ES2711455T3 - A method of therapeutic protein purification - Google Patents

Abstract

A method of reducing the level of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator in a solution comprising at least one protein selected from the group consisting of fibrinogen, the method comprising: (i) passing a raw material comprising fibrinogen through a hydrophobic charge induction chromatography resin under conditions selected so that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator present in the raw material is bound to the resin; and (ii) recovering a solution comprising fibrinogen that passes through the resin, reducing the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator in the solution by at least 50% in comparison with raw material; the hydrophobic charge induction chromatography resin being equilibrated to a pH of from about 6.5 to about 8.5 before passing the raw material and / or the solution comprising the fibrinogen through the induction chromatography resin of hydrophobic charge.

Description

DESCRIPTION

A method of therapeutic protein purification

Technical field

The present invention relates in general to a method of reducing the level of impurities in a solution containing at least one therapeutic protein. More specifically, to a method of reducing the level of plasminogen and / or tissue plasminogen activator in a raw material comprising fibrinogen.

Background

Current methods of purification of a therapeutic protein that is produced naturally or recombinantly from a solution comprising said protein usually carry at least some impurities to the final preparation. In some cases, the presence of impurities, such as proteases, will destabilize the therapeutic protein in solution, particularly during storage. For this reason, many therapeutic proteins are stored as lyophilized or frozen preparations.

Although the destabilizing levels of impurities can affect many different types of therapeutic proteins, they are particularly relevant to those used to maintain hemostasis. Hemostasis is an important physiological process that prevents bleeding after damage (eg, rupture) of blood vessels. There are three basic mechanisms that promote hemostasis: (i) vasoconstriction, (ii) platelet aggregation at the site of rupture; and (iii) coagulation. During coagulation, damaged endothelial cells release tissue factor (factor III), which in turn activates factor VII with the help of Ca2 +. Factor XII, which is released by activated platelets, activates factor XI. Activated factor VII and factor XI promote a cascade of enzymatic reactions that lead to the activation of factor X. Active factor X (factor Xa), together with factor III, factor V, Ca2 + and platelet thromboplastic factor (PF3) ), activate the prothrombin activator. The prothrombin activator converts prothrombin into thrombin, which converts fibrinogen (factor I) into fibrin, which forms an initial mesh over the site of damage. The initial mesh is then converted into a dense fibrin clot by factor XIII, sealing the break until the site is repaired. During the coagulation cascade, thrombin will also activate factor VIII, a glycoprotein pro-cofactor that in circulation is mainly complexed with von Willebrand factor (VWF). Factor VIII interacts with factor IXa to activate factor X in the presence of Ca + 2 and phospholipids.

A deficiency in the level of any one or more of the proteins involved in coagulation, including fibrinogen, factor VIII and / or von Willebrand factor (VWF), whether congenital or acquired, can lead to insufficient coagulation of the blood and at the risk of hemorrhage. The current treatment options are limited to the administration of a pharmaceutical preparation of one or more therapeutic proteins, with a view to restoring the endogenous levels of said proteins and maintaining hemostasis. However, existing pharmaceutical preparations, which are usually derived from donated blood plasma or a recombinant source, comprise zymogens and proteases (eg, prothrombin, plasminogen, tissue plasminogen activator (tPA) and / or other proteases), which can destabilize Therapeutic proteins, such as fibrinogen, factor VIII or VWF during storage. Accordingly, such preparations are relatively unstable in aqueous solution, with long-term storage limited to lyophilized or frozen preparations.

For classical applications, the fibrinogen is normally purified from human plasma, where it represents only about 2-5% (1.5-4.0 g / L) of the total plasma proteins. Traditionally, the purification of fibrinogen from plasma is carried out by fractionation of classical plasma, in which the fibrinogen is cryoprecipitated from the plasma followed by precipitation with either ethanol, or ammonium sulfate, or palanin / glycine, or either polymers (eg polyethylene glycol) or low ionic strength solutions. Such methods can achieve a relatively high yield and homogeneity. When a higher level of purity is required, chromatographic techniques are often employed. However, existing precipitation and chromatographic techniques amenable to commercial scale manufacturing processes typically produce fibrinogen preparations comprising contaminating proteins, such as zymogens or proteases (e.g., prothrombin, tissue plasminogen activator (tPA) and plasminogen), that can destabilize the fibrinogen in solution. For example, when prothrombin is present, it can be activated to give serine protease thrombin, which in turn will convert the fibrinogen into fibrin. Similarly, when both tPA and plasminogen are present, tPA can activate plasminogen to its active form plasmin, which in turn hydrolyzes the fibrinogen to fibrin. Accordingly, fibrinogen preparations are relatively unstable in aqueous solution, with long-term storage limited to lyophilized or frozen preparations.

Specific contaminants can be removed by absorption; for example, fibronectin in immobilized gelatin and plasminogen in immobilized lysine (Vuento et al., 1979, Biochem. J., 183 (2): 331-337). However, the use of specific affinity resins is not susceptible to large-scale commercial processes. The reasons for this include that the affinity resins themselves are not robust enough to be used repeatedly and generally significantly increase both time and processing costs.

EP1240200 (US6960463) relates to methods of purifying fibrinogen from a solution containing fibrinogen using ion exchange chromatography (IEX). In particular, the method involves applying a solution containing fibrinogen to an ion exchange matrix under conditions that allow the fibrinogen to bind to the matrix and then wash the ion exchange matrix with a solution comprising at least one omega-amino acid. This is done to promote the differential removal of plasminogen from the resin. The fibrinogen that has been bound to the matrix is then eluted from the matrix.

WO2012038410 provides a method of purifying fibrinogen using anion exchange resins containing a hydroxylated polymeric support grafted with tertiary or quaternary amines that bind to the fibrinogen.

EP1519944 teaches the use of an affinity chromatography matrix for immobilized metal ions under conditions in which fibrinogen and plasminogen are bound to the matrix, and the selective elution of the fibrinogen and plasminogen separately, so that the fraction of main fibrinogen contains approximately 600 ng of plasminogen per mg of protein.

US20050197493 relates to a process for purifying fibrinogen, comprising one or more process steps in which one or more contaminating proteins are depleted by negative chromatography and / or negative adsorption using a cation exchanger, hydrophobic gel and / or gel staining

The present invention provides a method of reducing the level of plasminogen and / or tissue plasminogen activator in a solution comprising fibrinogen. The purified protein (s) are stable during storage as liquid preparations and can be used for clinical or veterinary applications, including the treatment or prevention of conditions associated with a deficiency in the level of said protein (s). (s)

Summary of the invention

In one aspect of the present invention, there is provided a method of level reduction of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator in a solution comprising at least one protein selected from the group consisting of fibrinogen, the method comprising: (i) passing a raw material comprising at least one protein selected from the group consisting of fibrinogen through a hydrophobic charge induction chromatography resin under selected conditions so that at least one protein selected from the group which consists of plasminogen, tissue plasminogen activator present in the raw material is attached to the resin; Y

(ii) recovering a solution comprising the at least one protein selected from the group consisting of fibrinogen, which passes through the resin, reducing the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator in the solution in at least 50% compared to the raw material, the hydrophobic charge induction chromatography resin being equilibrated at a pH of from about 6.5 to about 8.5 before passing the raw material and / or the solution comprising the fibrinogen through the hydrophobic charge induction chromatography resin. In one example described herein, there is provided a method of level reduction of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease (s) in a solution comprising at least a protein selected from the group consisting of fibrinogen, factor VIII and von Willebrand factor (VWF), the method comprising:

(i) passing a raw material comprising at least one protein selected from the group consisting of fibrinogen, factor VIII and VWF through a first hydrophobic charge induction chromatography resin; (ii) recovering a solution comprising the at least one protein selected from the group consisting of fibrinogen, factor VIII and VWF which passes through the first hydrophobic charge induction chromatography resin;

(iii) passing the solution that has been recovered in step (ii) through a second hydrophobic charge induction chromatography resin; Y

(iv) recovering the solution comprising the at least one protein selected from the group consisting of fibrinogen, factor VIII and VWF which passes through the second hydrophobic charge induction chromatography resin;

the conditions of the chromatographic steps being such that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease (s) present in the raw material is attached to the first and / or second resin, and reducing the concentration of the at least one selected protein of the group consisting of plasminogen, tissue plasminogen activator and other protease (s) in the solution that has been recovered in step (iv) by at least 50% compared to the raw material.

In another example, a solution comprising at least one protein selected from the group consisting of fibrinogen, factor VIII and VWF recovered by the method described herein is provided.

In another example, a container containing at least 5 ml of a pharmaceutically acceptable fibrinogen solution is provided, the fibrinogen concentration being at least 20 mg / ml.

In another example, a pharmaceutical formulation comprising a solution comprising at least one protein selected from the group consisting of fibrinogen, factor VIII and VWF recovered by the method described herein, and a pharmaceutically acceptable carrier is provided.

In another example, a solution is provided comprising:

(a) at least 75% of the total fibrinogen protein;

(b) less than 50 pg / mg of the total tissue plasminogen activator protein; I

(c) less than 1 pg / mg of the total plasminogen protein.

In another example, a solution is provided comprising:

(a) at least 90% of the total fibrinogen protein;

(b) less than 50 pg / mg of the total tissue plasminogen activator protein; I

(c) less than 150 ng / mg of the total plasminogen protein.

In another example, a solution is provided comprising:

(a) at least 90% of the total fibrinogen protein;

(b) less than 20 pg / mg of the total tissue plasminogen activator protein; I

(c) less than 10 ng / mg of the total plasminogen protein.

In another example, a method of treating or preventing a condition associated with fibrinogen deficiency is provided, the method comprising administering to a subject in need thereof the pharmaceutical solution or formulation described herein.

In another example, the use of the solution described herein is provided in the manufacture of a medicament for treating or preventing a condition associated with fibrinogen deficiency.

In another example, a fibrin glue is provided comprising the solution described herein.

In another example, a method of producing a stable liquid fibrinogen solution is provided, the method comprising:

(i) passing a raw material comprising fibrinogen through a hydrophobic charge induction chromatography resin under selected conditions so that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other (s) protease (s) present in the raw material is attached to the resin; Y

(ii) recovering a solution comprising fibrinogen passing through the resin, reducing the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease (s) in the solution at least 50% compared to the raw material. In another example, a method of producing a stable liquid fibrinogen solution is provided, the method comprising:

(i) passing a raw material comprising fibrinogen through a first hydrophobic charge induction chromatography resin;

(ii) recovering a solution comprising fibrinogen passing through the first hydrophobic charge induction chromatography resin;

(iii) passing the solution that has been recovered in step (ii) through a second hydrophobic charge induction chromatography resin; Y

(iv) recovering a solution comprising fibrinogen passing through the second hydrophobic charge induction chromatography resin;

the conditions of the chromatographic steps being such that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease (s) present in the raw material is attached to the first and / or second resin, and reducing the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease (s) in the solution that has been recovered in step (iv) by at least 50% in comparison with the raw material.

In another example, a method for purifying fibrinogen is provided, the method comprising the steps of: (i) passing a solution comprising fibrinogen through an ion exchange chromatography resin under selected conditions such that fibrinogen monomer is attached to the resin;

(ii) eluting the fibrinogen monomer from the resin with an elution buffer; Y

(iii) filtering the fibrinogen monomer eluted from step (ii) through a filter having a pore size in the range of from about 15 nm to about 35 nm.

Brief description of the figures

Figure 1 shows the percent recovery of fibrinogen, plasminogen and t-PA from a fibrinogen solution over a range of pH levels when the solution is passed through an HEA Hypercel ™ in negative mode. regarding fibrinogen. The bars within each group represent (from left to right): fibrinogen recovery, plasminogen recovery and t-PA recovery.

Figure 2 shows the percent recovery of fibrinogen, plasminogen, t-PA and factor II from a fibrinogen solution over a range of pH levels when the solution is passed through a Hypercel ™ PPA in negative mode with respect to fibrinogen. The bars within each group represent (from left to right): fibrinogen recovery, plasminogen recovery, recovery of t-PA and factor II. Figure 3 shows the recovery percentage of fibrinogen, plasminogen, t-PA and factor II from a solution of fibrinogen over a range of pH levels when the solution is passed through a MEP Hypercel ™ in negative mode with respect to fibrinogen. The bars within each group represent (from left to right): fibrinogen recovery, plasminogen recovery, recovery of t-PA and factor II. Figure 4a shows the percent recovery of fibrinogen, plasminogen, t-PA and factor II from a solution containing fibrinogen over a range of pH levels when the solution is passed through an HEA Hypercel ™ in negative mode with respect to fibrinogen. The bars within each group represent (from left to right): fibrinogen recovery, plasminogen recovery, t-PA recovery and factor II recovery.

Figure 4b shows the stability of the fibrinogen-containing solution recovered from the coffee fraction through the HEA Hypercel ™ column (Figure 4a) for 6 days at room temperature (approximately 20 ° C), measured by the % of initial coagulable protein. The bars within each group represent (from left to right): T = 1 day, T = 3 days, T = 6 days.

Figure 4c shows the stability of the fibrinogen-containing solution recovered from the coffee fraction through the HEA Hypercel ™ column (Figure 4a) over 6 days at room temperature (approximately 20 ° C), measured by the % coagulable protein. The bars within each group represent (from left to right): T = 1, T = 3 days, T = 6 days.

Figure 5 shows the percentage of process recovery for fibrinogen, t-PA, plasminogen and factor II in fractions derived from a method according to an embodiment of the present invention, from the plasma cryoprecipitate to the MacroPrep ™ -HQ eluate. The bars within each group represent (from left to right): fibrinogen, fibronectin, plasminogen, t-PA and factor II.

Figure 6 shows the purity of monomeric fibrinogen that was recovered from the MacroPrep ™ -HQ chromatographic resin as measured by an HPLC chromatogram of exclusion by analytical size.

Figure 7 shows the filtration capacity of fibrinogen recovered from the MacroPrep ™ -HQ chromatography resin using elution buffers with different conductivities (190 mM NaCl, 21.5 mS / cm (diamonds), 200 mM NaCl, 22.5 mS / cm (square), 210 mM NaCl, 23.5 mS / cm (triangles), 1% (w / w) arginine / 200 mM NaCl, 25 mS / cm (cross)).

Figure 8 shows the fibrinogen activity as a percentage of the initial fibrinogen activity at t = 0 of liquid fibrinogen recovered from the MacroPrep ™ -HQ chromatography resin which is stored at or 2-8 ° C (diamonds) for a period of 9 days. weeks or 30 ° C (squares) for a period of 7 weeks.

Detailed description

Throughout this specification, unless the context requires otherwise, it will be understood that the word "understand", or variations such as "comprises" or "comprising", implies the inclusion of an element or whole number or group of elements or whole numbers expressed, but not the exclusion of any other element or whole number or group of elements or whole numbers.

The reference in this specification to any prior publication (or information derived therefrom), or to any known material, is not and should not be taken as an acknowledgment or admission or any form of suggestion that such prior publication (or information derived from the same) or known matter is part of the common general knowledge in the field of work to which this descriptive report refers.

It should be noted that, as used in the object descriptive memory, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, the reference to "one formulation" includes a single formulation, as well as two or more formulations.

In the absence of any indication to the contrary, the reference made to a content in "%" throughout this specification should be taken as meaning% p / p (weight / weight). For example, a solution comprising at least 80% of the total fibrinogen protein should be taken to mean a solution comprising fibrinogen at a concentration of at least 80% w / w of the total protein. This can be calculated, for example, by dividing the amount of fibrinogen derived from the coagulable protein assay by the amount of total protein derived from a conventional protein assay (eg, biuret) and multiplying by 100. In the coagulable protein assay, it is add thrombin to a sample to form a clot, which is almost all of fibrin. The coagulum can be separated from the supernatant containing non-coagulable proteins by centrifugation. Subsequently, the clot is washed and dissolved by alkaline urea or other substances and the protein concentration is determined by spectrophotomethane. Since the majority of the clot is fibrin, the protein concentration will be equivalent to the concentration of fibrinogen. Therefore, the amount of coagulable protein in a sample is equivalent to the difference between the total protein and the non-coagulable protein component of the sample.

The purification of fibrinogen and / or factor VIII and / or VWF from a raw material (eg, cell culture or plasma supernatant) is usually carried out by conventional fractionation, in which the fibrinogen and / or factor VIII and / or VWF is precipitated from the solution using, for example, ethanol, ammonium sulfate, p-alanine / glycine, polymers (eg, polyethylene glycol) and / or solutions of low ionic strength. Current plasma purification methods employing different chromatographic steps can yield a relatively high yield and homogenous preparations of a protein of interest. However, these methods usually result in preparations comprising residual amounts of impurities that may not be suitable for formulating a stable liquid preparation for classical applications. Impurities such as prothrombin, tissue plasminogen activator (tPA) and plasminogen are particularly problematic, since destabilizing levels of these impurities can hydrolyze the fibrinogen in aqueous solution, thus rendering the fibrinogen unstable, particularly during manufacture and / or storage. long-term.

The present invention is based, at least in part, on the finding that passing a raw material comprising fibrinogen through a hydrophobic charge induction chromatography resin (HCIC) and recovering the solution comprising fibrinogen passing through The resin is an efficient alternative to the existing purification processes to reduce the destabilizing level of plasminogen and / or tissue plasminogen activator in the solution.

Therefore, in one aspect of the present invention, there is provided a method of level reduction of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator in a solution comprising at least one protein selected from the group consisting of in fibrinogen, comprising the method:

(i) passing a raw material comprising at least one protein selected from the group consisting of fibrinogen through a hydrophobic charge induction chromatography resin under selected conditions so that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator present in the raw material is bound to the resin; Y

(ii) recovering a solution comprising the at least one protein selected from the group consisting of fibrinogen passing through the resin, reducing the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator in the dissolution in at least 50% in comparison with the raw material; the hydrophobic charge induction chromatography resin being equilibrated at a pH of from about 6.5 to about 8.5 before passing the raw material and / or the solution comprising the fibrinogen through the induction chromatography resin. hydrophobic charge. In one embodiment, the concentration of the plasminogen and / or tissue plasminogen activator in the recovered solution comprising fibrinogen is reduced by at least 60%, by at least 70%, by at least 80% or by at least 90%. % or at least 95% or at least 98% compared to the raw material.

In another example, a method of producing a stable liquid fibrinogen solution is provided, the method comprising:

(i) passing a raw material comprising fibrinogen through a hydrophobic charge induction chromatography resin under selected conditions so that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other (s) protease (s) present in the raw material is attached to the resin; Y

(ii) recovering a solution comprising fibrinogen passing through the resin, reducing the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease (s) in the solution at least 50% compared to the raw material. In one embodiment, the concentration of the plasminogen and / or tissue plasminogen activator and / or other protease (s) in the recovered solution comprising fibrinogen is reduced by at least 60%, by at least 70%, in at least 80% or at least 90% or at least 95% or at least 98% compared to the raw material.

The chromatographic processes normally employ a solid support, also referred to interchangeably herein as a resin or matrix. Suitable solid supports will be familiar to those skilled in the art. Examples include inorganic carriers, such as glass and silica gel, organic, synthetic or naturally occurring carriers, such as agarose, cellulose, dextran, polyamide, polyacrylamides, vinyl copolymers of bifunctional acrylates, and various hydroxylated monomers, and similar. Commercially available carriers are sold under the names of Sephadex ™, Sepharose ™, Hypercel ™, Capto ™, Fractogel ™, MacroPrep ™, Unosphere ™, GigaCap ™, Trisacryl ™, Ultrogel ™, Dynospheres ™, Macrosorb ™ and XAD ™ resins.

The chromatography steps will be carried out generally under non-denaturing conditions and at convenient temperatures in the range of about 10 ° C to 30 ° C, more usually at about ambient temperatures. The chromatographic steps may be carried out batchwise or continuously, as appropriate. Any convenient separation method may be employed, such as column, centrifugation, filtration, decantation, or the like.

Hydrophobic charge induction chromatography (HCIC), which is often also referred to as chromatography either mixed or multimodal, will be familiar to those skilled in the art. The HCIC uses binding residues coupled to a solid support, the binding residues may have specificity for one or more proteins that, according to the methods of the present invention, represent impurities in the raw material (for example, zymogens and proteases, such as prothrombin, tPA and plasminogen).

Any suitable HCIC resin known to those skilled in the art can be used. In one embodiment, the HCIC resin comprises a ligand selected from the group consisting of mercaptoethyl pyridine (4-mercaptoethyl pyridine, e.g., MEP Hypercel ™), n-hexylamine (e.g., HEA Hypercel ™) and phenylpropylamine (e.g., PPA Hypercel ™). In one embodiment, the HCIC resin comprises n-hexylamine.

HCIC ligands, such as HEA, MEP and PPA, have a window because they allow separation based on the surface hydrophobicity of proteins, but do not require the addition of lyotropic salts often seen in other processes for the purification of fibrinogen using hydrophobic chromatography. (for example, hydrophobic interaction chromatography, HIC). Unlike traditional hydrophobic interaction chromatography, HCIC is controlled on the basis of pH, instead of salt concentration. The HCIC resins also provide a high binding capacity and high flow rates, ideal for purification both on a laboratory scale and on an industrial scale.

The HCIC resins are often stacked in columns with bed heights of from about 2 cm to about 40 cm. On an industrial scale, the bed heights are usually at least 10 cm and are usually in the range of from about 15 cm to 25 cm. The column diameters of industrial columns can range between 20 cm and approximately 1.5 m. Such columns are operated at flow rates according to the HCIC resin manufacturing instructions with flow rates in the typical range of 100 100 cm / h. The upper flow rate limitation is partly due to the pressure restrictions of the HCIC resin. For the HEA resin, for example, the upper operating pressure limit is <3 bar (<300 kPa). The typical dynamic binding capacities (10% saturation of a binding protein) for HCIC resins are from about 20 to 30 mg of bound protein per ml of resin. For the present invention, this enables relatively large amounts of protein to be loaded into the HCIC column since abundant proteins, such as fibrinogen, can pass through the column, while less abundant proteins such as plasminogen and / or the tissue plasminogen activator and / or other protease (s) bind to the HCIC resin. This is advantageous for manufacturing on an industrial scale, since columns of smaller size and / or fewer column cycles are required to process a batch.

The present inventors have also found that the pH of the solution or feedstock comprising fibrinogen which is passed through the HCIC resin according to the methods of the present invention can be adjusted to control the recovery of the fibrinogen and the removal of impurities. Therefore, in an embodiment disclosed herein, the solution or raw material comprising fibrinogen which is passed through the HCIC resin has a pH of from about 6.5 to about 8.5. In particular examples, the solution or raw material comprising fibrinogen and / or factor VIII and / or VWF is passed through the HCIC resin, preferably at a pH of about 4.0, 5.0, 5.25, 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5 or 10.0. In a particular embodiment, the solution or raw material comprising fibrinogen which is passed through the HCIC resin has a pH of about 7.0. In particular examples, the HCIC resin is equilibrated before loading the solution or raw material at a pH of about 4.0, 5.0, 5.25, 5.5, 5.75, 6.0, 6.25 , 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5 or 10.0.

The method of the present invention may also employ the use of more than one additional chromatography step to remove additional impurities, if necessary, and thereby improve the purity of the final preparation. Additional chromatographic purification steps may be implemented either before or after the purification of fibrinogen through the HCIC resin according to the present invention. For example, the solution comprising fibrinogen that is recovered from the HCIC resin in step (ii) can be passed through another chromatographic resin.

The additional chromatographic purification steps can employ another HCIC resin. Thus, in another example, a method of level reduction of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease (s) in a solution comprising at least one protein is provided. selected from the group consisting of fibrinogen, factor VIII and von Willebrand factor (VWF), the method comprising:

(i) passing a raw material comprising at least one protein selected from the group consisting of fibrinogen, factor VIII and VWF through a first hydrophobic charge induction chromatography resin; (ii) recovering a solution comprising the at least one protein selected from the group consisting of fibrinogen, factor VIII and VWF which passes through the first hydrophobic charge induction chromatography resin;

(iii) passing the solution that has been recovered in step (ii) through a second hydrophobic charge induction chromatography resin; Y

(iv) recovering the solution comprising the at least one protein selected from the group consisting of fibrinogen, factor VIII and VWF which passes through the second hydrophobic charge induction chromatography resin;

the conditions of the chromatographic steps being such that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease (s) present in the raw material is attached to the first and / or second resin, and reducing the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease (s) in the solution that has been recovered in step (iv) by at least 50% in comparison with the raw material.

In one example, the concentration of the plasminogen and / or tissue plasminogen activator and / or other protease (s) in the solution comprising fibrinogen and / or factor VIII and / or VWF that has been recovered in the step (iv ) is reduced by at least 60%, by at least 70%, by at least 80% or by at least 90% or by at least 95% or by at least 98% compared to raw material .

In another example, a method of producing a stable Kquida fibrinogen solution is provided, the method comprising:

(i) passing a raw material comprising fibrinogen through a first hydrophobic charge induction chromatography resin;

(ii) recovering a solution comprising fibrinogen passing through the first hydrophobic charge induction chromatography resin;

(iii) passing the solution that has been recovered in step (ii) through a second hydrophobic charge induction chromatography resin; Y

(iv) recovering a solution comprising fibrinogen passing through the second hydrophobic charge induction chromatography resin;

the conditions of the chromatographic steps being such that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease (s) present in the raw material is attached to the first and / or second resin, and reducing the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease (s) in the solution that has been recovered in step (iv) by at least 50% in comparison with the raw material.

In one example, the concentration of the plasminogen and / or tissue plasminogen activator and / or other protease (s) in the solution comprising fibrinogen that has been recovered in step (iv) is reduced by at least 60% , in at least 70%, in at least 80% or in at least 90% or in at least 95% or in at least 98% in comparison with the raw material.

In an example disclosed herein, the second HCIC resin is different from the first HCIC resin. In another example disclosed herein, the first and second hydrophobic charge induction chromatography resin are the same. When the solution comprising fibrinogen and / or factor VIII and / or VWF which is recovered from the HCIC resin in step (ii) is passed through the same HCIC resin, it may be desirable to wash the HCIC resin after step (ii) and before passing the solution recovered through the HCIC resin in step (iii) again in order to remove any impurity that may be bound to the resin.

The additional chromatographic resin can also be an anion exchange chromatography resin. In anion exchange chromatography, negatively charged molecules are attracted by a solid, positively charged support. A positively charged solid support can be prepared by any means known to those skilled in the art and will usually involve the covalent coupling of a negatively charged ligand on a solid support. The appropriate negatively charged functional ligands will invariably depend on the molecule to be separated from the solution. Examples of suitable anion exchange resins are those comprising a quaternary amine group (Q) and / or a functional tertiary amine group (DEAE), or a diethylaminopropyl group (ANX). Commercially available anion exchange chromatography matrices include, but are not limited to, DEAE-cellulose, Poros ™ PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, MonoQ ™, MiniQ ™, Source ™ 15Q and 30Q, Q, DEAE and ANX Sepharose Fast Flow ™, Q Sepharose high Performance ™, QAE SEPHADEX ™ and FAST Q SEPHAROSE ™ by GE Healthcare, WP PEI ™, WP DEAM ™, WP QUAT ™ by JT Baker, Hydrocell ™ DEAE and Hydrocell ™ QA from Biochrom Labs Inc., UNOsphere ™ Q, Macro-Prep ™ DEAE and Macro-Prep ™ High Q from Biorad, Ceramic HyperD ™ Q, ceramic HyperD ™ DEAE, Q HyperZ ™, Trisacryl ™ M and LS ™ DEAE, Spherodex ™ LS DEAE, QMA Spherosil ™ LS, QMA Spherosil ™ M from Pall Technologies, DOWEX ™ Fine Mesh Strong Base Type I and Type II Anion Matrix and DOWEX ™ MONOSPHER E 77, weak base anion from Dow Liquid Separations, Matrex Cellufine ™ A200, A500, Q500 and Q800, from Millipore, Fractogel ™ EMD TMAE3 Fractogel ™ and Md DEAE and Fractogel ™ EMD DMAE from EMD, weak and strong anionic exchangers type I and II Amberlite ™, weak anionic exchangers and Strong Type I and II DOWEX ™, weak and strong anionic exchangers type I and II Diaion ™, Duolite ™ by Sigma-Aldrich, t Sk ™ gel Q and DEAE 5PW and 5PW-HR, Toyopearl ™ SuperQ-650S, 650M and 650C3 QAE - 26 - 550C and 650S, DEAE- 65OM and 650C from Tosoh, and QA52 ™, DE23 ™, DE32 ™, DE51 ™, DE52 ™, DE53 ™, Ex Press-Ion ™ D and Express-Ion ™ Q from Whatman. If desired, an anion exchange chromatography membrane can be used in place of an anion exchange chromatography matrix. Commercially available anion exchange membranes include, but are not limited to, Sartobind ™ Q from Sartorius, Mustang ™ Q from Pall Technologies, and Intercept ™ Q membrane from Millipore.

In an example disclosed herein, the anion exchange resin is a strong anion exchange resin. In another embodiment disclosed herein, the strong anion exchange resin comprises a quaternary amine functional ligand (e.g., -N + (CH3) 3 as seen, for example, in MacroPrep ™ -HQ; Rad Laboratories). In yet another example, the anion exchange resin are trimethylamine groups grafted to a hydroxylated metacrylic polymer by means of a linkage group, such as GigaCap Q-650M®.

In one example, the anion exchange chromatography is carried out in positive mode with respect to the fibrinogen and / or factor VIII and / or VWF. That is, the conditions used are such that, when the solution or raw material comprising fibrinogen and / or factor VIII and / or VWF is passed through the anion exchange chromatography resin, the fibrinogen and / or factor VIII and / or VWF joins the positively charged functional groups coupled to the resin, allowing impurities in the solution to pass through the resin in the fraction of flow through (through), when they can be discarded or recovered. for other purposes. Once the fraction of flow through passes through the resin, the anion exchange chromatography resin can be washed with a suitable washing buffer known to those skilled in the art. The constituents of the wash buffer and the conditions of the washing step will normally be selected to retain the fibrinogen and / or factor VIII and / or VWF bound to the resin during the washing step. The person skilled in the art will also recognize that reference to washing buffer, elution buffer or the like in relation to chromatography may include solutions having limited buffering capacity or no capacity.

In an example disclosed herein, before eluting the fibrinogen and / or factor VIII and / or VWF from the anion exchange chromatography resin, the resin is washed with a wash solution comprising the epsilon-aminocaproic acid (e-ACA). The addition of -ACA to the wash buffer can promote the elution of proteases (such as plasminogen) that can bind to the anion exchange chromatography resin during the first pass. An example of a suitable washing step is described in U.S. Patent 6,960,463.

To elute the fibrinogen and / or factor VIII and / or VWF remaining bound to the anion exchange chromatography resin, any suitable elution buffer known to those skilled in the art can be used. For the removal of plasminogen and / or t-PA and / or other protease (s) from a solution comprising fibrinogen, the present inventors have found that an elution buffer comprising NaCl from about 150 mM to about 300 mM allows fibrinogen monomers to be eluted from the anion exchange resin while minimizing the elution of aggregates of fibrinogen and / or other proteins (e.g., factor VIII, VWF, fibronectin or proteases) that may also be attached to the resin . Thus, in one example disclosed herein, the fibrinogen is eluted from the anion exchange resin with an elution buffer comprising NaCl from about 150 mM to about 300 mM. This corresponds to an elution buffer having a conductivity range of about 18 mS / cm (150 mM NaCl) at about 32 mS / cm (300 mM NaCl).

In another example, the fibrinogen is eluted from the anion exchange resin with an elution buffer comprising NaCl from about 150 mM to about 270 mM. This is equivalent to an elution buffer having a conductivity range of about 18 mS / cm (150 mM NaCl) to about 29 mS / cm (270 mM NaCl).

In another example, the fibrinogen is eluted from the anion exchange resin with an elution buffer comprising NaCl from about 170 mM to about 230 mM. This corresponds to an elution buffer having a conductivity range of about 19 mS / cm (170 mM NaCl) to about 25 mS / cm (230 mM NaCl).

In another example, the fibrinogen is eluted from the anion exchange resin with an elution buffer comprising NaCl from about 200 mM to about 220 mM. This corresponds to an elution buffer having a conductivity range of about 22 mS / cm (200 mM NaCl) to about 24 mS / cm (220 mM NaCl).

In another example, the fibrinogen is eluted from the anion exchange resin with an elution buffer comprising NaCl from about 190 mM to about 210 mM.

In another example, the fibrinogen is eluted from the anion exchange resin with an elution buffer comprising NaCl from about 150 mM to about 190 mM.

In another example, the elution buffer has a conductivity in the range of 18 to 32 mS / cm; or from 20 to 25 mS / cm; or from 21 to 23.5 mS / cm; or from 22 to 23 mS / cm. In a preferred example, the conductivity of the elution buffer is about 22.5 mS / cm.

In an example disclosed herein, the elution buffer comprises a free amino acid at a concentration that promotes the elution of fibrinogen monomer onto aggregates thereof. In another example, the elution buffer comprises a free amino acid at a concentration of about 0.5 to 10% (w / w). Any suitable free amino acid can be used in this capacity. In one example, the free amino acid is arginine. In another example, the elution buffer comprises arginine in the range of about 4 to about 10% (w / w).

In other examples, the elution buffer comprises 200 mM NaCl, 0.5% arginine (w / w); or 160 mM NaCl, 1% arginine (w / w).

In one example, the anion exchange chromatography is carried out in negative mode with respect to the fibrinogen and positive mode with respect to factor VIII and / or VWF. That is, the conditions used are such that, when the solution or raw material comprising fibrinogen and factor VIII and / or VWF is passed through the anion exchange chromatography resin, factor VIII and / or VWF binds (FIG. n) to the positively charged functional groups coupled to the resin, allowing the fibrinogen in the solution to pass through the resin in the fraction of flow through (through). Once the fraction of flow through which contains fibrinogen passes through the resin, the anion exchange chromatography resin can be washed with a suitable washing buffer known to those skilled in the art. The constituents of the wash buffer and the conditions of the washing step will normally be selected to retain factor VIII and / or VWF bound to the resin during the washing step.

In one example, the solution or raw material comprising fibrinogen and / or factor VIII and / or VWF is passed through the anion exchange chromatography resin in the presence of NaCl from about 150 mM to about 270 mM. This corresponds to a conductivity range of about 18 mS / cm (150 mM NaCl) to about 29 mS / cm (270 mM NaCl). Under these conditions, the fibrinogen, particularly the monomeric form, passes through the anion exchange chromatography resin while the aggregates containing fibrinogen and other impurities, such as IgG and fibronectin, are bound to the resin. In additional examples, the solution or raw material comprising fibrinogen and / or factor VIII and / or VWF is passed through the anion exchange chromatography resin in the presence of NaCl from about 170 mM to about 230 mM (about 19 mM). mS / cm at about 25 mS / cm) or NaCl from about 200 mM to about 220 mM (from about 22 mS / cm to about 24 mS / cm). In these types of conditions, it is expected that factor VIII and / or vWF will bind to the anion exchange chromatography resin. Factor VIII and / or VWF can be eluted from the anion exchange resin with an elution buffer comprising at least 300 mM of a salt, such as NaCl. In a particular embodiment factor VIII and / or VWF are eluted from the anion exchange resin with approximately 500 mM NaCl. When fibrinogen and factor VIII and / or VWF are bound to the anion exchange resin, the elution step can be performed so that the fibrinogen is initially eluted (e.g., using the conditions set forth above) and then factor VIII and / or VWF can be eluted using a higher salt concentration, such as 500 mM NaCl.

When an anion exchange chromatography step is employed, it can be performed either before and / or after passing the raw material comprising fibrinogen and / or factor VIII and / or VWF through the HCIC resin. In an example disclosed herein, the method further comprises passing the solution comprising fibrinogen and / or factor VIII and / or VWF which has been recovered in step (ii) through an exchange chromatography resin. anionic In another example, when first and second HCIC chromatography steps are employed, as described herein, the method further comprising passing the solution comprising fibrinogen and / or factor VIII and / or VWF that has been recovered in step (ii) and / or step (iv) through an anion exchange chromatography resin.

In an example disclosed herein, the method further comprises passing the raw material comprising fibrinogen and / or factor VIII and / or VWF through an anion exchange chromatography resin before step (i).

Those skilled in the art will understand that the number of additional chromatographic steps used according to the present invention will depend on the level of purity required in the final preparation. For example, the method of the present invention may comprise 2, 3, 4 or 5 chromatography steps, as disclosed herein. For example, when the method comprises 2 steps of chromatography, the sequence of steps will be HCIC / IEX or HCIC / HCIC or IEX / HCIC; When the method comprises 3 steps of chromatography, the sequence of steps will be HCIC / IEX / HCIC or HCIC / HCIC / IEX or HCIC / HCIC / HCIC or HCIC / IEX / IEX or IEX / HCIC / HCIC or IEX / HCIC / IEX or IEX / IEX / HCIC; When the method comprises 4 steps of chromatography, the sequence of steps will be HCIC / IEX / HCIC / HCIC or HCIC / HCIC / IEX / HCIC or HCIC / HCIC / HCIC / IEX or HCIC / HCIC / HCIC / HCIC or HCIC / IEX / IEX / HCIC or HCIC / IEX / IEX / IEX or HCIC / HCIC / IEX / IEX or HCIC / IEX / HCIC / IEX or IEX / HCIC / IEX / HCIC or IEX / HCIC / HCIC / IEX or IEX / HCIC / HCIC / HCIC or IEX / HCIC / IEX / IEX or IEX / IEX / HCIC / HCIC or IEX / IEX / HCIC / IEX or IEX / IEX / IEX / HCIC; etcetera (where "IEX" denotes anion exchange chromatography). The level of purity required may be dictated by the intended use of the solution (for example, for the treatment of a patient with a deficiency of fibrinogen and / or factor VIII and / or VWF) and / or when a storage period is required. more prolonged as aqueous preparation.

Chromatography can be performed using any means known to those skilled in the art. For example, the chromatography steps according to the present invention can use axial flow columns, such as those available from GE Healthcare, Pall Corporation and Bio-Rad, or radial flow columns, such as those available from Proxcys. The chromatography steps according to the present invention can also be carried out using expanded bed technologies.

In one example, the concentration of the plasminogen and / or activator of plasminogen tisulary / or other protease (s) in the recovered solution comprising fibrinogen and / or factor VIII and / or VWF is reduced by at least 60%, in at least 70%, in at least 80% or in at least 90% or in at least 95% in comparison with the raw material.

Methods that maximize the removal of impurities such as plasminogen and / or tissue plasminogen activator and / or other protease (s) are particularly advantageous, because the stability and efficacy of the fibrinogen and / or factor VIII are decisively improved and / or VWF in solution, particularly during long-term storage. Storage in liquid form is particularly advantageous for solutions comprising fibrinogen and / or factor VIII and / or VWF because immediate use in a patient is possible. This is in contrast to the use of lyophilized preparations of purified fibrinogen and / or factor VIII and / or VWF, which require the reconstitution of the lyophilized protein (s) in a suitable buffer and / or water for injection immediately before administration to a subject who needs it.

One advantage of depleting the proteases or their zymogens (such as plasminogen) from a solution comprising fibrinogen and / or factor VIII and / or VWF is that this minimizes the need to add antifibrinolytic agents to inhibit any residual protease and / or zymogen (eg. example, plasmin or plasminogen). Examples of such agents include aprotinin, a bovine protein inhibitor of plasmin; ortranexamic acid, a synthetic plasmin inhibitor also associated with neurotoxic side effects.

A further advantageous feature is that the plasminogen, which has been separated from the solution comprising fibrinogen and / or factor VIII and / or VWF by HCIC, can be further processed to produce a concentrate containing plasminogen for, for example, clinical use. Thus, HCIC can be used to prepare both plasminogen and solutions comprising fibrinogen and / or factor VIII and / or VWF from a single starting solution.

A further advantageous feature is that the costs of producing the HCIC resin are much cheaper than the cost of the lysine-Sepharose ™ or immobilized lysine resin which are used in affinity chromatography procedures.

A further advantageous feature is that the HCIC could be used to replace the aluminum hydroxide steps (eg, Alhydrogel ™) for the removal of proteases (eg, factor II). Alhydrogel ™ is currently widely used in the commercial production of factor VIII and VWF. However, the material is relatively expensive, usually using hundreds of kg per batch. In addition, alhydrogel ™ often requires manual manipulation and the material is discarded after a single use. On the contrary, the HCIC stages can be completely automated and the resin can be used in the manufacture of multiple batches.

Another advantageous feature is that the HCIC resin is compatible with 1 M NaOH, which can be used for the inactivation and elimination of pathogens, including viruses and prions during column cleaning and resin sanitation procedures.

Liquid preparations derived from the methods of the present invention also have advantages over the use of frozen preparations, which require expensive storage and transportation means and have to be thawed before their immediate use. Even when the fibrinogen and / or factor VIII and / or VWF is stored as lyophilized or frozen preparation, it is advantageous that the reconstituted or thawed protein be stable for a longer time. This is evident, for example, when material has been reconstituted as a precaution for a medical procedure, but its use is not required based on medical considerations. This material is normally discarded, since the fibrinogen is only stable over a short period of time due to the presence of prothrombin and / or t-PA and / or other protease (s).

In particular examples, liquid preparations containing fibrinogen and / or factor VIII and / or VWF are stored as a liquid or lyophilized or frozen preparation.

In another example, a method for purifying fibrinogen is provided, the method comprising the steps of: (i) passing a solution comprising fibrinogen through an ion exchange chromatography resin under selected conditions such that fibrinogen monomer is attached to the resin;

(ii) eluting the fibrinogen monomer from the resin with an elution buffer; Y

(iii) filtering the fibrinogen monomer eluted from step (ii) through a filter having a pore size in the range of from about 15 nm to about 35 nm.

In one example, the solution comprising fibrinogen (step (i)) is recovered after passing a raw material comprising fibrinogen through a hydrophobic charge induction chromatography resin in conditions selected such that the plasminogen and / or tissue plasminogen activator and / or other protease (s) binds to the resin and the fibrinogen passes through the resin.

In one example, the ion exchange chromatography resin is selected from an anion exchange chromatography resin or a cation exchange chromatography resin.

In one example, the anion exchange chromatography resin is a strong anion exchange chromatography resin or a weak anion exchange chromatography resin. In one example, the anion exchange chromatography resin comprises a quaternary amino group. Examples include quaternary alkylamine and quaternary alkyl alkanolamine, or amine, diethylamine, diethylaminopropyl, amino, trimethylammonioethyl, trimethylbenzyl ammonium, dimethylethanolbenzylammonium and polyamine. In some embodiments, the anion exchange chromatography resin is a polymeric support grafted with tertiary or quaternary amines or is a hydroxylated polymeric support grafted with tertiary or quaternary amines. In some embodiments, the anion exchange chromatography resin comprises a polymeric methacrylate support. In one example, the anion exchange chromatography resin is a MacroPrep ™ HQ. In another example, the anion exchange chromatography resin is a GigaCap ™ Q-650M. In other examples, the anion exchange chromatography resin is packaged in a column.

If desired, an anion exchange chromatography membrane can be used in place of an anion exchange chromatography resin. Commercially available anion exchange chromatography membranes include, but are not limited to, Sartobind ™ Q from Sartorius, Mustang ™ Q from Pall Technologies and Intercept ™ Q membrane from Millipore.

In one example, the cation exchange chromatography resin is a strong cation exchange chromatography resin or a weak cation exchange chromatography resin.

Commercially available cation exchange chromatography resins include, but are not limited to, for example, those having a sulfonate-based group (e.g., MonoS, MiniS, Source ™ 15S and 30S, SP Sepharose Fast Flow ™, SP Sepharose High Performance ™ from GE Healthcare, Toyopearl ™ SP-650S and SP-650M from Tosoh, Macro-Prep High ™ S from BioRad, Ceramic HyperD ™ S, Trisacryl ™ M and LS ™ SP and Spherodex ™ LS SP from Pall Technologies, ); a sulfoethyl-based group (e.g., Fractogel ™ SE from EMD, Poros ™ S-10 and S-20 from Applied Biosystems); a sulfopropyl-based group (e.g., TSK ™ Gel SP 5PW and SP-5PW-HR from Tosoh, Poros ™ HS-20 and HS 50 from Applied Biosystems); a sulfoisobutyl-based group (e.g., Fractogel ™ EMD SO3 "from EMD); a sulfoxyethyl group (e.g., SE52, SE53 and Express-Ion S from Whatman), a carboxymethyl-based group (e.g., CM Sepharose Fast Flow ™ from GE Healthcare, Hydrocell ™ CM from Biochrom Labs Inc., Macro-Prep ™ CM from BioRad, Ceramic HyperD ™ CM, Trisacryl ™ M CM, Trisacryl ™ LS CM from Pall Technologies, Matrex Cellufine ™ C500 and C200 from Millipore, CM52 ™, CM32 ™, CM23 ™ and Express ™ - Ion C from Whatman, Toyopearl ™ CM-650S, CM-650M and CM-650C from Tosoh); sulphonic and carboxylic acid based groups (for example, BAKERBOND ™ Carboxy-Sulfon from J.T. Baker); a group based on carboxylic acid (for example, Wp ™ CBX from JT Baker, DOWEX MAC-3 ™ from Dow Liquid Separations, Amberlite ™ Weak Cation Exchangers, DOWEX ™ Weak Cation Exchanger, and Diaion ™ Weak Cation Exchangers from Sigma-Aldrich and Fractogel ™ EMD COO- from EMD); a group based on sulfonic acid (for example, Hydrocell ™ SP from Biochrom Labs Inc., DOWEX ™ Fine Mesh Strong Acid Cation Matrix from Dow Liquid Separations, UNOsphere ™ S, WP Sulfonic from JT Baker, Sartobind ™ S membrane from Sartorius, Amberlite ™ Strong Cation Exchangers, DOWEX ™ Strong Cation and Diaion Strong Cation Exchanger from Sigma-Aldrich); and a group based on orthophosphate (for example, Pl 1 of Whatman). If desired, a cation exchange chromatography membrane can be used in place of a cation exchange matrix, eg, Sartobind ™ S (Sartorius; Edgewood, NY).

In one example, the solution comprising fibrinogen has a pH in the range of about pH 7 to pH 10. In one example, the pH of the solution comprising fibrinogen is about pH 8. In some examples, the chromatography resin of Anion exchange is pre-equilibrated and washed after loading the fibrinogen with buffer / buffers having a pH similar to that of the solution comprising the fibrinogen.

In one example, the elution buffer has a conductivity in the range of from about 18 to about 30 mS / cm. For example, the conductivity of the elution buffer may be in the range of about 18 to about 25 mS / cm; or from about 19 to about 24 mS / cm; or from about 20 to about 24 mS / cm; or from about 21 to about 23 mS / cm. In one example, the conductivity of the elution buffer is approximately 22 mS / cm. In other examples, the elution buffer comprises NaCl. In one example, the elution buffer comprises NaCl at a concentration in the range of about 180 mM to about 230 mM, or from about 190 mM to about 210 mM. In one example, the concentration of NaCl in the elution buffer is about 200 mM.

In one example, the elution buffer comprising the fibrinogen contains a protein concentration of about 0.5 to about 10 mg / ml. In some examples, the protein concentration of the elution buffer comprising the fibrinogen is in the range of from about 4 to about 8 mg / ml. In particular examples, the elution buffer comprising the fibrinogen is about 6 mg / ml.

In one example the eluent fibrinogen monomer is formulated with one or more amino acids before filtering (step (iii)). In one example, the amino acid is arginine or glycine or a combination thereof. In some examples, the concentration of the amino acid in the elution buffer comprising fibrinogen is in the range of from about 0.5 to about 10% (w / w). In one example, the concentration of the amino acid in the elution buffer comprising fibrinogen is from about 1% to about 6% (w / w), or from about 2% to about 6% (w / w) or from about 2% to about 5% (w / w). In one example, the elution buffer comprising the fibrinogen is formulated with about 2% or about 3% or about 4% or about 5% (w / w) of arginine.

In one example, the eluent fibrinogen monomer has a pH of from about pH 7 to about pH 9.

In one example, the stage filter (ii) has a pore size in the range of from about 15 nm to about 35 nm; or from about 15 nm to about 30 nm; or from about 15 nm to about 25 nm; or from about 15 nm to about 20 nm.

Virus filtration can be performed using either tangential flow filtration (TFF) or "terminal" filtration (also known as normal flow filtration). Virus filters were originally designed for use in TFF with the feed flowing adjacent to the upper thin layer of the asymmetric membrane. The TFF provides a high flux by sweeping the surface of the membrane to reduce fouling and polarization by concentration. However, the simplicity and lower capital cost of the terminal filtration has led to the widespread use of virus filters designed specifically for terminal filtering. Unlike TFF, these terminal filters are normally operated with the more open side of the membrane directed towards the feed stream, allowing protein aggregates and other large scale encrustants to be captured within the macroporous substructure thereby protecting the thin layer of virus retention. The advantages of using single-use terminal filters include that they simplify the design as well as the validation of the system, reducing labor and capital costs.

Terminal filtration usually involves using a single pump to force fluid through the membrane from the surface.

Tangential filtration generally requires a first pump to maintain a constant flow rate at the surface of the filter membrane and a second pump removes the protein through the membrane creating a negative pressure at the back of the membrane.

In one example, the filtering is done by terminal filtering.

In one example, the terminal filtering process is performed using either constant pressure filtration or constant velocity filtering. In one example, the terminal filtering process is performed using constant pressure filtration.

The filtration is usually performed with a filtration pressure that is equal to or is below the level that the membrane can withstand, depending on the material of a virus elimination membrane for use herein, for example, with pressures of about 0.2 to about 3.4 bar. In one embodiment, the filtration pressure is maintained between about 0.2 bar and about 3.4 bar. In another example, the filtration pressure is maintained at from about 1 to about 3 bar; or from about 1 to about 2 bar; or from about 1.2 to about 2 bar. In another example, the filtration pressure is maintained at from about 1.5 bar to about 1.9 bar.

The temperature can have an effect on the viscosity of a protein solution and on the flux after filtration with a virus elimination membrane. It will be understood by those skilled in the art that the solution to be used in the filtration step should normally have a temperature in the range of from about 0 ° C to the temperature at which the protein of interest is denatured. The temperature of the solution is suitably within the range of from about 10 ° C to about 50 ° C. In one embodiment, the temperature of the solution is within the range of from about 18 ° C to about 35 ° C. In another embodiment, the solution is filtered at room temperature from about 18 ° C to about 26 ° C.

In one example, the virus filter capacity is at least 0.20 kg or at least 0.50 kg or at least 0.75 kg or at least 1.00 kg or at least 1.25 kg or at least 1 , 50 kg or at least 2 kg of fibrinogen per m2 of filter surface area.

Optionally, a prefiltration or filtration step can be performed by clarification prior to virus filtration in order to eliminate macroscopic size paraffins. In one example, prefiltration is carried out with a prefilter comprising a membrane with a pore diameter larger than that of the virus elimination membrane. In one example, the prefilter is a membrane filter having a pore size of about 0.1 μm. In another example, the prefilter is selected from the Pall Nylon membrane filter (SKL 7002 NTP 0.1 pm or FTKNI), or other commercially available pre-filters that have similar properties for the removal of aggregates and / or protein particulates. The prefiltration can be carried out either in line with the virus filter or outside the line with respect to the virus filter. In one example, the prefiltration is carried out in line with respect to the virus filter.

Those skilled in the art will know filters suitable for the method of virus filtration according to this aspect of the invention. An example includes Planova BioEx ™, among others. Such filters are sometimes called "small virus" elimination filters.

In one example, the filter membrane is a flat sheet or a hollow fiber membrane. Examples of flat sheet membranes include hydrophilized PVDF filter membranes such as Pegasus ™ Grade SV4 Small Virus Removal Filters (Pall Corporation). In one example, the filter is the Pegasus ™ Grade SV4.

In other examples, the filter is a hollow fiber membrane. A hollow fiber membrane can contain a bundle of straw-shaped hollow fibers containing the wall of each hollow fiber a three-dimensional network structure of pores composed of gaps interconnected by fine capillaries. Examples of hollow fiber filters include Planova ™ BioEX ™ filters (Asahi Kasei Corporation) incorporating hydrophilic modified poly (vinylidene fluoride) (PVDF) in a hollow fiber membrane format. In one embodiment, the filter is the Planova ™ BioEX.

In one example, two or more small virus filters are used in series. In one embodiment, the filtration is performed using two series filters having a pore size in the range of from about 15 to about 20 nm. Such filtration steps have the potential to enable fibrinogen to be manufactured with at least LRV 6.9 log LRV for MVM similar to parvovirus.

In another example, the solution comprising fibrinogen is passed through an ion exchange chromatography resin under selected conditions such that the fibrinogen present in the solution passes through the resin. That is, the ion exchange chromatography is performed in negative mode with respect to fibrinogen under conditions such that, when the solution is passed through the resin, impurities such as aggregates of fibrinogen, plasminogen and fibronectin present in the solution are added. (n) to the charged functional groups coupled to the resin, allowing the fibrinogen present in the solution, particularly monomer of fibrinogen, to pass through the resin in the fraction of flow through (through). Once the fraction of flow through which contains fibrinogen passes through the resin, the ion exchange chromatography resin can be washed with a suitable washing buffer known to those skilled in the art. The constituents of the wash buffer and the conditions of the washing step will normally be selected to retain the impurities bound to the resin during the washing step.

In one example, the ion exchange chromatography resin is selected from an anion exchange chromatography resin or a cation exchange chromatography resin.

In one example, the solution comprising fibrinogen is passed through an anion exchange chromatography resin in the presence of NaCl from about 150 mM to about 270 mM. This corresponds to a conductivity range of about 18 mS / cm (150 mM NaCl) to about 29 mS / cm (270 mM NaCl). Under these conditions the fibrinogen, particularly the monomeric form, passes through the anion exchange chromatography resin while the aggregates containing fibrinogen and other impurities such as plasminogen and fibronectin are bound to the resin. In further examples, the solution comprising fibrinogen is passed through the anion exchange chromatography resin in the presence of NaCl from about 170 mM to about 230 mM (from about 19 mS / cm to about 25 mS / cm) or NaCl from about 200 mM to about 220 mM (from about 22 mS / cm to about 24 mS / cm). In these types of conditions, the impurities are expected to bind to the anion exchange chromatography resin, allowing the fibrinogen to pass through the resin in the flow fraction through.

The solution comprising fibrinogen and / or factor VIII and / or VWF recovered by the methods described herein are advantageous because they provide a preparation of fibrinogen and / or factor VIII and / or VWF which has greater stability than the existing lyophilized preparations. , even at room temperature. This can be particularly advantageous on long transport routes, where low temperatures, when appropriate, may not be guaranteed throughout transport and / or storage. The stable storage of fibrinogen and / or factor VIII and / or VWF in solution also facilitates, in many aspects, the production, utilization, transport and administration to a patient in need thereof. Due to the increased stability of the fibrinogen and / or factor VIII and / or VWF prepared according to the methods described herein, it is possible in Many pharmaceutical preparations dispense with the addition of stability agents, such as inhibitors of fibrinolysis or fibrinogenolysis that can, in some circumstances, lead to unwanted side effects or that should be avoided to reduce potential risks.

The term "stable", as used herein, means that there is little or no substantial loss of activity of the fibrinogen and / or factor VIII and / or VWF after a period of time in storage compared to the level of activity of fibrinogen and / or factor VIII and / or VWF before storage (eg, in comparison with the level of activity determined immediately upon recovery of the solution comprising fibrinogen and / or factor VIII and / or VWF according to the methods described in This document). In an example disclosed herein, the solution comprising fibrinogen and / or factor VIII and / or VWF retains at least 70% activity, preferably at least 80% activity, more preferably at least 90% of activity, even more preferably at least 95% activity and most preferably 100% activity after a period of time in storage at a temperature of from about 0 ° C to about 30 ° C. The person skilled in the art will understand that the activity of fibrinogen can be determined for the preparation of fibrinogen immediately before the start of the storage period and this initial value can be used to designate 100% activity from which the fibrinogen activity determined in Different time points during the storage period can be compared and expressed as a percentage of this initial value.

In an example disclosed herein, the fibrinogen recovered by the methods described herein retains from about 90% to 100% activity after at least 4 weeks in storage of the solution at a temperature of about 2. ° C at about 8 ° C, preferably retains about 90% activity after 4 weeks in storage of the solution at a temperature of about 2 ° C to about 8 ° C. In another example disclosed herein, fibrinogen retains from about 60% to about 80% activity after at least 4 weeks in storage of the solution at a temperature of about 30 ° C, preferably retained from about 60% up to about 70% activity after 5 weeks in storage of the solution at a temperature of about 30 ° C. The activity level of the fibrinogen and / or factor VIII and / or VWF can be determined by any means known to those skilled in the art. Examples of suitable methods for determining fibrinogen activity, for example, are summarized by Mackie et al. (British J. Haematol, 121: 396-404, 2003). Particular methods include Clauss (Clauss, 1957, Acta-Haematol, 17, 237-246) and / or coagulable protein (Jacobsson K., Scand J Clin Lab Invest 1955; 7 (supp 14): 1-54 or Fibrin sealant Ph. Eur. Monograph 903, 2012). The results can be reported as% coagulable protein; % of initial coagulable protein, and / or% of initial fibrinogen activity determined using the Clauss method or the like.

The person skilled in the art will understand that it is likely that the concentration of the plasminogen and / or tissue plasminogen activator and / or other protease (s) in the recovered solution comprising fibrinogen and / or factor VIII and / or VWF dictates the storage length and / or storage conditions (eg temperature). For example, it will be understood that a preparation in which the concentration of the plasminogen and / or tissue plasminogen activator and / or other protease (s) in the recovered solution is reduced by 80% in comparison with the raw material may stored for a longer period of time and / or at higher temperatures without significantly destabilizing the activity of the fibrinogen and / or factor VIII and / or VWF in comparison with a preparation in which the concentration of the plasminogen and / or tissue plasminogen activator and / or / or other protease (s) in the recovered solution comprising fibrinogen and / or factor VIII and / or VWF is reduced by only 50% in comparison with the raw material.

Although the methods of the present invention can be performed on a laboratory scale, they can be scaled up to industrial size without significant changes in conditions. Therefore, in an embodiment disclosed in the present document, the methods of the present invention are carried out on an industrial or commercial scale. Preferably, the methods of the invention are suitable for the manufacture on a commercial scale of fibrinogen. For example, when plasma fractions are used as starting material in the method of the invention, then manufacture on a commercial scale involves the use of a plasma fraction derived from at least about 500 kg of plasma. More preferably, the starting plasma fraction will be derived from at least about 5,000 kg, 7,500 kg, 10,000 kg and / or 15,000 kg of plasma per batch. In particular examples, pharmaceutical solutions and formulations comprising fibrinogen and / or factor VIII and / or VWF are manufactured on a commercial scale from a fraction of plasma or a recombinant raw material.

When a solution comprising fibrinogen and / or factor VIII and / or VWF is to be used for clinical or veterinary applications (eg, for administration to a subject with a deficiency of fibrinogen and / or factor VIII and / or VWF or for use as fibrin glue), those skilled in the art will understand that it may be desirable to reduce the level of content of active virus (virus titer) and other potential infectious agents (e.g., prions) in the solution. This may be desirable particularly when the raw material comprising fibrinogen and / or factor VIII and / or VWF (ie, the starting material) is derived from blood plasma. Those skilled in the art will know methods of reducing the virus toll in a solution. Examples include pasteurization (for example, incubating the solution at 60 ° C for 10 hours in the presence of high concentrations of stabilizers such as glycine (for example, 2.75 M) and sucrose (for example, 50%) and / or other selected salts or excipients), dry heat treatment, virus filtration (passing the solution through a nanofilter; for example, cut-off point of 20 nm) and / or by subjecting the solution to treatment with a suitable detergent or organic solvent for a period of time and under conditions to inactivate the virus in the solution. The solvent / detergent has been used for more than 20 years to inactivate enveloped viruses, particularly in plasma-derived products, including fibrinogen and factor VIII and / or VWF. Therefore, it can be carried out using various reagents and methods known in the art (see, for example, US 4540573 and US4764369 which are hereby incorporated by reference). Suitable solvents include tri-n-butyl phosphate (TnBP) and ether, preferably TnBP (typically at about 0.3%). Suitable detergents include polysorbate (Tween) 80, polysorbate (Tween) 20 and Triton X-100 (typically at about 0.3%). The selection of treatment conditions, including concentrations of solvent and detergent, depends in part on the characteristics of the raw material, generally requiring less pure raw materials higher concentrations of reagents and more extreme reaction conditions. A preferred detergent is polysorbate 80 and a particularly preferred combination is polysorbate 80 and TnBP. The raw material can be stirred with solvent and detergent reagents at a temperature and for a time sufficient to inactivate any enveloped viruses that may be present. For example, the solvent / detergent treatment can be carried out for about 4 hours at 25 ° C. The solvent / detergent chemicals are subsequently removed by, for example, adsorption on chromatographic media such as C-18 hydrophobic resins or by eluting them in the coffee fraction through ion exchange resins under conditions that adsorb the protein of interest.

The virus inactivation step can be performed at any stage of the methods disclosed herein. In one example, the raw material comprising fibrinogen and / or factor VIII and / or VWF is subjected to a viral inactivation step before step (i). In another embodiment, the solution comprising fibrinogen that is recovered from the hydrophobic charge induction chromatography resin (i.e., from steps (ii) and / or (iv)) is subjected to a viral inactivation step. In an example disclosed herein, the viral inactivation step comprises pasteurization or treatment with a detergent and organic solvent. In another example disclosed herein, the virus inactivation step comprises virus filtration. When using virus filtration, the inventors have found that the addition of a free amino acid (eg, arginine) prior to the filtration step can significantly improve the flow rate and the recovery of fibrinogen and / or factor VIII and / or VWF through the filter. An example of such a method is described in US7919592.

In an example disclosed herein, the raw material or solution comprising fibrinogen and / or factor VIII and / or VWF is subjected to a viral inactivation step before passing it through the anion exchange chromatography resin. . The advantage of employing a virus inactivation step such as solvent / detergent treatment before passing the solution or raw material treated through an anion exchange chromatography resin is that the anion exchange resin allows the removal of the detergent and organic solvent of the solution treated using conditions that promote the binding of the fibrinogen and / or factor VIII and / or VWF to the resin and the removal of the detergent and organic solvent with the fraction of flow through (through).

Pasteurization can generate protein aggregates and polymers, particularly in a solution comprising fibrinogen (also referred to herein as "fibrinogen solution"). Therefore, it may be desirable in some cases to reduce the level of aggregates / polymers in a pasteurized solution. This can be achieved by any means known to those skilled in the art, although conveniently it can be achieved by an additional chromatographic purification. In an example disclosed herein, the solution or pasteurized raw material is passed through an anion exchange chromatography resin in positive mode with respect to the fibrinogen and / or factor VIII and / or VWF so that eliminates any aggregate or polymer with the fraction of flow through (through).

The term "raw material" is used herein to designate any solution comprising fibrinogen and / or factor VIII and / or VWF. The raw material may also comprise other proteins (e.g., therapeutic proteins) known to those skilled in the art. Examples include proteins involved in the blood clotting cascade. In an embodiment disclosed in the present document, the raw material comprises fibrinogen.

Those skilled in the art will know a suitable raw material comprising fibrinogen and / or factor VIII and / or VWF. Examples include plasma or plasma fractions such as cryoprecipitate of solubilized plasma or solubilized fraction I paste derived from human or animal plasma or a plasma fraction, cell culture fractions of recombinant technology, fractions derived from the milk of transgenic animals, etc. Sources of recombinant fibrinogen proteins are also suitable for use as a raw material according to the present invention. When the raw material is plasma or a fraction of plasma, it can be either plasma donations combined or it can be from an individual donor. In an embodiment disclosed herein, the raw material comprising fibrinogen is a cryoprecipitate of solubilized plasma. This component, either derived from whole blood or collected by means of aferesis, is prepared by controlled thawing of freshly frozen plasma between 1-6 ° C and recovery of the precipitate. The insoluble precipitate in fno freezes again. A unit of cryoprecipitate is equivalent to approximately 2 units of cryoprecipitate derived from whole blood. It contains most of the fibrinogen, factor VIII and VWF along with other proteins such as factor XIII and fibronectin from freshly frozen plasma. An alternative source of fibrinogen is the fraction I precipitate that can be prepared from frozen plasma by thawing and removing the cryoprecipitate by either centrifugation or filtration. The resulting cryosefibrant is then mixed with ethanol to precipitate fraction I. For example, a fraction I precipitate can be obtained by adding ethanol at about 8% (v / v) to pH 7.2 and controlling the temperature to about -3 ° C. (Cohn, et al., 1946, J. Am. Chem. Soc. 62: 459-475). In one embodiment, the raw material comprising fibrinogen is cryoprecipitated.

The reference in the specification to "protease (s)" can be to any protease and / or its zymogen present in the raw material or solution comprising fibrinogen and / or factor VIII and / or VWF which, when exposed to a resin of HCIC, can be bound to HCIC resin under conditions in which the fibrinogen and / or factor VIII and / or VWF passes through the resin. The proteases can be of any type, including serine proteases (e.g., plasmin, thrombin, trypsin), threonine proteases, cysteine proteases (e.g., cathepsin B and cathepsin H), aspartate proteases (e.g., pepsin), metalloproteases (e.g. example, collagenases and gelatinases) and glutamic proteases. When the raw material comprising fibrinogen and / or factor VIII and / or VWF is derived from human or animal plasma, the proteases / zymogens may include plasminogen, tissue plasminogen activator (tPA), thrombin, elastase, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, factor XIIIa, plasma kallikremas and the like. With respect to solutions comprising fibrinogen and / or factor VIII and / or VWF, a particular preferred protease / zymogen to be removed is plasminogen. Other preferred proteases / zymogens to be removed from a solution comprising fibrinogen and / or factor VIII and / or VWF are t-PA, prothrombin and / or active thrombin (factor II / IIa). When the feedstock comprising fibrinogen and / or factor VIII and / or VWF is derived from cell culture supernatant, the protease / zymogen can include any host cell protease, such as serine proteases (eg, caseinases), metalloproteases (eg. example, gelatinases, matrix metalloproteases (MMP), including MMP3, MMP10 or MMP12), aspartic proteases (cathepsin D), acid proteases, among others.

In some cases, it may be desirable to eliminate or reduce the level of impurities in the raw material before passing the raw material through an HCIC resin in step (i). Eliminating or reducing the level of impurities in the raw material can reduce the load on the HCIC resin during the chromatographic purification and thus improve the efficiency of separation of plasminogen and / or activator of tissue plasminogen and / or other protease (s). ) of the raw material. The impurities can be eliminated or reduced, for example by precipitating fibrinogen and / or factor VIII and / or VWF from the raw material and recovering the precipitated protein (s). Those skilled in the art will know of suitable methods of precipitating fibrinogen and / or factor VIII and / or VWF from a feedstock comprising fibrinogen and / or factor VIII and / or VWF. An example includes adding suspension of aluminum hydroxide to the raw material, which is particularly useful for removing vitamin K-dependent proteins (e.g., coagulation factors II, VII, IX, and X) and other proteins having an affinity for binding by aluminum hydroxide, such as prothrombin (factor II) and t-PA, plasma or plasma cryoprecipitate.

Therefore, in an example disclosed herein, prior to step (i), the vitamin K dependent proteins are eliminated or reduced from the raw material. In a further example, the vitamin K dependent proteins are eliminated or reduced by the addition of aluminum hydroxide to the raw material. The aluminum hydroxide may be in the form of Alhydrogel®, added to the raw material to a final concentration of about 10% to about 80% w / w. In some examples, aluminum hydroxide is added to the raw material to a final concentration in the range of from about 10% to about 50% (w / w). In other examples, aluminum hydroxide is added to the raw material to a final concentration in the range of from about 10% to about 30% (w / w). In preferred examples, the concentration is from about 15% to about 30% (w / w). Most preferably, the aluminum hydroxide is added to the raw material at about 15% to about 25% (w / w) for optimal fibrinogen recovery and removal of impurities such as prothrombin. In another example, the vitamin K dependent proteins are removed from the raw material by batch adsorption using aluminum hydroxide.

In another example, a solution comprising fibrinogen and / or factor VIII and / or VWF is provided which is recovered by the method described herein. In one example, the level of plasminogen and / or tissue plasminogen activator and / or other protease (s) in the solution will be less than 20% of the total protein, preferably less than 10% of the total protein and more preferably less than 5% of the total protein, or less than 1% of the total protein or less than 0.1% of the total protein or less than 0.01% of the total protein or less than 0.001% of the protein total or less than 0.0001% of the total protein. The person skilled in the art will understand that the level of plasminogen and / or tissue plasminogen activator and / or other protease (s) that is present in the solution comprising fibrinogen and / or factor VIII and / or VWF may depend on the intended use of the solution or storage duration. For example, when the solution must be stored for at least 4 weeks at a temperature of about 0 ° C to about 8 ° C, it may be acceptable for the solution to comprise more than about 10% of plasminogen and / or tissue plasminogen activator and / or other protease (s) (of the total protein). When the solution should be stored for at least 4 weeks at a temperature of about 30 ° C, it may be desirable for the solution to comprise less than approximately 10% of plasminogen and / or tissue plasminogen activator and / or other protease (s) (of the total protein).

In an example disclosed herein, a solution comprising fibrinogen recovered by the method described herein is provided. In another example, the solution comprises at least 80% of the total fibrinogen protein.

In another example, a solution is provided comprising:

(a) at least 75% of the total fibrinogen protein;

(b) less than 50 pg / mg of the total tissue plasminogen activator protein; I

(c) less than 1 pg / mg of the total plasminogen protein.

In one example, the solution also comprises less than 1.5 x 10-5 U / mg of the total factor II protein.

In another example, a solution is provided comprising:

(a) at least 90% of the total fibrinogen protein;

(b) less than 50 pg / mg of the total tissue plasminogen activator protein; I

(c) less than 150 ng / mg of the total plasminogen protein.

In one example, the solution also comprises:

(a) less than 3.5 x 10-6 U / mg of the total factor II protein; I

(b) less than 150 pg / mg of the total fibronectin protein.

In another example, a solution is provided comprising:

(a) at least 90% of the total fibrinogen protein;

(b) less than 50 pg / mg of the total tissue plasminogen activator protein; I

(c) less than 10 ng / mg of the total plasminogen protein.

In another example, a solution is provided comprising:

(a) at least 90% of the total fibrinogen protein;

(b) less than 20 pg / mg of the total tissue plasminogen activator protein; I

(c) less than 10 ng / mg of the total plasminogen protein.

In one example, the solution also comprises:

(a) less than 2.7 x 10-6 U / mg of the total factor II protein; I

(b) less than 15 pg / mg of the total fibronectin protein.

The concentration of the fibrinogen and / or factor VIII and / or VWF in the solution recovered by the methods disclosed herein and the concentration of impurities (eg, plasminogen and / or tissue plasminogen activator and / or other (s) protease (s)) can be measured by any means known to those skilled in the art. Examples of suitable assays for measuring fibrinogen are described by Mackie et al. (Br J Haematol, May 2003; 121 (3): 396-404). The exclusion HPLC by size can also be used to measure the concentration of fibrinogen and / or factor VIII or an impurity in the solution comprising fibrinogen and / or factor VIII (for example, Cardinali et al., 2010, Arch. Biochem. Biphys. 493 (2): 157-168; and Kosloski et al., 2009, AAPS J.

11 (3); 424-431). HPLC also allows the person skilled in the art to discriminate between monomers and fibrinogen aggregates. In addition, the concentration of fibrinogen and / or factor VIII and / or VWF may differ depending on the sensitivity of the assay that is used. For example, the concentration of fibrinogen in a solution measured using the Clauss assay may be slightly lower than the concentration measured in the same solution by HPLC.

In an example disclosed herein, the concentration of monomeric fibrinogen in the solution is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of Total protein measured by HPLC exclusion by size.

In another example, a pharmaceutical formulation comprising a solution comprising fibrinogen and / or factor VIII and / or VWF recovered by the methods disclosed herein, and a pharmaceutically acceptable carrier is provided. Those skilled in the art will know suitable pharmaceutically acceptable carriers, including diluents and / or pharmaceutically acceptable excipients. Examples include solvents, dispersion media, antifungal and antibacterial agents, surfactants, isotonic and absorption agents and the like.

The pharmaceutical formulation can also be formulated by the addition of a combination of suitable stabilizers, for example, an amino acid, a carbohydrate, a salt and a detergent. In particular examples, the stabilizer comprises a mixture of a sugar alcohol and an amino acid. The stabilizer may comprise a mixture of a sugar (e.g., sucrose or trehalose), a sugar alcohol (e.g., mannitol or sorbitol), and an amino acid (e.g., proline, glycine and arginine). In a preferred example, the formulation comprises an amino acid such as arginine. In other examples, the formulation comprises divalent metal ions in a concentration of up to 100 mM and a complexing agent described in US7045601. Particular examples include formulations 1 to 7 described in Example 1 of US7045601. In particular examples, the formulation is formulated without the addition of any antifibrinolytic agent or stabilizing protein such as albumin. In examples where the formulation comprises fibrinogen, the pH is preferably from about 6.5 to 7.5 and the osmolality is at least 240 mOsmol / kg.

The pharmaceutical formulation can also be sterilized by filtration before dispensing and long-term storage. Preferably, the formulation will substantially retain its original stability characteristics for at least 2, 4, 6, 8, 10, 12, 18, 24, 36 or more months. For example, formulations stored at 2-8 ° C or 25 ° C can usually retain substantially the same molecular size distribution as measured by HPLC-SEC when stored for 6 months or more. Particular examples of the pharmaceutical formulation can be stable and suitable for commercial pharmaceutical use for at least 6 months, 12 months, 18 months, 24 months, 36 months or even more when stored at 2-8 ° C and / or room temperature .

The pharmaceutical solutions and formulations, as described herein, can be formulated into one of many possible dosage forms, such as injectable formulations. The formulations and their subsequent administration (dosing) are within the knowledge of those skilled in the art. The dosage depends on the response of the subject to the treatment, but will invariably last as long as the desirable effect is required (for example, a return to normal plasma levels of fibrinogen). Those of ordinary skill in the art can easily determine optimal dosages, dosing methodologies and repetition rates.

In an example disclosed herein, the pharmaceutical formulation has a volume of at least 5 ml and comprises at least 5 mg / ml of fibrinogen. In another example, the pharmaceutical formulation has a volume of at least 5 ml and comprises at least 20 mg / ml of fibrinogen. In particular embodiments, the pharmaceutical formulation has a volume of at least 5 ml and comprises fibrinogen at a concentration of about 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 90 mg / ml or 100 mg / ml. In another example, a container containing at least 5 ml of a pharmaceutically acceptable fibrinogen solution is provided, the fibrinogen concentration being at least 20 mg / ml.

In another example, a method of treating or preventing a condition associated with fibrinogen and / or factor VIII and / or VWF deficiency is provided, the method comprising administering to a subject in need thereof a solution comprising fibrinogen and / or factor. VIII and / or VWF recovered by the method, as disclosed herein, or the pharmaceutical formulation, as disclosed herein.

In another example, the use of a solution comprising fibrinogen and / or factor VIII and / or VWF recovered by the method, as disclosed herein, is provided in the manufacture of a medicament for treating or preventing a state associated with fibrinogen and / or factor VIII and / or VWF deficiency. Those skilled in the art will be familiar with the types of conditions associated with fibrinogen and / or factor VIII and / or VWF deficiency. In one example, the fibrinogen status is selected from the group consisting of afibrinogenemia, hypofibrinogenemia and dysfibrinogenemia. In one example, the factor VIII and / or VWF status is selected from the group consisting of hemophilia A, bleeding disorders (e.g., defective platelet function, thrombocytopenia or von Willebrand disease), vascular injury, bleeding from trauma or surgery, bleeding due to anticoagulant therapy, bleeding due to liver disease.

Other conditions that can be treated with a solution comprising fibrinogen and / or factor VIII and / or VWF recovered by the methods, as disclosed herein, include wounds. important and hemorrhages and severe burns. In cases of hypofibrinogenemia and afibrinogenemia, a solution comprising fibrinogen prepared by the methods described herein can be injected intravenously into the patient who needs it in order to compensate for the state of fibrinogen deficiency and the skilled in the art. technique can determine the dosages based on the degree of deficiency.

A solution comprising fibrinogen recovered by the methods disclosed herein also has advantages in the use of fibrin glue (also known as fibrin sealants) due to the absence of destabilizing levels of plasminogen and / or tissue plasminogen activator. and / or other protease (s). The tPA converts the plasminogen into its active form plasmin, which in turn digests the fibrin clot and thus reduces the formation of clots after topical application (for example, for hemostasis).

Fibrin glues typically comprise two components: (i) fibrinogen (often together with factor XIII and a fibrinolysis inhibitor such as aprotinin), and (ii) thrombin (often together with calcium ions). The two components are reconstituted in order to prepare the glue ready for use. Fibrin glue is used in clinical and veterinary applications to simulate the last coagulation step through the formation of cross-linked fibrin fibers using a combination of fibrinogen with thrombin in the presence of calcium and factor XIII. Fibrin glue has various applications in clinical and veterinary medicine, including hemostasis, wound closure, adhesion prophylaxis and wound healing. Fibrin glue can also be used to close skin wounds (including skin transplants), to seal sutures and to join connective tissues, such as bone, carotlage and tendons. Therefore, in another example disclosed herein, a fibrin glue comprising the solution comprising fibrinogen recovered by the methods is provided, as disclosed herein.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the invention includes all such variations and modifications that fall within the scope and scope. The invention also includes all the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Now certain embodiments of the invention will be described with reference to the following examples which are intended solely for the purpose of illustration and are not intended to limit the scope of the generality described hereinabove.

EXAMPLES

Example 1 - Purification of fibrinogen through hydrophobic charge induction chromatography resin (HCIC) HEA, PPA and MEP

Cryoprecipitate of human combined plasma was used as starting material (ie, raw material containing fibrinogen). Briefly, the combined plasma cryoprecipitate was solubilized in an extraction buffer containing 20 mM trisodium citrate, 200 mM epsilon-aminocaproic acid (e-ACA), 60 IU / ml heparin and 500 mM NaCl (pH 7.2 ± 2) at 31 ± 2 ° C for 30 minutes (1 g cryoprecipitate per 4 g of buffer). Then 2% (w / w) aluminum hydroxide was added to the cryoprecipitate solubilized at a concentration of 25% (w / w), after which the aluminum hydroxide gel was removed by either centrifugation or deep filtration and recovered the supernatant containing fibrinogen for further chromatographic purification through an HCIC chromatography resin.

The supernatant containing fibrinogen was applied to chromatography columns that had been packaged with 1.8 ml of resin or HEA, PPA or MEP Hypercel ™. The chromatography columns were pre-equilibrated in 25 mM Tris at a different pH, ranging from 6.5 to 8.5. The supernatant containing fibrinogen was loaded onto the chromatography column at a rate of approximately 11 ml / ml resin. The purification by HCIC was carried out in negative mode with respect to fibrinogen, in which the fibrinogen was allowed to fall through in the fraction of flow through unbound, while the majority of t-PA, plasminogen and factor II remained attached to the resin.

Figures 1 to 3 show the stepwise recovery of fibrinogen, plasminogen, t-PA and factor II after chromatographic purification using h Ea Hypercel ™, Hypercel ™ PPA and Hypercel ™ MEP. The results show that the pH has little or no effect on the binding of plasminogen to these resins, while the binding of t-PA to the resins seems to be most effective in the lower pH range. The HEA Hypercel ™ column showed the highest fibrinogen recovery in the coffee fraction through and the operating pH range tested had little effect on fibrinogen recovery compared to that observed for both PPA and MEP Hypercel ™ columns. The columns both PPA and MEP showed the highest fibrinogen recovery in the coffee fraction through pH 8.5.

Example 2 - Level of impurities in a reduced fibrinogen solution through HEA Hypercel Approximately 48.5 ml of solubilized cryoprecipitate containing fibrinogen prepared according to example 1 was applied on a 5 ml HEA Hypercel ™ column which had been pre-equilibrated in Tris. 25 mM at pH or 6.5, 7.0, 7.5, 8.0 or 8.5. Purification was performed by HCIC in negative mode with respect to fibrinogen, in which the fibrinogen was allowed to fall through in the fraction of flow through unbound, while the t-PA, plasminogen and factor II remained attached to the resin. Figure 4a shows the stepwise recovery of fibrinogen, plasminogen, t-PA and factor II during post-chromatographic purification using HEA Hypercel ™. The results show that the pH had little or no effect on the binding of plasminogen and factor II to the HCIC resin, whereas the binding of t-PA to the resin seemed to be most effective at the lower pH range of 6.5 -7.0. The fibrinogen recovery was greater than 90% in the coffee fraction through different pH conditions despite not performing any column washing. As shown in Figure 4a, these results demonstrate the effective removal of proteases in a crude fibrinogen-containing raw material, such as solubilized cryoprecipitate, by the HCIC resin. For example, the experimental condition performed at pH 7.0 showed a reduction of> 99.9% for factor II, 88.3% for t-PA and> 98.2% for plasminogen from the solution of cryoprecipitate solubilized.

The fibrinogen remained stable in solution for at least 6 days at room temperature (approximately 20 ° C). The results are presented in Figures 4b and 4c.

Example 3 - Level of impurities in a solution of purified fibrinogen through HEA Hypercel Approximately 500 ml of the supernatant containing fibrinogen obtained after the adsorption step of alhydrogel ™ generated according to example 1 was loaded onto an XK 16/30 column packed with 36 ml of HEA Hypercel ™ resin, pre-equilibrated in 25 mM Tris pH 7.0. The fraction of the pass through was collected to test the fibrinogen, plasminogen, t-PA and factor II. A summary of the results is given in Table 1 below.

Table 1

Example 4 - The effect of glycine precipitation on the level of impurities in a purified fibrinogen solution through HEA Hypercel

The supernatant containing fibrinogen after the adsorption step of alhydrogel generated according to Example 1 was subjected to an additional precipitation step adding a saline solution comprising 2.4 M glycine, 2.7 M NaCl, 22.1 mM CaCl and citrate 23 mM trisodium (pH 6.6-7.3). The solubilized precipitate was heated to 30 ° C before being added to the glycine buffer, which is also incubated up to 30 ° C, at a product ratio with respect to 1: 2 buffer. The mixture was allowed to stir for 10 minutes and the resulting precipitate was recovered from the liquid phase by centrifugation. The liquid phase, which predominantly comprises fibronectin and IgG, was discarded and the precipitate containing fibrinogen was collected and resuspended in a solubilization buffer containing 100 mM NaCl, 21.1 M CaCl, 10 mM trisodium citrate, Tris- (hydroxymethylmethylamine ) 10 mM and 4.5 mM sucrose (pH 7.0). The solubilized fibrinogen intermediate (250 ml) was clarified using a 1 μm filter before being passed through an XK 16/30 column packed with 36 ml of HEA Hypercel ™ resin pre-equilibrated in 25 mM Tris pH 7.0. The fraction of the pass through was collected to test the fibrinogen, plasminogen, t-PA and factor II and a summary of the results is given in table 2 below.

The results demonstrate that the binding of plasminogen, t-PA and factor II to the HEA Hypercel ™ resin was more effective under these processing conditions compared to that observed under the conditions described in example 2. The characterization results show a recovery by stages of approximately 93% for fibrinogen, 6% for plasminogen, 12% for t-PA and 5% for factor II.

Table 2

Example 5 - Preparation of purified fibrinogen from plasma cryoprecipitate

Process step 1 - solubilization of plasma cryoprecipitate;

Process step 2 - Adsorption of Alhydrogel ™ (aluminum hydroxide) (concentration of Alhydrogel ™: target of 15 to 20% w / w, ranging from 10 to 50% w / w) of solubilized plasma cryoprecipitate and recovery of supernatant containing fibrinogen using methods such as centrifugation or deep filtration in the presence of a filter aid. Alternatively, this step can be replaced by either a HCIC chromatography step in negative mode (pass through) with respect to fibrinogen or a combination of HCIC chromatography and anion exchange both in negative mode with respect to fibrinogen. If both HClC and anion exchange chromatography are used in combination, then process step 3 is optional;

Process step 3 - Precipitation of fibrinogen glycine from the supernatant containing fibrinogen from step 2. Alternatively, this step can be replaced by anion exchange chromatography in negative mode with respect to the fibrinogen;

Process step 4 - passing the solubilized glycine precipitate from step 3 through a HCIC chromatography resin in negative mode with respect to fibrinogen;

Process step 5 - treat the purified fibrinogen solution recovered in step 4 with solvent or detergent or pasteurization to inactivate pathogens;

Process step 6 - passing the treated solution of step 5 through an anion exchange chromatography resin in positive mode with respect to fibrinogen, washing weakly bound proteins from the resin and eluting the fibrinogen from the resin;

Process step 7 - subjecting the fibrinogen eluted from the anion exchange resin in step 6 to nanofiltration (35 nm or 20 nm or a combination of 35/20 nm); Y

Process step 8 - subjecting the filtered fibrinogen of step 7 to ultrafiltration (membrane filters of 50, 100, 200 and 300 kDa).

Example 6 - Preparation of purified fibrinogen by the combination of HCIC and anion exchange chromatography

Three laboratory-scale experiments were completed in which approximately 100 g of cryoprecipitate was prepared according to the steps described in Examples 1 to 3 to the mixed-mode chromatography step using an HEA Hypercel ™ column.

Slurry fraction through the HEA Hypercel ™ column, which predominantly contains fibrinogen, was subjected to a solvent / detergent treatment overnight for virus inactivation.

The inactivated solution for the viruses was then diluted to <10 mS / cm using 25 mM Tris (pH 8.0) before being loaded onto an anion exchange column (XK 50/30, GE Healthcare), packaged with approximately 412 ml of MacroPrep ™ -HQ resin that had been pre-equilibrated with 25 mM Tris (pH 8.0). The flow fraction was discarded and the MacroPrep ™ -HQ column was washed with 4 column volumes of a wash buffer containing 90 mM NaCl, 50 mM Tris, 20 mM EACA (pH 8.0). Under these chromatographic conditions, the initial flow fraction and the washing fractions predominantly contain plasminogen and t-PA, while the fibrinogen remains bound to the chromatographic resin. The monomeric form of fibrinogen was eluted selectively from the MacroPrep ™ -HQ column using an elution buffer comprising 200 mM NaCl, 10 mM Tris, 10 mM trisodium citrate, 46 mM sucrose and 21.1MM CaCl (pH 7.0) , leaving aggregates of fibrinogen and low molecular weight proteins bound to the MacroPrep ™ -HQ resin.

The product intermediates generated from the cryoprecipitate solubilized to the MacroPrep ™ -HQ chromatography elutant were characterized for the levels of fibrinogen, t-PA, plasminogen, fibronectin and factor II and the process stage recoveries for each of these proteins in the various phases of the process. The results are shown in Table 3, which represent an average value of three batches of consistency at independent laboratory scale. The overall process recovery for fibrinogen and purified proteins together from plasma cryoprecipitate to the MacroPrep ™ -HQ eluate is shown in Figure 5. The purity of fibrinogen that was recovered from the MacroPrep ™ -HQ chromatography resin was greater than 95% , as revealed by HPLC exclusion chromatography by analytical size. A representative of the HPLC profile of fibrinogen analyzed in TSKGel ™ G4000SWXL (Tosoh Corporation) is shown in Figure 6. As shown in Figure 6, the fibrinogen monomer is eluted at a retention time of approximately 17.4 minutes. and contributed to 96.6% of the total peak area, while the fibrinogen dimer and / or other high molecular weight proteins are eluted at a retention time of approximately 14.8 minutes.

Additional batches of fibrinogen were manufactured on a pilot scale (81 kg of plasma equivalent) following the process described in Examples 5 and 6. The characteristics of the fibrinogen preparations are given in Table 4.

Example 7 - Filtration of purified fibrinogen virus

The filtration capacity was examined through a 20 nm virus filter for fibrinogen preparations obtained following the method of Example 6, with the MacroPrep ™ -HQ elution step being used or 190 mM NaCl (21.5mS / cm) , 200 mM NaCl (22.5 mS / cm), 10 mM NaCl (23.5 mS / cm) or a 200 mM NaCl buffer containing 1% (w / w) of arginine (25 mS / cm).

The method involved formulating the preparations with 3% (w / w) of arginine at a pH of about 7.5 (the protein concentration of the samples was about 6 g / l). The formulated preparations were then filtered using a 0.1 μm filter before the virus filtration step. The virus filtration step was carried out using a 47 mm Pall SV4 ™ filter in terminal mode using a constant pressure of 1.8 bar. The results indicate that the fibrinogen preparations obtained from the MacroPrep ™ HQ column using either 190 mM, 200 mM or 210 mM resulted in similar filtration characteristics. In contrast, the preparation of fibrinogen eluted from the MacroPrep ™ HQ column using 200 mM NaCl buffer containing 1% (w / w) of arginine rapidly soiled the filter (Figure 7).

Example 8 - Stability study

The purified fibrinogen solution recovered by the method described in Example 5 above was subjected to sterile filtration and a stability study over a period of 9/7 weeks at 2 ° -8 ° C or 30 ° C. The preparation of liquid fibrinogen set at 2 ° -8 ° C retained approximately 90% of its original activity measured by the Clauss method after the storage period of 9 weeks. The preparation of liquid fibrinogen set at 30 ° C retained approximately 70% of its original activity after the storage period of 2 weeks and did not lose more activity for at least 5 weeks. A further reduction in activity to less than 60% was observed at week 7 of incubation at 30 ° C. The loss of fibrinogen activity at 30 ° C can probably be attributed to thermal denaturation, rather than to proteolytic degradation, since the addition of a protease inhibitor (C1 esterase) did not inhibit the loss of fibrinogen activity throughout of the storage period of 5 weeks. A summary of the stability data is provided in Figure 8.

Table 3

Table 4

Example 9 - Reduction of plasma protease levels in a solution containing factor VIII and / or VWF using HEA Hypercel

This example demonstrates that the HCIC chromatography step can also be used to reduce proteases in preparations containing factor VIII and / or VWF. The method involved clarifying the solution containing fibrinogen obtained from Example 4 using a deep filter before then passing the clarified solution through a second hydrophobic charge induction chromatography (HCIC) resin. The HCIC step was operated under conditions to allow proteases such as plasminogen to bind to the resin, while factor VIII and VWF could pass through the resin. In particular, an XK 50/30 column was packed with 340 ml of HEA Hypercel ™ resin. The column was pre-equilibrated in 50 mM Tris pH 6.7. The clarified fibrinogen solution prepared according to Example 4 was then loaded onto the column and the column washed with 50 mM Tris pH 6.7. The coffee fraction was collected through and the levels of factor VIII, VWF, plasminogen and t-PA were determined (VWF: RCo = von Willebrand factor-cofactor of ristocetin). A summary of the results is provided average obtained from 4 individual passes in Table 5. The results indicate that the HCIC chromatography step effectively removed proteases such as plasminogen and tPA from the fraction comprising factor VIII and VWF. In addition, good recoveries of factor VIII and VWF were observed.

Table 5

Example 10 - Comparative study of purified fibrinogen from different methods

The preparation of fibrinogen manufactured according to the method described in Example 6 was compared with fibrinogen preparations manufactured by the methods described in WO2001048016, WO2012038410 and WO2013135684.

(a) Preparation of fibrinogen manufactured by a preferred embodiment of the methods described in WO2001048016.

Briefly, the method involved suspending paste from fraction I in extraction buffer (0.8 M NaCl, 5 mM EACA (epsilon-aminocaproic acid), 20 mM Na citrate, 60 IU / ml heparin, pH 7.3) (1 g of fraction I with respect to 8.33 g of extraction buffer). The solution was then mixed at 37 ° C for 1.5 hours before adding 50 g of a 2% Al (OH) 3 (Alhydrogel) solution per gram of fraction I (10.8%). The mixture was stirred for 15 minutes at room temperature and then centrifuged at 5000 g for 10 minutes, discarding the pellet. To the supernatant treated with Alhydrogel was added a glycine / NaCl buffer (2.1 M glycine, 20 mM Na citrate, 3.6 M NaCl and 2.4 mM CaCl 2) (both solutions were pre-equilibrated to 30 ° C). The addition of the supernatant to the buffer was completed in about 4.5 minutes (1 part of supernatant with respect to 2.05 parts of buffer). The mixture was then stirred for 20 minutes at 30 ° C before being centrifuged for 10 minutes at 5010 g (the supernatant was discarded). Then the precipitate was solubilized in buffer D (100 mM NaCl, 1.1 mM CaCl2, 10 mM Na citrate, 10 mM tris, 45 mM sucrose, pH 6.9) at room temperature mixing for 2 hours (1/3 of the volume of the one used to resuspend fraction I) (example 1, sections 1.1.1 1.1.6, WO0148016). The solution containing resolubilized fibrinogen is then loaded onto a MacroPrep ™ HQ column (XK26 with a bed height of 20 cm). The column was pre-equilibrated with at least 1.5 column volumes (CV) of MQ buffer (50 mM Tris, 100 mM NaCl, 20 mM EACA, pH 8.0 at 10 ml / min (113 cm / h). equilibrated until the conductivity after the column was 90-110% of the prepared buffer.Then the fibrinogen solution was loaded in the column and the column was washed with 6 CV of MQ buffer.The fibrinogen eluted as a single peak using buffer ME (500 mM NaCl, 1.1 mM CaCl 2, 10 mM Na citrate, 10 mM Tris, and 45 mM sucrose, pH 7.0) The column can be regenerated using 2CV of 1 M NaCl (Example 2, WO2001048016).

(b) Preparation of fibrinogen manufactured by a preferred embodiment of the methods described above in WO2012038410 and WO2013135684.

The cryoprecipitate, produced from plasma by the established methods, was reconstituted or solubilized at approximately neutral pH, subjected to adsorption with Al (OH) 3 and the resulting gel was removed by centrifugation. The supernatant is then inactivated for virus by treatment with solvent / detergent (S / D). The compounds of S / D, Triton and TnBP were extracted with vegetable oil and the aqueous phase was contacted with Fractogel® EMD-TMAE. Chromatographic conditions (pH value of 6.9-7.1 and an osmolality of 570-610 mosmol / 1) were used in which the fibrinogen was not added to the gel and therefore was in the flow through or supernatant. The unbound fibrinogen solution was stirred for approximately 90 minutes after the addition of glycine (final concentration of 1 mol / l and pH = 7.4) in the presence of EDT ^at 20 mM to precipitate the fibrinogen. The precipitate containing fibrinogen was then separated by centrifugation, producing an intermediate fibrinogen paste. The intermediate fibrinogen paste was resuspended in 20 mM Tris buffer (pH = about 8.0). The obtained suspension was filtered and then subjected to ultra / diafiltration. The resulting fibrinogen containing solution was then loaded into GigaCap Q-650M® and the gel or chromatographic resin was pre-equilibrated with the same Tris buffer used for resuspension before applying the fibrinogen solution. The loosely bound substances were washed with the equilibration buffer followed by washing with a washing buffer (sodium citrate 1.5 g / 1, sodium chloride 6.0 g / 1, adjusted to approximately pH = 7.0 and a conductivity of approximately 12.0 mS / cm). Then the fibrinogen was eluted from the chromatographic column with a buffer of Elution (sodium citrate 1.5 g / 1 and glycine 10.0 g / 1, adjusted to the same pH as the washing buffer and adjusted with NaCl approximately 7.0 g / 1 to the conductivity of 13.1-15 mS / cm.).

The solutions containing fibrinogen obtained from the methods described in WO2001048016, WO2012038410 and WO2013135684 were tested to determine the levels of total protein (biuret), fibronectin and plasminogen. In addition, samples were placed in the stability test at 2-8 ° C and 30 ° C.

A comparison of the properties of the fibrinogen preparations is provided in Table 6. The results of the study indicate that the fibrinogen manufactured from the methods described herein contains lower levels of plasminogen in comparison with the other methods.

Table 6

Claims (10)

A method of reducing the level of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator in a solution comprising at least one protein selected from the group consisting of fibrinogen, the method comprising: (i) passing a raw material comprising fibrinogen through a hydrophobic charge induction chromatography resin under selected conditions so that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator present in the raw material it binds to the resin; and (ii) recovering a solution comprising the fibrinogen passing through the resin, reducing the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator in the solution by at least 50% in the comparison with the raw material; the hydrophobic charge induction chromatography resin being equilibrated at a pH of from about 6.5 to about 8.5 before passing the raw material and / or the solution comprising the fibrinogen through the induction chromatography resin. hydrophobic charge. 2. The method according to claim 1, further comprising passing the solution comprising the fibrinogen recovered in step (ii) through an anion exchange chromatography resin. 3. The method according to claim 2, wherein the solution comprising the fibrinogen is passed through the anion exchange chromatography resin in the presence of about 150 mM NaCl to about 270 mM. 4. The method according to any one of claims 2 to 3, wherein the fibrinogen is eluted from the anion exchange chromatography resin with an elution buffer comprising NaCl from about 150 mM to about 300 mM. 5. The method according to any one of claims 1 to 4, wherein the raw material is subjected to a viral inactivation step before step (i). 6. The method according to any one of claims 1 to 5, wherein the solution comprising the fibrinogen recovered from the hydrophobic charge induction chromatography resin is subjected to a viral inactivation step. 7. - The method according to any one of claims 1 to 6, wherein the raw material is a cryoprecipitate of solubilized plasma. 8. - The method according to any one of claims 1 to 7, wherein, prior to step (i), vitamin K-dependent proteins are eliminated or reduced from the raw material. 9. The method according to any one of claims 1 to 8, wherein the raw material or solution comprising the fibrinogen, which is passed through the hydrophobic charge induction chromatography resin has a pH of from about 6.5 to approximately 8.5. 10. The method according to any one of claims 1 to 9, wherein the hydrophobic charge induction chromatography resin comprises a ligand selected from the group consisting of mercaptoethyl pyridine, n-hexylamine and phenylpropylamine.