CN103351431A - Coagulation factor 8 and its mutant purifying method - Google Patents
Coagulation factor 8 and its mutant purifying method Download PDFInfo
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- CN103351431A CN103351431A CN2012104832968A CN201210483296A CN103351431A CN 103351431 A CN103351431 A CN 103351431A CN 2012104832968 A CN2012104832968 A CN 2012104832968A CN 201210483296 A CN201210483296 A CN 201210483296A CN 103351431 A CN103351431 A CN 103351431A
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Abstract
An antibody fragment (such as a single-stranded variable region scFv) is used to purify a coagulation factor 8, can be produced through bacterial expression, and is more specific than the full length molecule of the antibody, so the method reduces the production cost and improves the purity of the coagulation factor 8.
Description
Technical field
The present invention announces a kind of method with antibody fragment purifying blood coagulation eight factors.
Background technology
(coagulation factor 8 FVIII) is a kind of protein that plays an important role to blood coagulation eight factors in blood coagulation.Blood coagulation eight factor defectives can cause haemophilia A (Nature 312:342-347,1984; , Nature 312:337-342,1984).Hemophilia is a kind of serious inherited disease.The patient will occur hemorrhage from birth repeatedly.This hemorrhage, be not only when wound or operation occur, also can spontaneously appear at the parts such as joint, muscle, in the course of time, can make the patient chronic arthritis occur and also finally cause deformity.If in case occur intracranialing hemorrhage, throat is hemorrhage, direct threat to life.
The unique methods for the treatment of of haemophilia A is intravenous injection blood coagulation eight factors.Blood coagulation eight factors can be from blood plasma purification, also can produce with mammaliancellculture with the method for genetically engineered restructuring.Because the blood plasma raw material has the potential disease infectivity, restructuring blood coagulation eight factors are better.But, because blood coagulation eight factor molecules are huge, beyond expression of words, synthetic blood coagulation eight factors of cell are difficult to be secreted into the extracellular, and blood coagulation eight factors that are secreted in the nutrient solution of extracellular are easy to degraded, the productive rate of genetically engineered restructuring blood coagulation eight factors, than the low hundred times of productive rate of other molecules to several thousand times of (Human Gene Therapy 4:259-272,1993; Blood Cells, Molecules, and Diseases.28:234-248,2002; Blood.103:3412-3419,2004).Therefore, genetically engineered restructuring blood coagulation eight factor production costs are high, expensive.
In time in the past, there are many methods to attempt to increase blood coagulation eight factor productive rates.The method that increases blood coagulation eight factor output mainly contains two kinds of strategies.The firstth, the upstream increases the productive rate of Mammals production blood coagulation eight factors, and the secondth, the downstream increases purification efficiency.
A main policies that increases blood coagulation eight factor productive rates is with blood coagulation eight factors and vWF (von Willebrand factor) associating co expression (J.Biol.Chem.263,6352-6362,1988; Mol.Cell.Biol.9,1233-1242,1989; J.Biol.Chem.266,21948-21955,1991).Blood coagulation eight factors can also be united expression with other molecules, to increase expression amount.Such as Fontes etc. blood coagulation eight factors and P140K gene are united expression, increase the expression (Genet Mol Res.11 (1): 775-89,2012) of blood coagulation eight factors in the 293T cell.
The B structural domain of blood coagulation eight factors is inoperative in blood coagulation activity, removes the blood coagulation eight factor variation bodies of B structural domain, and molecule is less than full-length molecule, expression rate is wanted high (United States Patent (USP) number 5,661,008, WO-A-91/09122, United States Patent (USP) number 5,112,950, United States Patent (USP) number 7,041,635).
Adding some composition in substratum, increase the degraded of blood coagulation eight factor expressions or reduction blood coagulation eight factors, is another main policies that improves blood coagulation eight factor productive rates.The interpolation external source vWF factor can be combined with blood coagulation eight factors and be reduced blood coagulation eight factors degradeds (J.Biol.Chem.263:6352-6362,1988) in substratum.In substratum, add ortho-phospho-L-serine (OPLS), reduce the combination of blood coagulation eight factors and cytolemma and express (J.Biotechnol.151:357-62,2011) to increase.The dextran sulfate that adds high molecular in substratum also can reduce blood coagulation eight factors degradeds (U.S. Patent application US20100112641) of secretion.
Improving the purifying yield aspects, applying immobilized antibody purification antigen blood coagulation eight factors, to improve the purifying rate of recovery, it is strategy (the United States Patent (USP) number 5,470,954 of a widespread use, United States Patent (USP) number RE32011), compare with the ion exchange chromatography purification process, the rate of recovery of affinity chromatography is higher, and the lipidated protein that obtains is higher.All these antibody affinity chromatography methods all are to use the full length antibody molecule.The full length antibody molecule need to obtain with Hybridoma Cell Culture or other mammaliancellcultures.The cost compare for preparing the full length antibody molecule with mammalian cell culture processes is high, and this accounts for sizable ratio in blood coagulation eight factor total manufacturing costs.
This invention is with blood coagulation eight factors or vWF factor antibody sheet segment molecule, and ((single chainantibody fragment, scFv) molecule, being fixed is for separating of purifying blood coagulation eight factors in strand variable region commonly used.
Summary of the invention
This invention is that application antibody fragment rather than complete antibody molecule carry out the immunoaffinity purification of " blood coagulation eight factors ".
Antibody fragment scFv carries out the Expression product preparation with bacterium usually.And the antibody full-length molecule will carry out the Expression product preparation with mammaliancellculture usually.Compare with mammaliancellculture, produce with bacterium, can reduce production costs very significantly.
The antigen neutralization that many full length antibody molecules all have in various degree is active, can reduce or eliminate the activity of antigen.And many scFv do not possess in and the function of antigen, only have the function of conjugated antigen.Activity in " blood coagulation eight factors " purge process be can keep to greatest extent with the scFv purifying that does not possess the antigen neutralization function " blood coagulation eight factors ", thereby the activity of purifying " blood coagulation eight factors " medicine, corresponding raising drug quality improved.
ScFv does not have the Fc part in the antibody full-length molecule, can reduce the Fc non-specific adsorption, thereby improves the purity of " blood coagulation eight factors ", corresponding raising drug quality.
The general about 30KD of the molecular weight of scFv.The about 150KD of full length antibody molecular weight.Genetically engineered " blood coagulation eight factors " the about 300KD of molecular weight.In " blood coagulation eight factors " affinity purification technique, no matter be immobilization full length antibody molecule or scFv, in use all certain coming off can occur.The antibody that these come off or scFv are the impurity of medicine, remove in follow-up sieve chromatography.Sieve chromatography is to carry out separation and purification according to the big or small gap between the molecule.Because the scFv molecule is less, and is larger with " blood coagulation eight factors " molecular size gap, in sieve chromatography, can accesses more effectively and remove, thereby improve " blood coagulation eight factors " purity, corresponding raising drug quality.
The enforcement of this invention technology can decrease " blood coagulation eight factors " drug manufacture cost, significantly improves drug quality.
Description of drawings
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Embodiment
1. monoclonal antibody preparation
Preparation method for antibody can be the Mouse Hybridoma Cells method, and perhaps phage shows etc.The antigen of monoclonal antibody preparation can be the total length recombinant protein of plasma proteins, purifying of purifying or varient, synthetic polypeptide.
2. antibody fragment scFv preparation
The structure that antibody fragment scFv is commonly used is variable region of heavy chain (VH)-commissure district (Linker)-variable region of light chain (VL).The commissure district commonly uses (Gly4Ser) 3.The variable region of light chain of antibody and weight chain variabl area sequence can obtain with PCR method.When the known antibodies sequence, antibody fragment can obtain with synthetic method.Antibody fragment can be at bacterium, yeast, mammalian cell expression.Expression in escherichia coli commonly used.The purifying of antibody fragment can with a-protein or protein G or Protein L affinity purification, perhaps be used antigen blood coagulation eight factor affinity purifications, perhaps with method purifying such as ion-exchanges.
3. antibody fragment scFv is coupled solid dielectric
Solid substrate can be agarose, Sepharose, and perhaps other are used for matrix that part is coupled, the glass of size control for example, the materials such as silicon.The activation method of solid substrate can be CNBr activation, NHS activation, epoxy activation, and other activation methods.
4. immobilized antibody fragment scFv affinity purification technique
The immobilized antibody fragment is loaded onto chromatography column.The raw material that contains blood coagulation eight factors, for example blood plasma or recombinant cell lines nutrient solution pass through dilution or adjust potential of hydrogen, upper prop.The antibody fragment chromatography column washs with damping fluid.Can add salt and stain remover etc. in the damping fluid.Blood coagulation eight factors can be used buffer solution elution.
Claims (10)
1. method of using antibody fragment separation and purification blood coagulation eight factors and varient thereof.
2. according to claim 1, blood coagulation eight factors can be total length blood coagulation eight factors, the varient of B structural domain disappearance, point mutation varient, perhaps other varients.
3. according to claim 1, antibody fragment can be from the antibody of anticoagulation eight factors and varient thereof.
4. according to claim 1, in the situation that contain vWF in vWF coexpression or the raw material, antibody fragment can be from the antibody of vWF and varient thereof.
5. according to claim 1, antibody fragment can be scFv, perhaps other antibody fragment molecules.
6. according to claim 1, antibody fragment can produce in bacterium, yeast, mammalian cell, produces with bacterium to be more suitable for, and produces the most suitable with intestinal bacteria.
7. according to claim 1, antibody fragment can be people from source, mouse, rabbit, sheep.
8. the raw material that according to claim 1, contains blood coagulation eight factors can be blood plasma or restructuring blood coagulation eight factor cell culture fluids.
9. according to claim 1, when the raw material that contains blood coagulation eight factors was cell culture fluid, cell can be CHO, and BHK or other animals or human body cell, nutrient solution can be to contain serum or serum-free.
10. according to claim 1, the immobilized antibody fragment can replace immobilized antibody, perhaps as an additional technique, is further purified blood coagulation eight factors.
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| CN2012104832968A CN103351431A (en) | 2012-11-13 | 2012-11-13 | Coagulation factor 8 and its mutant purifying method |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1117526A (en) * | 1994-06-09 | 1996-02-28 | 遗传和生物工程研究中心 | Recombinant single chain Fv antibody fragment and its use in the immunopurification of the recombinant hepatitus B virus surface antigen |
| US5994310A (en) * | 1998-09-03 | 1999-11-30 | Bayer Corporation | Peptide ligands for affinity purification of human Factor VIII |
| CN1758961A (en) * | 2003-02-13 | 2006-04-12 | 贝克顿·迪金森公司 | Devices for component removal during blood collection, and uses thereof |
| CN101035439A (en) * | 2004-08-20 | 2007-09-12 | 普洛麦提生命科学有限公司 | Sequential protein isolation and purirication schemes by affinity chromatography |
| CN102532316A (en) * | 2010-12-24 | 2012-07-04 | 神州细胞工程有限公司 | Anti-vWF (von Willebrand factor) monoclonal antibody and application thereof |
-
2012
- 2012-11-13 CN CN2012104832968A patent/CN103351431A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1117526A (en) * | 1994-06-09 | 1996-02-28 | 遗传和生物工程研究中心 | Recombinant single chain Fv antibody fragment and its use in the immunopurification of the recombinant hepatitus B virus surface antigen |
| US5994310A (en) * | 1998-09-03 | 1999-11-30 | Bayer Corporation | Peptide ligands for affinity purification of human Factor VIII |
| CN1758961A (en) * | 2003-02-13 | 2006-04-12 | 贝克顿·迪金森公司 | Devices for component removal during blood collection, and uses thereof |
| CN101035439A (en) * | 2004-08-20 | 2007-09-12 | 普洛麦提生命科学有限公司 | Sequential protein isolation and purirication schemes by affinity chromatography |
| CN102532316A (en) * | 2010-12-24 | 2012-07-04 | 神州细胞工程有限公司 | Anti-vWF (von Willebrand factor) monoclonal antibody and application thereof |
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Application publication date: 20131016 |