

technical field [0001] The invention relates to the technical field of monoclonal antibodies, in particular to a method for preparing anti-canine distemper virus monoclonal antibodies using a bioreactor. Background technique [0002] Canine distemper is a highly contagious disease caused by canine distemper virus, commonly known as canine distemper. The infectiousness of this disease is extremely strong, and the mortality rate is as high as more than 80%. Vaccination against canine distemper is one of the most effective ways to prevent canine distemper. In recent years, while the canine distemper vaccine has been widely used to prevent and treat canine distemper, there are still reports of canine distemper virus infection in dogs or wild animals all over the world, which has caused epidemics of canine distemper, and the variation of canine distemper virus has made cured dogs Canine distemper virus can still be transmitted. [0003] Monoclonal antibodies have the characteri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/08
Inventor 李伟刘洁秦红刚漆世华温文生朱薇谢红玲韩兴王桢桢廖园园李建王威
Owner WUHAN CHOPPER BIOLOGY
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