WO2000049047A1

WO2000049047A1 - METHOD FOR PRODUCING A vWF PREPARATION

Info

Publication number: WO2000049047A1
Application number: PCT/AT2000/000039
Authority: WO
Inventors: Katalin Varadi; Peter Turecek; Hans-Peter Schwarz
Original assignee: Baxter Aktiengesellschaft
Priority date: 1999-02-19
Filing date: 2000-02-15
Publication date: 2000-08-24
Also published as: AU2645900A; AT407750B; ATA28399A

Abstract

The invention relates to a method for producing a von Willebrand factor (vWF) preparation from pro-von Willebrand factor (pro-vWF) which is characterized in that pro-vWF is treated with thrombin, whereby vWF is generated and a preparation containing vWF is obtained.

Description

Process for the preparation of a vWF preparation

The invention relates to a method for producing a von Willebrand factor (vWF) preparation.

Von Willebrand factor is a glycoprotein that circulates in the plasma as a series of multimers in the size range from about 500 to 20,000 kD. Multimeric forms of vWF consist of 250 kD polypeptide subunits that are connected by disulfide bridges. vWF mediates the initial platelet adhesion to the sub-endothelium of the damaged vessel wall, whereby only the larger multimers also have hemostatic activity. Endothelial cells are thought to secrete large polymeric forms of vWF, and those forms of vWF that have a low molecular weight (low molecular weight vWF) are formed by proteolytic cleavage. The multimers with large molar masses are stored in the Weibel-Pallade bodies of the endothelial cells and released when stimulated.

vWF is synthesized by endothelial cells and megakaryocytes as pre-pro-vWF, which consists largely of repeated domains. After cleavage of the signal peptide, pro-vWF dimerizes through disulfide bridges at its C-terminal region. The dimers serve as protromers for multimerization, which is controlled by free end disulfide bridges between the termini. The arrangement to multimers is followed by the proteolytic removal of the pro-peptide (Leyte et al., Biochem. J. 274 (1991), 257-261).

The entire length of vWF c-DNA was cloned. The pro-polypeptide corresponds to amino acid residues 23 to 764 of the pre-pro vWF of the entire length (Eikenboom et al., Haemophilia 1 (1995), 77 to 90).

It is shown that the pro-peptide of vWF (pp-vWF) is identical to that of Willebrand antigen II, the second identified antigen, which is absent in the plasma and in the platelets of patients with severe von Willebrand disease (vWD) is. pp-vWF is specifically synthesized in platelets and endothelial cells. The molecular ratio of pp-vWF to vWF in plasma is 1:10, the plasma concentration of pp-vWF is about 5 nmol / 1. As already known, pp-vWF is released from endothelial cells after activation by various agonists.

It is postulated that the physiological role of pp-vWF in controlling the arrangement of vWF multimers is either prior. or after the cleavage of pp-vWF molecules. (Takagi et al., JBC 264 (18) (1989) 10425-10430). Pp-vWF has also been shown to inhibit platelet collagen interaction activity (Takagi et al., JBC 264 (11) (1989) 6017-6020).

Von Willebrand's disease (vWD; von Willebrand syndrome; Willebrand-Jürgens syndrome) is generally characterized by a decrease or a structural defect in vWF, which can result in a lack of platelet aggregation and adhesion to the sub-endothelium and thus defects in primary hemostasis . These defects cause prolonged bleeding times in vWD patients.

For the treatment of vWD, attempts are usually made to compensate for the deficiency of functionally active vWF, for example by means of preparations which are also used for the therapy of hemophilia A, such as cryoprecipitate or factor VIII concentrates prepared therefrom, which contain complexes of factor VIII and vWF . However, since the preparations for the treatment of haemophilia A are becoming ever purer with regard to factor VIII due to improvements in the purification methods, and vWF is not or only contained in traces, such preparations for treatment in vWD patients are becoming increasingly unsuitable. It is therefore proceeding to specifically administer such vWF preparations to such patients. Attempts are also being made to keep such vWF preparations as factor-VHI-free as possible, since patients with von Willebrand syndrome do not actually need any supplementation with factor VIII and, when using factor VIII, there is always the risk of inducing inhibitory factor VIII antibodies in the patient can be given.

For the purification of vWF, a whole series of different, mostly chromatographic processes have been described. There the source material for the vWF preparation often contains a mixture of mature vWF and vWF precursors such as pro-vWF, or even contains only (pre-) pro-vWF (e.g. in the case of recombinantly produced preparations) it is necessary in the production of vWF - Preparations to convert the (pre-) ro-vWF into mature vWF. In the prior art, the treatment of pro-vWF with furin or PACE (see EP 0 775 750 A) is described as a suitable system to enable the splitting of pro-vWF into pp-vWF and mature vWF. Furin is an endoproteolytic enzyme that is anchored in the trans-Golgi network by a transmembrane domain. In the recombinant production of mature vWF, it has been proposed to co-express furin and vWF in order to bring about a suitable maturation of the vWF in situ (EP 0 775 750 A). However, this turned out to be extremely difficult and can be associated with a sensitive reduction in the level of expression. In addition, the problem arises in connection with the coexpression of furin with a pro protein in the industrial scale in cell culture that a high expression rate of the protease is toxic to the cells, which likewise leads to a low yield of furin and of mature protein .

In addition to furin or similar PACE enzymes from the group of subtilisin-like serine proteases, protease-processing proteases are also described in AT-PS 404 554 and AT-PS 404 359.

Thrombin (factor Ha of blood coagulation) is an enzyme from prothrombin in the blood plasma with a molecular weight of approximately 35,500. Thrombin converts the fibrinogen molecules into fibrin monomers by splitting off fibrinopeptides A and B, which spontaneously aggregate into fibrin polymers. Furthermore, factor XIII, factor VIII and protein C are activated by thrombin. Thrombin therefore finds a role as a proteolytic enzyme in the production of these activated proteins (cf. EP 0 416 890 A regarding the production of active recombinant human protein C by treatment with immobilized thrombin).

It can be seen that the methods for activating pro-vWF described in the prior art are either very cumbersome or were associated with low yields or could not be used on an industrial scale, or only to a limited extent, owing to the insufficient commercial or biological availability of the process components. The object of the present invention is therefore to avoid the disadvantages of the prior art and to provide a method for activating pro-vWF which is highly selective and can nevertheless be used on an industrial scale.

This object is achieved according to the invention by a method for producing a vWF preparation from pro-vWF, which is characterized in that pro-vWF is treated with thrombin, whereby vWF is generated and a preparation of mature vWF can thus be obtained.

In the context of the present invention, it was surprisingly found that not only furin is a selective enzyme for cleaving pro-vWF into pp-vWF and mature vWF, but also thrombin. It was shown that by using thrombin as a processing protease, a simple process for the maturation of pro-vWF could be provided, which - due to the established large-scale availability of thrombin - can also be used on an industrial scale.

In the method according to the invention, a solution containing pro-vWF is preferably treated with thrombin, in principle any solution containing pro-vWF is suitable. Pro-vWF can also be matured as an immobilized product on a solid support.

In the method according to the invention, pro-vWF is preferably treated with immobilized thrombin, since this enables the protease reaction to be controlled even more efficiently and the frequent use of the immobilizate and the continuous implementation of the treatment are made possible in a simple manner. Methods for immobilizing thrombin are well known in the art. The fact that the immobilization of thrombin leaves its pro-vWF-cleaving activity essentially unaffected presents another decisive advantage - 5 - part of this embodiment.

It is precisely the stability of immobilized thrombin and its unimpaired activity towards pro-vWF that make it possible to carry out the process according to the invention particularly preferably continuously, for example in a reactor containing a solid surface with immobilized thrombin, through which a solution containing pro-vWF is passed. so that more mature vWF is generated. The individual process parameters, such as flow rate, thrombin immobilized amount, reactor cross-section, residence time, reactor dimensions and control, etc., can easily be optimized by a person skilled in the art using conventional process engineering methods.

The starting material containing pro-vWF for the process according to the invention is not critical for the present invention. Possible starting materials are blood serum, blood fractions, plasma, colostrum or milk from transgenic animals or cell culture solutions, in particular cells that were generated using recombinant DNA technology (see, for example, FEBS letters 351 (1994) 345-348 or Blood 88 (8) 1996 2951 -2958).

The pro-vWF-containing starting material is preferably subjected to one or more purification steps before the thrombin treatment, so that the pro-vWF-containing solution contains pro-vWF in a purified form. Suitable pre-purification steps include precipitation (for example with ethanol, ammonium sulfate or PEG), adsorbing (for example treatment with aluminum hydroxide) or chromatographic processes (for example affinity, gel or ion exchange chromatography). Such pretreatment methods are particularly favorable if the source material containing pro-vWF contains substances which can interfere with the proteolytic processing of pro-vWF by thrombin, for example inhibitors of thrombin or the like. Although the presence of thrombin inhibitors in the starting material does not in principle have to hinder the successful implementation of the process according to the invention (since thrombin is generally prescribed in excess quantities anyway), such inhibitors can, for example, improve the yield of the process or reduce the lifespan of the thrombin immobilizate so that a simple pre-cleaning step often appears advisable (especially if thrombin inhibitors are present).

It is also possible for pro-vWF to be present in the vWF-containing solution which is subjected to the thrombin treatment in a mixture with other, preferably also at least partially purified proteins. This is particularly the case if pro-vWF has been separated from other proteins or components of the starting solution by partial purification, but other proteins, especially those that naturally have a certain affinity for pro-vWF or vWF, in the same way as with thrombin solution to be treated are available. Such proteins preferably include activators and inhibitors of thrombin, such as, for example, heparin, calcium, thrombomodulin, antithrombin III, peptide inhibitor, non-specific thrombin inhibitors, thrombin mutants, prothrombin, alpha ₂ -macroglobulin, factor VIII, collagen, surface proteins of platelets.

As a further possible embodiment of the method according to the invention, pro-vWF or the pro-vWF-containing solution can be bound to collagen and then processed with thrombin. It has been shown here that by binding the pro vWF to the collagen, the pro-peptide cleavage can take place with reduced concentrations of throbine or the processing process can be accelerated.

By using a matrix that binds vWF, the mature vWF no longer has to be separated from the pp-vWF by chromatographic methods after the vWF has been processed using thrombin.

However, if separation is advisable, pp-vWF can be separated using a suitable chromatography material, and it has been shown that the purification of pp-vWF using lentil-lectin-Sepharose is particularly advantageous.

According to a preferred embodiment of the method according to the invention, the pro-vWF-containing solution is produced by a biotechnological logical method, for example by a cell or tissue incubation method. It is particularly preferred that pro-vWF is expressed by a transformed cell. In the case of pro-vWF-containing solutions produced using biotechnology, pro-vWF is preferably expressed by a cell culture, in particular by a mammalian cell culture.

In contrast to furin, the co-expression of pro-vWF with thrombin is not a problem which could lead to a significant reduction in expression or the yield of pro-vWF, but nevertheless that in EP 0 775 750 A2 and EP 0 319 944 B1 disclosed advantages of in situ processing occur.

Depending on the degree of purification of the pro-vWF preparation subjected to the thrombin proteolysis step, it may also be advantageous to further purify the vWF solution obtained after proteolytic cleavage of pro-vWF in vWF, the provision of a chromatographic purification step for vWF being particularly preferred is. Such cleaning processes are, for example, in EP 0 503 991 A, in WO 89/12 065 A, in EP 0 469 985 A, in EP 0 383 234 A, WO 96/10 584 A, and EP 0 705 846 A and WO 98/38 219 A described.

With the present method it is also possible to obtain vWF and thrombin together, for example after co-expression or concerted purification and cleavage from a source material which contains both pro-vWF and thrombin, since it has been shown according to the invention that vWF formed is not further proteolytically degraded by the mere presence of thrombin. Obtaining vWF and thrombin in a combination preparation is therefore possible in a simple manner by not separating thrombin from the resulting vWF after proteolytic treatment to generate vWF. The process product - ie the mature vWF - with or without thrombin can optionally also contain a cleavage product of the pro-sequence of the vWF generated by the thrombin treatment. This fission product characterizes the new process product. It is also possible to obtain pp-vWF or its cleavage product, which arises from the treatment of pro-vWF with thrombin, from the proteolytically treated solution. For example, pp-vWF can be obtained by treatment with immobilized heparin. In this process, mature vWF and, if appropriate, also pro vWF and thrombin are bound to the immobilized material, while pp-vWF is obtained in the course of the run and is optionally further purified. If necessary, pp-vWF can be used in accordance with Austrian application A 917/97. Pp-vWF preparations as well as preparations that additionally contain about vWF, factor VIII, other activated blood coagulation factors, blood factors with FEIB activity and FEIBA can also be used for the treatment of patients at risk for blood coagulation disorders or for patients with vWD, phenotypic Hemophilia, Hemophilia A and factor VIII inhibitors can be used.

Furthermore, the present invention relates to a vWF preparation which, in addition to (mature) vWF, also contains thrombin. Such a preparation can be used, for example, for direct local application to wounds or bleeding tissue to improve or support primary hemostasis and wound healing.

Such a combination preparation of vWF and thrombin preferably also contains calcium ions, since the calcium ions present in the wound may not be sufficient for the thrombin or wound enzymes to function optimally.

The vWF preparation produced according to the invention, which can optionally also contain thrombin and pp-vWF, has preferably been made virus-safe by treatment for virus inactivation or removal.

The virus inactivation or removal treatment can be carried out by any treatment accepted as efficient. According to preferred embodiments of the present invention, the pharmaceutical composition is treated with surfactants and / or heat, for example by means of heat treatment in solid State, in particular by means of a steam treatment according to EP 0 159 311, EP 0 519 901 or EP 0 637 451, by means of a hydrolysis treatment according to EP 0 247 998, by means of a radiation treatment or by means of a treatment with chemical or chemical / physical processes , for example with chaotropic agents according to WO 94/13 329, by means of a treatment with organic solvents and / or surfactants according to EP 0 131 740 or by means of photo inactivation. Filtration techniques, such as depth filtration or nanofiltration, are also a preferred method for virus depletion in the context of the present invention.

The vWF (/ thrombin combination) preparation according to the invention is preferably provided as a pharmaceutical preparation and furthermore contains suitable pharmaceutically acceptable carriers and / or suitable buffer, auxiliary, preservation and / or stabilizing substances such as carbohydrates, salts or protease inhibitors - or co-factors. The pharmaceutical preparation is preferably made available by packaging for storage in the lyophilized or frozen state. In the manufacture of a pharmaceutical vWF preparation, further purification and virus inactivation steps are particularly preferred before the final formulation and packaging. According to a further aspect, the present invention relates to a set for producing the preparation according to the invention or a vWF preparation, which contains component pro-vWF and component B thrombin. To produce the preparation of vWF according to the invention, components A and B are combined, this combination being possible both by in vitro mixing of the two components, but also in situ, for example on a wound.

Accordingly, component A of the set according to the invention can further contain fibrinogen or (fibrin) adhesive protein.

The invention is explained in more detail with reference to the following examples and the drawing figures, to which it is of course not restricted. The figures show:

1: the pro-vWF processing by thrombin;

2: the pro-vWF processing by 200 nM / 1 thrombin using 1 Ag U / ml rp-vWF (recombinantly produced vWF containing the pro-peptide). pro-vWF: Ag (■), pp-vWF-Ag (±), total vWF: Ag (•) and collagen binding activity (CBA, O)

3: the in vitro processing of pro-vWF with purified human thrombin. The rp-vWF preparation (1 vW Ag U / ml) was treated with 10 (D), 20 (■), 50 (A), 100 (O), 200 (•), 500 (♦) and 1000 (Δ) nm thrombin incubated.

A) Measurement of the time-dependent disappearance of the pro-vWF: Ag using the pro-vWF ELISA.

B) Presentation of the changes in the pro-VWF monomer (upper bands) and mature vWF monomer (lower bands) by staining the immunoblots of the SDS-PAGE under reduction conditions with a polyclonal anti-vWF antibody.

4: the thrombin-mediated pro-vWF processing.

Immunoblots from SDS-PAGE with samples containing 1 Ag U / ml rp-vWF with 200 nM / 1 thrombin.

A) under reduction conditions: the detection was carried out with a polyclonal vWF antibody;

B) under non-reducing conditions: the detection was carried out with a monoclonal anti-vWF-pro-peptide antibody.

Here it can be seen that when developing with the anti-vWF-pro peptide antibody, the time-dependent increase in the pro-peptide band (lower band) correlates clearly with the disappearance of the pro-vWF (upper band). 5: the thrombin-mediated processing of collagen-bound rp-vWF. The rp-vWF preparation (1 vW Ag U / ml) was incubated with 1 (■), 5 (Δ), 10 (A), 25 (O), 50 (•) nM thrombin. The broken lines show the effect of 25 (❖) and 50 (♦) nM thrombin when rp-vWF is bound to immobilized polyclonal antibodies against human vWF.

A) pro-vWF: Ag

B) pp-vWF: Ag

Example 1: Pro vWF processing using thrombin

A human recombinant vWF preparation (rp-vWF) containing pro-vWF was made by expression in CHO cells. Rp-vWF was then purified from the cell culture supernatant by means of ion exchange chromatography and immunoaffinity chromatography and immobilized monoclonal antibodies against mature vWF (Mejan et al., Thromb. Haemost. 59: 364, 1998). The purified, recombinant vWF preparation (10 Ag U / ml) containing 50% pro-vWF and 50% mature vWF was incubated with human thrombin (20 NIH U / ml, Sigma) at 37 ° C and sub-samples were taken in different Time intervals subtracted to measure the changes in the various parameters.

A buffer containing 25 mM HEPES, 175 mM NaCl and 1 mg / ml BSA, pH 7.35 was used for the dilutions. The thrombin reaction was stopped by adding 450 μm PPACK and the samples were frozen at -80 ° C. for later analysis.

As shown in Figure 1, a time-dependent decrease in pro-vWF and an increase in pro-peptide was observed, while no changes were found in the total vWF antigen and collagen binding activity (CBA), indicating that it was not degradation, but a specific "maturation".

2 shows a similar result, here 1 Ag U / ml pro vWF was used. This was confirmed by immunoblots developed with a polyclonal anti-human vWF antibody after SDS-PAGE under reduction conditions, the gradual disappearance of the pro-vWF band being observed without any further significant degradation of the mature vWF band.

The dose-dependent processing of the pro-vWF by thrombin can be seen in FIG. 3. FIG. 3A shows the time curve of the thrombin-mediated disappearance of the pro-vWF antigen in an rp-vWF preparation in a pro-vWF ELISA. 1 Ag U / ml rp-vWF with different thrombin concentrations was incubated here. The changes in the band pattern of the pro-vWF and mature vWF are shown in Fig. 3B. Here the upper band represents the pro-vWF monomer, the lower band the mature vWF monomer.

4 also shows the result of a thrombin-mediated processing of the pro-vWF in SDS-PAGE immunoblots under reducing and non-reducing conditions.

Example 2: amino terminal analysis of the processing products

The time of 120 minutes of the processing mixture (rprovWF and thrombin) showed the SLS_RPPMV sequence of the mature vWF and the AEGT_G_SS sequence of the pro-peptide in approximately equimolar amounts in addition to the heavy and light chain sequences of thrombin, which were also present. Possible other degradation products, if present, were below the detection limit of the system, which confirms that thrombin specifically cleaves the pro-peptide from vWF.

Example 3: Thrombin-mediated processing of

Collagen-bound pro-vWF

Since vWF is an adhesive protein that can bind to different components of the extracellular matrix, it was tested whether the binding of vWF to a surface ligand leads to a change in the tertiary structure of the vWF multimers, and therefore by. the proteolytic cleavage sites are made more accessible to thrombin.

The influence of the pro-vWF-collagen interaction on the processing by thrombin was therefore tested. Rp-vWF was bound to solid phase collagen. There is an incubation with increasing amounts of thrombin, as described in Example 1. 5A and B show the thrombin concentration-dependent disappearance of bound pro-vWF and the simultaneous increase in pp-vWF in the supernatant. The total content of the antigen bound to the solid phase remained unchanged, which proves that the measured changes in the liquid phase were exclusively due to the pp-vWF. In contrast, rp-vWF, which is bound to a carrier under comparable conditions by means of a monoclonal vWF antibody, is processed only very slowly and an at least 10-fold higher thrombin concentration must be used in order to obtain the same processing effect as with collagen-bound vWF.

Example 4: Cleaning the pp-vWF

The solution containing pp-VWF was purified by applying the solution to a Lentil-lectin-Sepharose column. Pp-vWF binds to the column material. After washing the column and eluting the non-binding proteins, pp-vWF was eluted. The fractions obtained were concentrated, dialyzed against physiological buffer and frozen at -80 ° C.

Claims

Patent claims:

1. A method for producing a von Willebrand factor (vWF) - preparation from pro-von Willebrand factor (pro-vWF), characterized in that pro-vWF is treated with thrombin, generating vWF, and a vWF preparation is obtained.

2. The method according to claim 1, characterized in that a pro-vWF-containing solution is treated with thrombin.

3. The method according to claim 2, characterized in that the pro-peptide (pp-vWF) is obtained in addition to the vWF.

4. The method according to claim 1 or 2, characterized in that pro-vWF is treated with immobilized thrombin.

5. The method according to any one of claims 1 to 4, characterized in that the treatment with thrombin is carried out continuously.

6. The method according to any one of claims 1 to 5, characterized in that pro-vWF of biological origin is used.

7. The method according to any one of claims 1 to 6, characterized in that pro-vWF is used in a purified form.

8. The method according to any one of claims 1 to 7, characterized in that pro-vWF is used in a mixture with other proteins.

9. The method according to claim 8, characterized in that pro- vWF is used in a mixture with at least one further agent selected from the group consisting of activators and inhibitors of thrombin and factor VIII.

10. The method according to any one of claims 2 to 9, characterized in that a solution produced by biotechnological processes is used as the pro-vWF-containing solution.

11. The method according to claim 10, characterized in that pro-vWF is expressed by a transformed cell.

12. The method according to claim 3, characterized in that vWF is bound to immobilized heparin and pp-vWF is obtained in one pass.

13. The method according to claim 10 or 11, characterized in that pro-vWF is expressed in a cell culture, in particular in mammalian cells.

14. The method according to claim 13, characterized in that pro-vWF is processed with recombinant thrombin.

15. The method according to claim 14, characterized in that after the generation of vWF, the vWF and thrombin obtained are obtained together to form a combination preparation.

16. The method according to any one of claims 1 to 15, characterized in that a chromatographic purification step for vWF is further provided.

17. The method according to any one of claims 1 to 16, characterized in that the vWF preparation obtained is optionally completed with thrombin to a pharmaceutical preparation, wherein preferably further purification and virus inactivation steps are provided.

18. Preparation, obtainable according to claim 15, containing vWF and thrombin and optionally containing pp-vWF.

19. A preparation according to claim 18, characterized in that it further contains calcium ions.

20. A preparation according to claim 18 or 19, characterized in that it is pharmaceutically formulated and possibly virus inactivated.

21. Set for producing a preparation according to one of claims 18 to 20 containing a component A, which comprises pro-vWF and a component B, which comprises thrombin.

22. Set according to claim 21, characterized in that component A further contains fibrin glue protein.