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Factor X activation

A blood coagulation factor and anion technology, applied in biochemical equipment and methods, enzymes, peptidases, etc., can solve problems affecting the blood coagulation process, etc.

Active Publication Date: 2017-08-29
TAKEDA PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Although FX can be purified or recombinantly produced from human plasma, only its activated state (FXa) affects blood coagulation

Method used

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  • Factor X activation
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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] A cell line expressing recombinant factor X (rFX) was generated in the CHO-S cell line using a plasmid vector carrying cDNA encoding human FX and a neomycin resistance gene for antibiotic selection. Cell clones producing rFX were isolated by limiting dilution cloning and transfected with a vector encoding the endopeptidase furin. Furin-transfected cells producing rFX were cloned a second time. Producer clones were selected based on growth parameters and productivity of highly active rFX after activation by exogenous liquefying enzyme (tenase) and ruselquin viper venom (RVV), and coagulation assays in FX-deficient plasma ( The production clone was activated exogenously in the prothrombin time (PT) coagulation assay). Determination of impurities by FXa-specific chromogenic assay, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis for uncarboxylated rFX, unprocessed rFX forms (containing single chains or propeptides), and degradation products (SDS-PAGE) to ...

Embodiment 2

[0060] Cell lines expressing recombinant factor X (rFX) were generated in BHK cell lines using a plasmid vector carrying cDNA encoding human FX and a neomycin resistance gene for antibiotic selection. Cell clones producing rFX were isolated by limiting dilution cloning and transfected with a vector encoding the endopeptidase furin. Furin-transfected cells producing rFX were cloned a second time. Production clones were selected based on growth parameters and productivity of highly active rFX following activation of exogenous liquefaction enzymes and RVV, and were tested by exogenous pathway to activate the production clone. Protein quality was verified by determination of impurities by FXa-specific chromogenic assays and by SDS-PAGE for uncarboxylated rFX, unprocessed rFX forms (containing single chain or propeptide) and degradation products. rFX was produced in commercially available media in a 100 L bioreactor using clones meeting all quality criteria, and supernatant colle...

Embodiment 3

[0066]A cell line expressing recombinant factor X (rFX) was generated in 293 cell line using a plasmid vector carrying cDNA encoding human FX and an ampicillin resistance gene for antibiotic selection. Cell clones producing rFX were isolated by limiting dilution cloning and transfected with a vector encoding the endopeptidase furin. Furin-transfected cells producing rFX were cloned a second time. Production clones were selected based on growth parameters and productivity of highly active rFX following activation of exogenous liquefaction enzymes and RVV, and by the exogenous route in a coagulation assay in FX-deficient plasma (prothrombin time (PT) coagulation assay) to activate the production clone. Protein quality was verified by determination of impurities by FXa-specific chromogenic assays and by SDS-PAGE for uncarboxylated rFX, unprocessed rFX forms (containing single chain or propeptide) and degradation products. rFX was produced in commercially available media in a 50...

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Abstract

Methods and systems for activating Factor X are disclosed

Description

Background technique [0001] Coagulation factor X, also known as FX, F10, Eponym St-P factor, and thromboplastin, is a vitamin K-dependent plasma protease that activates thrombin. FX, initially synthesized in the liver as a single-chain precursor, is activated by intrinsic and extrinsic pathways to form activated FX (FXa) consisting of disulfide-linked light and heavy chains. The light chain contains a gamma-carboxyglutamate (Gla) domain and two epidermal growth factor-like (EGF-like) domains, while the heavy chain corresponds to a serine protease domain. FXa contributes to the function of the prothrombin complex concentrate and has been identified as the active component in factor 8 bypass activity (FEIBA). FEIBA can be used to treat patients with hemophilia who develop inhibitory antibodies against FVIII or FIX. [0002] Although FX can be purified from human plasma or produced recombinantly, only its activated state (FXa) affects the blood coagulation process. The activat...

Claims

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Application Information

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IPC IPC(8): C12N9/64
CPCC12N9/6432C12Y304/21006
Inventor M.哈斯莱彻T.加特尼格C.菲德勒E.玻姆M.多克卡尔F.霍林
Owner TAKEDA PHARMA CO LTD