Methods for purifying therapeutic proteins

Abstract

To provide: a solution having at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases; and a pharmaceutical formulation comprising the solution.SOLUTION: A solution comprises: (a) fibrinogen in an amount of at least 90% of the total protein; (b) a tissue plasminogen activator in an amount of less than 50 pg / mg of the total protein; and (c) plasminogen in an amount less than 10 ng / mg of the total protein. The fibrinogen retains (i) about 90% to 100% activity after the solution is stored for at least 4 weeks at a temperature of about 0°C to about 8°C, and / or (ii) about 60% to about 70% activity after the solution is stored for 5 weeks at a temperature of about 30°C.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates generally to methods for reducing the level of impurities in solutions containing at least one therapeutic protein and to the resulting therapeutic protein-containing solutions. More specifically, the present invention relates to methods for reducing the levels of plasminogen and / or tissue plasminogen activator and / or other proteases in raw materials containing fibrinogen and / or Factor VIII and / or von Willebrand factor (VWF). The present invention also relates generally to solutions and pharmaceutical formulations containing fibrinogen and / or Factor VIII and / or VWF recovered by such methods and their uses. [Background technology]

[0002] Current methods for purifying naturally occurring or recombinant therapeutic proteins from solutions containing the proteins typically result in at least some impurities being carried over into the final preparation. In some cases, the presence of impurities such as proteases can compromise the stability of the therapeutic protein in solution, particularly during storage. For this reason, many therapeutic proteins are stored as lyophilized or frozen preparations.

[0003] Stability-compromising levels of impurities can affect many different types of therapeutic proteins, and this is particularly problematic for therapeutic proteins used to maintain hemostasis. Hemostasis is an important physiological process that prevents bleeding after injury to a blood vessel (e.g., rupture). There are three basic mechanisms that promote hemostasis: (i) vasoconstriction, (ii) platelet aggregation at the site of rupture; and (iii) blood coagulation. During blood clotting, damaged endothelial cells release tissue factor (factor III), which in turn induces Ca 2+Factor XII, released by activated platelets, activates factor VII with the help of factor Xa. Activated factor XII and factor XI promote a cascade of enzymatic reactions that leads to the activation of factor X. Active factor X (factor Xa) activates factor III, factor V, and Ca. 2+ and platelet thromboplastin factor (PF3), activates prothrombin activators. Prothrombin activators convert prothrombin to thrombin, which converts fibrinogen (factor I) to fibrin, which forms the initial meshwork that covers the injury site. The initial meshwork is then converted by factor XIII into a dense fibrin clot, sealing the tear until the site is repaired. In the blood coagulation cascade, thrombin also activates factor VIII, a glycoprotein cofactor precursor that forms a complex primarily with von Willebrand factor (VWF) in the circulation. Factor VIII interacts with factor IXa to induce Ca +2 and activates factor X in the presence of phospholipids.

[0004] Deficiencies in the levels of one or more proteins associated with blood coagulation, including fibrinogen, factor VIII, and / or von Willebrand factor (VWF), whether congenital or acquired, can lead to insufficient blood clotting and the risk of bleeding. Current treatment options are limited to administering pharmaceutical preparations of one or more therapeutic proteins to restore endogenous levels and maintain hemostasis. However, existing pharmaceutical preparations, typically obtained from donated plasma or recombinant sources, contain proenzymes and proteases (e.g., prothrombin, plasminogen, tissue plasminogen activator (tPA), and / or other proteases), which can impair the stability of therapeutic proteins such as fibrinogen, factor VIII, or VWF during storage. As a result, such preparations are relatively unstable in aqueous solution, and long-term storage is limited to lyophilized or frozen preparations.

[0005] For clinical applications, fibrinogen is typically purified from human plasma, where fibrinogen accounts for only approximately 2–5% (1.5–4.0 g / L) of total plasma protein. Traditionally, fibrinogen purification from plasma is performed by classical plasma fractionation methods, in which fibrinogen is cryoprecipitated from the plasma and then precipitated with either ethanol, ammonium sulfate, β-alanine / glycine, polymers (e.g., polyethylene glycol), or low ionic strength solutions. Such methods can achieve relatively high yields and homogeneity. When higher purity is required, chromatographic techniques are often used. However, existing precipitation and chromatographic techniques suitable for commercial-scale manufacturing typically produce fibrinogen preparations that contain contaminating proteins, such as enzyme precursors or proteases (e.g., prothrombin, tissue plasminogen activator (tPA), and plasminogen), which can compromise fibrinogen stability in solution. For example, when prothrombin is present, it can be activated to the serine protease thrombin, which can then convert fibrinogen to fibrin. Similarly, when both tPA and plasminogen are present, tPA can activate plasminogen to its active form, plasmin, which can then hydrolyze fibrinogen to fibrin. As a result, fibrinogen preparations are relatively unstable in aqueous solution, and long-term storage is limited to lyophilized or frozen preparations.

[0006] Certain contaminants, such as fibronectin, can be absorbed onto immobilized gelatin, and plasminogen onto immobilized lysine (Non-Patent Document 1). However, the use of certain affinity resins is not suitable for large-scale commercial processes, as the affinity resins themselves are not robust enough for repeated use and typically significantly increase both processing time and costs.

[0007] U.S. Patent No. 5,999,499 is directed to a method for purifying fibrinogen from a fibrinogen-containing solution using ion exchange (IEX) chromatography. Specifically, the method involves adding the fibrinogen-containing solution to an ion exchange matrix under conditions that allow the fibrinogen to bind to the matrix, followed by washing the ion exchange matrix with a solution containing at least one omega amino acid. This is done to differentiate plasminogen from the resin and facilitate its removal. The matrix-bound fibrinogen is then eluted from the matrix.

[0008] US Pat. No. 5,629,999 provides a method for purifying fibrinogen using an anion exchange resin containing a hydroxylated polymer support conjugated with tertiary or quaternary amines that bind fibrinogen.

[0009] US Patent No. 5,999,499 teaches the use of a metal-immobilized ion affinity chromatography matrix under conditions that allow fibrinogen and plasminogen to bind to the matrix and selectively elute fibrinogen and plasminogen separately, such that the major fibrinogen fraction contains approximately 600 ng of plasminogen per mg of protein.

[0010] The present invention provides a method for reducing the levels of plasminogen and / or tissue plasminogen activator and / or other proteases in a solution containing fibrinogen and / or factor VIII and / or VWF. The purified proteins are stable during storage as liquid preparations and can be used in clinical or veterinary applications, including treating or preventing conditions associated with deficiencies in the levels of said proteins. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] European Patent No. 1240200 (U.S. Patent No. 6960463) [Patent Document 2] International Publication No. 2012038410 [Patent Document 3] European Patent No. 1519944 [Non-patent literature]

[0012] [Non-Patent Document 1] Vuento et al., 1979, Biochem. J., 183(2):331-337 Summary of the Invention [Means for solving the problem]

[0013] In one aspect of the invention, there is provided a method for reducing the level of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases in a solution comprising at least one protein selected from the group consisting of fibrinogen, factor VIII and von Willebrand factor (VWF), comprising: (i) passing a feedstock containing at least one protein selected from the group consisting of fibrinogen, factor VIII, and VWF through a hydrophobic charge induction chromatography resin under conditions selected to bind at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator, and other proteases present in the feedstock; and (ii) recovering a solution containing at least one protein selected from the group consisting of fibrinogen, factor VIII, and VWF that has passed through the resin; wherein the concentration of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases in the solution is reduced by at least 50% compared to the starting material.

[0014] In another aspect of the present invention, there is provided a method for reducing the level of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases in a solution containing at least one protein selected from the group consisting of fibrinogen, factor VIII and von Willebrand factor (VWF), comprising: (i) passing a feedstock containing 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 containing at least one protein selected from the group consisting of fibrinogen, factor VIII, and VWF that has passed through the first hydrophobic charge induction chromatography resin; (iii) passing the solution recovered in step (ii) through a second hydrophobic charge induction chromatography resin; and (iv) recovering the solution containing at least one protein selected from the group consisting of fibrinogen, factor VIII, and VWF that has passed through the second hydrophobic charge induction chromatography resin; wherein the chromatography conditions are such that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases present in the raw material binds to the first and / or second resin, and the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases in the solution recovered in step (iv) is reduced by at least 50% compared to the raw material. .

[0015] In another aspect, there is provided a solution comprising at least one protein selected from the group consisting of fibrinogen, Factor VIII and VWF recovered by the method of the invention as described herein.

[0016] In another embodiment, there is provided a container containing at least 5 mL of a stable, pharmaceutically acceptable fibrinogen solution, wherein the concentration of fibrinogen is at least 20 mg / mL.

[0017] In another aspect, there is provided 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 methods of the invention as described herein, and a pharmaceutically acceptable carrier.

[0018] In another aspect of the present invention: (a) fibrinogen at least 75% of the total protein; (b) less than 50 pg tissue plasminogen activator per mg of total protein; and / or (c) Less than 1 μg of plasminogen per mg of total protein A solution comprising:

[0019] In another aspect of the present invention: (a) At least 90% fibrinogen of the total protein; (b) less than 50 pg tissue plasminogen activator per mg of total protein; and / or (c) Less than 150 ng of plasminogen per mg of total protein A solution comprising:

[0020] In another aspect of the present invention: (a) At least 90% fibrinogen of the total protein; (b) less than 20 pg tissue plasminogen activator per mg of total protein; and / or (c) Less than 10 ng of plasminogen per mg of total protein A solution comprising:

[0021] In another aspect, there is provided a method for treating or preventing a condition associated with fibrinogen deficiency, the method comprising administering to a subject in need thereof a solution or pharmaceutical formulation of the invention as disclosed herein.

[0022] In another aspect, there is provided the use of a solution of the invention as described herein in the manufacture of a medicament for treating or preventing a condition associated with fibrinogen deficiency.

[0023] In another aspect, there is provided a fibrin glue comprising a solution of the invention as described herein.

[0024] In another embodiment, there is provided a method for producing a stable liquid fibrinogen solution, comprising: (i) passing a raw material containing fibrinogen through a hydrophobic charge induction chromatography resin under conditions selected to bind at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases present in the raw material; and (ii) recovering the solution containing fibrinogen that has passed through the resin; wherein the concentration of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases in the solution is reduced by at least 50% compared to the starting material.

[0025] In another embodiment, there is provided a method for producing a stable liquid fibrinogen solution, comprising: (i) passing a raw material containing fibrinogen through a first hydrophobic charge induction chromatography resin; (ii) recovering the solution containing fibrinogen that has passed through the first hydrophobic charge induction chromatography resin; (iii) passing the solution recovered in step (ii) through a second hydrophobic charge induction chromatography resin; and (iv) recovering the solution containing fibrinogen that has passed through the second hydrophobic charge induction chromatography resin; wherein the chromatography conditions are such that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases present in the raw material binds to the first and / or second resin, and the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases in the solution recovered in step (iv) is reduced by at least 50% compared to the raw material.

[0026] In another aspect of the invention there is provided a method for purifying fibrinogen, comprising: (i) passing a solution containing fibrinogen through an ion exchange chromatography resin under conditions selected to cause fibrinogen monomers to bind to the resin; (ii) eluting the fibrinogen monomer from the resin with an elution buffer; and (iii) filtering the eluted fibrinogen monomer from step (ii) through a filter having a pore size in the range of about 15 nm to about 35 nm. The method includes: [Brief explanation of the drawings]

[0027] [Figure 1] 1 shows the recovery of fibrinogen, plasminogen, and t-PA from a fibrinogen solution at various pH levels when the solution is passed through a HEA Hypercel™ in negative mode for fibrinogen. Each group of bars represents (from left to right): fibrinogen recovery, plasminogen recovery, and t-PA recovery. [Figure 2]Figure 1 shows the recovery of fibrinogen, plasminogen, t-PA and Factor II from fibrinogen solutions at various pH levels when the solutions are passed through PPA Hypercel™ in negative mode for fibrinogen. Each group of bars represents (from left to right): fibrinogen recovery, plasminogen recovery, t-PA recovery and Factor II. [Figure 3] Figure 1 shows the recovery of fibrinogen, plasminogen, t-PA, and Factor II from fibrinogen solutions at various pH levels when the solutions are passed through MEP Hypercel™ in negative mode for fibrinogen. Each group of bars represents (from left to right): fibrinogen recovery, plasminogen recovery, t-PA recovery, and Factor II. [Figure 4a] 1 shows the recovery of fibrinogen, plasminogen, t-PA, and Factor II from a fibrinogen-containing solution at various pH levels when the solution is passed through a HEA Hypercel™ in negative mode for fibrinogen. Each group of bars represents (from left to right): fibrinogen recovery, plasminogen recovery, t-PA recovery, and Factor II recovery. [Figure 4b] Figure 4a shows the stability of fibrinogen-containing solutions recovered from the flow-through of a HEA Hypercel™ column (Fig. 4a) over 6 days at room temperature (approximately 20°C), as measured by % of initial clottable protein. Each group of bars represents (from left to right): T = 1 day, T = 3 days, T = 6 days. [Figure 4c] Figure 4a shows the stability of fibrinogen-containing solutions recovered from the flow-through of a HEA Hypercel™ column (Fig. 4a) over 6 days at room temperature (approximately 20°C), as measured by % of clot protein. Each group of bars represents (from left to right): T=1, T=day 3, T=day 6. [Figure 5]FIG. 1 shows the process recovery of fibrinogen, t-PA, plasminogen and Factor II in fractions obtained from a method according to one embodiment of the present invention from plasma cryoprecipitate to MacroPrep™-HQ eluate. Each group of bars represents (from left to right): fibrinogen, fibronectin, plasminogen, t-PA and Factor II. [Figure 6] FIG. 1 shows the purity of monomeric fibrinogen recovered from MacroPrep™-HQ chromatography resin as determined by analysis of size-exclusion HPLC chromatograms. [Figure 7] FIG. 1 shows the filterability of fibrinogen recovered from MacroPrep™-HQ chromatography resin using elution buffers of various conductivities: 190 mM NaCl, 21.5 mS / cm (diamonds); 200 mM NaCl, 22.5 mS / cm (squares); 210 mM NaCl, 23.5 mS / cm (triangles); 1% (w / w) arginine / 200 mM NaCl, 25 mS / cm (crosses). [Figure 8] FIG. 1 shows the fibrinogen activity of liquid fibrinogen recovered from MacroPrep™-HQ chromatography resin stored at either 2-8°C (diamonds) for 9 weeks or at 30°C (squares) for 7 weeks, as a percentage of the initial fibrinogen activity at t=0. DETAILED DESCRIPTION OF THE INVENTION

[0028] Throughout this specification, unless the context otherwise requires, the words "comprise" or variations such as "comprises" and "comprising" should be understood to mean the inclusion of a stated element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.

[0029] Reference herein to any prior publication (or information derived from a prior publication) or to any publicly known matter is not, and should not be construed as, an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from a prior publication) or publicly known matter forms part of the common general knowledge in the field of development to which this specification pertains.

[0030] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "formulation" includes a single formulation as well as two or more formulations.

[0031] Unless indicated to the contrary, references throughout this specification to "%" content are intended to mean % w / w (weight / weight). For example, a solution containing at least 80% fibrinogen of total protein is intended to mean a solution containing fibrinogen at a concentration of at least 80% w / w of total protein. This may be achieved, for example, by comparing the amount of fibrinogen obtained from a clotting protein assay with a standard protein assay (e.g., bilirubin). The amount of coagulation protein can be calculated by dividing by the total protein obtained from the uret (thrombin) and multiplying by 100. In a clotting protein assay, thrombin is added to a sample, forming a clot, which is almost entirely fibrin. The clot can be centrifuged from the supernatant, which contains non-clotting proteins. The clot is then washed, dissolved with alkaline urea or other substances, and the protein concentration is measured spectrophotometrically. Because the majority of the clot is fibrin, the protein concentration corresponds to the fibrinogen concentration. Therefore, the amount of coagulation protein in a sample corresponds to the difference between the total protein and non-clotting protein components of the sample.

[0032] Purification of fibrinogen and / or factor VIII and / or VWF from raw materials (e.g., plasma or cell culture supernatant) is typically carried out by conventional fractionation methods, where fibrinogen and / or factor VIII and / or VWF are precipitated from solution using, for example, ethanol, ammonium sulfate, β-alanine / glycine, polymers (e.g., polyethylene glycol), and / or low ionic strength solutions. Current plasma purification methods using various chromatographic steps can achieve relatively high yields and homogeneous preparations of the protein of interest. However, these methods typically result in preparations containing residual impurities in amounts unsuitable for formulating stable liquid preparations for clinical applications. Impurities such as prothrombin, tissue plasminogen activator (tPA), and plasminogen are particularly problematic because destabilizing levels of these impurities can hydrolyze fibrinogen in aqueous solution, thus destabilizing fibrinogen, especially during manufacturing and / or long-term storage.

[0033] The present invention is based, at least in part, on the discovery that passing a raw material containing fibrinogen and / or Factor VIII and / or VWF through a hydrophobic charge induction chromatography (HCIC) resin and recovering a solution containing fibrinogen and / or Factor VIII and / or VWF that has passed through the resin is an effective alternative to existing purification methods for reducing the level of destabilizing plasminogen and / or tissue plasminogen activator and / or other proteases in the solution.

[0034] Thus, one aspect of the present invention provides a method for reducing the level of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases in a solution containing at least one protein selected from the group consisting of fibrinogen, factor VIII and von Willebrand factor (VWF), comprising: (i) passing a feedstock containing at least one protein selected from the group consisting of fibrinogen, factor VIII, and VWF through a hydrophobic charge induction chromatography resin under conditions selected to bind at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator, and other proteases present in the feedstock; and (ii) recovering a solution containing at least one protein selected from the group consisting of fibrinogen, factor VIII, and VWF that has passed through the resin; wherein the concentration of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases in the solution is reduced by at least 50% compared to the starting material.

[0035] In one embodiment, the concentration of plasminogen and / or tissue plasminogen activator and / or other proteases in the recovered solution containing fibrinogen and / or factor VIII and / or VWF is reduced by at least 60%, at least 70%, at least 80%, or at least 90%, or at least 95%, or at least 98% compared to the raw material.

[0036] In another embodiment, there is provided a method for producing a stable liquid fibrinogen solution, comprising: (i) passing a raw material containing fibrinogen through a hydrophobic charge induction chromatography resin under conditions selected to bind at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases present in the raw material; and (ii) recovering the solution containing fibrinogen that has passed through the resin; wherein the concentration of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases in the solution is reduced by at least 50% compared to the starting material.

[0037] In one embodiment, the concentration of plasminogen and / or tissue plasminogen activator and / or other proteases in the recovered solution containing fibrinogen is reduced by at least 60%, at least 70%, at least 80%, or at least 90% or at least 95% or at least 98% compared to the raw material.

[0038] Chromatography methods typically use a solid support, also referred to interchangeably herein as a resin or matrix. Suitable solid supports are well known to those skilled in the art. Examples include inorganic supports such as glass and silica gel, and synthetic or naturally occurring organic supports such as agarose, cellulose, dextran, polyamide, polyacrylamide, bifunctional vinyl copolymers of acrylic acid, and various hydroxylated monomers. Commercially available supports are sold under the names Sephadex™, Sepharose™, Hypercel™, Capto™, Fractogel™, MacroPrep™, Unosphere™, GigaCap™, Trisacryl™, Ultrogel™, Dynospheres™, Macrosorb™, and XAD™ resins.

[0039] Chromatographic steps are typically carried out under non-denaturing conditions at convenient temperatures ranging from about +10° C. to +30° C., more typically at ambient temperature. Chromatographic steps may be carried out batchwise or continuously, as appropriate. Any convenient separation method may be used, such as columns, centrifugation, filtration, decantation, etc.

[0040] Hydrophobic charge induction chromatography (HCIC), often referred to as mixed-mode or multimode chromatography, is well known to those skilled in the art. HCIC utilizes binding moieties linked to a solid support, which, in accordance with the methods of the present invention, may be specific for one or more proteins representative of impurities in the raw material (e.g., proenzymes and proteases such as prothrombin, tPA, and plasminogen).

[0041] 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 mercaptoethylpyridine (4-mercaptoethylpyridine, 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.

[0042] HCIC ligands, such as HEA, MEP, and PPA, allow separation based on the surface hydrophobicity of proteins, but have the advantage of not requiring the addition of lyotropic salts, which is often present in other methods for purifying fibrinogen using hydrophobic chromatography (e.g., hydrophobic interaction chromatography; HIC). In contrast to traditional hydrophobic interaction chromatography, HCIC relies on pH rather than salt concentration. HCIC resins also offer high binding capacities and fast flow rates, making them ideal for laboratory and industrial-scale purification.

[0043] HCIC resins are often packed into columns with bed heights ranging from about 2 cm to about 40 cm. At industrial scale, bed heights are usually at least 10 cm, typically ranging from about 15 cm to 25 cm. Industrial column diameters can range from 20 cm to about 1.5 m. Such columns are operated at flow rates specified in the HCIC resin manufacturer's instructions, with typical flow rates ranging from 50 to 100 cm / hr. The upper flow rate limit is due in part to pressure limitations imposed by HCIC resins. For HEA resins, for example, the upper operating pressure limit is <3 bar (<300 kPa). Typical dynamic binding capacities (at which 10% of the bound protein breaks through) for HCIC resins are approximately 20 to 30 mg of bound protein per mL of resin. In the present invention, it is possible to load relatively large amounts of protein onto the HCIC column, as abundant proteins such as fibrinogen are able to pass through the column, while less abundant proteins such as plasminogen and / or tissue plasminogen activator and / or other proteases bind to the HCIC resin. This is advantageous for industrial-scale production, as batch processing requires either smaller column sizes and / or fewer column runs.

[0044] The inventors have also discovered that the pH of a solution or feedstock containing fibrinogen and / or Factor VIII and / or VWF passed through an HCIC resin by the methods of the present invention can be adjusted to control the recovery of fibrinogen and / or Factor VIII and / or VWF and the removal of impurities. Thus, in embodiments disclosed herein, the pH of a solution or feedstock containing fibrinogen and / or Factor VIII and / or VWF passed through an HCIC resin is from about 6.0 to about 9.5. In certain embodiments, the solution or feedstock containing fibrinogen and / or Factor VIII and / or VWF is passed over the HCIC resin at a pH of preferably 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 certain embodiments, the pH of the solution or feedstock containing fibrinogen and / or Factor VIII and / or VWF passed over the HCIC resin is about 7.0. In certain embodiments, the HCIC resin is equilibrated to 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 prior to adding the solution or raw materials.

[0045] The method of the present invention can also employ multiple additional chromatographic steps, if necessary, to remove other impurities and thus increase the purity of the final preparation. Additional chromatographic purification steps can be performed either before or after the purification of fibrinogen and / or factor VIII and / or VWF on the HCIC resin according to the present invention. For example, the solution containing fibrinogen and / or factor VIII and / or VWF recovered from the HCIC resin in step (ii) can be passed through another chromatographic resin.

[0046] Further chromatographic purification steps may use different HCIC resins. Accordingly, in another aspect of the present invention, there is provided a method for reducing the level of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases in a solution containing at least one protein selected from the group consisting of fibrinogen, factor VIII and von Willebrand factor (VWF), comprising: (i) at least one selected from the group consisting of fibrinogen, factor VIII, and VWF; passing a feedstock containing at least one protein through a first hydrophobic charge induction chromatography resin; (ii) recovering a solution containing at least one protein selected from the group consisting of fibrinogen, factor VIII, and VWF that has passed through the first hydrophobic charge induction chromatography resin; (iii) passing the solution recovered in step (ii) through a second hydrophobic charge induction chromatography resin; and (iv) recovering the solution containing at least one protein selected from the group consisting of fibrinogen, factor VIII, and VWF that has passed through the second hydrophobic charge induction chromatography resin; wherein the chromatography conditions are such that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases present in the raw material binds to the first and / or second resin, and the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases in the solution recovered in step (iv) is reduced by at least 50% compared to the raw material.

[0047] In one embodiment, the concentration of plasminogen and / or tissue plasminogen activator and / or other proteases in the solution containing fibrinogen and / or Factor VIII and / or VWF recovered in step (iv) is reduced by at least 60%, at least 70%, at least 80%, or at least 90%, or at least 95%, or at least 98% compared to the starting material.

[0048] In another embodiment, there is provided a method for producing a stable liquid fibrinogen solution, comprising: (i) passing a raw material containing fibrinogen through a first hydrophobic charge induction chromatography resin; (ii) recovering the solution containing fibrinogen that has passed through the first hydrophobic charge induction chromatography resin; (iii) passing the solution recovered in step (ii) through a second hydrophobic charge induction chromatography resin; and (iv) recovering the solution containing fibrinogen that has passed through the second hydrophobic charge induction chromatography resin; wherein the chromatography conditions are such that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases present in the raw material binds to the first and / or second resin, and the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other proteases in the solution recovered in step (iv) is reduced by at least 50% compared to the raw material.

[0049] In one embodiment, the concentration of plasminogen and / or tissue plasminogen activator and / or other proteases in the fibrinogen-containing solution recovered in step (iv) is reduced by at least 60%, at least 70%, at least 80%, or at least 90%, or at least 95%, or at least 98% compared to the starting material.

[0050] In an embodiment disclosed herein, the second HCIC resin is different from the first HCIC resin. In another embodiment disclosed herein, the first and second hydrophobic charge induction chromatography resins are the same. The fibrinogen recovered from the HCIC resin in step (ii) If a solution containing factor VIII and / or VWF is passed over the same HCIC resin, it may be desirable to wash the HCIC resin after step (ii) and before passing the solution recovered in step (iii) over the HCIC resin again to remove any impurities that may bind to the resin.

[0051] The additional chromatography resin may also be an anion exchange chromatography resin. In anion exchange chromatography, negatively charged molecules are attracted to positively charged solid supports. Positively charged solid supports can be prepared by any means known to those skilled in the art, and typically require covalent attachment of negatively charged functional ligands to the solid support. Suitable negatively charged functional ligands depend largely on the molecules to be separated from the solution. Examples of suitable anion exchange resins include resins containing functional quaternary amine groups (Q) and / or tertiary amine groups (DEAE) or diethylaminopropyl groups (ANX). Commercially available anion exchange chromatography matrices include, but are not limited to, DEAE cellulose, Applied Biosystems' Poros™ PI20, PI50, HQ10, HQ20, HQ50, D50, GE. MonoQ™, MiniQ™, Source™ 15Q and 3OQ, Q, DEAE and ANX Sepharose Fast Flow™, Q Sepharose high Performance™, QAE SEPHADEX™ and FAST Q SEPHAROSE™ from Healthcare; WP PEI™, WP DEAM™, WP QUAT™ from JT Baker; Biochrom Labs Inc.Hydrocell™ DEAE and Hydrocell™ QA from BioRad; 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 E77, weak base anions from Dow Liquid Separations; Matrex Cellufine™ A200, A500, Q500 and Q800 from Millipore; Fractogel™ EMD from EMD. TMAE3 Fractogel™ EMD DEAE and Fractogel™ EMD DMAE, Amberlite™ weak and strong anion exchangers type I and II manufactured by Sigma-Aldrich, DOWEX™ weak and strong anion exchangers type I and II, Diaion™ weak and strong anion exchangers type I and II, Duolite™, TSK™ Gel Q and DEAE manufactured by Tosoh. These include Toyopearl™ SuperQ-650S, 650M and 650C, 3QAE-26-550C and 650S, DEAE-65OM and 650C, and Whatman™ QA52™, DE23™, DE32™, DE51™, DE52™, DE53™, Express-Ion™ D and Express-Ion™ Q.

[0052] If desired, an anion exchange chromatography membrane may be used in place of the 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 membranes from Millipore.

[0053] In one embodiment disclosed herein, the anion exchange resin is a strong anion exchange resin. In another embodiment disclosed herein, the strong anion exchange resin contains a quaternary amine functional ligand (e.g., -N as found in MacroPrep™-HQ; Bio-Rad Laboratories). + (CH3)3). In yet another embodiment, the anion exchange resin is a trimethylamine group grafted to a hydroxylated methacrylic acid polymer via a bridging group, such as GigaCap Q-650M®.

[0054] In one embodiment, anion exchange chromatography is performed in a positive mode with respect to fibrinogen and / or factor VIII and / or VWF. That is, conditions are used such that when a solution or raw material containing fibrinogen and / or factor VIII and / or VWF passes through the anion exchange chromatography resin, fibrinogen and / or factor VIII and / or VWF bind to the positively charged functional groups linked to the resin, and impurities in the solution pass from the resin into a flow-through fraction that can be collected for disposal or other purposes. Once the flow-through fraction has passed through the resin, the anion exchange chromatography resin can be washed with an appropriate wash buffer known to those skilled in the art. The composition of the wash buffer and the conditions of the wash step are typically selected so that the fibrinogen and / or factor VIII and / or VWF bound to the resin are retained during the wash step. Those skilled in the art will also recognize that when referring to wash buffers, elution buffers or similar buffers for chromatography can include solutions with limited or no buffer capacity.

[0055] In one embodiment disclosed herein, prior to eluting fibrinogen and / or Factor VIII and / or VWF from the anion exchange chromatography resin, the resin is washed with a wash solution containing epsilon-aminocaproic acid (ε-ACA). The addition of ε-ACA to the wash buffer can facilitate the elution of proteases (such as plasminogen) that may bind to the anion exchange chromatography resin during the first pass. An example of a suitable wash step is described in U.S. Patent No. 6,960,463.

[0056] Any suitable elution buffer known to those skilled in the art can be used to elute fibrinogen and / or factor VIII and / or VWF remaining bound to the anion exchange chromatography resin. To remove plasminogen and / or t-PA and / or other proteases from a solution containing fibrinogen, the inventors have discovered that an elution buffer containing about 150 mM to about 300 mM NaCl elutes fibrinogen monomers from the anion exchange resin while minimizing the elution of fibrinogen aggregates and / or other proteins (e.g., factor VIII, VWF, fibronectin, or proteases) that may also bind to the resin. Therefore, in one embodiment disclosed herein, fibrinogen is eluted from the anion exchange resin with an elution buffer containing about 150 mM to about 300 mM NaCl. This is equivalent to an elution buffer with a conductivity range of about 18 mS / cm (150 mM NaCl) to about 32 mS / cm (300 mM NaCl).

[0057] In another embodiment, fibrinogen is eluted from the anion exchange resin with an elution buffer containing about 150 mM to about 270 mM NaCl, which is equivalent to an elution buffer with a conductivity range of about 18 mS / cm (150 mM NaCl) to about 29 mS / cm (270 mM NaCl).

[0058] In another embodiment, fibrinogen is eluted from the anion exchange resin with an elution buffer containing about 170 mM to about 230 mM NaCl, which is equivalent to an elution buffer with a conductivity range of about 19 mS / cm (170 mM NaCl) to about 25 mS / cm (230 mM NaCl).

[0059] In another embodiment, fibrinogen is eluted from the anion exchange resin with an elution buffer containing about 200 mM to about 220 mM NaCl, which is equivalent to an elution buffer with a conductivity range of about 22 mS / cm (200 mM NaCl) to about 24 mS / cm (220 mM NaCl).

[0060] In another embodiment, the fibrinogen is eluted from the anion exchange resin with an elution buffer comprising about 190 mM to about 210 mM NaCl.

[0061] In another embodiment, the fibrinogen is eluted from the anion exchange resin with an elution buffer comprising about 150 mM to about 190 mM NaCl.

[0062] In other embodiments, the conductivity of the elution buffer ranges from 18 to 32 mS / cm; or 20 to 25 mS / cm; or 21 to 23.5 mS / cm; or 22 to 23 mS / cm. In a preferred embodiment, the conductivity of the elution buffer is about 22.5 mS / cm.

[0063] In embodiments disclosed herein, the elution buffer contains a concentration of free amino acid that promotes elution of fibrinogen monomers over fibrinogen aggregates. In another embodiment, the elution buffer contains 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 concentration range. In one embodiment, the free amino acid is arginine. In another embodiment, the elution buffer contains arginine in the range of about 4 to about 10% (w / w).

[0064] In other embodiments, the elution buffer comprises 200 mM NaCl, 0.5% (w / w) arginine; or 160 mM NaCl, 1% (w / w) arginine.

[0065] In one embodiment, anion exchange chromatography is performed in negative mode for fibrinogen and in positive mode for factor VIII and / or VWF. That is, the conditions used are such that when a solution or raw material containing fibrinogen and factor VIII and / or VWF is passed through the anion exchange chromatography resin, factor VIII and / or VWF binds to the positively charged functional groups linked to the resin, and fibrinogen in the solution passes through the resin into the flow-through fraction. Once the flow-through fraction containing fibrinogen has passed through the resin, the anion exchange chromatography resin can be washed with an appropriate wash buffer known to those skilled in the art. The composition of the wash buffer and the conditions of the wash step are typically selected so that the factor VIII and / or VWF bound to the resin is retained during the wash step.

[0066] In one embodiment, a solution or raw material containing fibrinogen and / or factor VIII and / or VWF is passed through an anion exchange chromatography resin in the presence of about 150 mM to about 270 mM NaCl. 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, fibrinogen, particularly in its monomeric form, passes through the anion exchange chromatography resin, while fibrinogen containing aggregates and other impurities, such as IgG and fibronectin, binds to the resin. In other embodiments, a solution or raw material containing fibrinogen and / or factor VIII and / or VWF is passed through the anion exchange chromatography resin in the presence of about 170 mM to about 230 mM NaCl (about 19 mS / cm to about 25 mS / cm) or about 200 mM to about 220 mM NaCl (about 22 mS / cm to about 24 mS / cm). Under these types of conditions, factor VIII and / or VWF are expected to bind to the anion exchange chromatography resin. It is measured.

[0067] Factor VIII and / or VWF can be eluted from the anion exchange resin with an elution buffer containing at least 300 mM of a salt, such as NaCl. In a specific embodiment, Factor VIII and / or VWF is eluted from the ion exchange resin with about 500 mM NaCl. If fibrinogen and Factor VIII and / or VWF bind to the anion exchange resin, the elution step can be performed so that fibrinogen elutes first (e.g., using the conditions set forth in the previous embodiment), and then Factor VIII and / or VWF can be eluted using a higher concentration of salt, such as 500 mM NaCl.

[0068] If an anion exchange chromatography step is used, it can be performed either before and / or after passing the raw material containing fibrinogen and / or Factor VIII and / or VWF through the HCIC resin. In one embodiment disclosed herein, the method further comprises passing the solution containing fibrinogen and / or Factor VIII and / or VWF recovered in step (ii) through an anion exchange chromatography resin. In another embodiment, when first and second HCIC chromatography steps are used as described herein, the method further comprises passing the solution containing fibrinogen and / or Factor VIII and / or VWF recovered in step (ii) and / or step (iv) through an anion exchange chromatography resin.

[0069] In one embodiment disclosed herein, the method further comprises, prior to step (i), passing the raw material containing fibrinogen and / or Factor VIII and / or VWF through an anion exchange chromatography resin.

[0070] Those skilled in the art will understand that the number of additional chromatography steps used in accordance with the present invention will depend on the level of purity required for the final preparation. For example, the methods of the present invention may include two, three, four, or five chromatography steps as disclosed herein. For example, if the method includes two chromatography steps, the order of steps is HCIC / IEX or HCIC / HCIC or IEX / HCIC; if the method includes three chromatography steps, the order of steps is 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; if the method includes four chromatography steps, the order of steps is HCIC / IEX / HCIC / HCIC or HCIC / HCIC / IEX / 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 / HCIC or HCIC / IEX / IEX / IEX or HCIC / HCIC / IEX / IEX or HCIC / IEX / HCIC / IEX or IEX / HCIC / HCIC / 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; etc. ("IEX" refers to anion exchange chromatography). The required purity level is determined by the intended use of the solution (e.g., for treating patients with fibrinogen and / or factor VIII and / or VWF deficiencies) and / or if a longer shelf life is required as an aqueous preparation.

[0071] Chromatography can be performed using any means known to those skilled in the art. For example, the chromatography step according to the present invention can be performed using the chromatographic methods available from GE Healthcare, Pall Axial flow columns such as those available from Bio-Rad and Bio-Rad Laboratories, Inc. Alternatively, a radial flow column such as those available from Proxcys may be used. The chromatography step according to the invention may also be carried out using expanded bed technology.

[0072] In one embodiment, the concentration of plasminogen and / or tissue plasminogen activator and / or other proteases in the recovered solution containing fibrinogen and / or factor VIII and / or VWF is reduced by at least 60%, at least 70%, at least 80%, or at least 90% or at least 95% compared to the raw material.

[0073] Methods that maximize the removal of impurities such as plasminogen and / or tissue plasminogen activator and / or other proteases are particularly advantageous, as they clearly improve the stability and efficacy of fibrinogen and / or factor VIII and / or VWF in solution, especially during long-term storage. Storage in liquid form is particularly advantageous for solutions containing fibrinogen and / or factor VIII and / or VWF, as they can be used immediately in patients. This contrasts with the use of lyophilized preparations of purified fibrinogen and / or factor VIII and / or VWF, which require the reconstitution of the lyophilized proteins with an appropriate buffer and / or water for injection immediately before administration to a subject in need thereof.

[0074] An advantage of removing proteases or their zymogens (such as plasminogen) from solutions containing fibrinogen and / or factor VIII and / or VWF is that it minimizes the need to add antifibrinolytic agents to inhibit remaining proteases and / or zymogens (e.g., plasmin or plasminogen). Examples of such agents include aprotinin, a bovine protein inhibitor of plasmin; and tranexamic acid, a synthetic plasmin inhibitor that is also associated with neurotoxic side effects.

[0075] Another advantageous property is that plasminogen separated by HCIC from a solution containing fibrinogen and / or Factor VIII and / or VWF can be further processed, for example to produce a plasminogen-containing concentrate for clinical use. Thus, HCIC can be used to prepare both plasminogen and fibrinogen and / or Factor VIII and / or VWF-containing solutions from a single starting solution.

[0076] Another advantageous property is that the production cost of HCIC resin is much more economical than the cost of lysine-Sepharose™ or immobilized lysine resins used in affinity chromatography methods.

[0077] Another advantageous property is that HCIC can be used to replace aluminum hydroxide (e.g., Alhydrogel™) processes to remove proteases (e.g., factor II). Alhydrogel™ is currently widely used in the commercial production of factor VIII and VWF. However, this material is quite expensive at the 100 kg typically used per batch. Furthermore, Alhydrogel™ often requires manual handling, and the material is discarded after a single use. In contrast, the HCIC process can be fully automated, and the resin can be used in the manufacture of multiple batches.

[0078] Another advantageous property is that the HCIC resin is compatible with NaOH 1M, which can be used to inactivate and remove pathogens, including viruses and prions, during column washing and resin sanitization procedures.

[0079] The liquid preparations obtained by the methods of the present invention are also advantageous over frozen preparations, which require costly storage and transportation and must be thawed immediately before use. Storing fibrinogen and / or factor VIII and / or VWF as lyophilized or frozen preparations is also advantageous because the reconstituted or thawed proteins are stable for longer periods. This is evident, for example, when material is reconstituted as a precaution for a medical procedure, but medical considerations do not dictate its use. Such material is typically discarded because the fibrinogen is only stable for a short period of time due to the presence of prothrombin and / or t-PA and / or other proteases.

[0080] In certain embodiments, the liquid preparations of the invention containing fibrinogen and / or Factor VIII and / or VWF are stored as liquid or lyophilized or frozen preparations.

[0081] In another aspect of the invention there is provided a method for purifying fibrinogen, comprising: (i) passing a solution containing fibrinogen through an ion exchange chromatography resin under conditions selected to cause fibrinogen monomers to bind to the resin; (ii) eluting the fibrinogen monomer from the resin with an elution buffer; and (iii) filtering the eluted fibrinogen monomer from step (ii) through a filter having a pore size in the range of about 15 nm to about 35 nm. The method includes:

[0082] In one embodiment, the fibrinogen-containing solution (step (i)) is recovered after passing a source material containing fibrinogen through a hydrophobic charge induction chromatography resin under conditions selected such that plasminogen and / or tissue plasminogen activator and / or other proteases bind to the resin and fibrinogen passes through the resin.

[0083] In one embodiment, the ion exchange chromatography resin is selected from an anion exchange chromatography resin or a cation exchange chromatography resin.

[0084] In one embodiment, the anion exchange chromatography resin is a strong anion exchange chromatography resin or a weak anion exchange chromatography resin. In one embodiment, the anion exchange chromatography resin contains a quaternary amino group. Examples include quaternary alkylamines and quaternary alkylalkanolamines, or amines, diethylamine, diethylaminopropyl, amino, trimethylammoniumethyl, trimethylbenzylammonium, dimethylethanolbenzylammonium, and polyamines. In some embodiments, the anion exchange chromatography resin is a tertiary or quaternary amine-conjugated polymeric support or a tertiary or quaternary amine-conjugated hydroxylated polymeric support. In some embodiments, the anion exchange chromatography resin contains a methacrylic acid polymeric support. In one embodiment, the anion exchange chromatography resin is MacroPrep™ HQ. In another embodiment, the anion exchange chromatography resin is GigaCap™ Q-650M. In other embodiments, the anion exchange chromatography resin is packed in a column.

[0085] If desired, an anion exchange chromatography membrane can be used in place of the anion exchange chromatography resin. Commercially available anion exchange chromatography membranes include, but are not limited to, Sartobind™ Q from Sartorius, Pall Technologies' Mustang™ Q and Millipore's Intercept™ Q membranes.

[0086] In one embodiment, the cation exchange chromatography resin is a strong cation exchange chromatography resin or a weak cation exchange chromatography resin.

[0087] Commercially available cation exchange chromatography resins include, but are not limited to, sulfonic acid-based groups (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); sulfoethyl-based groups (e.g., Fractogel™ SE from EMD, Poros™ S-10 and S-20 from Applied Biosystems); sulfopropyl-based groups (e.g., TSK™ Gel SP 5PW and SP-5PW-HR from Tosoh, Applied Biosystems, Poros™ HS-20 and HS 50 from Biosystems; sulfoisobutyl-based groups (e.g., Fractogel™ EMD SO3" from EMD); sulfoxyethyl-based groups (e.g., SE52, SE53, and Express-Ion S from Whatman), carboxymethyl-based groups (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 S from Whatman), C, Toyopearl™ CM-650S, CM-650M, and CM-650C from Tosoh Corporation; sulfonic acid and carboxylic acid based groups (e.g., JTBAKERBOND™ Carboxy-Sulfon from JT Baker; carboxylic acid-based groups (e.g., WP™ CBX from JT Baker, DOWEX MAC-3™ from Dow Liquid Separations, Amberlite™ weak cation exchanger, DOWEX™ weak cation exchanger, and Diaion™ weak cation exchanger from Sigma-Aldrich, and Fractogel™ EMD COO- from EMD); sulfonic acid-based groups (e.g., Hydrocell™ SP from Biochrom Labs Inc., DOWEX™ Fine Mesh Strong Acid Cation Matrix from Dow Liquid Separations, UNOsphere™ S from JT Baker, WP Sulfonic, Sartobind™ S membrane from Sartorius, Amberlite™ Strong Cation Exchanger, DOWEX™ Strong Cation, and Diaion Strong Cation Exchanger from Sigma-Aldrich). Exchanger); as well as those with orthophosphate-based groups (e.g., P1 1 from Whatman). If desired, a cation exchange chromatography membrane, e.g., Sartobind™ S (Sartorius; Edgewood, NY), can be used in place of the cation exchange matrix.

[0088] In one embodiment, the pH of the fibrinogen-containing solution ranges from about pH 7 to pH 10. In one embodiment, the pH of the fibrinogen-containing solution is about pH 8. In some embodiments, the anion exchange chromatography resin is pre-equilibrated with a buffer having a pH similar to that of the fibrinogen-containing solution and washed after the addition of fibrinogen.

[0089] In one embodiment, the conductivity of the elution buffer ranges from about 18 to about 30 mS / cm. For example, the conductivity of the elution buffer may range from 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 embodiment, the conductivity of the elution buffer is about 22 mS / cm. In other embodiments, the elution buffer comprises NaCl. In one embodiment, the elution buffer comprises NaCl at a concentration ranging from about 180 mM to about 230 mM, or from about 190 mM to about 210 mM. In one embodiment, the NaCl concentration of the elution buffer is about 200 mM.

[0090] In one embodiment, the fibrinogen-containing elution buffer contains a protein concentration of about 0.5 to about 10 mg / mL. In some embodiments, the protein concentration of the fibrinogen-containing elution buffer ranges from about 4 to about 8 mg / mL. In a specific embodiment, the fibrinogen-containing elution buffer is about 6 mg / mL.

[0091] In one embodiment, one or more amino acids are blended with the eluted fibrinogen monomer prior to filtration (step (iii)). In one embodiment, the amino acid is arginine or glycine or a combination thereof. In some embodiments, the concentration of the amino acid in the elution buffer containing fibrinogen ranges from about 0.5 to about 10% (w / w). In one embodiment, the concentration of the amino acid in the elution buffer containing fibrinogen is about 1% to about 6% (w / w), or about 2% to about 6% (w / w), or about 2% to about 5% (w / w). In one embodiment, the elution buffer containing fibrinogen is blended with about 2%, or about 3%, or about 4%, or about 5% (w / w) arginine.

[0092] In one embodiment, the pH of the eluted fibrinogen monomer is from about pH 7 to about pH 9.

[0093] In one embodiment, the pore size of the filter in step (ii) ranges 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.

[0094] Virus filtration can be performed using either tangential flow filtration (TFF) or "dead-end" filtration (also known as normal flow filtration). Virus filters were originally designed for use in TFF, with the feed stream adjacent to the upper skin layer of an asymmetric membrane. TFF achieves high flow rates by sweeping the membrane surface to reduce concentration bias and fouling. However, the simplicity and low cost of dead-end filtration have led to the popularity of virus filters specifically designed for dead-end filtration. In contrast to TFF, these dead-end filters are typically operated with the more open side of the membrane facing the feed stream, trapping protein aggregates and other fouling within the macroporous base, thereby protecting the virus-retaining skin layer. Advantages of using disposable dead-end filters include simplified system design and validation, reducing labor and expense.

[0095] Dead-end filtration typically requires the use of a single pump to direct fluid from the surface into the membrane.

[0096] Tangential filtration typically involves the use of a filter membrane to maintain a constant flow rate across the membrane. A first pump is required to draw the protein into the membrane, and a second pump is required to draw the protein into the membrane by creating a negative pressure behind the membrane.

[0097] In one embodiment, the filtration is performed by dead-end filtration.

[0098] In one embodiment, the dead-end filtration method is carried out using either constant pressure filtration or constant rate filtration. In one embodiment, the dead-end filtration method is carried out using constant pressure filtration.

[0099] Filtration is typically carried out at a filtration pressure that is equal to or lower than the level that the virus removal membrane can withstand, depending on the material of the membrane used herein, for example, a pressure 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 embodiment, the filtration pressure is maintained at about 1 to about 3 bar; or about 1 to about 2 bar; or about 1.2 to about 2 bar. In another embodiment, the filtration pressure is maintained at about 1.5 bar to about 1.9 bar.

[0100] Temperature can affect the viscosity of the protein solution and the flow rate of filtration through the virus removal membrane. Those skilled in the art will understand that the solution used in the filtration process is typically at a temperature ranging from about 0°C to the temperature at which the protein of interest denatures. The solution temperature is suitably within the range of about 10°C to about 50°C. In one embodiment, the solution temperature is within the range of 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.

[0101] In one embodiment, the virus filter capacity is determined by the filter size and filter surface area. 2 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 1000 mg of fibrinogen.

[0102] Optionally, a pre-filtration or clarifying filtration step can be used before virus filtration to remove larger particles. In one embodiment, pre-filtration is performed with a pre-filter containing a membrane with a larger pore size than the virus removal membrane. In one embodiment, the pre-filter is a membrane filter with a pore size of approximately 0.1 μm. In another embodiment, the pre-filter is selected from Pall nylon membrane filters (SKL 7002 NTP 0.1 μm or FTKNI) or other commercially available pre-filters with similar properties for removing protein aggregates and / or particulates. Pre-filtration can be performed with or without a virus filter. In one embodiment, pre-filtration is performed with a virus filter.

[0103] Filters suitable for virus filtration according to this aspect of the invention will be known to those skilled in the art. One example includes, among others, Planova BioEx™. Such filters are sometimes referred to as "small virus" removal filters.

[0104] In one embodiment, the filter membrane is a flat sheet or hollow fiber membrane. Examples of flat sheet membranes include hydrophilized PVDF filter membranes, such as the Pegasus™ SV4 grade small virus removal filter (Pall Corporation). In one embodiment, the filter is a Pegasus™ SV4 grade.

[0105] In other embodiments, the filter membrane is a hollow fiber membrane. A hollow fiber membrane may contain a bundle of straw-shaped hollow fibers, where the wall of each hollow fiber contains a three-dimensional web structure of pores made up of tiny capillaries and interconnected voids. Examples of hollow fiber filters include Included are Planova™ BioEX™ filters (Asahi Kasei Corporation) that incorporate hydrophilically modified polyvinylidene fluoride (PVDF) in hollow fiber membrane form. In one embodiment, the filter is Planova™ BioEX.

[0106] In one embodiment, two or more small virus filters are used in series. In one embodiment, filtration is performed using two filters in series with pore sizes ranging from about 15 to about 20 nm. Such a filtration process potentially allows for the production of fibrinogen with an LRV of at least 6.9 log LRV for parvovirus-like MVM.

[0107] In another embodiment of the present invention, a solution containing fibrinogen is passed through an ion exchange chromatography resin under conditions selected to allow the fibrinogen present in the solution to pass through the resin. That is, ion exchange chromatography is performed in a negative mode with respect to fibrinogen under conditions such that, as the solution passes through the resin, impurities present in the solution, such as fibrinogen aggregates, plasminogen, and fibronectin, bind to charged functional groups linked to the resin, and fibrinogen, particularly fibrinogen monomers, present in the solution pass through the resin into the flow-through fraction. Once the fibrinogen-containing flow-through fraction has passed through the resin, the ion exchange chromatography resin can be washed with an appropriate wash buffer known to those skilled in the art. The composition of the wash buffer and the conditions of the wash step are usually selected so that the bound impurities are retained on the resin during the wash step.

[0108] In one embodiment, the ion exchange chromatography resin is selected from an anion exchange chromatography resin or a cation exchange chromatography resin.

[0109] In one embodiment, a solution containing fibrinogen is passed through an anion exchange chromatography resin in the presence of about 150 mM to about 270 mM NaCl. This equates to a conductivity ranging from about 18 mS / cm (150 mM NaCl) to about 29 mS / cm (270 mM NaCl). Under these conditions, fibrinogen, particularly in its monomeric form, passes through the anion exchange chromatography resin, while fibrinogen containing aggregates and other impurities, such as plasminogen and fibronectin, binds to the resin. In other embodiments, a solution containing fibrinogen is passed through the anion exchange chromatography resin in the presence of about 170 mM to about 230 mM NaCl (about 19 mS / cm to about 25 mS / cm) or about 200 mM to about 220 mM NaCl (about 22 mS / cm to about 24 mS / cm). Under these conditions, impurities are expected to bind to the anion exchange chromatography resin, while fibrinogen passes through the resin into the flow-through fraction.

[0110] Solutions containing fibrinogen and / or factor VIII and / or VWF recovered by the methods of the present invention are advantageous because they form preparations of fibrinogen and / or factor VIII and / or VWF that are more stable than existing lyophilized preparations, even at room temperature. This can be particularly advantageous for long-term transportation routes where low temperatures cannot be ensured during transportation and / or storage, as required. Stable storage of fibrinogen and / or factor VIII and / or VWF in solution also facilitates in many ways the manufacture, use, transportation, and administration to patients in need thereof. The high stability of fibrinogen and / or factor VIII and / or VWF prepared by the present invention allows for the elimination of the addition of stabilizers, such as fibrinolysis or fibrinogenolysis inhibitors, in many pharmaceutical preparations, which may lead to undesirable side effects in some circumstances or should be avoided to reduce potential risks. is possible.

[0111] As used herein, the term "stable" means that there is little or substantially no loss of fibrinogen and / or factor VIII and / or VWF activity after a storage period, compared to the activity level of fibrinogen and / or factor VIII and / or VWF before storage (e.g., compared to the activity level measured immediately after collection of a solution comprising fibrinogen and / or factor VIII and / or VWF according to the present invention). In one embodiment disclosed herein, a 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% activity, even more preferably at least 95% activity, and most preferably 100% activity, after a storage period at a temperature of about 0°C to about 30°C. Those skilled in the art will understand that fibrinogen activity can be measured in a fibrinogen preparation immediately prior to the start of the storage period, and this initial value can be used, designated as 100% activity, relative to which fibrinogen activity measured at various times during the storage period can be expressed as a percentage of this initial value.

[0112] In one embodiment disclosed herein, fibrinogen recovered by the methods of the present invention retains about 90% to 100% activity after at least four weeks of storage in solution at a temperature of about 2°C to about 8°C, preferably about 90% activity after four weeks of storage in solution at a temperature of about 2°C to about 8°C. In another embodiment disclosed herein, fibrinogen retains about 60% to 80% activity after at least four weeks of storage in solution at a temperature of about 30°C, preferably about 60% to 70% activity after five weeks of storage in solution at a temperature of about 30°C. Fibrinogen and / or Factor VIII and / or VWF activity levels can be measured by any means known to those skilled in the art. Examples of suitable methods for measuring fibrinogen activity are summarized, for example, by Mackie et al. (British J. Haematol. 121:396-404, 2003). Specific methods include the Clauss method (Clauss, 1957, Acta-Haematol. 17, 237-246) and / or clotting protein (Jacobsson K., Scand J Clin Lab Invest 1955;7(Suppl 14):1-54 or Fibrin sealant Ph. Eur. Monograph 903, 2012). Results can be reported as % clotting protein; % initial clotting protein and / or % initial fibrinogen activity measured using the Clauss method or similar methods.

[0113] Those skilled in the art will appreciate that the concentration of plasminogen and / or tissue plasminogen activator and / or other proteases in a collection solution containing fibrinogen and / or factor VIII and / or VWF may dictate the length of storage and / or storage conditions (e.g., temperature). For example, it will be appreciated that a preparation in which the concentration of plasminogen and / or tissue plasminogen activator and / or other proteases in the collection solution is reduced by 80% compared to the starting material can be stored for a longer period of time and / or at a higher temperature without significantly destabilizing the activity of fibrinogen and / or factor VIII and / or VWF compared to a preparation in which the concentration of plasminogen and / or tissue plasminogen activator and / or other proteases in the collection solution is reduced by only 50% compared to the starting material.

[0114] While the method of the present invention can be carried out on a laboratory scale, it is possible to scale up to an industrial scale without significant changes in conditions. Thus, in one embodiment disclosed herein, the method of the present invention is carried out on an industrial or commercial scale. Preferably, the method of the present invention The method is suitable for commercial-scale production of fibrinogen and / or Factor VIII and / or VWF. For example, when a plasma fraction is used as the starting material for the method of the present invention, commercial-scale production will require the use of a plasma fraction obtained from at least about 500 kg of plasma. More preferably, the starting plasma fraction is obtained from at least about 5,000 kg, 7,500 kg, 10,000 kg, and / or 15,000 kg of plasma per batch. In certain embodiments, solutions and pharmaceutical formulations containing fibrinogen and / or Factor VIII and / or VWF of the present invention are produced on a commercial scale from plasma fractions or recombinant raw materials.

[0115] Those skilled in the art will understand that when a solution containing fibrinogen and / or factor III and / or VWF is used for clinical or veterinary applications (e.g., for administration to subjects deficient in fibrinogen and / or factor VIII and / or VWF, or for use as a fibrin glue), it may be desirable to reduce the active viral content (viral titer) and the level of other potentially infectious agents (e.g., prions) in the solution. This may be particularly desirable when the raw material (i.e., starting material) containing fibrinogen and / or factor VIII and / or VWF is obtained from plasma. Methods for reducing viral titer in a solution are known to those skilled in the art. Examples include pasteurization (e.g., incubating the solution at 60°C for 10 hours in the presence of high concentrations of stabilizers, such as glycine (e.g., 2.75M) and sucrose (e.g., 50%), and / or other selected excipients or salts), dry heat treatment, virus filtration (passing the solution through a nanofilter; e.g., a 20nm cutoff), and / or treating the solution with an appropriate organic solvent and detergent for a period and under conditions that inactivate the virus in the solution. Detergent solvents have been used for 20 years to inactivate enveloped viruses, particularly in plasma-derived products containing fibrinogen and factor VIII and / or VWF. Accordingly, inactivation can be performed using a variety of reagents and methods known in the art (see, e.g., U.S. Pat. Nos. 4,540,573 and 4,764,369, which are incorporated herein by reference). Suitable solvents include tri-n-butyl phosphate (TnBP) and ethers, preferably TnBP (usually about 0.3%). Suitable surfactants include polysorbate (Tween) 80, polysorbate (Tween) 20, and Triton X-100 (usually about 0.3%). The selection of processing conditions, including solvent and surfactant concentration, depends in part on the characteristics of the raw materials; less pure raw materials generally require higher concentrations of reagents and more extreme reaction conditions.A preferred surfactant is polysorbate 80, with a particularly preferred combination being polysorbate 80 and TnBP. The raw material can be stirred with the solvent and surfactant reagent at a temperature and for a time sufficient to inactivate any enveloped viruses that may be present. For example, the solvent-surfactant treatment can be carried out at 25°C for about 4 hours. The solvent-surfactant chemical is then removed, for example, by adsorption onto a chromatography medium such as a C-18 hydrophobic resin, or by elution into the flow-through of an ion-exchange resin under conditions that adsorb the protein of interest.

[0116] The virus inactivation step can be carried out at any appropriate stage of the methods disclosed herein. In one embodiment, prior to step (i), a virus inactivation step is carried out on the raw material containing fibrinogen and / or Factor VIII and / or VWF. In another embodiment, a virus inactivation step is carried out on the solution containing fibrinogen and / or Factor VIII and / or VWF recovered from the hydrophobic charge induction chromatography resin (i.e., steps (ii) and / or (iv)). In one embodiment disclosed herein, the virus inactivation step comprises pasteurization or treatment with organic solvents and detergents. In another embodiment disclosed herein, the virus inactivation step comprises virus filtration. When virus filtration is used, the inventors have found that the free amino acids (e.g., amino acids) are removed prior to the filtration step. We have found that the addition of fibrinogen and / or factor VIII and / or VWF to the filter significantly improves the flux and recovery of fibrinogen and / or factor VIII and / or VWF from the filter. An example of such a method is described in U.S. Patent No. 7,919,592.

[0117] In one embodiment disclosed herein, a raw material or solution containing fibrinogen and / or Factor VIII and / or VWF is subjected to a viral inactivation step before the solution is passed through an anion exchange chromatography resin. The advantage of using a viral inactivation step such as a solvent detergent treatment before passing the treated solution or raw material through an anion exchange chromatography resin is that the anion exchange resin allows for the removal of organic solvents and detergents from the treated solution by utilizing conditions that promote binding of fibrinogen and / or Factor VIII and / or VWF to the resin and removal of the organic solvent and detergent with the flow-through fraction.

[0118] Pasteurization can result in protein aggregates and polymers, particularly in solutions containing fibrinogen (also referred to herein as "fibrinogen solutions"). Therefore, in some cases, it may be desirable to reduce the level of aggregates / polymers in the pasteurized solution. This can be done by any means known to those skilled in the art, but can be conveniently achieved by further chromatographic purification. In one embodiment disclosed herein, the pasteurized solution or raw material is passed through an anion exchange chromatography resin in positive mode for fibrinogen and / or Factor VIII and / or VWF, such that any aggregates or polymers are removed in the flow-through fraction.

[0119] The term "raw material" is used herein to refer to any solution containing fibrinogen and / or Factor VIII and / or VWF. The raw material may also include other proteins (e.g., therapeutic proteins) known to those skilled in the art. Examples include proteins involved in the blood coagulation cascade. In one embodiment disclosed herein, the raw material includes fibrinogen.

[0120] Suitable sources containing fibrinogen and / or factor VIII and / or VWF are known to those skilled in the art. Examples include plasma or plasma fractions such as solubilized plasma cryoprecipitate, or solubilized fraction I pastes obtained from human or animal plasma or plasma fractions, recombinant cell culture fractions, and fractions obtained from the milk of transgenic animals. Recombinant sources of fibrinogen and / or factor VIII and / or VWF protein are also suitable for use as raw materials in the present invention. When the raw material is plasma or a plasma fraction, it may be pooled transfusion plasma or obtained from individual donors. In one embodiment disclosed herein, the source containing fibrinogen and / or factor VIII and / or VWF is solubilized plasma cryoprecipitate. This component, obtained from whole blood or collected by apheresis, is prepared by controlled thawing of freshly frozen plasma between 1 and 6°C and recovery of the precipitate. The cryo-insoluble precipitate is then refrozen. One unit of cryoprecipitate apheresis is roughly equivalent to two units of cryoprecipitate obtained from whole blood. It contains most of the fibrinogen, factor VIII, and VWF, along with other proteins such as factor XIII and fibronectin, derived from freshly frozen plasma. Another source of fibrinogen is fraction I precipitate, which can be prepared from frozen plasma by thawing and removing the cryoprecipitate by centrifugation or filtration. The resulting cryosupernatant is then mixed with ethanol to precipitate fraction I. For example, fraction I precipitate can be obtained by adding approximately 8% (v / v) ethanol at pH 7.2 and controlling the temperature at approximately -3°C (Cohn et al., 1946, J. Am. Chem. Soc. 62:459-475). In one embodiment, the raw material containing fibrinogen and / or factor VIII and / or VWF is cryoprecipitated. It's raining.

[0121] As used herein, the term "protease" refers to any protease and / or proenzyme thereof present in a source material or solution containing fibrinogen and / or factor VIII and / or VWF that, when exposed to the HCIC resin, can bind to the resin under conditions that allow the fibrinogen and / or factor VIII and / or VWF to pass through the resin. The protease may be of any type, including serine proteases (e.g., plasmin, thrombin, trypsin), threonine proteases, cysteine ​​proteases (e.g., cathepsin B and cathepsin H), aspartic acid proteases (e.g., pepsin), metalloproteases (e.g., collagenase and gelatinase), and glutamic acid proteases. When the fibrinogen and / or factor VIII and / or VWF-containing raw material is obtained from human or animal plasma, the proteases / proenzymes may include plasminogen, tissue plasminogen activator (tPA), thrombin, elastase, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, factor XIIIa, plasma kallikrein, etc. For solutions containing fibrinogen and / or factor VIII and / or VWF, a particularly preferred protease / proenzyme to be removed is plasminogen. Other proteases / proenzymes that are preferred to be removed from solutions containing fibrinogen and / or factor VIII and / or VWF are t-PA, pro- and / or activated thrombin (factor II / IIa). When the raw material containing fibrinogen and / or factor VIII and / or VWF is obtained from cell culture supernatant, the proteases / proenzymes may include any host cell proteases, such as serine proteases (e.g., caseinases), metalloproteases (e.g., gelatinases, matrix metalloproteases (MMPs) including MMP3, MMP10 or MMP12), aspartic acid proteases (cathepsin D), among others.

[0122] In some cases, it may be desirable to remove impurities or reduce the level of impurities from the raw material before passing the raw material through the HCIC resin in step (i). Removing impurities or reducing the level of impurities from the raw material can reduce the load on the HCIC resin during chromatographic purification and thus improve the separation efficiency of plasminogen and / or tissue plasminogen activator and / or other proteases from the raw material. Impurities may be removed or reduced, for example, by precipitating fibrinogen and / or factor VIII and / or VWF from the raw material and recovering the precipitated proteins. Suitable methods for precipitating fibrinogen and / or factor VIII and / or VWF from raw materials containing fibrinogen and / or factor VIII and / or VWF are known to those skilled in the art. One example involves adding an aluminum hydroxide suspension 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 with binding affinity for aluminum hydroxide, such as prothrombin (factor II) and t-PA, from plasma or plasma cryoprecipitate.

[0123] Thus, in one embodiment disclosed herein, vitamin K-dependent proteins are removed or reduced from the raw material prior to step (i). In other embodiments, vitamin K-dependent proteins are removed or reduced by adding aluminum hydroxide to the raw material. Aluminum hydroxide may be added to the raw material in the form of Alhydrogel® to a final concentration of about 10% to about 80% w / w. In some embodiments, aluminum hydroxide is added to the raw material to a final concentration ranging from about 10% to about 50% (w / w). In other embodiments, aluminum hydroxide is added to the raw material to a final concentration of about 10% In a preferred embodiment, the concentration is from about 15% to about 30% (w / w). Most preferably, for optimal fibrinogen recovery and removal of impurities such as prothrombin, aluminum hydroxide is added to the raw material at about 15% to about 25% (w / w). In another embodiment, vitamin K-dependent proteins are removed from the raw material by batch adsorption using aluminum hydroxide.

[0124] Another aspect of the present invention provides a solution containing fibrinogen and / or factor VIII and / or VWF recovered by the method of the present invention as described herein. In one embodiment, the level of plasminogen and / or tissue plasminogen activator and / or other proteases in the solution is less than 20% of the total protein, preferably less than 10% of the total protein, 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 total protein, or less than 0.0001% of the total protein. Those skilled in the art will understand that the level of plasminogen and / or tissue plasminogen activator and / or other proteases present in a solution containing fibrinogen and / or factor VIII and / or VWF may depend on the intended use or length of storage of the solution. For example, it may be acceptable for the solution to contain more than about 10% (of total protein) of plasminogen and / or tissue plasminogen activator and / or other proteases when stored at room temperature of about 0° C. to about 8° C. for at least four weeks. It may be desirable for the solution to contain less than about 10% (of total protein) of plasminogen and / or tissue plasminogen activator and / or other proteases when stored at a temperature of about 30° C. for at least four weeks.

[0125] In one embodiment disclosed herein, there is provided a solution comprising fibrinogen recovered by the methods of the present invention, hi another embodiment, the solution comprises at least 80% fibrinogen of total protein.

[0126] In another aspect of the present invention: (a) fibrinogen at least 75% of the total protein; (b) less than 50 pg tissue plasminogen activator per mg of total protein; and / or (c) Less than 1 μg of plasminogen per mg of total protein A solution containing to provide.

[0127] In one embodiment, the solution contains 1.5 x 10 per mg of total protein. -5 It further contains less than U of Factor II.

[0128] In another aspect of the present invention: (a) At least 90% fibrinogen of the total protein; (b) less than 50 pg tissue plasminogen activator per mg of total protein; and / or (c) Less than 150 ng of plasminogen per mg of total protein A solution comprising:

[0129] In one embodiment, the solution further comprises: (a) 3.5 × 10 per mg of total protein -6 Factor II less than U; and / or (b) less than 150 μg fibronectin per mg of total protein Includes.

[0130] In another aspect of the present invention: (a) At least 90% fibrinogen of the total protein; (b) less than 50 pg tissue plasminogen activator per mg of total protein; and / or (c) providing a solution containing less than 10 ng of plasminogen per mg of total protein;

[0131] In another aspect of the present invention: (a) At least 90% fibrinogen of the total protein; (b) less than 20 pg tissue plasminogen activator per mg of total protein; and / or (c) providing a solution containing less than 10 ng of plasminogen per mg of total protein;

[0132] In one embodiment, the solution further comprises: (a) 2.7 × 10 per mg of total protein -6 Factor II less than U; and / or (b) less than 15 μg fibronectin per mg of total protein Includes:

[0133] The concentrations of fibrinogen and / or factor VIII and / or VWF and impurities (e.g., plasminogen and / or tissue plasminogen activator and / or other proteases) in the solution recovered by the methods disclosed herein can be measured by any means known to those skilled in the art. Examples of suitable assays for measuring fibrinogen are described in Mackie et al. (Br J. Haematol. 2003 May;121(3):396-404). Size-exclusion HPLC can also be used to measure the concentrations of fibrinogen and / or factor VIII or impurities in a solution containing fibrinogen and / or factor VIII (e.g., 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 those skilled in the art to distinguish between fibrinogen monomers and aggregates. Furthermore, the concentrations of fibrinogen and / or factor VIII and / or VWF may vary depending on the sensitivity of the assay 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.

[0134] In one embodiment 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 the total protein as measured by size exclusion HPLC.

[0135] Another aspect of the present invention provides a pharmaceutical formulation comprising a solution containing fibrinogen and / or Factor VIII and / or VWF recovered by the methods disclosed herein and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers, including pharmaceutically acceptable diluents and / or excipients, are known to those skilled in the art. Examples include solvents, dispersion media, antifungal and antibacterial agents, surfactants, isotonic and absorption agents, etc.

[0136] The pharmaceutical formulation may also contain suitable stabilizers, such as amino acids, carbohydrates, salts and surfactants. The formulation can be prepared by adding a combination of: In certain embodiments, the stabilizer includes a mixture of a sugar alcohol and an amino acid. The stabilizer can include 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 embodiment, the formulation includes an amino acid such as arginine. In other embodiments, the formulation includes a divalent metal ion at a concentration of up to 100 mM and a complexing agent such as those described in U.S. Pat. No. 7,045,601. Certain embodiments include formulations 1 to 7, such as those described in Example 1 of U.S. Pat. No. 7,045,601. In certain embodiments, the formulation is formulated without the addition of any antifibrinolytic agent or stabilizing protein such as albumin. In embodiments, when the formulation includes fibrinogen, the pH is preferably about 6.5 to 7.5, and the osmolality is at least 240 mosmol / kg.

[0137] Pharmaceutical formulations may also be sterilized by filtration prior to dispensing and long-term storage. Preferably, the formulations substantially retain their 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 typically retain substantially the same molecular size distribution, as measured by HPLC-SEC, even after storage for 6 months or more. Certain embodiments of pharmaceutical formulations may be stable and suitable for commercial pharmaceutical use when stored at 2-8°C and / or room temperature for at least 6, 12, 18, 24, 36, or more months.

[0138] The solutions and pharmaceutical formulations of the present invention as described herein can be formulated into any of many possible dosage forms, such as injectable formulations. Formulation and subsequent administration (dosing) are within the skill of those in the art. Dosing will depend on the responsiveness of the subject being treated, but will continue as long as necessary to achieve the desired effect (e.g., restoration of normal plasma levels of fibrinogen). Those skilled in the art can easily determine the optimal dosage, method of administration, and number of repetitions.

[0139] In one embodiment disclosed herein, the pharmaceutical formulation of the invention has a volume of at least 5 mL and contains at least 5 mg / mL of fibrinogen. In another embodiment, the pharmaceutical formulation has a volume of at least 5 mL and contains at least 20 mg / mL of fibrinogen. In certain embodiments, the pharmaceutical formulation has a volume of at least 5 mL and contains 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 aspect, a container containing at least 5 mL of a stable, pharmaceutically acceptable fibrinogen solution is provided, wherein the concentration of fibrinogen is at least 20 mg / mL.

[0140] In another aspect, the present invention provides a method for treating or preventing a condition associated with fibrinogen and / or factor VIII and / or VWF deficiency, 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 of the present invention as disclosed herein or a pharmaceutical formulation of the present invention as disclosed herein.

[0141] In another aspect of the present invention, there is provided the use of a solution comprising fibrinogen and / or factor VIII and / or VWF recovered by the method of the present invention as disclosed herein in the manufacture of a medicament for treating or preventing a condition 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 embodiment, the fibrinogen condition is selected from the group consisting of afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia. In one embodiment, the Factor VIII and / or VWF condition is selected from the group consisting of bleeding disorders hemophilia A (e.g., platelet function defect, thrombocytopenia, or von Willebrand's disease), bleeding due to vascular injury, trauma, or surgery, bleeding due to anticoagulant therapy, and bleeding due to liver disease.

[0142] As disclosed herein, other conditions that can be treated with solutions containing fibrinogen and / or Factor VIII and / or VWF recovered by the methods of the present invention include major injuries and severe bleeding and burns. In cases of hypofibrinogenemia and afibrinogenemia, fibrinogen-containing solutions prepared according to the present invention can be administered intravenously to patients in need thereof to compensate for the fibrinogen deficiency, with dosages that can be determined by those skilled in the art based on the degree of deficiency.

[0143] Solutions containing fibrinogen recovered by the methods of the present invention are also advantageous for use in fibrin glues (also known as fibrin sealants) because they do not contain destabilizing levels of plasminogen and / or tissue plasminogen activator and / or other proteases. tPA converts plasminogen to active plasmin, which then digests fibrin clots, thereby reducing clot formation in topical applications (e.g., hemostasis).

[0144] Fibrin glue typically contains two components: (i) fibrinogen (often with factor XIII and a fibrinolysis inhibitor such as aprotinin) and (ii) thrombin (often with calcium ions). The two components are reconstituted to prepare a ready-to-use adhesive. Fibrin glue is used in clinical and veterinary applications to stimulate the final step of blood clotting by forming crosslinked fibrin fibers using a combination of fibrinogen and thrombin in the presence of calcium and factor XIII. Fibrin glue has a variety of applications in clinical and veterinary medicine, including hemostasis, wound closure, adhesion prevention, and wound healing. Fibrin glue can also be used to close skin wounds (including skin grafts), for sealing sutures, and to connect connective tissues such as bone, cartilage, and tendon. Accordingly, another aspect disclosed herein provides a fibrin glue comprising a solution containing fibrinogen recovered by the methods of the present invention, as disclosed herein.

[0145] Those skilled in the art will recognize that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications that fall within its spirit and scope. The invention also includes all steps, features, compositions, and compounds described or illustrated herein, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0146] Certain embodiments of the present invention will now be described with reference to the following examples, which are intended for illustrative purposes only and are not intended to limit the scope of the general principles set forth above. [Example]

[0147] Example 1 Purification of fibrinogen with HEA, PPA, and MEP hydrophobic charge induction chromatography (HCIC) resins Pooled human plasma cryoprecipitate was used as the starting material (i.e., fibrinogen-containing raw material). Briefly, pooled plasma cryoprecipitate was diluted with 20 mM citrate and 3% Fibrinogen. The fibrinogen was solubilized in an extraction buffer containing sodium, 200 mM epsilon-aminocaproic acid (ε-ACA), 60 IU / mL heparin, and 500 mM NaCl (pH 7.2 ± 2) for 30 minutes at 31 ± 2°C (1 g of cryoprecipitate per 4 g of buffer). Next, 2% (w / w) aluminum hydroxide was added to the solubilized cryoprecipitate at a concentration of 25% (w / w). The aluminum hydroxide gel was then removed by either centrifugation or depth filtration, and the fibrinogen-containing supernatant was collected for further purification by chromatography on HCIC chromatography resin.

[0148] The fibrinogen-containing supernatant was loaded onto a chromatography column packed with 1.8 mL of either HEA, PPA, or MEP Hypercel™ resin. The chromatography column was pre-equilibrated with 25 mM Tris at various pHs ranging from 6.5 to 8.5. The fibrinogen-containing supernatant was loaded onto the chromatography column at a ratio of approximately 11 mL / mL resin. HCIC purification was performed in negative mode with respect to fibrinogen, allowing it to pass through in the unbound flow-through fraction, while most of the t-PA, plasminogen, and factor II remained bound to the resin.

[0149] Figures 1 through 3 show the step recoveries after postchromatographic purification of fibrinogen, plasminogen, t-PA, and factor II using HEA Hypercel™, PPA Hypercel™, and MEP Hypercel™. The results indicate that pH has little or no effect on plasminogen binding to these resins, while t-PA binding to the resin appears most effective in the low pH range. The HEA Hypercel™ column exhibited the highest fibrinogen recovery in the flow-through fraction and appeared to have little effect on fibrinogen recovery over the operating pH range tested, compared to the recoveries observed with both the PPA and MEP Hypercel™ columns. Both the PPA and MEP columns exhibited the highest fibrinogen recovery in the flow-through fraction at pH 8.5.

[0150] Example 2 Impurity levels in fibrinogen solutions reduced by HEA Hypercel Approximately 48.5 mL of solubilized cryoprecipitate containing fibrinogen prepared according to Example 1 was loaded onto a 5 mL HEA Hypercel™ column pre-equilibrated with 25 mM Tris at either pH 6.5, 7.0, 7.5, 8.0, or 8.5. HCIC purification was performed in negative mode for fibrinogen, allowing it to pass through in the unbound flow-through fraction, while t-PA, plasminogen, and factor II remained bound to the resin. Figure 4a shows the step recoveries of fibrinogen, plasminogen, t-PA, and factor II after post-chromatographic purification using the HEA Hypercel™. The results indicate that pH has little or no effect on the binding of plasminogen and factor II to the HCIC resin, while t-PA binding to the resin appears most effective in the low pH range of 6.5 to 7.0. The fibrinogen recovery rate exceeded 90% in the flow-through fraction under various pH conditions, even without column washes. As shown in Figure 4a, these results demonstrate that the HCIC resin effectively removes proteases from crude fibrinogen-containing raw materials, such as solubilized cryoprecipitate. For example, experimental conditions performed at pH 7.0 showed a reduction of ≥99.9% of factor II, ≥88.3% of t-PA, and ≥98.2% of plasminogen from the solubilized cryoprecipitate solution.

[0151] Fibrinogen remained stable in solution for at least 6 days at room temperature (approximately 20° C.) The results are shown in Figures 4b and 4c.

[0152] Example 3 Impurity levels in fibrinogen solutions purified by HEA Hypercel Approximately 500 mL of fibrinogen-containing supernatant obtained after the Alhydrogel™ adsorption step produced according to Example 1 was loaded onto an XK16 / 30 column packed with 36 mL of HEA Hypercel™ resin pre-equilibrated with 25 mM Tris pH 7.0. The flow-through was collected for testing of fibrinogen, plasminogen, t-PA, and Factor II. A summary of the results is shown in Table 1 below.

[0153] [Table 1]

[0154] Example 4 Effect of glycine precipitation on the levels of impurities in fibrinogen solutions purified by HEA Hypercel. A further precipitation step was carried out by adding saline (pH 6.6-7.3) containing 2.4 M glycine, 2.7 M NaCl, 2.1 mM CaCl2, and 23 mM trisodium citrate to the fibrinogen-containing supernatant obtained after the alhydrogel adsorption step, as described in Example 1. The solubilized precipitate was warmed to 30°C, and then glycine buffer, also incubated at 30°C, was added in a 1:2 ratio of product to buffer. The mixture was stirred for 10 minutes, and the resulting precipitate was recovered from the liquid phase by centrifugation. The liquid phase, which primarily contained fibronectin and IgG, was discarded, and the fibrinogen-containing precipitate was collected and resuspended in solubilization buffer (pH 7.0) containing 100 mM NaCl, 1.1 mM CaCl2, 10 mM trisodium citrate, 10 mM Tris-(hydroxymethylmethylamine), and 4.5 mM sucrose. The solubilized fibrinogen intermediate (250 mL) was clarified using a 1 μm filter and then passed through an XK16 / 30 column packed with 36 mL of HEA Hypercel™ resin and pre-equilibrated with 25 mM Tris pH 7.0. The flow-through was collected for testing of fibrinogen, plasminogen, t-PA, and Factor II, and the results are summarized in Table 2 below.

[0155] The results indicate that binding of plasminogen, t-PA, and Factor II to the HEA Hypercel™ resin is more effective under these processing conditions than that observed under the conditions described in Example 2. Analytical results showed that the process recoveries were approximately 93% fibrinogen, 6% plasminogen, 12% t-PA, and 5% Factor II.

[0156] [Table 2]

[0157] Example 5 Preparation of purified fibrinogen from plasma cryoprecipitate Processing step 1 - solubilization of plasma cryoprecipitate; Process step 2 - Alhydrogel™ (aluminum hydroxide) adsorption of the solubilized plasma cryoprecipitate (Alhydrogel™ concentrate: target 15-20% w / w, range 10-50% w / w) and recovery of the fibrinogen-containing supernatant using methods such as centrifugation or depth filtration in the presence of a filter aid. Alternatively, this step can be replaced by an HCIC chromatography step in negative mode (flow-through) for fibrinogen or a combination of HCIC and anion exchange chromatography, both in negative mode for fibrinogen. If both HCIC and anion exchange chromatography are used in combination, process step 3 is selected; Process step 3 - glycine precipitation of fibrinogen from the fibrinogen-containing supernatant of step 2. Alternatively, this step can be replaced by anion exchange chromatography in negative mode with respect to fibrinogen; Process step 4 - The solubilized glycine precipitate from step 3 is passed through HCIC chromatography resin in negative mode with respect to fibrinogen; Processing step 5 - Treating the purified fibrinogen solution recovered in step 4 with a solvent or detergent or pasteurizing it to inactivate pathogens; Processing step 6 - passing the processing solution from step 5 through an anion exchange chromatography resin in positive mode with respect to fibrinogen, washing weakly bound proteins from the resin, and eluting fibrinogen from the resin; Process step 7 - subjecting the fibrinogen eluted from the anion exchange resin of step 6 to nanofiltration (35 nm or 20 nm or a combination of 35 / 20 nm); Process Step 8 - The fibrinogen filtered in step 7 is ultrafiltered (50, 100, 200 and 300 kDa membrane filters).

[0158] Example 6 Preparation of purified fibrinogen by a combination of HCIC and anion exchange chromatography Three laboratory-scale experiments were completed in which approximately 100 g of cryoprecipitate was prepared by the process described in Examples 1-3 in a mixed chromatography process using a HEA Hypercel™ column.

[0159] The flow-through fraction from the HEA Hypercel™ column, which contained primarily fibrinogen, was subjected to an overnight solvent / detergent treatment for viral inactivation.

[0160] The virus-inactivated solution was then diluted to ≦10 mS / cm with 25 mM Tris (pH 8.0) and loaded onto an anion exchange column (XK50 / 30, GE Healthcare) packed with approximately 412 mL of MacroPrep™-HQ resin pre-equilibrated with 25 mM Tris (pH 8.0). The flow-through fraction was discarded, and the MacroPrep™-HQ column was washed with four column volumes of wash buffer (pH 8.0) containing 90 mM NaCl, 50 mM Tris, 20 mM EACA. Under these chromatographic conditions, the initial flow-through and wash fractions contained primarily plasminogen and t-PA, while fibrinogen remained bound to the chromatographic resin. Monomeric fibrinogen was selectively eluted from the MacroPrep™-HQ column using an elution buffer containing 200 mM NaCl, 10 mM Tris, 10 mM trisodium citrate, 46 mM sucrose, and 1.1 mM CaCl2 (pH 7.0), while fibrinogen aggregates and low molecular weight proteins remained bound to the MacroPrep™-HQ resin.

[0161] The product intermediates resulting from the solubilized cryoprecipitate that were passed through to the MacroPrep™-HQ chromatography eluate were characterized by the levels of fibrinogen, t-PA, plasminogen, fibronectin, and Factor II, as well as the process step recoveries of each of these proteins at various processing stages. The results shown in Table 3 represent the average values ​​of three separate batches run consistently at laboratory scale. The recoveries of fibrinogen and co-purified proteins throughout the process, starting from the plasma cryoprecipitate to the MacroPrep™-HQ eluate, are shown in Figure 5.

[0162] The purity of fibrinogen recovered from the MacroPrep™-HQ chromatography resin was greater than 95%, as evidenced by analysis of the size-exclusion HPLC chromatogram. A representative example of the HPLC profile of fibrinogen analyzed on a TSKGel™ G4000SWXL (Tosoh Corporation) is shown in Figure 6. As shown in Figure 6, fibrinogen monomer eluted at a retention time of approximately 17.4 minutes, accounting for 96.6% of the total peak area, while fibrinogen dimers and / or other high molecular weight proteins eluted at a retention time of approximately 14.8 minutes.

[0163] Other fibrinogen batches were produced at pilot scale (81 kg plasma equivalent) according to the methods described in Examples 5 and 6. The characteristics of the fibrinogen preparation are shown in Table 4.

[0164] Example 7 Viral filtration of purified fibrinogen The resulting fibrinogen preparations were tested for filterability through 20 nm virus filters according to the method of Example 6, using either 190 mM NaCl buffer (21.5 mS / cm), 200 mM NaCl buffer (22.5 mS / cm), 210 mM NaCl buffer (23.5 mS / cm) or 200 mM NaCl buffer containing 1% (w / w) arginine (25 mS / cm) for the MacroPrep™-HQ elution step.

[0165] This method required the preparation to be formulated with 3% (w / w) arginine at a pH of approximately 7.5 (the protein concentration of the sample was approximately 6 g / L). The formulated preparation was then filtered using a 0.1 μm filter prior to the viral filtration step, which was performed using a 47 mm Pall SV4™ filter in a dead-end mode, using a constant pressure of 1.8 bar. Results are shown for fibrinogen preparations obtained from MacroPrep™ HQ columns using either 190 mM, 200 mM, or 210 mM. In contrast, fibrinogen preparations eluted from a MacroPrep™ HQ column using 200 mM NaCl buffer containing 1% (w / w) arginine adhered rapidly to the filter (Figure 7).

[0166] Example 8 Stability testing Purified fibrinogen solutions recovered by the method described in Example 5 above were sterile filtered and stability tested at 2°C-8°C or 30°C for 9 / 7 weeks. Liquid fibrinogen preparations stored at 2°C-8°C retained approximately 90% of their original activity after 9 weeks of storage, as measured by the Clauss method. Liquid fibrinogen preparations stored at 30°C retained approximately 70% of their original activity after 2 weeks of storage, with no further loss of activity for at least 5 weeks. A further decrease in activity below 60% was observed after 7 weeks of incubation at 30°C. The loss of fibrinogen activity at 30°C is likely due to thermal denaturation rather than proteolysis, as the addition of a protease inhibitor (C1 esterase) did not inhibit the loss of fibrinogen activity over 5 weeks of storage. A summary of the stability data is shown in Figure 8.

[0167] [Table 3]

[0168] [Table 4]

[0169] Example 9 Reduction of plasma protease levels in solutions containing factor VIII and / or VWF using HEA Hypercel This example demonstrates that an HCIC chromatography step can also be used to reduce proteases in factor VIII- and / or VWF-containing preparations. This method involves clarifying the fibrinogen-containing solution obtained from Example 4 using a depth filter and then passing the clarified solution through a second hydrophobic charge induction chromatography (HCIC) resin. The HCIC step was operated under conditions that allowed proteases, such as plasminogen, to bind to the resin while allowing factor VIII and VWF to pass through. Specifically, an XK50 / 30 column was packed with 340 mL of HEA Hypercel™ resin. The column was pre-equilibrated with 50 mM Tris pH 6.7. The clarified fibrinogen solution prepared according to Example 4 was then applied to the column, and the column was washed with 50 mM Tris pH 6.7. The flow-through fraction was collected, and the levels of factor VIII, VWF, plasminogen, and t-PA were measured (VWF: RCo = von Willebrand ristocetin cofactor). A summary of the average results from four individual experiments is shown in Table 5. The results indicate that the HCIC chromatography step effectively removed proteases such as plasminogen and tPA from the fraction containing factor VIII and VWF. Furthermore, good recoveries of factor VIII and VWF were observed.

[0170] [Table 5]

[0171] Example 10 Comparative study of fibrinogen purified by various methods. As described in Example 6, fibrinogen preparations produced by the methods of the present invention were compared to fibrinogen preparations produced by the methods described in WO2001048016, WO2012038410 and WO2013135684.

[0172] (a) A fibrinogen preparation produced by a preferred embodiment of the method described in WO2001048016.

[0173] Briefly, this method involves suspending Fraction I paste 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 Fraction I to 8.33 g Extraction Buffer). This solution was then mixed at 37°C for 1.5 hours, after which 50 g of 2% Al(OH)3 (Alhydrogel) solution was added to 1 g Fraction I (10.8%). This mixture was stirred for 15 minutes at room temperature and then centrifuged at 5000g for 10 minutes, and the pellet was discarded. To the Alhydrogel-treated supernatant, glycine / NaCl buffer (2.1 M glycine, 20 mM Na citrate, 3.6 M NaCl, and 2.4 mM CaCl2) was added (both solutions were pre-equilibrated to 30°C). Addition of the supernatant to the buffer was completed in approximately 4.5 minutes (1 part supernatant to 2.05 parts buffer). The mixture was then stirred at 30°C for 20 minutes before being centrifuged at 5010g for 10 minutes (the supernatant was discarded). The precipitate was then redissolved in Buffer D (100 mM NaCl, 1.1 mM CaCl, 10 mM Na-citrate, 10 mM Tris, 45 mM sucrose, pH 6.9) by mixing at room temperature for 2 hours (1 / 3 the volume used to resuspend Fraction I) (WO 0148016, Example 1, sections 1.1.1-1.1.6). The solution containing the redissolved fibrinogen was then loaded onto a MacroPrep™ HQ column (XK26 with a 20 cm bed height). The column was filled with at least 1.5 column volumes (CV) of MQ buffer (50 mM Tris, 100 mM The column was pre-equilibrated with NaCl, 20 mM EACA, pH 8.0, at 10 mL / min (113 cm / hr). Equilibration was continued until the conductivity after the column was 90-110% of that of the prepared buffer. Next, the fibrinogen solution was applied to the column, and the column was washed with 6 CV of MQ buffer. Fibrinogen was then dissolved in ME buffer (500 mM NaCl, 1.1 mM EACA). The column eluted as a single peak using 10 mM CaCl, 10 mM Na citrate, 10 mM Tris, and 45 mM sucrose, pH 7.0. The column could be regenerated using 2 CV of 1 M NaCl (WO 2001048016, Example 2).

[0174] (b) A fibrinogen preparation produced by preferred embodiments of the methods previously described in WO2012038410 and WO2013135684.

[0175] Cryoprecipitate produced from plasma by established methods was reconstituted or solubilized at near-neutral pH, adsorbed with Al(OH)3, and the resulting gel was removed by centrifugation. The virus in the supernatant was then inactivated by solvent / detergent (S / D) treatment. S / D compounds, Triton, and TnBP were extracted with vegetable oil, and the aqueous phase was contacted with Fractogel® EMD-TMAE. Chromatographic conditions (pH 6.9-7.1 and osmolality 570-610 mosmol / L) were used such that fibrinogen did not bind to the gel and was therefore found in the flow-through or supernatant. After the addition of glycine (final concentration 1 mol / L, pH 7.4), the unbound fibrinogen solution was stirred for approximately 90 minutes in the presence of 20 mM EDTA to precipitate fibrinogen. The fibrinogen-containing precipitate was then separated by centrifugation to produce an intermediate fibrinogen paste. The intermediate fibrinogen paste was resuspended in 20 mM Tris buffer (pH = approximately 8.0). The resulting suspension was then filtered and subjected to ultrafiltration / diafiltration. The resulting fibrinogen-containing solution was then added to a GigaCap Q-650M®, and the chromatography gel or resin was pre-equilibrated with the same Tris buffer used for resuspension before adding the fibrinogen solution. Loosely bound material was washed with equilibration buffer and then with wash buffer (1.5 g / L sodium citrate, 6.0 g / L sodium chloride, adjusted to pH = approximately 7.0 and conductivity approximately 12.0 mS / cm). Fibrinogen was then eluted from the chromatography column with elution buffer (NaCl, adjusted to the same pH as the wash buffer, approximately 7.0 g / L, 1.5 g / L sodium citrate, adjusted to a conductivity of approximately 13.1-15 mS / cm, 10.0 g / L glycine).

[0176] Fibrinogen-containing solutions obtained from the methods described in WO 2001048016, WO 2012038410, and WO 2013135684 were tested for total protein (biuret), fibronectin, and plasminogen levels. Additionally, stability tests of the samples were performed at 2-8 °C and 30 °C.

[0177] A comparison of the properties of the fibrinogen preparations is shown in Table 6. The results of the tests show that fibrinogen produced from the method of the present invention contains lower levels of plasminogen compared to other methods.

[0178] [Table 6]

Claims

1. (a) at least 90% fibrinogen of the total protein; (b) less than 50 pg tissue plasminogen activator per mg of total protein; (c) less than 10 ng plasminogen per mg of total protein; and (d) 2.7 × 10 per mg of total protein -6 Factor II less than U; A solution comprising: The solution is derived from plasma, The fibrinogen is (i) about 90% to 100% activity after at least 4 weeks of storage in solution at a temperature of about 0°C to about 8°C; and / or (ii) The solution retains about 60% to about 70% of its activity after 5 weeks of storage at a temperature of about 30°C.

2. 10. The solution of claim 1 comprising less than 20 pg tissue plasminogen activator per mg of total protein.

3. 3. The solution of claim 1 or 2, further comprising less than 15 μg of fibronectin per mg of total protein.

4. 4. The solution of claim 1, which comprises at least 80% monomeric fibrinogen of total protein.

5. A pharmaceutical formulation comprising the solution of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

6. 6. The pharmaceutical formulation of claim 5, having a volume of at least 5 mL and containing at least 5 mg / mL of fibrinogen.

7. having a volume of at least 5 mL and containing at least 20 mg / mL of fibrinogen; The pharmaceutical formulation of claim 5.

8. A solution according to any one of claims 1 to 4 or a pharmaceutical formulation according to any one of claims 5 to 7 for use in a method for treating or preventing conditions associated with fibrinogen deficiency.

9. 9. The solution or pharmaceutical formulation of claim 8, wherein the condition is selected from the group consisting of afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia.

10. A fibrin glue comprising the solution according to any one of claims 1 to 4.

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