CN102776260A - Method for effectively expressing recombinant human coagulation factor VIII - Google Patents
Method for effectively expressing recombinant human coagulation factor VIII Download PDFInfo
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- CN102776260A CN102776260A CN2012102620936A CN201210262093A CN102776260A CN 102776260 A CN102776260 A CN 102776260A CN 2012102620936 A CN2012102620936 A CN 2012102620936A CN 201210262093 A CN201210262093 A CN 201210262093A CN 102776260 A CN102776260 A CN 102776260A
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Abstract
The invention relates to the technical field of biological engineering, in particular to a process method for effectively expressing a recombinant human coagulation factor VIII by utilizing mammalian cell culture. The method specifically comprises the following steps: adding an exogenous angiohemophilia factor to cell culture liquid for expressing the recombinant human coagulation factor VIII, and controlling an active ratio of the angiohemophilia factor to the human coagulation factor VIII in the cell culture liquid to be 1-10: 1 in the culture process. According to the invention, by utilizing a VWF (Von Willebrand Factor) molecule partner, which is necessary in an expression process of the recombinant human coagulation factor VIII, the newly generated human coagulation factor VIII is stabilized, the accumulating effect of the human coagulation factor VIII is improved, the technical difficulty is reduced, and the expression effect of a target protein is improved.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of mammalian cell that utilizes and cultivate the process method that efficiently expresses reorganization eight factors.
Background technology
Natural human blood coagulation eight factors (FVIII) are a kind of macromole gp, by heavy chain and light chain be combined into, and total molecular weight 330KD, the about 0.2mg/L of content in the blood plasma exists with the form that combines vWF ELISA (VWF).In the blood agglutination reaction, activatory FVIII makes the raising greatly alive of the enzyme of FIX, the activation of catalysis FX, and combine to form a complex body, catalysis aggegation Kettenreaktion goes on.The disappearance or the shortage of FVIII molecule cause haemophilia A, and being supplemented with active FVIII is the effective measure of treatment haemophilia A.
Recombinant human FVIII has identical physiology with natural FVIII, pharmacology and immune characteristic, and stop HIV, and HDV, the advantage that pathogenies such as prion are propagated, therefore, the research of reorganization FVIII protein expression becomes an important trend of haemophilia A treatment.First recombinant human blood coagulation eight factor in the world, promptly reorganization eight factors of the first-generation were just gone on the market in nineteen ninety.Eight factors in the whole world are sold and year have been reached 7,000,000,000 ius at present, and wherein 60% is genetically engineered reorganization blood coagulation eight factors.
Natural FVIII always is combined into mixture and stable existence with vWF.When reorganization FVIII secreted to the serum free medium of cell, molecule was easy to disintegrate, and is degraded, and the FVIII of expression can not get accumulation.Utilize vWF and FVIII coexpression system can solve problems such as the instability of reorganization FVIII in emiocytosis expression process and easy degraded well.People's such as Kaufman research shows, can significantly improve the expression level of FVIII behind FVIII and the VWF coexpression.In addition, having under the culture condition of serum, the VWF in the serum can combine newly-generated VIII, forms more stable mixture, reaches FVIII cumulative effect in cell culture fluid.
In utilizing cell large scale to cultivate to produce reorganization eight factor processes; In order to improve the expression amount of eight factors; Utilization has blood serum medium perhaps cotransfection FVIII and VWF gene in mammalian cell, the expression of having stablized eight factors, but all there is limitation separately in these two kinds of methods.For blood serum medium is arranged, because in the mammalian cell culturing process, external source is added animal serum and has been brought the risk of viral potentially contaminated.Therefore, utilize mammalian cell to produce at present to tend in the pharmaceutical grade protein serum-free culture to substitute the serum cell cultures is arranged.And for the coexpression of VWF and FVIII gene; People find in research recombinant human blood coagulation eight factor expressions; VWF and FVIII coexpression; The expression amount of eight factors is lower, and the structure of cell strain is more complicated also, and the expression amount of high expression level eight factor cell strains in serum free medium that filters out is generally in 0.1-0.5 iu/sky/10
6Cell, this expression level does not reach the requirement of industrialization far away.It is thus clear that, be badly in need of a kind of new cultural method at present and come express recombinant eight factors, can under the serum-free culture condition, realize the suitability for industrialized production of reorganization eight factors, make the expression of eight factors simple, efficient, virus-free pollution.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, provide a kind of mammalian cell that utilizes to cultivate the process method that efficiently expresses reorganization eight factors.
The present invention at first discloses a kind of method of efficiently expressing recombinant human blood coagulation eight factors; Add to for vWF ELISA in the cell culture fluid of express recombinant people blood coagulation eight factors, and the specific activity of control cell culture fluid medium vessels property christmas factor and people's blood coagulation eight factors is 1～10:1 in culturing process external source.
More excellent, the cell culture fluid of said express recombinant people's blood coagulation eight factors is a serum-free cell culture medium.
The cell of the people of express recombinant described in the present invention blood coagulation eight factors is the cell strain of single expression reorganization FVIII, and VWF prepares in addition, as the allogenic material in the extra cell culture fluid that adds express recombinant people blood coagulation eight factors to of Chaperones Molecular.
Technical scheme provided by the invention is that VWF is added in the cell culture fluid of reorganization eight factors according to certain ratio, can improve the expression amount of reorganization eight factors greatly, and this method is that the production of industriallization eight factors provides a kind of simple and practical realizing route.Method of the present invention does not need eight factors and VWF gene co-transfection host cell have been simplified the cell strain building process, has improved the high yielding cell sarain screening efficiency, is particularly suitable for large-scale industrialization production recombinant human blood coagulation eight factors.
More excellent, the method concrete steps of said efficiently expressing recombinant human blood coagulation eight factors are following:
1) preparation of express cell: the recovery of the freeze-stored cell of the structure of the cell strain of expressing human blood coagulation eight factors or expressing human blood coagulation eight factors;
2) the expansion kind of express cell is cultivated: with step 1) make up or people's blood coagulation eight factor expression cell inoculations of recovery in serum free medium, shaking culture 3～4 days reaches 1～2 * 10 to cell density
6Cells/ml, passage, vibration training amplification is supported to cell density and is reached 1～2 * 10
6Cells/ml obtains seed liquor;
3) seed liquor that cell reactor cultivation: with step 2) obtains is inoculated in the serum free medium of cell reactor, cultivates 4～7 days, makes the cell density of enchylema reach 1～5 * 10
6Cells/ml; In enchylema, add external source VWF and pair cell liquid then and carry out the feed supplement cultivation, obtain cell stoste; In the said feed supplement culturing process, the VWF activity is 1～10:1 with the FVIII specific activity in the control enchylema;
4) preparation of blood coagulation eight factor products: pair cell stoste is carried out centrifugal, filtration, purification process, obtains recombinant human blood coagulation eight factor products.
The cell strain of said expressing human blood coagulation eight factors of step 1) or the freeze-stored cell of expressing human blood coagulation eight factors are the cell strain of single expression people's blood coagulation eight factors of making up, and the FVIII that expresses comprise total length FVIII with the B district FVIII fragment of disappearance is arranged.Construction process is conventional molecular biology recombinant protein preparation method.
More excellent, step 2) make up in or people's blood coagulation eight factor expression cells of recovery with 1～9 * 10
5The inoculum size of cells/ml is inoculated.The expansion kind of express cell cultivate can with make up or people's blood coagulation eight factor expression cell inoculations of recovery in triangular flask, through shake-flask culture, go down to posterity, shake-flask culture obtains seed liquor.
More excellent, step 2) passage described in is that the ratio of going down to posterity with 1:2～4 goes down to posterity.
More excellent, step 2) passage described in is that the ratio of going down to posterity with 1:3 goes down to posterity.
More excellent, step 2) condition of shaking culture is in: 60～150 rev/mins of rotating speeds, 32～38 ℃ of culture temperature.
More excellent, the inoculum size that the step 3) seed liquor is inoculated into cell reactor is 5～10v/v%.
More excellent, the said feed supplement culture condition of step 3) is: control dissolved oxygen 40～80% in the feed supplement culturing process, pH6.8～7.2; 40～90 rev/mins of mixing speed; 32～38 ℃ of culture temperature are carried out feed supplement according to the consumption of glucose, make glucose concn maintain 0.5～1.5g/L.
More excellent, the said feed supplement culture condition of step 3) is: control dissolved oxygen 40～80% in the feed supplement culturing process, pH6.8～7.2; 40～90 rev/mins of mixing speed; 37 ℃ of culture temperature are carried out feed supplement according to the consumption of glucose, make glucose concn maintain 1.0g/L.
More excellent, the cell density that the step 3) reactor drum is cultivated the said cell stoste that obtains is 1～2 * 10
7Cells/ml.
That the preparation of said recombinant human blood coagulation eight factor products of step 4) is adopted is centrifugal, filtration, purification process are conventional separation and purification of protein step, and VWF both can remove also and can still keep among the FVIII of the higher degree of acquisition.
Step 2 of the present invention) cultivation of the expansion kind of express cell and step 3) cell reactor are cultivated and all can be adopted the existing conventional serum-free cell culture medium to carry out cell cultures.
The present invention overcome in the prior art for obtain stable blood coagulation eight factors must be in same cell strain the difficult point of coexpression eight factors and VWF.The present invention proposes a kind of effective cell cultures approach; Be controlled in certain optimization range through content FVIII and VWF; The expression that makes FVIII is by a large amount of accumulations, and preparation technology's express cell of the present invention structure difficulty is little, is particularly suitable for suitability for industrialized production recombinant human blood coagulation eight factors.
Effect of the present invention comprises: what 1) working method of the present invention adopted is single expression eight factor cell strains, does not need eight factors and VWF cotransfection cell strain, and the cell strain structure and the screening process in early stage are simple; 2) cell cultivation process has added the VWF factor, has improved the accumulation of eight factors and stable; 3) be fit to suitability for industrialized production, can improve eight factor expression amounts very simply, also amplify easily.
Description of drawings
Fig. 1: the commercial scale reactor culture process flow process of eight factors of recombinating
Fig. 2: the canonical plotting that blood coagulation eight factor actives detect
Embodiment
Further set forth the present invention below in conjunction with embodiment.Should be understood that embodiment only is used to explain the present invention, and unrestricted scope of the present invention.The reagent of the experimental technique of unreceipted actual conditions and undeclared prescription is according to normal condition among the embodiment, like works such as ［ U.S.A ］ Sambrook.J; Huang Peitang etc. translate.The molecular cloning test guide, the third edition.Beijing: the condition of the condition described in the Science Press 2002 or manufacturers's suggestion is carried out or is disposed.
Embodiment one
1. experimental technique
1) will be domesticated for eight factor expression cells (the structure reference of eight factor expression cells " expression study of thrombin FVIII in mammalian cell ", Wang Qi etc., medicine biotechnology, 2002,9 (5): 251 ~ 255 of serum-free culture; Cell strain serum-free acclimation method can be with reference to the cell cultures handbook, like cell cultures function part in the invitrogen Company products handbook appendix), with 9 * 10
5Cells/ml inoculation triangle shakes bottle, 32 ℃ of culture temperature, and shaking bottle rotating speed is 60 rev/mins; Serum-free culture after about 3 days cell density reach 2 * 10
6Cells/ml goes down to posterity with the ratio of 1:2, shake-flask culture, and 38 ℃ of culture temperature, 60 rev/mins of rotating speeds reach 1～2 * 10 to cell density
6Cells/ml.Serum-free cell culture medium is a commercialization SIGMA Company products SFMII302 nutrient solution.
2) with containing different activities reorganization VWF (the proteic preparation reference of reorganization VWF Recombinant von Willebrand factor:Potential Therapeutic Use; BernhardE.Fischer; Journal of Thrombosis and Thrombolysis; Volume8, Number3 (1999), nutrient solution 197-205) change the cell (detecting the active test kit of VWF available from Shanghai Sun Bio-Tech Co., Ltd.) of liquid culture expression eight factors; Cultivate after 24 hours, the collecting cell nutrient solution carries out eight factor active analyses (seeing table 1).
The serum-free cell culture medium that present embodiment adopted is a commercialization SIGMA Company products SFMII302 nutrient solution.
3) eight factor active analytical procedures
Adopt human blood coagulation factor VII I assay method (first phase method), can participate in three appendix XN of Chinese Pharmacopoeia human blood coagulation factor VII I assay method, concrete steps are following:
A. reagent:
⑴ 3.8% Sodium Citrate: get Sodium Citrate 9.5g, be dissolved in water and be diluted to 250ml.
⑵ imidazole buffer (pH7.3): get imidazoles 0.68g and sodium-chlor 1.17g, add water and make and be dissolved into 100ml, add 0.1mol/L hydrochloric acid soln 42.2ml, thin up promptly gets to 200ml again.
⑶ diluent: 3.8% the Sodium Citrate of getting a volume adds 5 volume imidazole buffers and mixes, and adding an amount of 20% rHSA to final concentration is 1%.
⑷ activated partial thromboplastin (APTT) reagent.
⑸ human blood coagulation factor VII I lacks blood plasma: be lower than 1% human plasma or artificial substratum blood plasma for people's blood coagulation factor VIII content.
⑹ 0.05mol/L calcium chloride solution: get calcium chloride (CaCl
22H2O) 147g is dissolved in water and is diluted to 1000ml, is mixed with the calcium chloride stock solution of 1mol/L, with 20 times of preceding dilute with waters, is mixed with the 0.05mol/L calcium chloride solution.
B. the preparation of human blood coagulation factor VII I reference liquid:
The personnel selection blood coagulation factor VIII lacks blood plasma with standard substance (human blood coagulation factor VII I standard substance; Available from French Stago company) be diluted to every 1ml and contain the 1IU blood coagulation factor VIII; Carry out 10 times, 20 times, 40 times and 80 times of dilutions respectively with diluent again, it is for use to put ice bath.
C. the preparation of testing sample solution:
With human blood coagulation factors VIII shortage blood plasma detected sample is diluted to every 1ml and contains the 1IU platelet cofactor, carry out 10 times and 20 times or 40 times of dilutions with diluent again, it is for use to put ice bath.
D. the detection of sample:
⑴ get activated partial thromboplastin reagent 0.1ml; Put 37 ℃ of water bath heat preservation certain hours (general 4min); Add platelet cofactor and lack blood plasma 0.1ml, different dilution human blood coagulation factors VIII standardized solution 0.1ml, mixing is put 37 ℃ of water bath heat preservation certain hours (general 5min); Add and be preheated to 37 ℃ of 0.05mol/L calcium chloride solution 0.1ml, the record setting time.The tire logarithm of (IU/ml) of standardized solution human blood coagulation factors VIII is carried out straight-line regression to the logarithm of its corresponding setting time (second) and handles, obtain linear regression equation, regressive linearity curve is as shown in Figure 2.
⑵ get activated partial thromboplastin reagent 0.1ml; Put 37 ℃ of water bath heat preservation certain hours (general 4min); Add platelet cofactor and lack blood plasma 0.1ml, testing sample solution 0.1ml, mixing is put 37 ℃ of water bath heat preservation certain hours (general 5min); Add and be preheated to 37 ℃ of 0.05mol/L calcium chloride solution 0.1ml, the record setting time.
⑶ calculate the testing sample solution human blood coagulation factors VIII and tire, and multiply by extension rate again, is testing sample human blood coagulation factors VIII tire (IU/ml).
The VWF of the different additions of table 1 is to the influence of eight factor actives
Detected result is seen table 1, and detected result is presented in the cultivation of the different VWF of interpolation concentration, cell stand density basically identical, and error is no more than 15% each other, but eight factor actives are because the interpolation of VWF has obtained the raising of 100-300%.Explain that the interpolation of external source VWF has improved the expression of recombinant human blood coagulation eight factors greatly.And we also find the addition (when VWF is 2:1 with eight factor actives ratio) when 5IU of VWF, and effect is the most obvious.The VWF that optimizes adds concentration also to be needed according to different cell densities, and results rhythm etc. is optimized.
Embodiment two
1) structure of the cell strain of expressing human blood coagulation eight factors
The preparation method of the cell strain of expressing human blood coagulation eight factors is with embodiment 1.
2) eight factor expression cells expand kind of a cultivation
With 1 * 10
5Cells/ml inoculation triangle shakes bottle, 37 ℃ of serum-free culture after about 4 days cell density reach 1～2 * 10
6Cells/ml; Go down to posterity with the 1:3 ratio of going down to posterity, 37 ℃ are continued shake-flask culture to cell density and reach 1～2 * 10
6Cells/ml obtains seed liquor; Shaking bottle rotating speed is 150 rev/mins.
3) eight factor expression cell reactors are cultivated
After in the serum free medium of cell reactor, inoculating seed liquor with 10% inoculum size, last jar is cultured to cell density about 2 * 10
6Cells/ml, pair cell liquid carries out the feed supplement cultivation then; The feed supplement cultivation stage; Detecting the activity of eight factors in the cell culture fluid of different incubation times through eight factor active analytical procedures, is that the ratio of 3:1 adds VWF (preparation method of VWF detects with embodiment 1 with active) in cell culture fluid according to the specific activity of VWF and FVIII; And feed supplement cultivation stage control enchylema dissolved oxygen 40%, pH7.2,90 rev/mins of mixing speed, 37 ℃ of culture temperature, according to cell stand density and glucose consumption situation, glucose concn maintains 1g/L, gradually feed supplement.Feed supplement is cultivated and to be reached 1～2 * 10 to cell density in 10 days
7Cells/ml obtains cell stoste, as experimental group; And every day, the cell platform expected that blue dyeing counting viable count should reach more than 95%.
4) pair cell stoste carry out that albumen is centrifugal, filtration, purification process, obtain blood coagulation eight factors of purifying.
People's blood coagulation eight factor protein activation analysis methods are seen embodiment one, adopt human blood coagulation factor VII I assay method (first phase method), can participate in three appendix X of Chinese Pharmacopoeia N human blood coagulation factor VII I assay method.
Experimentation is provided with control group, and the substratum of control group is consistent with experimental group, does not just add VWF in the serum-free cell culture medium of control group.The cell of control experiment in reactor drum reaches 5.0 * 10
6Begin during cells/ml to carry out.Time this moment, every day, timing sampling was analyzed, successive analysis 4 days in 0 hour.The experimental result of gained is following,
The time dependent influence of table 2 eight factor actives
Can know by table 2; When the specific activity of the feed supplement cultivation stage control VWF and eight factors is 3:1; Eight factors of the experimental group of interpolation VWF obviously strengthen with prolongation eight factor actives of incubation time; And the analytical results of each time point shows to be compared with the control group that does not add VWF, and the adding of VWF can significantly strengthen the activity of eight factors, and eight factors are efficiently expressed.The expression amount of recombinant human blood coagulation eight factors that obtain through the inventive method reaches as high as 10 ~ 20 iu/sky/10
6Cell.
The above; Being merely preferred embodiment of the present invention, is not to any formal and substantial restriction of the present invention, should be understood that; For those skilled in the art; Under the prerequisite that does not break away from the inventive method, also can make some improvement and replenish, these improvement and replenish and also should be regarded as protection scope of the present invention.Allly be familiar with the professional and technical personnel, under the situation that does not break away from the spirit and scope of the present invention, the technology contents that is disclosed more than capable of using and a little change of making, modify the equivalent variations with differentiation, be equivalent embodiment of the present invention; Simultaneously, the change of any equivalent variations that all foundations essence technology of the present invention is done the foregoing description, modify and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (10)
1. the method for efficiently expressing recombinant human blood coagulation eight factors; Add to for vWF ELISA in the cell culture fluid of express recombinant people blood coagulation eight factors, and the specific activity of control cell culture fluid medium vessels property christmas factor and people's blood coagulation eight factors is 1～10:1 in culturing process external source.
2. cultural method as claimed in claim 1 is characterized in that, the cell culture fluid of said express recombinant people's blood coagulation eight factors is a serum-free cell culture medium.
3. the method for claim 1 is characterized in that, concrete steps are following:
1) preparation of express cell: the recovery of the freeze-stored cell of the structure of the cell strain of expressing human blood coagulation eight factors or expressing human blood coagulation eight factors;
2) the expansion kind of express cell is cultivated: with step 1) make up or people's blood coagulation eight factor expression cell inoculations of recovery in serum free medium, shaking culture 3～4 days reaches 1～2 * 10 to cell density
6Cells/ml, passage, shaking culture is expanded to cell density and reaches 1～2 * 10
6Cells/ml obtains seed liquor;
3) seed liquor that cell reactor cultivation: with step 2) obtains is inoculated in the serum free medium of cell reactor, cultivates 4～7 days, makes the cell density of enchylema reach 1～5 * 10
6Cells/ml; In enchylema, add external source VWF and pair cell liquid then and carry out the feed supplement cultivation, obtain cell stoste; In the said feed supplement culturing process, the VWF activity is 1～10:1 with the FVIII specific activity in the control enchylema;
4) preparation of blood coagulation eight factor products: pair cell stoste is carried out centrifugal, filtration, purification process, obtains recombinant human blood coagulation eight factor products.
4. preparation technology as claimed in claim 3 is characterized in that step 2) in make up or people's blood coagulation eight factor expression cells of recovery with 1～9 * 10
5The inoculum size of cells/ml is inoculated.
5. preparation technology as claimed in claim 3 is characterized in that step 2) described in passage be that the ratio of going down to posterity with 1:2～4 goes down to posterity.
6. preparation technology as claimed in claim 3 is characterized in that step 2) in the condition of shaking culture be: 60～150 rev/mins of rotating speeds, 32～38 ℃ of culture temperature.
7. preparation technology as claimed in claim 3 is characterized in that, the inoculum size that the step 3) seed liquor is inoculated into cell reactor is 5～10v/v%.
8. preparation technology as claimed in claim 3; It is characterized in that the said feed supplement culture condition of step 3) is: control dissolved oxygen 40～80% in the feed supplement culturing process, pH6.8～7.2; 40～90 rev/mins of mixing speed; 32～38 ℃ of culture temperature are carried out feed supplement according to the consumption of glucose, make glucose concn maintain 0.5～1.5g/L.
9. preparation technology as claimed in claim 3 is characterized in that, the cell density that the step 3) reactor drum is cultivated the said cell stoste that obtains is 1～2 * 10
7Cells/ml.
10. preparation technology as claimed in claim 3 is characterized in that, step 3) is controlled cell culture fluid medium vessels property christmas factor and people's blood coagulation eight factors in culturing process specific activity is 2～3:1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630131A (en) * | 2015-01-15 | 2015-05-20 | 上海交通大学 | CHO (Chinese hamster ovary) cell strain for stably expressing human clotting factor VIII and application thereof |
| CN107236036A (en) * | 2016-03-29 | 2017-10-10 | 上海美迪西生物医药股份有限公司 | A kind of method of the factor of Prepare restructuring people blood coagulation eight |
| CN107287265A (en) * | 2016-03-31 | 2017-10-24 | 正大天晴药业集团南京顺欣制药有限公司 | A kind of method of Prepare restructuring human blood coagulation factors VIII |
| CN108611342A (en) * | 2018-03-27 | 2018-10-02 | 成都蓉生药业有限责任公司 | A kind of fed-batch fermentation method of recombinant human blood coagulation factor IX bioactive molecules |
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| EP0934748A2 (en) * | 1997-12-15 | 1999-08-11 | HemaSure Denmark A/S | Von Willenbrand factor (vWF)- containing preparation, process for preparing vWF-containing preparations, and use of such preparations |
| WO2010045321A2 (en) * | 2008-10-15 | 2010-04-22 | Baxter International Inc. | Pegylation of recombinant blood coagulation factors in the presence of bound antibodies |
| EP2310043B1 (en) * | 2008-07-10 | 2012-09-19 | CSL Behring GmbH | Von willebrand factor or factor viii and von willebrand factor for the treatment of coagulopathy induced by inhibitors of thrombocytes |
| WO2012171031A1 (en) * | 2011-06-10 | 2012-12-13 | Baxter International Inc. | Treatment of coagulation disease by administration of recombinant vwf |
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Patent Citations (4)
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|---|---|---|---|---|
| EP0934748A2 (en) * | 1997-12-15 | 1999-08-11 | HemaSure Denmark A/S | Von Willenbrand factor (vWF)- containing preparation, process for preparing vWF-containing preparations, and use of such preparations |
| EP2310043B1 (en) * | 2008-07-10 | 2012-09-19 | CSL Behring GmbH | Von willebrand factor or factor viii and von willebrand factor for the treatment of coagulopathy induced by inhibitors of thrombocytes |
| WO2010045321A2 (en) * | 2008-10-15 | 2010-04-22 | Baxter International Inc. | Pegylation of recombinant blood coagulation factors in the presence of bound antibodies |
| WO2012171031A1 (en) * | 2011-06-10 | 2012-12-13 | Baxter International Inc. | Treatment of coagulation disease by administration of recombinant vwf |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630131A (en) * | 2015-01-15 | 2015-05-20 | 上海交通大学 | CHO (Chinese hamster ovary) cell strain for stably expressing human clotting factor VIII and application thereof |
| CN104630131B (en) * | 2015-01-15 | 2017-08-15 | 上海交通大学 | A kind of the Chinese hamster ovary celI strain and its application of the stable factor of expression people blood coagulation eight |
| CN107236036A (en) * | 2016-03-29 | 2017-10-10 | 上海美迪西生物医药股份有限公司 | A kind of method of the factor of Prepare restructuring people blood coagulation eight |
| CN107287265A (en) * | 2016-03-31 | 2017-10-24 | 正大天晴药业集团南京顺欣制药有限公司 | A kind of method of Prepare restructuring human blood coagulation factors VIII |
| CN107287265B (en) * | 2016-03-31 | 2018-09-14 | 正大天晴药业集团南京顺欣制药有限公司 | A kind of method of Prepare restructuring human blood coagulation factors VIII |
| CN108611342A (en) * | 2018-03-27 | 2018-10-02 | 成都蓉生药业有限责任公司 | A kind of fed-batch fermentation method of recombinant human blood coagulation factor IX bioactive molecules |
| CN108611342B (en) * | 2018-03-27 | 2022-03-01 | 成都蓉生药业有限责任公司 | Feeding fermentation method of recombinant human coagulation factor IX active molecules |
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| CN102776260B (en) | 2015-04-29 |
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