CN102776260B - Method for effectively expressing recombinant human coagulation factor VIII - Google Patents
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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 mammaliancellculture high expression that utilizes and recombinate the process of eight factors.
Background technology
Natural human blood coagulation eight factor (FVIII) is a kind of large glycoproteins, and be combined into by heavy chain and light chain, total molecular weight 330KD, in blood plasma, content is about 0.2mg/L, exists with the form in conjunction with vWF ELISA (VWF).In blood coagulation reaction, the FVIII of activation makes the work of the enzyme of FIX greatly improve, the activation of catalysis FX, and in conjunction with composition complex, catalysis aggegation chain reaction goes on.Disappearance or the shortage of FVIII molecule cause haemophilia A, supplement the effective measures that activated FVIII is treatment haemophilia A.
The advantage that the pathogenies such as recombined human FVIII has identical physiology with natural FVIII, pharmacology and immune characteristic, and stops HIV, HDV, prion are propagated, therefore, the research of restructuring FVIII protein expression becomes an important trend of haemophilia A treatment.First recombined human blood coagulation eight factor in the world, namely restructuring eight factor of the first generation was just gone on the market in nineteen ninety.Eight factors in the current whole world are sold and are reached 7,000,000,000 international units year, and wherein 60% is genetic engineering restructuring blood coagulation eight factor.
Natural FVIII is always combined into compound and stable existence with vWF.When restructuring FVIII secretes the serum free medium to cell, molecule is easy to disintegrate, and is degraded, and the FVIII of expression can not get accumulation.Utilize vWF and FVIII coexpression system can solve the instability of restructuring FVIII in emiocytosis expression process and the easy problem such as degraded well.The research of the people such as Kaufman shows, can significantly improve the expression of FVIII after FVIII and VWF coexpression.In addition, under the condition of culture having serum, the VWF in serum in conjunction with newly-generated VIII, can form more stable compound, reaches the effect that FVIII accumulates in cell culture fluid.
Restruction eight is carried out because of in subprocess utilizing cell large-scale culture, in order to improve the expression of eight factors, utilization has blood serum medium or cotransfection FVIII and VWF gene in mammalian cell, stabilizes the expression of eight factors, but these two kinds of methods all exist respective limitation.For there being blood serum medium, due in mammalian cell culture process, external source is added animal blood serum and brings the risk of viral potential pollution.Therefore, utilize mammalian cell to produce in pharmaceutical grade protein at present to tend to free serum culture to substitute and have serum cell chulture.And for the coexpression of VWF and FVIII gene, people find in research recombined human blood coagulation eight factor expression, VWF and FVIII coexpression, the expression of eight factors is lower, the structure also more complicated of cell line, the expression of high expressed eight factor cell line filtered out in serum free medium is generally in 0.1-0.5 international unit/sky/10
6cell, this expression does not reach the requirement of industrialization far away.Visible, be badly in need of a kind of new cultural method at present and express restructuring eight factor, the suitability for industrialized production of restructuring eight factor can be realized under serum-free culturing conditions, make simple, efficient, the virus-free pollution of the expression of eight factors.
Summary of the invention
The object of the invention is to the defect overcoming prior art, provide a kind of mammaliancellculture high expression that utilizes to recombinate the process of eight factors.
First the present invention discloses a kind of method of efficiently expressing recombinant human blood coagulation eight factor, for being added to by the vWF ELISA of external source in the cell culture fluid of expression recombined human blood coagulation eight factor, and the specific activity controlling cell culture fluid medium vessels christmas factor and people's blood coagulation eight factor in incubation is 1 ~ 10:1.
Preferably, the cell culture fluid of described expression recombined human blood coagulation eight factor is serum-free cell culture medium.
The cell of expressing recombined human blood coagulation eight factor described in the present invention is the cell line of single expression restructuring FVIII, and VWF is preparation in addition, additionally adds the allogenic material in the cell culture fluid of expressing recombined human blood coagulation eight factor to as molecular chaperones.
Technical scheme provided by the invention is added to according to certain ratio in the cell culture fluid of restructuring eight factor by VWF, greatly can improve the expression of restructuring eight factor, and this method is that the production of industrialization eight factor provides a kind of simple and practical realizing route.Method of the present invention does not need, by eight Summing Factor VWF gene co-transfection host cells, to simplify cell line building process, improve high yielding cell sarain screening effeciency, is particularly suitable for industrialization large-scale production recombined human blood coagulation eight factor.
Preferably, the method concrete steps of described efficiently expressing recombinant human blood coagulation eight factor are as follows:
1) preparation of cell is expressed: the recovery of the structure of expressing the cell line of people's blood coagulation eight factor or the freeze-stored cell of expressing people's blood coagulation eight factor;
2) expanding species expressing cell is cultivated: be inoculated in serum free medium by people's blood coagulation eight factor expression cell of step 1) structure or recovery, shaken cultivation 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) cell reactor is cultivated: by step 2) seed liquor that obtains is inoculated in the serum free medium of cell reactor, and cultivate 4 ~ 7 days, make the cell density of cell liquid reach 1 ~ 5 × 10
6cells/ml; Then in cell liquid, add external source VWF and feed-batch culture is carried out to cell liquid, obtaining cell stoste; In described feed-batch culture process, controlling VWF in cell liquid active is 1 ~ 10:1 with FVIII specific activity;
4) preparation of blood coagulation eight Factor products: to cell stoste carry out centrifugal, filter, purification process, obtain recombined human blood coagulation eight Factor products.
The cell line of expressing people's blood coagulation eight factor described in step 1) or the freeze-stored cell of expressing people blood coagulation eight factor are the cell line of single expression people blood coagulation eight factor built, and the FVIII expressed comprise total length FVIII have the FVIII fragment of disappearance with B district.Construction method is conventional molecular biological recombinant protein preparation method.
Preferably, step 2) in build or recovery people's blood coagulation eight factor expression cell with 1 ~ 9 × 10
5the inoculum concentration of cells/ml is inoculated.Express cell expanding species cultivate can by build or recovery people's blood coagulation eight factor expression cell be inoculated in triangular flask, through Shaking culture, go down to posterity, Shaking culture obtain seed liquor.
Preferably, step 2) described in passage be go down to posterity with the ratio of going down to posterity of 1:2 ~ 4.
More excellent, step 2) described in passage be go down to posterity with the ratio of going down to posterity of 1:3.
Preferably, step 2) in the condition of shaken cultivation be: rotating speed 60 ~ 150 revs/min, cultivation temperature 32 ~ 38 DEG C.
Preferably, step 3) seed liquor is inoculated into the inoculum concentration of cell reactor is 5 ~ 10v/v%.
Preferably, the condition of feed-batch culture described in step 3) is: control dissolved oxygen 40 ~ 80%, pH6.8 ~ 7.2 in feed-batch culture process, speed of agitator 40 ~ 90 revs/min, cultivation temperature 32 ~ 38 DEG C, feed supplement is carried out in the consumption according to glucose, makes concentration of glucose maintain 0.5 ~ 1.5g/L.
More excellent, the condition of feed-batch culture described in step 3) is: control dissolved oxygen 40 ~ 80%, pH6.8 ~ 7.2 in feed-batch culture process, speed of agitator 40 ~ 90 revs/min, cultivation temperature 37 DEG C, feed supplement is carried out in the consumption according to glucose, makes concentration of glucose maintain 1.0g/L.
Preferably, the cell density of described cell stoste that step 3) bioreactor culture obtains is 1 ~ 2 × 10
7cells/ml.
That described in step 4), the preparation of recombined human blood coagulation eight Factor products adopts is centrifugal, filter, purification process is conventional separation and purification of protein step, and in the FVIII of the higher degree of acquisition, VWF both can remove and also can still retain.
Step 2 of the present invention) express cell expanding species cultivate and step 3) cell reactor cultivate existing conventional serum-free cell culture medium all can be adopted to carry out cell chulture.
Instant invention overcomes in prior art in order to obtain stable blood coagulation eight factor must in same cell line the difficult point of coexpression eight Summing Factor VWF.The present invention proposes a kind of effective cell chulture approach, by the content of FVIII and VWF is controlled in certain optimization range, the expression of FVIII is accumulated in a large number, and preparation technology of the present invention to express cell construction difficulty little, be particularly suitable for suitability for industrialized production recombined human blood coagulation eight factor.
Effect of the present invention comprises: what 1) production method of the present invention adopted is single expression eight factor cell line, does not need eight factors and VWF cotransfection cell line, the cell line structure in early stage and screening process simple; 2) cell cultivation process with the addition of the VWF factor, and the Cumulate Sum that improve eight factors is stablized; 3) be applicable to suitability for industrialized production, eight factor expression amounts can be improved very simply, also easily amplify.
Accompanying drawing explanation
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 active detects
Detailed description of the invention
The present invention is set forth further below in conjunction with embodiment.Should be understood that embodiment only for illustration of the present invention, but not limit the scope of the invention.In embodiment, the experimental technique of unreceipted actual conditions and the reagent of undeclared formula are conveniently condition, as works such as ［ beautiful ］ Sambrook.J; Huang Peitang etc. translate.Molecular cloning texts guide, the third edition.Beijing: the condition of the condition described in Science Press 2002 or manufacturer's suggestion is carried out or configures.
Embodiment one
1. experimental technique
1) the eight factor expression cells (the structure bibliography " expression study of clotting factor FVIII in mammalian cell " of eight factor expression cells, Wang Qi etc., Pharmaceutical Biotechnology, 2002,9(5) of free serum culture will be domesticated for: 251 ~ 255; Cell line serum-free acclimation method can refer to cell chulture handbook, as cell culture operations part in invitrogen Products handbook annex), with 9 × 10
5cells/ml inoculates triangle shaking flask, cultivation temperature 32 DEG C, and shaking flask rotating speed is 60 revs/min; Free serum culture after about 3 days cell density reach 2 × 10
6cells/ml, goes down to posterity with the ratio of 1:2, Shaking culture, cultivation temperature 38 DEG C, and rotating speed 60 revs/min, reaches 1 ~ 2 × 10 to cell density
6cells/ml.Serum-free cell culture medium is commercialization SIGMA Products SFMII302 nutrient solution.
2) with containing different activities VWF (VWF albumen prepare bibliography Recombinant von Willebrand factor:Potential Therapeutic Use, BernhardE.Fischer, Journal of Thrombosis and Thrombolysis, Volume8, Number3 (1999), nutrient solution 197-205) changes the cell (detecting the kit of VWF activity purchased from Shanghai Sun Bio-Tech Co., Ltd.) of liquid culture expression eight factor, cultivate after 24 hours, collecting cell nutrient solution carries out eight factor active analyses (see table 1).
The serum-free cell culture medium that the present embodiment adopts is commercialization SIGMA Products SFMII302 nutrient solution.
3) eight factor active analytical methods
Adopt human blood coagulation factor VII I determination method (first phase method), can participate in Chinese Pharmacopoeia three annex XN human blood coagulation factor VII I determination methods, concrete steps are as follows:
A. reagent:
(1) 3.8% sodium citrate: get sodium citrate 9.5g, is dissolved in water and is diluted to 250ml.
(2) imidazole buffer (pH7.3): get imidazoles 0.68g and sodium chloride 1.17g, add water and make to be dissolved into 100ml, adds 0.1mol/L hydrochloric acid solution 42.2ml, then is diluted with water to 200ml, to obtain final product.
(3) dilution: the sodium citrate getting 3.8% of a volume adds 5 volume imidazole buffer mixing, and adding appropriate 20% human serum albumin to final concentration is 1%.
(4) partial thromboplastin (APTT) reagent activated.
Human blood coagulation factor VII I lack blood plasma: for people's blood coagulation factor VIII content lower than 1% human plasma or artificial substratum blood plasma.
(6) 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 storage liquid of 1mol/L, with front dilute with water 20 times, is mixed with 0.05mol/L calcium chloride solution.
B. the preparation of human blood coagulation factor VII I titer:
Employment blood coagulation factor VIII lacks blood plasma by standard items (human blood coagulation factor VII I standard items, purchased from French Stago company) be diluted to every 1ml containing 1IU blood coagulation factor VIII, carry out 10 times, 20 times, 40 times and 80 times of dilutions respectively with dilution again, put ice bath stand-by.
C. the preparation of testing sample solution:
Lack blood plasma with human blood coagulation factors VIII and detected sample is diluted to every 1ml containing 1IU platelet cofactor Ⅰ, then carry out 10 times and 20 times or 40 times of dilutions with dilution, put ice bath stand-by.
D. the detection of sample:
(1) get the partial thromboplastin reagent 0.1ml of activation, put 37 DEG C of water bath heat preservation certain hours (general 4min), add platelet cofactor Ⅰ and lack blood plasma 0.1ml, different dilution human blood coagulation factors VIII standard liquid 0.1ml, mixing, put 37 DEG C of water bath heat preservation certain hours (general 5min), add and be preheated to 37 DEG C of 0.05mol/L calcium chloride solution 0.1ml, record setting time.The logarithm of logarithm to its corresponding setting time (second) of (IU/ml) of being tired by standard liquid human blood coagulation factors VIII carries out linear regression process, and obtain linear regression equation, the linearity curve of recurrence as shown in Figure 2.
(2) get the partial thromboplastin reagent 0.1ml of activation, put 37 DEG C of water bath heat preservation certain hours (general 4min), add platelet cofactor Ⅰ and lack blood plasma 0.1ml, testing sample solution 0.1ml, mixing, put 37 DEG C of water bath heat preservation certain hours (general 5min), add and be preheated to 37 DEG C of 0.05mol/L calcium chloride solution 0.1ml, record setting time.
(3) calculate testing sample solution human blood coagulation factors VIII to tire, then be multiplied by extension rate, be testing sample human blood coagulation factors VIII and tire (IU/ml).
The VWF of table 1 Different adding amount is on the impact of eight factor actives
Testing result is in table 1, and testing result is presented at different interpolation in the cultivation of VWF concentration, and cell density is basically identical, and mutual error is no more than 15%, but eight factor actives are because the interpolation of VWF obtains the raising of 100-300%.Illustrate that the interpolation of external source VWF substantially increases the expression of recombined human blood coagulation eight factor.And we also find the addition of VWF (VWF and eight factor actives are than during for 2:1) when 5IU, and effect is the most obvious.The VWF optimized adds concentration also to be needed according to different cell density, and results rhythm etc. is optimized.
Embodiment two
1) structure of the cell line of people's blood coagulation eight factor is expressed
Express the preparation method of the cell line of people's blood coagulation eight factor with embodiment 1.
2) eight factor expression cell expanding species are cultivated
With 1 × 10
5cells/ml inoculates triangle shaking flask, 37 DEG C of free serum 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 DEG C are continued Shaking culture and reach 1 ~ 2 × 10 to cell density
6cells/ml, obtains seed liquor; Shaking flask rotating speed is 150 revs/min.
3) eight factor expression cell reactors are cultivated
After inoculating seed liquor with 10% inoculum concentration in the serum free medium of cell reactor, upper tank is cultured to cell density about 2 × 10
6cells/ml, then carries out feed-batch culture to cell liquid; The feed-batch culture stage, detected the activity of eight factors in the cell culture fluid of different incubation time by eight factor active analytical methods, the ratio being 3:1 according to the specific activity of VWF and FVIII adds the preparation method of VWF(VWF and Activity determination with embodiment 1 in cell culture fluid); And feed-batch culture stage control cell liquid dissolved oxygen 40%, pH7.2, speed of agitator 90 revs/min, cultivation temperature 37 DEG C, according to cell density and glucose consumption situation, concentration of glucose maintains 1g/L, gradually feed supplement.Feed-batch culture reaches 1 ~ 2 × 10 to cell density in 10 days
7cells/ml, obtains cell stoste, as experimental group; And every day, cell platform expected that blue dyeing counting viable count should reach more than 95%.
4) to cell stoste carry out albumen centrifugal, filter, purification process, obtain blood coagulation eight factor of purifying.
People's blood coagulation eight factor protein activity assays is shown in embodiment one, adopts human blood coagulation factor VII I determination method (first phase method), can participate in Chinese Pharmacopoeia three annex X N human blood coagulation factor VII I determination methods.
Experimentation arranges control group, and the culture medium of control group is consistent with experimental group, is not add VWF in the serum-free cell culture medium of control group.Control experiment cell in the reactor reaches 5.0 × 10
6start during cells/ml to carry out.Now the time was in 0 hour, and every day, timing sampling was analyzed, and analyzed 4 days continuously.The experimental result of gained is as follows,
The time dependent impact of table 2 eight factor active
As shown in Table 2, when the specific activity of feed-batch culture stage control VWF and eight factors is 3:1, eight factors of adding the experimental group of VWF obviously strengthen with prolongation eight factor active of incubation time, and the display of the analysis result of each time point is compared with the control group not adding VWF, VWF adds the activity that significantly can strengthen eight factors, makes eight factors obtain high expression.The expression of recombined human blood coagulation eight factor obtained by the inventive method reaches as high as 10 ~ 20 international units/sky/10
6cell.
The above; be only preferred embodiment of the present invention; not to any formal and substantial restriction of the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the inventive method, also can make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.All those skilled in the art, without departing from the spirit and scope of the present invention, a little change made when utilizing disclosed above technology contents, the equivalent variations of modifying and developing, be Equivalent embodiments of the present invention; Meanwhile, all according to substantial technological of the present invention to the change of any equivalent variations that above-described embodiment is done, modify and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (9)
1. the method for efficiently expressing recombinant human blood coagulation eight factor, for being added to by the vWF ELISA of external source in the cell culture fluid of expression recombined human blood coagulation eight factor, and the specific activity controlling cell culture fluid medium vessels christmas factor and people's blood coagulation eight factor in incubation is 1 ~ 10:1; The concrete steps of the method are as follows:
1) preparation of cell is expressed: the recovery of the structure of expressing the cell line of people's blood coagulation eight factor or the freeze-stored cell of expressing people's blood coagulation eight factor; The cell line of described expression people blood coagulation eight factor or the freeze-stored cell of expressing people's blood coagulation eight factor are the cell line of single expression people blood coagulation eight factor built, and the FVIII expressed comprise total length FVIII have the FVIII fragment of disappearance with B district;
2) express cell expanding species cultivate: by step 1) build or recovery people's blood coagulation eight factor expression cell be inoculated in serum free medium, shaken cultivation 3 ~ 4 days, reaches 1 ~ 2 × 10 to cell density
6cells/ml, passage, shaken cultivation is expanded to cell density and reaches 1 ~ 2 × 10
6cells/ml, obtains seed liquor;
3) cell reactor is cultivated: by step 2) seed liquor that obtains is inoculated in the serum free medium of cell reactor, and cultivate 4 ~ 7 days, make the cell density of cell liquid reach 1 ~ 5 × 10
6cells/ml; Then in cell liquid, add external source VWF and feed-batch culture is carried out to cell liquid, obtaining cell stoste; In described feed-batch culture process, controlling VWF in cell liquid active is 1 ~ 10:1 with FVIII specific activity; The condition of described feed-batch culture is: control dissolved oxygen 40 ~ 80% in feed-batch culture process, pH 6.8 ~ 7.2, speed of agitator 40 ~ 90 revs/min, cultivation temperature 32 ~ 38 DEG C, feed supplement is carried out in consumption according to glucose, makes concentration of glucose maintain 0.5 ~ 1.5g/L;
4) preparation of blood coagulation eight Factor products: to cell stoste carry out centrifugal, filter, purification process, obtain recombined human blood coagulation eight Factor products.
2. the method for efficiently expressing recombinant human blood coagulation eight factor as claimed in claim 1, it is characterized in that, the cell culture fluid of described expression recombined human blood coagulation eight factor is serum-free cell culture medium.
3. the method for efficiently expressing recombinant human blood coagulation eight factor as claimed in claim 1, is characterized in that, step 2) in build or people's blood coagulation eight factor expression cell of recovery with 1 ~ 9 × 10
5the inoculum concentration of cells/ml is inoculated.
4. the method for efficiently expressing recombinant human blood coagulation eight factor as claimed in claim 1, is characterized in that, step 2) described in passage be go down to posterity with the ratio of going down to posterity of 1:2 ~ 4.
5. the method for efficiently expressing recombinant human blood coagulation eight factor as claimed in claim 1, is characterized in that, step 2) in the condition of shaken cultivation be: rotating speed 60 ~ 150 revs/min, cultivation temperature 32 ~ 38 DEG C.
6. the method for efficiently expressing recombinant human blood coagulation eight factor as claimed in claim 1, is characterized in that, step 3) the seed liquor inoculum concentration that is inoculated into cell reactor is 5 ~ 10v/v%.
7. the method for efficiently expressing recombinant human blood coagulation eight factor as claimed in claim 1, it is characterized in that, step 3) condition of described feed-batch culture is: control dissolved oxygen 40 ~ 80% in feed-batch culture process, pH 6.8 ~ 7.2, speed of agitator 40 ~ 90 revs/min, cultivation temperature 32 ~ 38 DEG C, feed supplement is carried out in the consumption according to glucose, makes concentration of glucose maintain 0.5 ~ 1.5g/L.
8. the method for efficiently expressing recombinant human blood coagulation eight factor as claimed in claim 1, is characterized in that, step 3) cell density of described cell stoste that obtains of bioreactor culture is 1 ~ 2 × 10
7cells/ml.
9. the method for efficiently expressing recombinant human blood coagulation eight factor as claimed in claim 1, is characterized in that, step 3) specific activity that controls cell culture fluid medium vessels christmas factor and people's blood coagulation eight factor in incubation is 2 ~ 3:1.
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| 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 |
| 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 |
| 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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|---|---|---|---|---|
| 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)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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