DK1037923T4 - A process for the filtration to produce a solution with regard to virus-safe factor VIII - Google Patents

Description

1

The present invention relates to a process for the filtration to produce a factor VIII solution which is safe with respect to viruses and substantially free of the von Willebrand factor (vWF), high molecular weight, from a solution of the factor VIII of large or very high purity and containing factor VIII-vWF complexes with high molecular weight.

Factor VIII is a protein component in the blood which for many years has been used to treat individuals suffering from hemophilia A which is a congenital disease caused by a deficiency or absence of factor VIII in the blood. There has long been used plasma concentrates enriched in factor VIII for the treatment of patients.

The most commonly used concentrates and cryoprecipitate was the purified concentrates obtained from cryoprecipitate. Cryoprecipitate is normally used for a precipitate that is obtained from a human plasma is frozen at a plasma fractionation technique at low temperature. The frozen plasma is thawed at a temperature of about -5 ° C-15 ° C and then heated slowly with stirring at a temperature not exceeding 3 ° C. Under these conditions, the frozen plasma melts partially, resulting in a liquid phase and a solid phase, wherein the solid phase is then isolated by centrifugation and provides cryoprecipitate which can then be purified in order to obtain sufficiently pure factor VIII preparations. This kryopræcipiterede fraction consists essentially of fibrinogen, fibronectin, Factor VIII and von Willebrand factor (vWF). In this kryopræcipiterede fraction, factor VIII is generally associated with vWF which stabilizes the factor VIII by complex formation. For a long time the purification steps is substantially directed towards to separate the unwanted proteins, such as especially fibrinogen and fibronectin, Factor VIII.

We know now, however, that one of the major problems associated with the production of factor VIII from plasma consists in the necessity of inactivating-vere / eliminate the virus originally in the blood in a satisfactory way. 2

Although it is difficult to establish an exhaustive list of these viruses may be mentioned in particular the various hepatitis viruses, hepatitis A, hepatitis B, hepatitis C, hepatitis G, or even different types of AIDS virus (HIV).

Thus, there are numerous techniques have been developed for viral inactivation such as dry heating, pasteurization, solvent detergent treatment. All of these techniques are relatively effective against envelope viruses, but the inactivation or elimination of naked viruses, in particular small viruses such as parvovirus B19 or hepatitis A virus, is still one of the main problems. In more recent technologies utilized skill virusretention of membranes with small pore size. These technologies have in fact a considerable efficacy against small viruses such as parvovirus B19 or hepatitis A virus, and may be applied to low molecular weight proteins. However, making the applied cut-off values ​​that are less than 900 kD, it is not possible to envisage filtration of proteins or protein complexes of high molecular weight, such as factor VIII, without great loss in yield. Thus, for example, published patent application W096 / 00237 a method for improving protein filterability in a solution containing at least one macromolecule, said method consists of using a solution in which the total salt content varies from 0.2 M to saturation of the solution with the the salt. Josic et al., J. Chromatogr. B. Biomed. Appl .; Vol 662, no. 2, 1994 discloses the use of Ca 2+ to separate factor VIII from vWF factor. This high salt content helps to increase filtration yields. The filtration step is preferably carried out at a time when the specific activity of the macromolecule of interest is already very large so that it is possible to use a filter of very fine structure and eliminate very small virus. This document is intended, however, not filtering the solution containing molecules with very high molecular weight, such as factor VIII and factor VIII in particular of plasma origin which is generally associated with vWF, as they form complexes with high molecular weight. This document mentions the example of a deleted form of recombinant factor VIII with an average molecular weight of about 3 170 kD and completely vWF. All of the examples relates to factor IX whose average molecular weight is about 55 kD, and in this document it is emphasized that the described method is particularly suitable for this size molecule, said filter cut-off value, moreover, must be slightly higher than the size of the molecule to be filtered. Thus, the filter used for the Factor IX, a cut off of 70 kD.

As noted previously, Factor VIII, however, depending on its origin is present in a complexed form which gives it a significant size that can reach 20,000 kD in molecular weight and several tens of nanometers with respect to the particle size (> 106 nm for the molecule's main axis, Eppel et al., Langmuir 1993, 9, 2281-88, American Chemical Society). There is currently no means available to satisfactorily filter this type of molecules and at the same time eliminate the small viruses such as parvovirus B19, for example, having a size of about 18-20 nm.

The inventors have now surprisingly found that it was possible by filtration to obtain a factor VIII solution which is safe with respect to viruses and substantially free of vWF high molecular weight, from a solution containing factor VIII of high or very high purity containing factor VIII-vWF complexes with high molecular weight.

Therefore, the invention relates to a process for the filtration to produce a virus with regard to secure factor VIII solution, according to which method: - preparing a solution of high or very high purity, which comprises the factor VIII protein complex; - Performing the step a) enabling dissociation of factor VIII-vWF complexes with high molecular weight by means of a chaotropic ion in an amount that is sufficient to allow for dissociation, and a solution is obtained which is essentially free of Factor VIII associated with vWF high molecular weight, - performing a step b) to filtration of the solution on a hydrophilic filter having a pore size of 15 nm.

The production of a Factor VIII solution by filtration makes it advantageously possible to obtain a solution which is substantially free of vWF high molecular weight, in particular 4 vWF having a degree of polymerization greater than or equal to 15, that is, where the vWF both qualitatively and quantitatively controlled. The method according to the invention also makes it possible to increase the filtration yields significantly due to a decrease in volume of the protein complexes of high molecular weight and to obtain a solution having a satisfactory degree of purity, while viruselimineringen of human pathogenic viruses with a size of> 15 nm is ensured.

The dissociation of the FVIII-vWF complex is obtained by means of at least one chaotropic ion is present in a sufficient amount to allow the dissociation. All the ions which are known to have a chaotrope activity, can be used.

It is preferably in the divalent ions are added factor VIII solution in the form ofa 0.2M saline solution until saturated with the salts.

There can, as non-limiting example include CaCl MgCl or · ·. Preferably a CaCl2 solution. Concentration of the solution will preferably be about 0.35 M.

It has been found that in addition to the filtering conditions, then the conditions used for the dissociation, an influence on a subsequent filtration yield. The parameters that define all of these conditions is below dissocieringstrinnet the nature and concentration of the salts, and during the filtration step the pressure and temperature.

Filtration yields are surprisingly significantly improved when the transmembrane pressure during the filtration is lowered to very low values ​​below the limits recommended by the filter manufacturer.

The temperature also has a significant influence on the filtration yields as too high or too low temperature values ​​helps to increase the number of multimeric-for-mer of vWF. There is advantageously a temperature of the order of 35 + 5 ° C.

Among the virus filters available on the market or under development, there are for example Planova® 15N membrane sold by the company 5

Asahi Chemical Industry. In this case, the filter is preferably at a pressure below 0.3 bar, preferably below 0.2 bar.

There can be used various filtering techniques. The most common techniques are the techniques of tangential filtration, frontal filtration or can be used with the same type of filters.

Factor VIII starting solution, has previously been purified, can be prepared in various ways, for example, from a plasma fraction, such as the kryopræcipite-dried fraction of the plasma, or by recombinant means. All process conditions known to the skilled person, may be used. In the non-limiting manner the following may be mentioned purification methods allowing to obtain a pre-purified factor VIII solution which can be used to practice the method of the invention:. Ion exchange chromatography, for example, according to one of the variants disclosed in the patent documents EP-B-359 593, EP 0343275, US 4743680, WO 97/17370 and EP 0 173 242.. Immunoaffinity chromatography, for example according to the variant described in patent application WO 97/39033, EP 0286323 or in the document Zimmerman and Fulcher, Thrombosis Res., Suppl. VII, p 58, 1987; Berntorp and Nilson, Thrombosis Res., Suppl. VII, p 60, 1987. Filtreringskromatografi the gel optionally in dissociating medium as described by Fay PJ (PJ Fay et al. Proc. Nat. Acad. Sci. USA vol 79 pp 7200-7204, 1982). . Affinity chromatography on immobilized heparin, as described in patent application WO 93/22337.

Purification by ion exchange chromatography from a plasma fraction, such as the kryopræcipiterede plasma fraction typically comprises a virus inactivation step which allows the inactivation of enveloped viruses. There can be used various chromatographic systems, since the conditions for adsorption and then eluting fraction is concentrated factor VIII may subsequently have an impact on the process 6 subsequent dividends. It may be the nature of the matrix and the ion exchanger can vary. Thus, there may be used a chromatography system with weak ion exchangers, such as, for example, the gel Toso Haas DEAE-Toyopearl 650M, or a chromatography system with strong ion exchangers, such as, for example, the gel Q-Sepharose Fast Flow (Pharmacia Biotech). When the starting solution is prepared by ion exchange purification, it contains a significant amount of factor VIII with vWF associated with high molecular weight, and step a) is necessary.

The dissociation in step a) can be carried out simultaneously with the elution or according to another aspect for the elution. An elution is carried out in the presence of a chaotropic salt, helps to increase the elution yield compared to an elution is carried out in the presence of a salt such as NaCl, while the dissociation safeguarded, which is necessary to carry out the subsequent filtration step under the required conditions.

According to a preferred embodiment, the eluted solution of Factor VIII concentrate obtained after purification by ion exchange chromatography, the following dissocieringsbetingelserne of step a), i.e., in the presence of a chaotropic ion.

The pre-purification of factor VIII-starting solution may also be obtained by a precipitation technique with heparin. In this case, for example, a kryopræcipiteret adsorbed plasma fraction in an aluminum hydroxide gel in the presence of heparin under cooling to a temperature of about 14 ° C to about 19 ° C and centrifugation. A first step of virus inactivation carried out on udfældningssupernatanten, may advantageously be conducted at a solvent-detergent treatment as disclosed in published European Patent Application EP-A-0,343,275th Udfældningssupernatantens pH and osmolality is then adjusted before the ion exchange chromatography step.

Finally, it is proposed to start with a recombinant factor VIII solution, which may require a further step for virus inactivation. It will not be necessary to practice step a).

Factor VIII starting solution will preferably have a specific activity at least equal to 50 IU / mg, preferably at least equal to 100 IU / mg, the filterability of the solution increases with the factor VIII specific activity. 7

The mean specific activity prior to the optional addition of albumin for stabilization of factor VIII.

Are used more particularly to a starting solution wherein the concentration of Factor VIII: C is between about 2 and about 100 U / ml, preferably about To about 10 50 U / ml.

The protein content of the Factor VHI-starting solution will advantageously comprise from about 0.05 to about 0.5 mg / ml, preferably from about To about 0.1 0.5 mg / ml.

The protein content is determined by the Bradford technique for the measurement of proteins (measurement kit sold by the company Pierce). Once the filtration is completed, the annealed the Factor VIII and the von Willebrand factor, which has been filtered in the form of complexes, after elimination of dissociating agents, for example, by dialysis, and after an optional lyophilization step is isolated solution of Factor VIII that is formulated for a commercial application.

The invention also relates to a factor VIII solution which is safe with respect to viruses and substantially free of high molecular weight vWF, obtainable by the process according to the invention.

The invention finally relates to the obtained solutions of the invention as a medicament, more particularly for the treatment of hemophilia A.

The invention will be described in more detail by way of the following examples which illustrate the invention without, however, limiting the scope of it. The examples are accompanied by Figure 1 which shows:. Figure 1: a graph showing the variation in the filterability of the factor VIII of Pla-15 N NoVa® the membrane as a function of the system at the pressure applied. Out of the abscissa is the pressure, expressed in bar, and out of the ordinate stands filtration yield expressed as a percentage (the ratio of FVIIIC activity expressed in IU / ml of the filtrate, and the output of the product activity). 8 Example 1: Preparation of a Factor VIII solution by filtration kryopræcipiteret from a plasma fraction.

Manufacture of factor VHI-output resolution is carried out in accordance with the information in patent FR 2,632,309 whose contents are hereby incorporated by reference in its entirety should be seen as part of this application. 835 g cryoprecipitate, constituting 113.5 liters of plasma, resolubilized by stirring in a solution of water with heparin (3 IU / mL) at ambient temperature for 30 minutes. 4217 ml of solution enriched with factor VIII proteins and clarified by adsorption of 90 g of aluminum hydroxide gel and by acid precipitation (pH 6.50) and lowering the temperature to between 15 and 19 ° C. A precipitate enriched with the fibrinogen and fibronectin is separated by centrifugation, which makes it possible to obtain a clear factor VIII solution having the purity of the solution are inactivated in terms of enveloped viruses by the addition of Polysorbate 80 and tri-n-butyl-phosphate (respectively in solution qs 1% and 0.3%) for a minimum of 6 hours at pH 7.1. 5172 ml of factor VIII solution is virus inactivated with respect to the enveloped virus is adsorbed onto 560 ml of weak anion exchange kromatografigel (TosoHaas Toyopearl DEAE-650M) previously equilibrated in buffered saline. After 2 hours adsorption, the gel is washed with a brine solution osmolality of 390 mOsm / kg and buffered at pH 7.00. The fraction not adsorbed on the gel, is rich in fibrinogen. The gel is then eluted a fraction enriched with von Willebrand factor in the increase of the osmolality to 452 mOsm / kg. The concentrated fraction of very high-purity factor VIII is then eluted by changing the pH to 6.0 and increasing the ionic strength. The eluted fraction is then adjusted in terms of CaCl2 to a concentration of 0.35 M and an osmolality of 1300 ± 100 mOsm / kg. This fraction consists of a mixture of factor VIII and von Willebrand factor in the dissociated form, thanks to the action of the high calcium content. 1260 ml of solution which is stable at + 4 ° C, is heated instantaneously to + 35 ° C to be subjected to a virus removal step by filtration by means of a BMM Pla-NoVa® 15N filter with a porøsitetsgrænse of 15 nanometers and a surface of the 0 , 12 m2. The flow rate is maintained throughout the filtration in such a way that the nine transmembrane pressure all the time is under 0.2 bar. After filtration of factor VIII filtered 210 ml buffered saline with osmolality 1300 mOsm / kg, then the membrane to isolate the 1470 ml of factor VIII solution, without pathogenic virus. The buffer solution makes it possible to equilibrate the filters in terms of osmolality and pH, and is used to flush the filters after filtration of factor VIII. The resulting FVIII solution is dilute with respect to von Willebrand factor of very high degree of polymerization (> 15), but contains sufficient von Willebrand factor having a polymerization degree> 5 and> 10 to turn to complex with factor VIII after dialysis.

results:

Table 1 lists the different steps in the method of the invention the obtained amounts of factor VIII, and the specific activity (SA), the protein content and yield in that step. TABLE 1

Example 2: The conditions are the same as those in Example 1 except for the use of the 10000 g cryoprecipitate, constituting 1330 liters of plasma. 13,700 ml of factor VIII solution is virus inactivated with regard to envelope virus filtered. After filtration of factor VIII is filtered 2 liters of buffer solution having osmolality 1300 mOsm / kg order that isolate 15,700 ml of factor VIII solution free from pathogenic viruses. The filtration membrane used is a membrane BBM Planova® 15 N and a surface area of ​​1.0 m2. 10

Table 2 below indicates a filtering of an equivalent of 1330 liters of plasma of BBM Planova® 15N membrane having an area of ​​1.0 m2 of the obtained amounts of factor VIII in the different steps of the filtration process, and the specific activity and the yield of the step . TABLE 2

Example 3: Preparation of a Factor VIII solution by filtration from a fraction obtained from plasma and cryoprecipitate is pre-purified by heparin-like precipitation.

Manufacture of factor VHI-output resolution is carried out in accordance with the information in the patent US 4,743,680 the contents of which hereby incorporated by reference in its entirety should be seen as part of this application. 678 g cryoprecipitate, constituting 92.2 liters of plasma, resolubilized by stirring in a solution of water with heparin (3 IU / mL) at ambient temperature for 30 minutes. 3424 mL solution that is rich in factor VIII and proteins, as done in Example 1.

4200 ml of factor VIII solution is virus inactivated with respect to the enveloped virus, under the same conditions as in Example 1, adsorbed after acidification to pH 11 6.50 to 300 ml of strong anion exchange kromatografigel (Pharmacia Biotech Q-Sepharose Fast Flow) pre- equilibrated in buffered saline. After 2 hours and 30 minutes adsorption, the gel is washed with a salt solution having osmolality of 450 mOsm / kg and buffered at pH 6.50. The fraction not adsorbed on the gel, is rich in fibrinogen. The gel is then eluted a fraction enriched with von Willebrand factor by increasing the osmolality to 581 mOsm / kg. The concentrated fraction of very high-purity factor VIII is then eluted by changing the pH to 6.0 and increasing the ionic strength. The eluted fraction is adjusted in respect of CaCl to a concentration of 0.35 M and an osmolality of 1300 ± 100 mOsm / kg. This fraction consists of a mixture of factor VIII and von Willebrand factor in the dissociated form, thanks to the action of the high calcium content. 1280 mL solution that is stable at + 4 ° C, is heated instantaneously to + 35 ° C to be subjected to a virus removal step by filtration in the same manner as in Example 1 on a Planova® 15 N membrane with a porøsitetsgrænse of 15 nanometers and a surface of 0.12 m2. A volume of 180 ml buffer solution having osmolality 1300 mOsm / kg is then filtered to isolate the 1460 ml of factor VIII solution free from pathogenic viruses. The factor VIII solution is dilute with respect to von Willebrand factor of very high degree of polymerization (> 15), but contains sufficient von Willebrand factor with a degree of polymerization of> 5 and> 10 to turn to complex with factor VIII after dialysis.

results:

Table 3 lists the different steps of the filtration process the obtained amounts of factor VIII, the total amount of proteins and the specific activity (SA) and the output of that stage. 12 TABLE 3

Example 4: Variation in filterability by a factor VIII in a solution Planova® 15 N membrane as a function of the nature and concentration of the salts used for the dissociation.

results:

Table 4 below shows the variations in the filtration yield as a function of these various parameters, the method other operating conditions are the same as defined in Example 1. TABLE 4

The use of dissociating agents makes it possible to increase the filterability of factor VIII significantly. The elimination of these agents by dialysis after filtration induces a reassociation of factor VIII-factor von Willebrand complexes. The analysis of the products obtained show a good ability of von Willebrand factor to bind factor VIII. 13

Example 5: Variation in the filterability of a factor VIII in a solution Planova®-15N membrane as a function of the parameters of temperature and pressure.

Table 5 shows, while the dissociation of salt and its concentration remains unchanged, the resulting filtration yield by allowing the pressure and temperature vary. When the temperature is lowered at a given pressure, a considerable decrease in filtration yield.

Figure 1 further shows clearly that the lowering of the transmembrane pressure at very low values ​​to allow a significant improvement in the yield. TABLE 5

Example 6: Study of virus-retention capability of the filtration system during the filtration conditions according to Example 1.

As virus model used phage island x 174, the size can be determined to 25-30 nm. The membrane and the working conditions are similar to those described in Examples 1 and 2.

IN

results:

The results in Table 6 below shows a fully satisfactory virusretentionsevne. 14 TABLE 6

Example 7: Effect of the method of the invention the vWF content of factor VIII solution. vWF content and vWF multimer profile bya factor VIII solution as described in Example 1 was compared before and after carrying out the method according to the invention.

The results obtained are shown in Table 7 below. TABLE 7

Thus making the performance of the method of the invention makes it possible to obtain a factor VIII solution which is substantially free of vWF high molecular weight, particularly substantially free of multimeric forms having a polymerization degree> 15th

Claims (21)

1. A process for the filtration to produce a virus with regard to the safe solution of Factor VIII and vWF substantially free of high molecular weight, according to which method: - preparing a solution of high or very high purity and comprising factor VIII-vWF -proteinkompleks; - Performing a step of enabling dissociation of factor VIII-vWF complexes with high molecular weight by means of a chaotropic ion in an amount that is sufficient to allow the dissociation, and the yield ofa solution which is essentially free of Factor VIII associated with vWF with high molecular weight, - performing a step of filtering the solution on a hydrophilic filter having a pore size of 15 nm.

2. The method of claim 1, characterized in that said chaotropic ion is a divalent ion.

3. A method according to claim 2, characterized in that the divalent ion is a Ca2 + ion.

4. A method according to any one of claims 1-3, characterized in that the divalent ion is added in the form ofa 0.2M saline solution until saturated with the salts.

5. The method according to claim 4, characterized in that the solution is a CaCl solution.

6. A method according to one of claims 4 and 5, characterized in that the Ca 2+ ion is added in the form ofa 0.35 M CaCl solution up to saturation.

7. A method according to claim 6, characterized in that the filter is a Planova® 15 N membrane used at a pressure below 0.3 bar, preferably below 0.2 bar.

8. A method according to any one of claims 1-7, characterized in that step b) is carried out at a temperature of about 35 + 5 ° C.

9. A method according to any one of claims 1-8, characterized in that the starting solution is obtained by purification of a plasma fraction, in particular the kryop afraid Pite prepared plasma fraction by ion exchange chromatography.

10. The method of claim 9, characterized in that the concentrated factor VIII fraction obtained after purification by ion exchange chromatography, eluted dissocieringstrinnets dissocieringsbetingelser.

11. A method according to any one of claims 1-10, characterized in that the factor VHI-starting solution is achieved by pre-purification of a plasma fraction, in particular the kryopræcipiterede plasma fraction by precipitation with heparin.

12. A method according to any one of claims 1-11, characterized in that the factor VHI-starting solution is partially viral inactivated by solvent-detergent treatment.

13. A method according to any one of claims 1-12, characterized in that the factor VHI-starting solution comprises immunopurified factor VIII.

14. A method according to any one of claims 1-12, characterized in that the factor VHI-starting solution comprises recombinant factor VIII.

15. A method according to any one of claims 1-12, characterized in that the starting solution is of plasma origin.

16. A method according to any one of claims 1-15, characterized in that the factor VIII in the starting solution has a specific activity which is at least equal to 50 IU / mg, preferably at least equal to 100 IU / mg.

17. A method according to any one of claims 1-16, characterized in that the concentration of Factor VIII in Factor VIII C-starting solution is between 2 and 100 U / ml, preferably of between 10 to 50 U / ml.

18. A method according to any one of claims 1-17, characterized in that the protein content of factor VHI-starting solution is from about 0.05 to about 0.5 mg / ml, preferably from about To about 0.1 0.5 mg / ml.

19. A with respect to virus-safe factor VIII solution which can be obtained by the method according to any one of claims 1-18.

20. A solution according to claim 19 as a medicament.

21. A solution according to claim 20 as a medicament for the treatment of hemophilia A.