WO2008135568A1 - Chromatography process for obtaining a fviii/vwf complex with different ratios between the two proteins, for use in haemophilia a treatment and in von willebrand disease - Google Patents
Chromatography process for obtaining a fviii/vwf complex with different ratios between the two proteins, for use in haemophilia a treatment and in von willebrand disease Download PDFInfo
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- WO2008135568A1 WO2008135568A1 PCT/EP2008/055526 EP2008055526W WO2008135568A1 WO 2008135568 A1 WO2008135568 A1 WO 2008135568A1 EP 2008055526 W EP2008055526 W EP 2008055526W WO 2008135568 A1 WO2008135568 A1 WO 2008135568A1
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- 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
- FVIII Factor VIII
- vWF a 256 KDa glycoprotein which circulates in plasma (0.1 ⁇ g/ml) bound to vWF.
- This glycoprotein being very sensitive to proteolysis which causes its activation or degradation, plays a key role as co-factor for FIXa in the coagulation cascade.
- the formation of the complex with vWF protects FVIII from its proteolytic inactivation.
- a deficiency or an anomaly of FVIII leads to the pathology known as haemophilia A, characterized by repeated haemorrhagic episodes due to blood coagulation problems.
- VWF is very important in the haemostatic process and in this respect, in addition to carrying out its function as a carrier protein for FVIII, it is essential for adhesion of thrombocytes to damaged endothelium/subendothelium.
- vWF acts as a bridge linking specific receptors on thrombocyte surfaces (gplb, gpllb/llla) and endothelial components. Changes in vWF levels can also cause serious haemostatic anomalies.
- Von Willebrand disease vWD is an autosomal hereditary disease of the blood, mainly caused by a deficiency or an abnormal multimeric composition of vWF.
- vWF vWF
- platelet adhesion to the damaged endothelium is less efficient and hence bleeding time is increased.
- vWF plasma half- life is reduced, with similar or more serious symptomatic manifestations than those of haemophilia A.
- the incidence of vWD is at least 0.8% in the world population, not accounting for differences between races or geographical distribution.
- DDAVP desmopressin
- FVIII/vWF concentrates Concentrates of FVIII/vWF are very important for the treatment of both haemophilia A and von Willebrand disease.
- a vWF concentrate on its own is unable to restore FVIII content to normal levels in the first stage of bleeding, and in some cases can actually lead to thrombosis.
- a FVIII/vWF concentrate must however possess a vWF with a multimeric pattern similar to that of plasma. (Haemostasis 1994; 24: 285-288).
- stabilizers e.g. albumin
- EP 0468181 describes a method for purifying FVIII from human plasma by ion exchange chromatography, eluting the FVIII at high ionic strength and at acidic pH, then collecting the eluate in the presence of a stabilizer such as heparin, albumin and PEG and lysine or histidine as antiproteases.
- a stabilizer such as heparin, albumin and PEG and lysine or histidine as antiproteases.
- albumin addition decreases the specific activity from 300-1200 U/mg protein to 18-24 U/mg protein.
- US 4361509 describes the purification of FVIII by means of monoclonal antibodies which bind to the FVIII/vWF complex, then dissociating the FVIII from vWF by CaCI 2 .
- the FVIII obtained, devoid of vWF, is however stable only after albumin addition.
- a FVIII/vWF complex is purified by means of anion exchange chromatography, but in this case also the addition of heparin and albumin are needed to stabilize the product.
- the purity of the FVIII/vWF complex is also very important, in which respect by reducing as far as possible the content of contaminating proteins, such as fibrinogen, fibronectin and IgG, allergic reactions or antibodies against the product itself occur less frequently.
- Some preparations obtained by affinity chromatography (EP 90308104) have a low FVIII specific activity (66 U/mg).
- the aim of the present invention is to provide a method for purifying a FVIII/vWF complex in which the vWF content can be easily modulated by suitably varying certain parameters of the chromatography process.
- a chromatography method By this chromatography method various FVIII/vWF complexes can be obtained for use in different pathologies.
- FVIIkvWF 1.25
- the product can be used both for haemophilia A and for type 3 von Willebrand disease. Indeed, in this case, a product without FVIII may not be sufficient to reestablish initial levels of FVIII.
- VWF concentrations of about 1.5 times greater than FVIII could instead be optimal for the various types of vWD.
- Figure 1 shows the influence of wash buffer and column bed washings on the vWF recovery.
- the present invention enables a FVIII concentrate to be obtained by means of an efficient, high yield method which can be easily scaled-up to industrial levels, and in which the vWF concentration can be modulated by using a single chromatography system and modifying only certain parameters of the buffers used for the chromatography.
- Chromatographic separation is carried out on a synthetic hydrophilic support on which are attached long polymer chains (tentacle-like structure) at whose ends positively charged groups (TMAE - trimethylaminoethyl), suitable for strong anion exchange, are present; an example of a resin of this type is known as Fractogel EMD TMAE.
- resins also enables high molecular weight proteins (>300 KDa), or adhesive proteins such as vWF, to easily bind to charged groups disposed at the ends of the long polymer chain, hence reducing interactions with the matrix which could result in a lower recovery of the protein and/or its inactivation.
- high molecular weight proteins >300 KDa
- adhesive proteins such as vWF
- the biological fraction that contains the FVIII to be purified by chromatography can be obtained: from human plasma, from cryoprecipitate or from a plasma fraction obtained by a pre-purification treatment, such as absorption on aluminium hydroxide.
- a chromatography column containing a resin of the aforesaid type is equilibrated with a buffer selected in the group consisting of citrate, phosphate and tris, having a pH comprised between 6.8 and 7.4, preferably 7.0, and containing NaCI at a concentration between 0.10 M and 0.15 M, preferably 0.12 M, and optionally comprising other substances such as glycine, calcium chloride.
- the column equilibrated with the aforesaid buffer, is loaded with the biological fraction containing the FVIII to be purified.
- the FVIII and the vWF bind preferentially to the resin while the contaminating proteins are for the most part eluted.
- the first chromatography step consists of washing the resin with the equilibration buffer described above, so as to remove excess contaminating proteins which do not interact with the resin.
- the preceding buffer is used though increasing its ionic strength (the equilibration buffer modified in this manner is known hereinafter as the “wash buffer”), so as to elute the weakly bound contaminating proteins and to partially elute the vWF.
- the FVIII/vWF complex is eluted using the same buffer while further increasing the conductivity (this buffer is known hereinafter as the “elution buffer”) by means of NaCI or another salt at a concentration between 0.4 and 1.0 M, preferably 0.45 M.
- the quantity of buffer used for washing the resin (normally comprised between 5 and 15 CV).
- NaCI molarity during the aforesaid wash is between 0.2 and 0.25 in order to obtain, after subsequent elution, a final product with a FVIII/vWF ratio between about 0.68 and 2.47 (see table 1 ).
- the presence of calcium ions at a suitable concentration in the wash buffer enables to obtain a FVIII/vWF concentrate with a very high R; the use of 8 mM CaCI 2 in the 0.25 M NaCI buffer enables to obtain a FVIIkvWF ratio of about 1 :0.5 without leading to a significant reduction in FVIII yield in the final product, which remains in the order of 85-90%. (Table 2).
- the purification method of the present invention is highly efficient, reproducible, can be scaled-up to industrial levels and provides a complex between FVIII and vWF with high yields.
- the product obtained has a high specific activity of FVIII, with low concentrations of the main contaminants such as fibrinogen, fibronectin, IgG, IgM, and the vWF exhibits a multimeric pattern with good percentages of high molecular weight multimers.
- the present chromatography process leads to an enrichment of high molecular weight multimers in the finished product, while reducing the concentration of low molecular weight multimers in the step preceding elution of the FVIII/vWF complex.
- FVIII/vWF complex purification according to the process of the present invention are given hereinafter.
- the contaminating proteins were removed by washing the resin with the same buffer then treating the resin with about 10 CV of 10 mM citrate buffer at pH 7.0 with 0.25 M of NaCI, whereas the FVIII/vWF complex was eluted with the same buffer but with a 0.45 M NaCI concentration.
- the supernatant (S1 ) obtained as in example 1 was loaded onto the Fractogel EMD TMAE resin equilibrated and washed with a 10 mM citrate buffer at pH 7.0 with NaCI at a 0.12 M concentration.
- the contaminating proteins were removed by treating the resin with about 10 CV of the same buffer containing 0.26 M of NaCI, whereas the FVIII/vWF complex was eluted using NaCI at a 0.45 M concentration.
- the activities of FVIIhC and vWF:Ag are summarized in Table C below.
- the supernatant (S1 ) obtained as in example 1 was loaded onto the Fractogel EMD TMAE resin equilibrated and washed with a 10 mM citrate buffer at pH 7.0 with NaCI at a 0.12 M concentration.
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Abstract
A process is described enabling FVIII/vWF complexes to be obtained with different ratios between said two proteins by acting only on the wash and elution buffers of the chromatography system.
Description
CHROMATOGRAPHY PROCESS FOR OBTAINING A FVIII/vWF COMPLEX WITH DIFFERENT RATIOS BETWEEN THE TWO PROTEINS, FOR USE IN HAEMOPHILIA A TREATMENT AND IN VON WILLEBRAND DISEASE Field of the invention The invention relates to the field of human blood protein purification. State of the art
Factor VIII (FVIII) is a 256 KDa glycoprotein which circulates in plasma (0.1 μg/ml) bound to vWF. This glycoprotein, being very sensitive to proteolysis which causes its activation or degradation, plays a key role as co-factor for FIXa in the coagulation cascade. The formation of the complex with vWF protects FVIII from its proteolytic inactivation. A deficiency or an anomaly of FVIII leads to the pathology known as haemophilia A, characterized by repeated haemorrhagic episodes due to blood coagulation problems. VWF is very important in the haemostatic process and in this respect, in addition to carrying out its function as a carrier protein for FVIII, it is essential for adhesion of thrombocytes to damaged endothelium/subendothelium. In the first stage of haemostasis, i.e. adhesion, vWF acts as a bridge linking specific receptors on thrombocyte surfaces (gplb, gpllb/llla) and endothelial components. Changes in vWF levels can also cause serious haemostatic anomalies. Von Willebrand disease (vWD) is an autosomal hereditary disease of the blood, mainly caused by a deficiency or an abnormal multimeric composition of vWF. Such deficiency has repercussions on vWF main functions: platelet adhesion to the damaged endothelium is less efficient and hence bleeding time is increased. Furthermore, without the protective action of vWF on FVIII the latter's plasma half- life is reduced, with similar or more serious symptomatic manifestations than those of haemophilia A. The incidence of vWD is at least 0.8% in the world population, not accounting for differences between races or geographical distribution. In von Willebrand disease the principle aim of the treatment is to correct bleeding time and FVIII deficiency. The drug of choice is desmopressin (DDAVP), a synthetic molecule. There are patients, however, who do not respond to desmopressin treatment or who exhibit side-effects from its use; in these cases recourse is normally made to replacement therapy with FVIII/vWF concentrates.
Concentrates of FVIII/vWF are very important for the treatment of both haemophilia A and von Willebrand disease. A vWF concentrate on its own is unable to restore FVIII content to normal levels in the first stage of bleeding, and in some cases can actually lead to thrombosis. To enable it to be also used for Von Willebrand disease, a FVIII/vWF concentrate must however possess a vWF with a multimeric pattern similar to that of plasma. (Haemostasis 1994; 24: 285-288). The various methods so far devised for preparing the FVIII/vWF complex have been aimed solely at improving FVIII purification, hence the concentrates obtained always have a precise and well defined ratio between the two proteins. This is true for US 5252709 which describes a method for separating FVIII, vWF and fibrinogen by anion exchange chromatography (Fractogel TSK-DEAE 650 M). In this case a product with a good yield is obtained but only with a FVIII/vWF ratio equal to 3. In US 6228613 which describes the purification of a stable FVIII/vWF complex, the FVIII/vWF ratio also remains at about 2.
In US 5679776, a FVIII/vWF complex is purified by means of an anion exchange resin to obtain a product with a FVIII/vWF ratio equal to 1.
To avoid a loss of biological activity of the FVIII/vWF complex over time, as occurs in the viral inactivation step, stabilizers (e.g. albumin) are often added. The addition of stabilizers can be found in many patents: EP 0468181 describes a method for purifying FVIII from human plasma by ion exchange chromatography, eluting the FVIII at high ionic strength and at acidic pH, then collecting the eluate in the presence of a stabilizer such as heparin, albumin and PEG and lysine or histidine as antiproteases. Naturally in this case albumin addition decreases the specific activity from 300-1200 U/mg protein to 18-24 U/mg protein.
US 4361509 describes the purification of FVIII by means of monoclonal antibodies which bind to the FVIII/vWF complex, then dissociating the FVIII from vWF by CaCI2. The FVIII obtained, devoid of vWF, is however stable only after albumin addition. In EP 0600480 a FVIII/vWF complex is purified by means of anion exchange chromatography, but in this case also the addition of heparin and albumin are needed to stabilize the product.
The purity of the FVIII/vWF complex is also very important, in which respect by reducing as far as possible the content of contaminating proteins, such as fibrinogen, fibronectin and IgG, allergic reactions or antibodies against the product itself occur less frequently. Some preparations obtained by affinity chromatography (EP 90308104) have a low FVIII specific activity (66 U/mg).
With the present invention an efficient, high yield method has been devised that can be easily scaled-up to industrial levels, enabling a FVIII concentrate to be obtained in which the vWF content can be modulated by using a single chromatography system and modifying only the ionic strength and composition of the buffers used for the chromatography. Summary of the invention
The aim of the present invention is to provide a method for purifying a FVIII/vWF complex in which the vWF content can be easily modulated by suitably varying certain parameters of the chromatography process. By this chromatography method various FVIII/vWF complexes can be obtained for use in different pathologies.
For example in haemophilia A, a FVIII concentrate is normally needed in which the vWF content is the minimum required for stabilizing the molecule (e.g. FVIIkvWF = 1.25). By increasing the vWF content in the aforecited complex (e.g. FVIIkvWF = 1 ) the product can be used both for haemophilia A and for type 3 von Willebrand disease. Indeed, in this case, a product without FVIII may not be sufficient to reestablish initial levels of FVIII.
VWF concentrations of about 1.5 times greater than FVIII could instead be optimal for the various types of vWD. Brief description of the drawing
Figure 1 shows the influence of wash buffer and column bed washings on the vWF recovery.
Detailed description of the invention
The present invention enables a FVIII concentrate to be obtained by means of an efficient, high yield method which can be easily scaled-up to industrial levels, and in which the vWF concentration can be modulated by using a single chromatography system and modifying only certain parameters of the buffers used
for the chromatography.
Chromatographic separation is carried out on a synthetic hydrophilic support on which are attached long polymer chains (tentacle-like structure) at whose ends positively charged groups (TMAE - trimethylaminoethyl), suitable for strong anion exchange, are present; an example of a resin of this type is known as Fractogel EMD TMAE.
The use of these resins also enables high molecular weight proteins (>300 KDa), or adhesive proteins such as vWF, to easily bind to charged groups disposed at the ends of the long polymer chain, hence reducing interactions with the matrix which could result in a lower recovery of the protein and/or its inactivation.
According to the present invention the biological fraction that contains the FVIII to be purified by chromatography can be obtained: from human plasma, from cryoprecipitate or from a plasma fraction obtained by a pre-purification treatment, such as absorption on aluminium hydroxide. According to the present invention a chromatography column containing a resin of the aforesaid type is equilibrated with a buffer selected in the group consisting of citrate, phosphate and tris, having a pH comprised between 6.8 and 7.4, preferably 7.0, and containing NaCI at a concentration between 0.10 M and 0.15 M, preferably 0.12 M, and optionally comprising other substances such as glycine, calcium chloride.
The column, equilibrated with the aforesaid buffer, is loaded with the biological fraction containing the FVIII to be purified.
The FVIII and the vWF bind preferentially to the resin while the contaminating proteins are for the most part eluted. The first chromatography step consists of washing the resin with the equilibration buffer described above, so as to remove excess contaminating proteins which do not interact with the resin.
In the second chromatography step, the preceding buffer is used though increasing its ionic strength (the equilibration buffer modified in this manner is known hereinafter as the "wash buffer"), so as to elute the weakly bound contaminating proteins and to partially elute the vWF. Finally in the third step the FVIII/vWF complex is eluted using the same buffer
while further increasing the conductivity (this buffer is known hereinafter as the "elution buffer") by means of NaCI or another salt at a concentration between 0.4 and 1.0 M, preferably 0.45 M.
By suitably modulating the physicochemical properties of the wash buffer in the second chromatography step, different recoveries of vWF in the FVIII/vWF complex eluted in the third step are possible.
Modulation of the physicochemical properties of the wash buffer indicated above essentially involves three factors:
- variation of the molar concentration of NaCI (or other salt) in the buffer (from 0.2 to 0.27 M);
- possible addition of CaCI2 at various concentrations (from 5 to 10 mM) into the wash buffer.
- the quantity of buffer used for washing the resin (normally comprised between 5 and 15 CV). In particular, NaCI molarity during the aforesaid wash is between 0.2 and 0.25 in order to obtain, after subsequent elution, a final product with a FVIII/vWF ratio between about 0.68 and 2.47 (see table 1 ).
Increasing ionic strength promotes vWF elution during the wash, hence resulting in a lower vWF concentration in the finished product and an increased FVIII/vWF ratio (R) as shown in table 1.
Table 1
Moreover, the presence of calcium ions at a suitable concentration in the wash
buffer enables to obtain a FVIII/vWF concentrate with a very high R; the use of 8 mM CaCI2 in the 0.25 M NaCI buffer enables to obtain a FVIIkvWF ratio of about 1 :0.5 without leading to a significant reduction in FVIII yield in the final product, which remains in the order of 85-90%. (Table 2).
Table 2
Finally, recovery of FVIII is also influenced by the number of column bed washings (CV = column volume) with the wash buffer. This is particularly evident where a 0.25 M buffer is used: passing from 5 to 10 CV leads to a percentage vWF recovery of from 27.2% to 20.3% in the final product and reaches 16.9% with 15 CV (see table 3 and figure 1 ), bringing the FVIII/vWF ratio from a value of 0.88 to 1.2 then to 1.52.
Table 3
In addition to enabling the vWF content to be modulated, the purification method of the present invention is highly efficient, reproducible, can be scaled-up to industrial levels and provides a complex between FVIII and vWF with high yields. The product obtained has a high specific activity of FVIII, with low concentrations of the main contaminants such as fibrinogen, fibronectin, IgG, IgM, and the vWF exhibits a multimeric pattern with good percentages of high molecular weight multimers. In particular, the present chromatography process leads to an enrichment of high molecular weight multimers in the finished product, while reducing the concentration of low molecular weight multimers in the step preceding elution of the FVIII/vWF complex. In order to further clarify the invention some examples of FVIII/vWF complex purification according to the process of the present invention are given hereinafter. Example 1
Purification of the FVIII/vWF complex with a ratio of about 0.7, by means of anion exchange chromatography After dissolving 65 g of cryoprecipitate in 10 volumes of H2O, aluminium hydroxide
at pH 6.5 at a temperature of 15QC was added to the solution. After centrifugation the supernatant (S1 ) was subjected to a viral inactivation step by addition of a solvent and detergent (Tween/TNBP), then loaded onto the Fractogel EMD TMAE resin equilibrated with 10 mM citrate buffer at pH 7.0 and NaCI at a 0.12 M concentration.
The contaminating proteins were removed by washing with the same buffer then treating the resin with 10 mM citrate buffer at pH 7.0 with 0.20 M NaCI. Finally, the FVIII/vWF complex was eluted with the same buffer containing NaCI at a 0.45 M concentration. The activities of FVIIkC and vWF:Ag are summarized in Table A below.
Table A
Activities of FVIIhC and vWF:Ag in the different fractions of the chromatography process Sample volume (ml) FVIIhC (U/ml) vWF:Ag(U/ml) FVIII/vWF
Supernatant 300 8^30 35.92 O23
0.20 M 294 23.17
0.45 M 90 27.64 40.65 0.68
Example 2
Purification of the FVIII/vWF complex with a 1.2 ratio, by means of anion exchange chromatography
The same supernatant (S1 ) as obtained in example 1 was loaded onto the Fractogel EMD TMAE equilibrated with a 10 mM citrate buffer at pH 7.0 containing NaCI at a 0.12 M concentration.
The contaminating proteins were removed by washing the resin with the same buffer then treating the resin with about 10 CV of 10 mM citrate buffer at pH 7.0 with 0.25 M of NaCI, whereas the FVIII/vWF complex was eluted with the same buffer but with a 0.45 M NaCI concentration.
The activities of FVIIhC and vWF:Ag are summarized in Table B below.
Table B
Activities of FVIIhC and vWF:Ag in the different fractions of the chromatography process
Sample volume (ml) FVIII: :C (U/ml) vWF:Ag (U/ml) FVIII/vWF Supernatant 290 7.36 38.74 0.19
0.20 M 288 0.69 33.17 0.02
0.45 M 90 20.88 17.40 1.20
Example 3 Purification of the FVIII/vWF complex with a ratio of about 1.5, by means of anion exchange chromatography
The supernatant (S1 ) obtained as in example 1 was loaded onto the Fractogel EMD TMAE resin equilibrated and washed with a 10 mM citrate buffer at pH 7.0 with NaCI at a 0.12 M concentration. The contaminating proteins were removed by treating the resin with about 10 CV of the same buffer containing 0.26 M of NaCI, whereas the FVIII/vWF complex was eluted using NaCI at a 0.45 M concentration. The activities of FVIIhC and vWF:Ag are summarized in Table C below.
Table C
Activities of FVIIhC and vWF:Ag in the different fractions of the chromatography process
Sample volume (ml) FVIII: :C (U/ml) vWF:Ag (U/ml) FVIII/vWF
Supernatant 286 8.19 34.16 0.24
0.26 M 210 2.28 40.64 0.06
0.45 M 96 19.29 12.86 1.50
Example 4 Purification of the FVIII/vWF complex with a > 2.0 ratio, by means of anion exchange chromatography
The supernatant (S1 ) obtained as in example 1 was loaded onto the Fractogel
EMD TMAE resin equilibrated and washed with a 10 mM citrate buffer at pH 7.0 with NaCI at a 0.12 M concentration.
The contaminating proteins and most of the vWF were removed by treating the resin with about 10 CV of the same buffer containing 0.25 M NaCI + 8 mM CaCI2, whereas the FVIII/vWF complex was eluted using NaCI at a 0.45 M concentration. The activities of FVIIkC and vWF:Ag are summarized in Table D.
Table D
Activities of FVIIhC and vWF:Ag in the different fractions of the chromatography process
Sample volume (ml) FVIII: C (U/ml) vWF:Ag (U/ml) FVIII/vWF
Supernatant 288 7.21 36.05 o.: 2
0.25 M + 200 3.28 47.75 0 .07
8 mM CaCI2 0.45 M 95 14.86 6.19 2 .4
Claims
1. A process for preparing FVIII/vWF concentrates at varying ratios from a biological fraction by chromatographic purification wherein an anion exchange resin with tentacle-like structure is used.
2. A process according to claim 1 wherein said biological fraction, which contains the FVIII and vWF to be purified, is obtained: from human plasma, from cryoprecipitate or from a plasma fraction obtained by a pre-purification treatment.
3. A process according to claims 1 and 2 wherein said resin is equilibrated with a buffer having a pH comprised between 6.8 and 7.4, containing NaCI at a concentration between 0.10 M and 0.15 M and optionally comprising glycine or calcium chloride, then loaded with the biological fraction containing the FVIII to be purified.
4. A process according to claim 3 wherein the resin loaded with the biological fraction containing the FVIII to be purified is washed with a wash buffer in which the molar concentration of NaCI is comprised between 0.2 and 0.27 M and in which CaCI2 is optionally present in concentrations comprised between 5 and 10 mM.
5. A process according to claims 3 and 4 wherein between 5 and 15 bed washings are used.
6. A process according to claim 1 wherein the FVIII/vWF ratio of the final product is comprised between about 0.7 and 2.5.
7. A process according to claim 4 wherein the buffer contains 8 mM CaCI2 and 0.25 M NaCI.
8. A process according to claim 1 wherein the wash buffer has a pH of 7.0 and 0.27 M NaCI and the FVIII/vWF ratio obtained is 2.47.
9. A process according to claim 1 wherein the wash buffer has a pH of 7.0 and 0.26 M NaCI and the FVIII/vWF ratio obtained is 1.50.
10. A process according to claim 1 wherein the wash buffer has a pH of 7.0 and 0.25 M NaCI and the FVIII/vWF ratio obtained is 1.20.
1 1. A process according to claim 4 wherein the wash buffer has a pH of 7.0, 0.25 M NaCI and 5 bed washings are used, and wherein the FVIII/vWF ratio obtained is 0.88.
12. A process according to claim 4 wherein the wash buffer has a pH of 7.0, 0.25 M NaCI and 8 mM CaCI2, and the FVIII/vWF ratio obtained is 2.40.
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EP08750078A EP2155784A1 (en) | 2007-05-07 | 2008-05-06 | Chromatography process for obtaining a fviii/vwf complex with different ratios between the two proteins, for use in haemophilia a treatment and in von willebrand disease |
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IT000007A ITLU20070007A1 (en) | 2007-05-07 | 2007-05-07 | CHROMATOGRAPHIC PROCESS FOR OBTAINING A FVIII / VWF COMPLEX WITH DIFFERENT RELATIONSHIPS BETWEEN THE TWO PROTEINS TO BE USED IN THE HEMOPHILIA A THERAPY AND IN VON WILLEBRAND'S DISEASE. |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010082184A1 (en) | 2009-01-19 | 2010-07-22 | Kedrion S.P.A. | New process for highly selective purification of two plasma proteins: von willebrand factor (vwf) and fibronectin (fn) |
US20130034876A1 (en) * | 2011-08-02 | 2013-02-07 | Sylvain Roy | Systems and Methods to Increase Protein Yield from Recombinant Manufacturing Processes |
US11352388B2 (en) | 2015-04-10 | 2022-06-07 | Cytiva Bioprocess R&D Ab | Method for chromatography |
US11542300B2 (en) | 2015-04-10 | 2023-01-03 | Cytiva Bioprocess R&D Ab | Chromatographic methods for purification of proteins from plasma |
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EP0503991A1 (en) * | 1991-03-08 | 1992-09-16 | Centre Regional De Transfusion Sanguine De Lille | Process for preparing a highly purified standardized concentrate of human von Willebrand factor on industrial scale, appropriate for therapeutic use |
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 |
EP1148063A1 (en) * | 2000-04-18 | 2001-10-24 | Octapharma AG | Composition containing hemostatic activ vWF and process for its preparation |
EP1500661A1 (en) * | 1998-06-01 | 2005-01-26 | Genetech, Inc. | Separation of protein monomers from aggregates by use of ion-exchange chromatography |
US20050209154A1 (en) * | 1996-03-15 | 2005-09-22 | Baxter Aktiengesellschaft | Stable factor VIII/von Willebrand factor complex |
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2007
- 2007-05-07 IT IT000007A patent/ITLU20070007A1/en unknown
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2008
- 2008-05-06 EP EP08750078A patent/EP2155784A1/en not_active Withdrawn
- 2008-05-06 WO PCT/EP2008/055526 patent/WO2008135568A1/en active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0503991A1 (en) * | 1991-03-08 | 1992-09-16 | Centre Regional De Transfusion Sanguine De Lille | Process for preparing a highly purified standardized concentrate of human von Willebrand factor on industrial scale, appropriate for therapeutic use |
US20050209154A1 (en) * | 1996-03-15 | 2005-09-22 | Baxter Aktiengesellschaft | Stable factor VIII/von Willebrand factor complex |
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 |
EP1500661A1 (en) * | 1998-06-01 | 2005-01-26 | Genetech, Inc. | Separation of protein monomers from aggregates by use of ion-exchange chromatography |
EP1148063A1 (en) * | 2000-04-18 | 2001-10-24 | Octapharma AG | Composition containing hemostatic activ vWF and process for its preparation |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010082184A1 (en) | 2009-01-19 | 2010-07-22 | Kedrion S.P.A. | New process for highly selective purification of two plasma proteins: von willebrand factor (vwf) and fibronectin (fn) |
US20130034876A1 (en) * | 2011-08-02 | 2013-02-07 | Sylvain Roy | Systems and Methods to Increase Protein Yield from Recombinant Manufacturing Processes |
US9534037B2 (en) * | 2011-08-02 | 2017-01-03 | Baxalta GmbH | Systems and methods to increase protein yield from recombinant manufacturing processes |
US11352388B2 (en) | 2015-04-10 | 2022-06-07 | Cytiva Bioprocess R&D Ab | Method for chromatography |
US11542300B2 (en) | 2015-04-10 | 2023-01-03 | Cytiva Bioprocess R&D Ab | Chromatographic methods for purification of proteins from plasma |
US11807666B2 (en) | 2015-04-10 | 2023-11-07 | Cytiva Bioprocess R&D Ab | Chromatographic methods for purification of proteins from plasma |
Also Published As
Publication number | Publication date |
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EP2155784A1 (en) | 2010-02-24 |
ITLU20070007A1 (en) | 2008-11-08 |
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