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WO2014075205A1 - Procédé de purification de facteur viii de coagulation du sang et variantes - Google Patents

Procédé de purification de facteur viii de coagulation du sang et variantes Download PDF

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Publication number
WO2014075205A1
WO2014075205A1 PCT/CN2012/001548 CN2012001548W WO2014075205A1 WO 2014075205 A1 WO2014075205 A1 WO 2014075205A1 CN 2012001548 W CN2012001548 W CN 2012001548W WO 2014075205 A1 WO2014075205 A1 WO 2014075205A1
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WO
WIPO (PCT)
Prior art keywords
coagulation factor
antibody
factor viii
variants
antibody fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2012/001548
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English (en)
Chinese (zh)
Inventor
蔡胜和
赵昕
刘瑾
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to PCT/CN2012/001548 priority Critical patent/WO2014075205A1/fr
Publication of WO2014075205A1 publication Critical patent/WO2014075205A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)

Definitions

  • the present invention discloses a method for purifying coagulation factor by antibody fragment.
  • Coagulation factor 8 FVIII is a protein that plays an important role in blood coagulation. Defects in coagulation factor eight cause hemophilia A (Nature 312: 342-347, 1984; Nature 312: 337-342, 1984). Hemophilia is a serious genetic disease. From the birth of the patient, bleeding will occur repeatedly. This type of bleeding, not only in the case of trauma or surgery, but also spontaneously occurs in the joints, muscles, etc. Over time, it can cause chronic arthritis and eventually lead to disability. If intracranial hemorrhage or laryngeal bleeding occurs, it will directly endanger life.
  • the only treatment for hemophilia A is intravenous injection of eight factors.
  • the coagulation factor can be purified from plasma or can be produced by mammalian cell culture by genetic engineering. Recombinant coagulation factor VIII is better because plasma materials are potentially infectious.
  • the coagulation factor VIII is large and difficult to express, the coagulation factor of the cell synthesis is difficult to secrete extracellularly, and the coagulation factor released into the extracellular culture medium is easily degraded, and the yield of genetically engineered coagulation factor VIII is higher than that.
  • the yield of other molecules is several hundred to several thousand times lower (Human Gene Therapy 4: 259-272, 1993; Blood Cells, Molecules, and Diseases. 28: 234-248, 2002; Blood.
  • coagulation factor VIII co-express coagulation factor VIII with vWF (von Willebrand factor)
  • vWF von Willebrand factor
  • Coagulation factor can also be expressed in combination with other molecules to increase expression.
  • Fontes et al. combined the expression of coagulation factor VIII with the P140K gene to increase the expression of coagulation factor VIII in 293T cells (Genet Mol Res. 11 (1): 775-89, 2012).
  • the B domain of coagulation factor VIII does not play a role in clotting activity, and the coagulation octa-factor variant of B domain is removed, and the molecule is smaller than the full-length molecule, and the expression rate is high (US Patent No. 5, 661, 008, W0- A-91/09122, U.S. Patent No. 5,112,950, U.S. Patent No. 7,041,635.
  • Adding certain components to the culture medium, increasing the expression of coagulation factor or reducing the degradation of coagulation factor VIII is another major strategy for increasing the yield of octafactors.
  • the addition of exogenous vWF factor to the culture medium can reduce the coagulation factor VIII degradation in combination with coagulation factor (J. Biol. Chem.
  • Antibody Fragments scFvs are usually prepared by expression production using bacteria.
  • the full length of the antibody is usually prepared by mammalian cell culture for expression production. Compared to mammalian cell culture, production with bacteria can significantly reduce production costs. Book
  • scFvs do not have the function of neutralizing antigens, but only have the function of binding antigens. Purification of the "coagulation factor” by scFv without antigen neutralization can maximize the activity of the "coagulation factor” purification process, thereby improving the activity of purifying the "coagulation eight factor” drug and improving the drug quality accordingly.
  • the scFv has no Fc portion in the full-length molecule of the antibody, which can reduce the non-specific adsorption of Fc, thereby improving the purity of the "coagulation factor” and correspondingly improving the quality of the drug.
  • the molecular weight of scFv is generally about 30KD.
  • the full length antibody has a molecular weight of approximately 150 kD.
  • the genetic engineering "coagulation factor” molecular weight is approximately 300KD.
  • coagulation factor affinity purification process, whether it is immobilized full-length antibody molecule or scFv, a certain shedding occurs during use.
  • These exfoliated antibodies or scFv are impurities in the drug and are removed in subsequent molecular sieve chromatography.
  • Molecular sieve chromatography is isolated and purified according to the size difference between molecules.
  • the difference in size from the "coagulation factor” molecule can be more effectively removed in molecular sieve chromatography, thereby improving the purity of the "coagulation eight factor” and correspondingly improving the quality of the drug.
  • the implementation of the technology of the present invention can greatly reduce the production cost of the "coagulation eight factor” drug and significantly improve the quality of the drug.
  • the antibody preparation method may be a mouse hybridoma method, or a phage display or the like.
  • the antigen prepared by the monoclonal antibody may be a purified plasma protein, a purified full-length recombinant protein or variant, or a synthetic polypeptide.
  • Antibody Fragments The commonly used structure of scFv is the heavy chain variable region (VH) - Linker - Light Chain Variable Region (VL). Commonly used in the junction area (Gly4Ser) 3.
  • the light chain variable region and heavy chain variable region sequences of the antibody can be obtained by PCR. When an antibody sequence is known, antibody fragments can be obtained synthetically.
  • Antibody fragments can be expressed in bacterial, yeast, and mammalian cells. It is commonly expressed in E. coli. Purification of the antibody fragment can be affinity-purified with protein A or protein G or protein L, or affinity-purified with antigen coagulation factor VIII, or purified by ion exchange or the like.
  • the solid substrate can be agarose, Sepharose, or other matrix for ligand coupling, such as size controlled glass, silicon, and the like.
  • the activation method of the solid substrate may be CNBr activation, NHS activation, epoxy activation, and other activation methods.
  • the immobilized antibody fragment was loaded onto a chromatography column.
  • Raw materials containing coagulation factor VIII such as plasma or recombinant cell line culture medium, are diluted or adjusted for pH and applied to the column.
  • the antibody fragment chromatography column was washed with a buffer. Salts, detergents, etc. may be added to the buffer. Coagulation factor VIII can be eluted with buffer.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de purification de facteur VIII de coagulation du sang à l'aide de fragments d'anticorps. Les fragments d'anticorps peuvent être des fragments variables à chaîne unique et peuvent être produits par expression bactérienne.
PCT/CN2012/001548 2012-11-15 2012-11-15 Procédé de purification de facteur viii de coagulation du sang et variantes Ceased WO2014075205A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2012/001548 WO2014075205A1 (fr) 2012-11-15 2012-11-15 Procédé de purification de facteur viii de coagulation du sang et variantes

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2012/001548 WO2014075205A1 (fr) 2012-11-15 2012-11-15 Procédé de purification de facteur viii de coagulation du sang et variantes

Publications (1)

Publication Number Publication Date
WO2014075205A1 true WO2014075205A1 (fr) 2014-05-22

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2012/001548 Ceased WO2014075205A1 (fr) 2012-11-15 2012-11-15 Procédé de purification de facteur viii de coagulation du sang et variantes

Country Status (1)

Country Link
WO (1) WO2014075205A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7214785B2 (en) * 2001-06-12 2007-05-08 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Human-type anti-blood coagulation factor VIII antibody
CN102532316A (zh) * 2010-12-24 2012-07-04 神州细胞工程有限公司 一种抗vWF单克隆抗体及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7214785B2 (en) * 2001-06-12 2007-05-08 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Human-type anti-blood coagulation factor VIII antibody
CN102532316A (zh) * 2010-12-24 2012-07-04 神州细胞工程有限公司 一种抗vWF单克隆抗体及其应用

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