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Preprartion method of improved hepatoma murine monoclonal antibody

A monoclonal antibody, mouse-derived technology, applied in biochemical equipment and methods, microbial determination/inspection, fermentation, etc., can solve the problems of difficult quality control, standardization and unsafety.

Inactive Publication Date: 2002-09-04
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At that time, the mouse-derived monoclonal antibodies produced by various countries in the world, whether they were whole molecules or small molecules, if the serum-free and protein-free culture medium was not used as the culture substrate, no matter what production method was used, the obtained products contained different levels of Such endogenous immunoglobulins (mouse or bovine), such products containing endogenous immunoglobulins are not only difficult to control and standardize in quality, but also contain several unsafe factors

Method used

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  • Preprartion method of improved hepatoma murine monoclonal antibody
  • Preprartion method of improved hepatoma murine monoclonal antibody
  • Preprartion method of improved hepatoma murine monoclonal antibody

Examples

Experimental program
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Embodiment 1

[0123] Example 1 Preparation of liver cancer mouse monoclonal antibody Hepama-1 without endogenous immunoglobulin: Prepare 10 liters of modified DXL serum-free culture medium: 3 mg of adenine, 70 mg of alanine, and 0.007 mg of aluminum chloride are required , Ammonium Metavanadate 0.007mg, Arginine 3g, Asparagine 300mg, Aspartic Acid 150mg, Barium Chloride 0.025mg, Biotin 2.5mg, Calcium Chloride 500mg, Choline Chloride 500mg , chromium potassium sulfate 0.025 mg, citric acid 300 mg, citrulline 50 mg, cobalt chloride 0.025 mg, copper sulfate 0.05 mg, cysteine ​​500 mg, double linoleic acid lecithin 5 mg, distearic acid egg Phospholipids 5mg, Ethanolamine 50mg, EDTA 70mg, Polyethylene Glycol 20mg, Ferrous Sulfate 25mg, Atomic Iron 25mg, Flavin Adenine Dinucleotide 0.25mg, Folic Acid 25mg , germanium dioxide 0.005 mg, glutamic acid 250 mg, glutamine 2.5 g, glycine 50 mg, glucose 60 g, histidine 500 mg, hypoxanthine 50 mg, isoleucine 3 g, leucine 3 gram, linoleic acid 0.5mg, lith...

Embodiment 2

[0127] Using the liver cancer murine monoclonal antibody Hepama-1 without endogenous immunoglobulins prepared in Example 1 as a raw material, the small molecule monoclonal antibody sHepama-1 (Fab) was prepared by conventional enzyme digestion method:

[0128] First prepare a digestive solution containing 0.1 mg / ml papain (0.02 mol edetate disodium, 0.02 mol cysteine), and mix the newly prepared digestive solution containing 0.1 mg / ml papain with the same volume of Integral monoclonal antibody of endogenous immunoglobulin to hepatic carcinoma was reacted at 37°C for 8 hours, then iodoacetamide was added to a final concentration of 0.3 moles to terminate the enzyme digestion reaction, and then dialyzed with pH 8.0 phosphate buffer at 4°C for 15 hours , and finally separated and purified by HPLC to obtain the small molecule monoclonal antibody sHepama-1 (Fab) without endogenous immunoglobulin.

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Abstract

The present invention relates to an improved preparation method of hepatoma murine monoclonal antibody. This invented method uses the improved DXL serum-free medium as culture medium, i.e. various growth factors, such as neural cell growth factor, epidermal growth factor, fibroblast grwoth factor or embyro growth factor are added in the DXL serum-free proteinless medium, and the content is 0.01-0mg / L. This invention adopts packed bed bioreactor to make high-density hybridoma cell continuous culture, and its technological conditions are as follows: temp. is 30-40 deg.c, rotating speed is 300-480 rpm, pH is 6.8-7.25, PO2 is 0.5-1.95 bar, PCO2 is 0.5-1.95 bar, PN2 is 0.5-1.95 bar, air flow rate is 0.1-2 hr. and pressure is 2-5 bar. Its cell density is upt o 10 to the eight power / ml, and theyield of monoclonal antibody of each litre of culture medium can be up to 1000-1500 mg.

Description

technical field [0001] The present invention relates to the field of monoclonal antibody production, in particular to a preparation method of liver cancer mouse-derived monoclonal antibody by changing the working mode and process of a bioreactor and improving the medium formula. Background technique [0002] The applicant of the present invention and Shanghai Meishan Biotechnology Co., Ltd. have applied for a patent on the product, method and application of endogenous immunoglobulin-free liver cancer monoclonal antibody on June 25, 1999. The patent application number is 99113824.4 , the name of the invention is: liver cancer mouse monoclonal antibody without endogenous immunoglobulin, preparation method and application thereof. [0003] At that time, the mouse-derived monoclonal antibodies produced by various countries in the world, whether they were whole molecules or small molecules, if the serum-free and protein-free culture medium was not used as the culture substrate, n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/18C12P21/08C12Q3/00
Inventor 李隽喆谢弘褚云芳来文王放王根凤陈中健朱海颖王泰安强家模
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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