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Method of producing antithrombin iii composition

A technology for antithrombin and manufacturing method, which is applied in the directions of botanical equipment and method, biochemical equipment and method, drug combination, etc., and can solve the problems of unable to successfully manufacture recombinant antithrombin III, etc.

Active Publication Date: 2007-01-03
KYOWA HAKKO KIRIN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many efforts have been made to reduce the proportion of complex-type N-glycoside-linked sugar chains in which fucose is incorporated in the sugar chains by various means such as improving the culture method, but it has not been successful in making sugar chain structures with natural anticoagulant Enzyme III identical recombinant antithrombin III

Method used

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  • Method of producing antithrombin iii composition
  • Method of producing antithrombin iii composition
  • Method of producing antithrombin iii composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0433] Construction of a CHO / DG44 cell line in which both alleles of α1,6-fucosyltransferase (FUT8) in the genome have been disrupted

[0434] A CHO / DG44 cell line in which the genomic region of α1,6-fucosyltransferase containing both alleles of the translation initiation codon (hereinafter referred to as FUT8) was deleted was constructed according to the following procedure.

[0435] 1. Construction of the vector plasmid pKOFUT8Neo targeting the Chinese hamster FUT8 gene containing exon 2

[0436] Using the vector plasmid pKOFUT8Puro targeting exon 2 of the Chinese hamster FUT8 gene constructed by the method described in WO02 / 31140 Example 13-1 and the plasmid pKOSelectNeo (manufactured by Lexicon), the plasmid pKOFUT8Neo was constructed in the following manner.

[0437] Using 16 units of restriction endonuclease AscI (New England Biolabs), 1.0 µg of plasmid pKOSelectNeo (manufactured by Lexicon) was reacted at 37°C for 2 hours. The reaction solution was subjected to agarose...

Embodiment 2

[0491] Expression of recombinant antithrombin III by FUT8 gene-double knockout cells:

[0492] A FUT8 gene-double knockout cell line expressing recombinant antithrombin III was prepared by the method shown below.

[0493] 1. Polymerase Chain Reaction (PCR)

[0494] Two primers (SEQ ID NO: 28 and 29) were prepared from the sequence of the human antithrombin III gene (UniGene: Hs.75599) for PCR as follows. That is, 20 µl consisting of Pyrobest(R) DNA polymerase (manufactured by Takara Bio), 10×Pyrobest(R) buffer, 0.2 mmol / l dNTP mixture, and 0.5 µmol / l of the above gene-specific primers (SEQ ID NOs: 28 and 29) was prepared, A reaction mixture containing human liver-derived cDNA (manufactured by Invitrogen) as a template was heated at 94°C for 1 minute, followed by 30 PCR cycles of 30 seconds at 94°C and 1 minute at 55°C. minutes and react at 74°C for 2 minutes. After PCR, the reaction mixture was subjected to 1.5% (w / v) agarose gel electrophoresis to confirm that a DNA fragme...

Embodiment 3

[0508] Expression of recombinant antithrombin III by CHO / DG44 cells:

[0509] 1. Introducing human antithrombin III expression plasmid into CHO / DG44 cell line

[0510] First, 600 µl of a reaction mixture containing 60 µl of NEBuffer 3 (manufactured by New England Biolabs) and 120 units of restriction endonuclease MluI (manufactured by New England Biolabs) was prepared, and 100 µg of the plasmid pKAN-ATIII prepared in Example 2-3 was linearized. The digestion reaction was carried out at 37°C for 5 hours. After the reaction, the reaction mixture was purified by phenol / chloroform extraction treatment and ethanol precipitation, thereby recovering a linear plasmid.

[0511] Next, the CHO / DG44 cell line [Proc.Natl.Acad.Sci.USA, 77 , 4216(1980)] suspended in K-PBS buffer (137mmol / l KCl, 2.7mmol / l NaCl, 8.1mmol / l NaCl 2 HPO4 , 1.5mmol / l KH 2 PO 4 , 4.0mmol / l MgCl 2 ), reaching 8×10 7 Density of cells / ml. Next, 200 μl of cell suspension (1.6×10 6 cells) were mixed with 9 μg of...

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Abstract

The invention provides a process for producing an antithrombin III composition comprising a genetically modified antithrombin III molecule having an N-glycoside-binding complex sugar chain, which has an N-glycoside-binding complex sugar chain having no fucose attached to N-acetylglucosamine at the reducing end of the sugar chain.

Description

technical field [0001] The present invention relates to a method for producing an antithrombin III composition comprising an antithrombin III molecule having a complex-type N-glycoside-linked sugar chain having an antithrombin III molecule in the sugar chain Structure of unbound fucose on N-acetylglucosamine at the reducing end. Background technique [0002] Thrombus formation poses the risk of interruption of blood flow. Since the interruption of blood flow caused by thrombosis becomes a lethal factor, the living body can control and regulate blood coagulation through several mechanisms. That is, direct inactivation of activated coagulation factors by serine proteases [The Thrombin, Volume I (Ed. Machovich R.), pP.1-21, CRC Press, Boca Raton (1982)], based on degradation of coagulation factor V by activation of protein C and the regulatory mechanism of coagulation factor VIII [Progress in Hemostasis and Thrombosis, Volume 7 (Spaet T.H. editor-in-chief), pp.25-54, Grune & ...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K14/81C12N15/12C12N5/10C12N1/19C12P21/02A61K38/57A61P7/02
CPCC12Y204/01068C07K14/8128C12N9/1051C12N9/90C12N9/88C12Y402/01047C12Y501/03018C12P21/02A61P15/00A61P43/00A61P7/02A61P9/10A61P9/14C07K14/81C07K14/47C12N15/11
Inventor 山田刚士佐藤光男神田丰山野和也
Owner KYOWA HAKKO KIRIN CO LTD
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