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Recombinant or transgenic factor VII compound having a majority of glycan, biantennary, bisialylated and non-fucosylated forms

A technology of non-fucosylation and sialylation, which is applied in the direction of drug combination, coagulation/fibrinolytic factors, medical preparations containing active ingredients, etc., and can solve the problem of lower efficiency

Inactive Publication Date: 2013-11-20
分馏和生物工艺法国实验室公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Currently, existing recombinant or transgenic Factor VII exhibits different glycosylation from human plasma FVII due to its expression in different systems than humans, which can lead to an increase in antibodies against the recombinant protein, resulting in It is less efficient than human FVII purified from human plasma

Method used

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  • Recombinant or transgenic factor VII compound having a majority of glycan, biantennary, bisialylated and non-fucosylated forms
  • Recombinant or transgenic factor VII compound having a majority of glycan, biantennary, bisialylated and non-fucosylated forms
  • Recombinant or transgenic factor VII compound having a majority of glycan, biantennary, bisialylated and non-fucosylated forms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0177] Example 1: Production of human FVII protein in milk of transgenic female rabbits

[0178] First, by introducing the WAP gene sequence (described in the document Devinoy et al, Nucleic Acids Research, vol.16, no.16, 25August1988, p.8180) into the polylinker of the p-poly III-I vector (in Plasmid p1 was prepared as described in the document Lathe et al, Gene (1987) 57, 193-201).

[0179] Plasmid p2, obtained from plasmid p1, contains the rabbit WAP gene promoter and the human FVII gene.

[0180] Transgenic female rabbits were obtained by traditional microinjection technique (Brinster et al, Proc. Natl. Acad. Sci. USA (1985) 82, 4438-4442). 1-2p1, which contains 500 copies of the gene, was injected into the male pronuclei of mouse embryos. The Not 1-Not 1 fragment of this plasmid containing the recombinant gene was microinjected. Afterwards, the embryos are transferred into the fallopian tubes of hormone-prepared succession females. About 10 percent of the manipulated ...

Embodiment 2

[0182] Example 2: Extraction and purification of obtained FVII

[0183] a) Extraction of FVII

[0184] Take 500ml whole raw milk and dilute it with 9 times the volume of 0.25M sodium phosphate with pH8.2. After stirring at room temperature for 30 minutes, the FVII-rich aqueous phase was centrifuged at 10000 g for 1 hour at 15° C. (centrifuge Sorvall Evolution RC-6700 rev / min-rotor SLC-6000). 6 cans of about 835ml are required.

[0185] After centrifugation, three phases are present: a lipid phase at the surface (milk fat), a clear FVII-rich aqueous non-lipid phase (major phase) and a remaining white solid phase (precipitated insoluble casein and calcium compounds).

[0186] The FVII-enriched aqueous non-lipid phase was collected by a peristaltic pump until the lipid phase. The lipid phase is recovered separately. The solid phase (precipitate) was removed.

[0187] However, the aqueous non-lipid phase still contained a very small amount of lipid, which was filtered through...

Embodiment 3

[0233] Example 3: Characterization of Glycosylation Sites and Glycopeptides by MS-ESI

[0234] The N-glycosylation sites of FVII-Tg, FVIIa,p (plasma FVII) and FVIIa,r were identified by LC-ESIMS( / MS), confirmed by MALDI-TOFMS, and each site was analyzed by LC-ESIMS The relative proportions of different glycans were determined.

[0235] figure 2 Deconvoluted ESI spectra of glycopeptides containing two glycosylated asparagine residues are depicted. The positions of glycosylation sites were confirmed by MALDI-TOF ( / TOF) and Edman sequencing.

[0236] Pairs exhibiting N-glycosylation site Asn 145 and Asn 322 Glycopeptide of FVIIa, p [D 123 -R 152 ] and [K 31.6 -R 353 ] showed the presence of a biantennary, disialo, unfucosylated structure (A2) (the observed Asn-containing 145 glycopeptide mass: 5563.8 Da) and the fucosylated structure (A2F) (observed glycopeptide mass with Asn145: 5709.8 Da). Note also that Asn 145 Triantennary, trisialyl, unfucosylated (A3) (observed ...

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Abstract

The present invention concerns a recombinant or transgenic factor VII compound, each factor VII molecule of the compound having glycan forms linked to N-glycosylation sites, wherein among all the factor VII molecules in said compound, glycan, biantennary, bisialylated and non-fucosylated forms are in the majority. The invention also concerns such a compound for use as a medication, and a method for preparing said compound, among others.

Description

[0001] The application date is July 31, 2007, the application number is 200780027968.1, and the title of the invention is "recombinant or transgenic factor VII synthesis with mostly biantennary, disialic acid and non-fucosylated glycan structures" The divisional application of the invention patent application. Background technique [0002] Factor VII (FVII) is a vitamin K-dependent glycoprotein involved in coagulation in its activated form (FVIIa), activating factor X and factor IX in the presence of calcium and tissue factor. FVII is secreted in the form of a single peptide chain with 406 amino acid residues, and its molecular weight is about 50 kDa. FVII contains four distinct domains: an N-terminal gamma-carboxyl domain (Gla), two "epidermal growth factor-like (EGF)" domains, and a serine protease domain. Activation of FVII to FVIIa is by arginine 152 - Isoleucine 153 Cleavage of the domain (arginine 152-isoleucine 153) linkage is characterized. Thus, FVIIa consists of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64A61K38/48A61P7/04
CPCC12N9/50C12N9/647A01K2267/01A01K2217/05C12N9/6437C12Y304/21021A61K38/00A01K2227/107C07K14/745A61P7/04C12N9/64A61K38/36C12P21/00
Inventor 阿卜杜勒萨塔尔·萨米·什图鲁埃马纽埃尔·诺尼尼古拉·比奥罗
Owner 分馏和生物工艺法国实验室公司
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