Manufacturing method of fibrinogen preparation

Abstract

The present invention relates to a method for producing a fibrinogen preparation from a plasma-derived fibrinogen-containing source. The method includes the steps of providing a liquid phase containing plasma fibrinogen; contacting the liquid phase with a cation exchange chromatography material under conditions conducive to binding of fibrinogen, wherein the liquid phase has a pH in the range of pH 5.6 to pH 7.0, which is close to or higher than the pI of fibrinogen; optionally washing unbound compounds from the cation exchange chromatography material; and eluting fibrinogen from the cation exchange material. The method of the present invention is also suitable for the reduction of von Willebrand factor.

Description

[Technical Field]

[0001] The present invention relates to the production of fibrinogen preparations from plasma-derived fibrinogen-containing sources. [Background technology]

[0002] Fibrinogen is the major structural protein in blood involved in the formation of blood clots, which is of great importance for the treatment of hemostatic disorders.

[0003] An important source of fibrinogen is its isolation from plasma, especially human plasma. During this purification, other components of the plasma or of the manufacturing process must be removed in order to obtain a specific pure coagulated blood product. These other components are usually other plasma proteins, especially von Willebrand factor (vWF).

[0004] Purification of fibrinogen from plasma is usually performed by a combination of precipitation followed by chromatographic methods and viral inactivation steps such as solvent / detergent (S / D) and / or viral removal steps such as filtration. Therefore, it is important to remove not only other plasma proteins but also reagents or additives used in previous process steps.

[0005] WO00 / 17234A1 / EP1115742B1 discloses the purification of recombinant fibrinogen from the milk of transgenic animals using cation exchange chromatography (CEX) to remove the milk protein casein from the preparation.

[0006] WO98 / 38219A1 discloses the purification of vWF (isoelectric point 5.5-6) ​​using a negatively charged gel matrix for cation exchange chromatography: vWF is bound to the cation exchanger at low salt concentrations and eluted with buffers having a pH in the range of 5.0-8.5.

[0007] WO91 / 01808A1 discloses the selective removal of LDL, fibrinogen, and / or urea from aqueous liquids such as whole blood, plasma, or serum. This method uses an adsorption material (tentacle cation exchange material) based on Fractogel® material (Merck, Germany) modified with polymer chains of monomers containing sulfonate groups to form graft copolymers. The specific adsorption properties of the cation exchange material depend on the presence of tentacle-like ligand structures.

[0008] EP2267025A2 describes the use of CEX for the purification of fibrinogen. Fibrinogen does not bind to the CEX matrix. Summary of the Invention

[0009] The object of the present invention is to provide a method for producing a fibrinogen preparation from a plasma-derived fibrinogen-containing source, which allows for an easy and efficient purification of fibrinogen, preferably with improved properties.

[0010] The object is achieved by a method for producing a fibrinogen preparation from a plasma-derived fibrinogen-containing source, comprising the following steps: a) providing a liquid phase comprising plasma fibrinogen; b) contacting the liquid phase with a cation exchange chromatography material under conditions that result in binding of fibrinogen; wherein the liquid phase has a pH in the range of pH 5.6 to pH 7.0, which is close to or higher than the pI of fibrinogen; c) optionally washing unbound compounds from the cation exchange chromatography material; d) Eluting the fibrinogen from the cation exchange material. DETAILED DESCRIPTION OF THE INVENTION

[0011] The object of the invention is also achieved by the method described below. In the following, the individual steps of the method are described in more detail. The steps do not necessarily have to be performed in the order described herein. Also, further steps not explicitly described may form part of the method.

[0012] As used herein, the term "fibrinogen" refers to the major structural protein present in plasma involved in clot formation, preferably the glycoprotein form of fibrinogen as a whole. Preferably, it refers to plasma fibrinogen, i.e., fibrinogen derived from plasma. More preferably, it is human plasma fibrinogen.

[0013] The pI or isoelectric point (IEP) of a protein is the pH value at which the protein has no net charge. At pH values ​​higher than the pI, the protein has a net negative charge, and at pH values ​​lower than the pI, the protein has a net positive charge. In this specification, the pI of fibrinogen is pH 5.5, as described, for example, in WO 00 / 17234 A1. According to some other publications, the pI of fibrinogen ranges from approximately pH 5.5 to 6.0, depending on the cleavage of polar residues during the clotting reaction by thrombin (Guo et al. Nature Nanotechnology 2016, 11, 817-824).

[0014] As used herein, a "cation exchange chromatography (CEX) material" is a solid phase containing negatively charged groups. Proteins are separated based on the interaction between the negatively charged groups on the resin and the positively charged groups on the proteins. The strength of the interaction also depends on the ionic strength (i.e., conductivity) of the buffer. Elution is usually achieved by increasing the ionic strength of the buffer so that the charged sites on the cation exchange material compete with the proteins. Another method for eluting proteins is to change the pH, thereby changing the charge of the protein. The change in conductivity and / or pH can be gradual or stepwise.

[0015] The charge of the CEX material can be provided by, for example, covalently binding one or more charged ligands to the solid phase. A preferred CEX material in the present invention is a strong CEX material, which maintains a negative charge on the solid phase over a wide pH range. It typically contains a sulfonic acid derivative, such as a sulfoethyl, sulfopropyl, sulfobutyl, or sulfoisobutyl group, as a functional group (e.g., sulfonate, S-type, or sulfopropyl group, SP-type).

[0016] Commercially available cation exchange materials include carboxy-methyl-cellulose, BAKERBOND ABX™, sulfopropyl (SP)-immobilized agarose (e.g., SP-SEPHAROSE FAST FLOW™, SP-SEPHAROSE FAST FLOW XL™, or SP-SEPHAROSE HIGH PERFORMANCE™, GE Healthcare), CAPTO S™ (GE Healthcare), sulfonyl-immobilized agarose (e.g., S-SEPHAROSE FAST FLOW™, GE Healthcare), and SUPER SP™ (Tosoh Biosciences). Preferred cation exchange materials in the present invention include cross-linked poly(styrene-divinylbenzene) flow-through particles (solid phases) coated with a polyhydroxylated polymer functionalized with sulfopropyl groups (e.g., POROS™ 50 HS chromatography resin, Thermo Fisher Scientific), or methacrylic copolymers with sulfonic acid groups (e.g., Macro-Prep® High S, Bio-Rad). Particularly preferred CEX materials are those having pores with an average pore size of 50 nm or more, preferably 100 nm or more, for example, an average pore size of 160 nm.

[0017] According to conventional processes, the skilled artisan uses a CEX substrate such that the pH of the medium for carrying out the separation is lower than the pI of the protein of interest, so that the protein of interest binds to the CEX substrate.

[0018] "Solid phase" refers to a non-aqueous matrix to which one or more charged ligands can be attached. The solid phase can be a purification column (including, but not limited to, expanded-bed and packed-bed columns), a discontinuous phase of individual particles, a membrane, or a filter. Examples of materials that form solid phases include polysaccharides (e.g., agarose and cellulose) and other mechanically stable matrices such as silica (e.g., controlled-pore glass), poly(styrene-divinylbenzene), methacrylate copolymers, polyacrylamide, ceramic particles, and derivatives of any of the above, preferably poly(styrene-divinylbenzene) and methacrylate copolymers.

[0019] As used herein, the term "liquid phase comprising plasma fibrinogen" refers to the composition that is loaded onto the cation exchange material. Preferably, the cation exchange material is equilibrated with an equilibration buffer prior to loading of the composition to be purified.

[0020] A "buffer" is a solution that resists changes in pH by the action of its acid-base conjugate components. A variety of buffers can be used depending on the desired buffer pH.

[0021] "Equilibration buffer" is a buffer used to equilibrate the cation exchange material prior to loading of the liquid phase containing fibrinogen. The composition of the equilibration buffer depends on the CEX material used.

[0022] As used herein, the term "wash buffer" refers to a buffer that is run over a cation exchange material after loading a composition and before eluting the fibrinogen bound to the CEX material. The wash buffer can serve to remove one or more contaminants from the cation exchange material without substantially eluting the bound fibrinogen. One or more wash buffers can be used before eluting the bound fibrinogen.

[0023] An "elution buffer" is used to elute fibrinogen from the solid phase. In the present invention, the elution buffer has a substantially higher conductivity and / or a higher pH than the final wash buffer so that fibrinogen is eluted from the cation exchange material. Preferably, the conductivity and / or pH of the elution buffer is substantially higher than that of the liquid phase containing fibrinogen and the previously used buffers, i.e., the equilibration buffer and all wash buffers used. "Substantially higher conductivity" means, for example, that the buffer has a conductivity that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 conductivity units (mS / cm) higher than the comparable composition or buffer. "Substantially higher pH" means, for example, that the buffer has a pH that is at least 0.2, 0.3, 0.4, or 0.5 pH units higher than the comparable composition or buffer. The conditions for elution depend on the CEX material used.

[0024] A "regeneration buffer" can be used to regenerate the cation exchange material so that it can be reused. The regeneration buffer has the conductivity and / or pH necessary to remove substantially all contaminants and any residual fibrinogen from the cation exchange material.

[0025] The term "conductivity" refers to the ability of an aqueous solution to pass an electric current between two electrodes. In a solution, electric current flows through the transport of ions. Therefore, the greater the amount of ions present in an aqueous solution, the higher the conductivity the solution may exhibit. The basic unit of conductivity measurement is siemens per meter (S / m), usually measured in mS / cm, and may be measured using a conductivity meter. Because conductivity is the ability of ions in a solution to pass an electric current, the conductivity of a solution may be changed by changing the concentration of ions therein. For example, to achieve a desired conductivity, the concentration of a buffer and / or the concentration of a salt (e.g., sodium chloride, sodium acetate, or potassium chloride) in the solution may be altered. Preferably, the salt concentration of various buffers is adjusted to achieve the desired conductivity. Additionally, organic cations, preferably salts of cationic amino acids, preferably arginine, may contribute to conductivity, especially when present as the hydrochloride salt.

[0026] The term "plasma-derived fibrinogen-containing source" specifies that the source of fibrinogen is plasma or a plasma fraction, preferably human plasma. In particular, it is not recombinant or synthetic fibrinogen. Furthermore, it is not produced by recombinant production in other organisms or fluids. A plasma source also specifies the possible presence of associated contaminants, such as albumin, fibronectin, IgG, vWF, or fibrinopeptide A.

[0027] The method for producing a fibrinogen preparation according to the present invention allows for the purification of the fibrinogen product with improved purity and activity profile. This method is particularly useful for separating vWF from a fibrinogen preparation, thereby reducing the vWF content in the resulting composition. Preferably, in step b), where the pH of the liquid phase is between 5.6 and 7.0, preferably between 6.0 and 6.9, and more preferably between 6.3 and 6.9, both fibrinogen and vWF bind to the cation exchange chromatography material. When fibrinogen is eluted from the material in step d), vWF remains on the cation exchange material, while fibrinogen is selectively eluted. For example, elution is performed at pH 7.0, which maintains the binding of vWF to the chromatography matrix and allows for the selective elution of fibrinogen.

[0028] Generally speaking, according to the methodological theory of cation exchange chromatography, fibrinogen should not bind to a cation exchange chromatography material at a pH higher than the pI of the protein of interest. Thus, for example, EP 2267025 A2 describes the application of a cation exchange chromatography step in a fibrinogen purification method, but fibrinogen does not bind to the matrix. Surprisingly, the inventors have found that fibrinogen binds to the chromatography material under the conditions used in the present invention, particularly at pH values ​​higher than the pI of fibrinogen. Furthermore, vWF also binds to the chromatography material, despite its isoelectric point being very close to that of fibrinogen. According to WO 98 / 38219 A1, the isoelectric point of vWF is in the range of 5.5 to 6. Therefore, it is surprising, firstly, that both proteins bind to the material, and secondly, that both proteins, especially fibrinogen, can be selectively eluted despite their very close isoelectric points. The inventors have found that under certain conditions, vWF binds slightly more strongly than fibrinogen. Therefore, the method of the present invention allows fibrinogen to be selectively eluted from the column, and the chromatography step can be applied to effectively reduce the amount of vWF in a fibrinogen preparation. Therefore, the present invention also relates to a method for reducing the amount of vWF in a composition containing fibrinogen by using the cation exchange chromatography described herein.

[0029] Thus, in a preferred embodiment of the present invention, the fibrinogen-containing liquid phase further contains vWF. The method of the present invention can be used with a liquid phase in which vWF is present in an amount of at least 0.3 U / mg total protein, e.g., at least 0.4 U / mg total protein, as measured by OD280 (e.g., VWF level assessed by VWF antigen). In a preferred embodiment, the vWF content in the liquid phase is about 0.4-1.0 U / mg total protein. The vWF content depends on the source of fibrinogen and on any preceding purification steps. Thus, the vWF content can be higher.

[0030] In a preferred embodiment, the liquid phase loaded onto the CEX material is a partially purified fibrinogen preparation. The partially purified fibrinogen preparation may contain 90% or more, preferably 95% or more, fibrinogen. In a preferred embodiment, the liquid phase contains proteins other than fibrinogen, preferably other plasma proteins, in amounts less than 10%, preferably less than 5%. The amount of vWF in the liquid phase loaded onto the CEX material may be, for example, 0.4 to 1.0 U / mg total protein. Preferably, the vWF content of the load material is at least 0.5 U / mg total protein, preferably 0.5 to 1.0 U / mg total protein.

[0031] Furthermore, the methods of the present invention are particularly useful for reducing the amount of prions in fibrinogen preparations. The inventors have been able to demonstrate that the cation exchange chromatography step described herein effectively removes prions to below the detection limit. To demonstrate this effect, hamster prions, a well-established test model for variant Creutzfeldt-Jakob disease, are used in spiking experiments. Thus, the present invention also relates to a method for removing or reducing prions in a composition containing fibrinogen by using the cation exchange chromatography described herein.

[0032] The liquid phase containing fibrinogen is preferably an aqueous phase. The liquid phase may further contain one or more surfactants, such as polysorbate 80, and / or solvents, such as tributyl phosphate (TnBP). Such surfactants and solvents may be present due to a previous S / D treatment for virus removal. A major advantage of the method of the present invention is that it also allows for the removal of such other undesirable compounds in the formulation that may result from previous manufacturing steps, such as the removal of surfactants and / or solvents from a previous virus inactivation step, such as a previous S / D treatment.

[0033] In a preferred embodiment, the fibrinogen-containing source is subjected to a virus inactivation process, such as a solvent detergent process (S / D treatment).

[0034] In a preferred embodiment, the liquid phase is prepared by resuspending the precipitate of the glycine precipitate.

[0035] The liquid phase is contacted with the cation exchange chromatography material under conditions that result in binding of fibrinogen, wherein the liquid phase has a pH of 5.6 to 7.0. The pH and / or conductivity of the liquid phase may need to be further adjusted to corresponding conditions prior to loading. The liquid phase is then loaded onto the cation exchange chromatography material.

[0036] In a particularly preferred embodiment, the liquid phase has a pH in the range of 6.0 to 6.9, more preferably 6.3 to 6.9, even more preferably 6.4 to 6.8, and most preferably 6.5 to 6.7, upon contact with the cation exchange chromatography material.

[0037] The ionic strength of the liquid phase upon contact with the cation exchange chromatography material is preferably in the range of 5 to 15 mS / cm, more preferably 7 to 11 mS / cm, especially 9.0±1.5 mS / cm, preferably 9.0±1.0 mS / cm.

[0038] In a preferred embodiment, the liquid phase has a pH in the range of 5.6 to 7.0 and an ionic strength in the range of 5 to 15 mS / cm, more preferably a pH in the range of 6.0 to 6.9 and an ionic strength in the range of 5 to 15 mS / cm, even more preferably a pH in the range of 6.3 to 6.9 and an ionic strength in the range of 7 to 11 mS / cm, most preferably a pH in the range of 6.4 to 6.8 and an ionic strength in the range of 9.0±1.5 mS / cm, more preferably a pH in the range of 6.4 to 6.8 and an ionic strength in the range of 9.0±1.0 mS / cm, even more preferably a pH in the range of 6.5 to 6.7 and an ionic strength in the range of 9.0±1.0 mS / cm.

[0039] In a preferred embodiment, the pH of the liquid phase is stabilized by the use of a buffer system, which can be a citrate, phosphate, or acetate buffer. A preferred buffer is a citrate buffer, more preferably a trisodium citrate buffer.

[0040] In a preferred embodiment, the buffer concentration is less than 50 mM, more preferably less than 30 mM, depending on the ionic strength to be achieved. The buffer concentration is preferably greater than 5 mM, more preferably greater than 10 mM. In a preferred embodiment, the buffer concentration is 15 mM.

[0041] If necessary, the ionic strength may be adjusted by the addition of one or more salts, preferably halide salts soluble in the liquid phase, more preferably sodium chloride.

[0042] Preferably, the protein load is less than 22 g / L of cation exchange chromatography material, preferably less than 21 g / L, more preferably less than 20 g / L. In a preferred embodiment, the protein load is between 5 and 22 g / L, preferably between 10 and 21 g / L, more preferably between 10 and 20 g / L.

[0043] The cation exchange chromatography process is preferably carried out at a temperature between 16°C and 28°C, more preferably between 18°C ​​and 26°C, more preferably at 22°C±4°C.

[0044] The cation exchange chromatography material is preferably a strong CEX material.

[0045] The CEX material is preferably a material containing particles with pores. Preferably, the CEX material is a macroporous material, particularly a macroporous polymer material. In a preferred embodiment, the CEX material contains particles with pore diameters ranging from 50 nm to 1000 nm, preferably 100 to 1000 nm. Preferably, the average pore size is 160 nm. In a particularly preferred embodiment, the chromatography material contains small diffusion pores so that diffusion is no longer limiting, and also relatively large flow pores that allow a small percentage of convection through the particles. In such materials, the chromatography material preferably contains particles with large pores with diameters of 200 nm to 1000 nm and small pores with diameters of 5 to 30 nm, preferably large pores with diameters of 250 nm to 600 nm, and small pores with diameters of 5 to 20 nm (measurements are based on the disclosure of Pirrung SM et al. Biotechnol. Prog. 2018, 34(4), 1006-1018).

[0046] In a preferred embodiment, the CEX material comprises particles having an average particle size of 30-100 μm, preferably 30-70 μm, more preferably about 50 μm.

[0047] In a preferred embodiment, the CEX material contains functional groups, particularly sulfonate functional groups (-SO 3- ) Preferably, the sulfonate groups are attached to straight or branched alkyl groups having 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, more preferably ethyl, propyl, butyl groups, more preferably ethyl, n-propyl and isobutyl, and even more preferably sulfopropyl (preferably corresponding to n-propyl). In a preferred embodiment, the sulfonate groups on the cation exchange material are attached to the polymer chains of the CEX material.

[0048] In another preferred embodiment, the CEX material is not a tentacle-type cation exchange material (e.g., Fractogel® from Merck Millipore). 3-Such CEX materials tend to bind fibrinogen and similar proteins too strongly for the purposes of the present invention.

[0049] In a preferred embodiment, the material comprises a polyhydroxyl surface coating with sulfopropyl groups. The resin backbone is preferably formed from cross-linked polystyrene divinylbenzene with sulfonate functional groups attached as sulfopropyl groups through the polyhydroxyl surface. An example of a preferred CEX material is POROS™ chromatography resin, more preferably POROS™ 50 HS (ThermoFisher, USA). This material also comprises particles with large and small pores as described above.

[0050] In another embodiment of the present invention, the CEX material is a methacrylate copolymer with sulfonate functional groups. In this embodiment, the average particle size can be 50 μm and the average pore diameter can be 100 nm. An example of a preferred CEX material in this embodiment is Macro-Prep®, more preferably Macro-Prep® High S (BioRad, USA).

[0051] In a preferred embodiment, the CEX material is equilibrated before the fibrinogen-containing liquid phase is added to the CEX material. This is done using an equilibration buffer. The pH range of the equilibration buffer is preferably 5.6 to 7.0, more preferably 6.0 to 6.9, even more preferably 6.3 to 6.9, even more preferably 6.4 to 6.8, and even more preferably 6.5 to 6.7.

[0052] The ionic strength of the equilibration buffer is preferably 5 to 15 mS / cm, more preferably 7 to 12 mS / cm, and particularly 9.0±1.5 mS / cm or 9.0±1 mS / cm.

[0053] In a preferred embodiment, the equilibration buffer has an ionic strength of 5 to 15 mS / cm at a pH range of 5.6 to 7.0, more preferably an ionic strength of 5 to 15 mS / cm at a pH range of 6.0 to 6.9, even more preferably an ionic strength of 7 to 11 mS / cm at a pH range of 6.3 to 6.9, most preferably an ionic strength of 9.0 ± 1.5 mS / cm at a pH range of 6.4 to 6.8, more preferably an ionic strength of 9.0 ± 1.0 mS / cm at a pH range of 6.4 to 6.8, and even more preferably an ionic strength of 9.0 ± 1.0 mS / cm at a pH range of 6.5 to 6.7.

[0054] In a preferred embodiment, the pH of the equilibration buffer is stabilized by the use of a buffer system. Preferred buffer systems are based on phosphate, acetate or citrate buffers, preferably citrate buffers, more preferably trisodium citrate.

[0055] In a preferred embodiment, the buffer concentration is less than 50 mM, more preferably less than 30 mM, depending on the ionic strength to be achieved. The buffer concentration is preferably greater than 5 mM, more preferably greater than 10 mM. In a preferred embodiment, the buffer component is at a concentration of 15 mM.

[0056] In a preferred embodiment, the equilibration buffer contains a salt, preferably sodium chloride. The salt concentration is preferably less than 100 mM, more preferably less than 80 mM, and particularly less than 70 mM. The lower range of the salt concentration is preferably greater than 40 mM, more preferably greater than 50 mM, and even more preferably greater than 60 mM, with the upper limits as described above, preferably 40 mM to 80 mM, more preferably 60 mM to 80 mM, and more preferably 65 mM ± 1 to 2 mM. The buffer system and the amount of salt are adjusted to obtain an equilibration buffer with a desired ionic strength.

[0057] Equilibration is preferably performed by washing the CEX material with at least two column volumes of equilibration buffer.

[0058] After loading the CEX material with the liquid phase according to step b) of the method of the present invention, an optional next step is to wash unbound compounds from the CEX material. For such a step, the CEX material is washed with a wash buffer. Preferably, the pH of the wash buffer is in the range of 5.6 to 7.0, more preferably 6.0 to 6.9, even more preferably 6.3 to 6.9, even more preferably 6.4 to 6.8, and most preferably 6.5 to 6.7.

[0059] The ionic strength of the washing buffer is preferably 5 to 15 mS / cm, more preferably 7 to 11 mS / cm, and particularly 9.0±1.5 mS / cm or 9.0+1.0 mS / cm.

[0060] In a preferred embodiment, the wash buffer has an ionic strength of 5 to 15 mS / cm at a pH range of 5.6 to 7.0, more preferably an ionic strength of 5 to 15 mS / cm at a pH range of 6.0 to 6.9, even more preferably an ionic strength of 7 to 11 mS / cm at a pH range of 6.3 to 6.9, most preferably an ionic strength of 9.0 ± 1.5 mS / cm at a pH range of 6.4 to 6.8, more preferably an ionic strength of 9.0 ± 1.0 mS / cm at a pH range of 6.4 to 6.8, and even more preferably an ionic strength of 9.0 ± 1.0 mS / cm at a pH range of 6.5 to 6.7.

[0061] In a preferred embodiment, the pH of the wash buffer is stabilized by the use of a buffer system. Preferred buffer systems are based on phosphate, acetate or citrate buffers, preferably citrate buffers, more preferably trisodium citrate.

[0062] In a preferred embodiment, the concentration of the wash buffer is less than 50 mM, more preferably less than 30 mM, depending on the ionic strength to be achieved, and the concentration of the buffer is greater than 5 mM, preferably greater than 10 mM, in a preferred embodiment 15 mM of buffer components.

[0063] In a preferred embodiment, the wash buffer contains a salt, preferably a chloride salt, more preferably sodium chloride. The salt concentration is preferably less than 100 mM, more preferably less than 80 mM, and particularly less than 70 mM. The lower range of the salt concentration is preferably greater than 40 mM, more preferably greater than 50 mM, and even more preferably greater than 60 mM, with the upper limits as described above, preferably 40 mM to 80 mM, more preferably 60 mM to 80 mM, and more preferably 65 mM ± 1 to 2 mM. The buffer system and the amount of salt are adjusted to obtain a wash buffer with the desired ionic strength.

[0064] In a preferred embodiment, after loading the CEX material is washed with at least 1 column volume of wash buffer, preferably with at least 2 column volumes of wash buffer.

[0065] In a preferred embodiment of the present invention, the equilibration buffer and the wash buffer have at least the same pH and / or ionic strength, more preferably the same pH and the same ionic strength. In an even more preferred embodiment, both buffers have the same composition.

[0066] After loading and preferably washing according to steps b) and c) of the method of the present invention, in the next step fibrinogen is eluted from the cation exchange material according to step d) of the method of the present invention. For this step, an elution buffer is passed through the CEX material. The elution buffer has a higher conductivity and / or a higher pH compared to the final wash buffer, or the liquid phase if no wash buffer is used, so that fibrinogen is selectively eluted from the CEX material, and preferably vWF remains on the column. The conditions may depend on the CEX material used.

[0067] Preferably, the elution buffer has a pH at least 0.2, 0.3, 0.4, or 0.5 units higher than the conditions used for binding fibrinogen to the CEX material. In preferred embodiments, the pH is 0.2 to 1 pH unit higher, particularly 0.2 to 0.5 units higher, than the binding conditions. In preferred embodiments, the pH of the elution buffer is 7.0±0.1.

[0068] The ionic strength of the elution buffer is preferably at least 2, 3, 4, or 5 mS / cm higher than the conditions used for binding fibrinogen to the CEX material, more preferably at least 5 mS / cm higher, even more preferably 5 to 15 mS / cm higher, and particularly 10±1.5 mS / cm higher. In a preferred embodiment, the ionic strength is at least 1.5 to 2.5 times, more preferably 1.8 to 2.2 times, the ionic strength of the conditions used for binding. In a preferred embodiment, the ionic strength is 19.5±1.5 mS / cm. All values ​​are at 20°C.

[0069] In a preferred embodiment, the elution buffer has a pH at least 0.2, 0.3, 0.4 or 0.5 units higher than the conditions used to bind fibrinogen to the CEX material and an ionic strength at least 5 mS / cm higher than the conditions used to bind fibrinogen to the CEX material, preferably a pH 0.2 to 1 pH unit higher and an ionic strength 5 to 15 mS / cm, even more preferably a pH 0.2 to 0.5 unit higher and an ionic strength 10±1.5 mS / cm higher than the conditions used for binding fibrinogen.

[0070] In a preferred embodiment, the pH of the elution buffer is stabilized by the use of a buffer system. Preferred buffers are phosphate, acetate or citrate buffers, preferably citrate buffers, more preferably those based on trisodium citrate.

[0071] In a preferred embodiment, the concentration of the elution buffer system is less than 30 mM, more preferably less than 10 mM, depending on the ionic strength to be achieved. The buffer concentration is preferably 5 mM to 30 mM, more preferably 5 mM to 10 mM. In a preferred embodiment, the buffer concentration is 7.5 mM.

[0072] In a preferred embodiment, the elution buffer contains a salt, preferably sodium chloride. The salt concentration of the elution buffer is preferably greater than 100 mM, more preferably greater than 110 mM, particularly greater than 120 mM, and most preferably 150 mM. The concentration is preferably less than 350 mM, more preferably less than 250 mM. The buffer system and the amount of salt are adjusted to obtain an equilibration buffer with a desired ionic strength.

[0073] In a preferred embodiment, the elution buffer contains one or more pharmaceutical formulation compounds, such as amino acids, e.g., glycine, histidine, alanine, and arginine, preferably cationic amino acids, preferably arginine. The amino acids can be added as hydrochlorides. In a preferred embodiment, the elution buffer contains more than 50 mM of the pharmaceutical formulation compounds. The salt concentration in the elution buffer can be reduced, especially if the pharmaceutical formulation compounds contribute to the ionic strength. Therefore, it is particularly preferred to use, for example, 50-100 mM of the pharmaceutical formulation compounds, preferably 70-80 mM, more preferably 75±2 mM, in combination with 100-200 mM sodium chloride, preferably 150±5 mM, in the elution buffer to provide sufficient ionic strength for fibrinogen elution from the column material. This avoids excessive salt concentrations, which may be detrimental to the final pharmaceutical product. Furthermore, by using components of the pharmaceutical formulation for elution purposes, an additional buffer exchange step can be avoided.

[0074] In a preferred embodiment, the elution buffer comprises 50-100 mM arginine (preferably added as the hydrochloride salt) and 100-200 mM sodium chloride, preferably 70-80 mM arginine and 100-200 mM sodium chloride, more preferably 75±2 mM arginine and 150±5 mM sodium chloride.

[0075] Elution is carried out until fibrinogen is eluted from the column, which can be detected by measuring UV absorption.

[0076] When the method of the present invention is used as one of the final steps of a purification process, a diafiltration step to remove excess salts or other undesirable compounds can be omitted. It may be sufficient to use an ultrafiltration step to concentrate the protein prior to preparation of a pharmaceutical composition from the product of the method of the present invention.

[0077] After elution of fibrinogen, the column may be regenerated using a regeneration buffer. During this regeneration step, vWF may be stripped from the column. The regeneration buffer may have a pH of 5.5 to 7.5, preferably 6.0 to 7.0, more preferably 6.5 to 6.7. The ionic strength is preferably greater than 50 mS / cm, more preferably greater than 100 mS / cm. In a preferred embodiment, the ionic strength is 50 to 150 mS / cm, more preferably 100 mS / cm to 130 mS / cm. The regeneration buffer may contain, for example, 1.5 M NaCl (high salt concentration).

[0078] In a preferred embodiment, the column material may be further washed using a sodium hydroxide (NaOH) solution, preferably at a concentration of at least 0.1 M, more preferably between 0.5 M and 1.5 M sodium hydroxide, and most preferably 1 M sodium hydroxide. After washing, the chromatographic material may be stored, for example in 0.1 M NaOH, until further use.

[0079] The method of the present invention is particularly useful for reducing the amount of vWF in compositions, particularly plasma-derived compositions. This is particularly surprising because vWF and fibrinogen have similar pI characteristics. Nevertheless, as the inventors have shown, it is possible to selectively elute fibrinogen from a cation exchange material, where vWF remains on the column. Thus, the method of the present invention is capable of separating vWF from fibrinogen. Preferably, the cation exchange chromatography step according to the method of the present invention can reduce the amount of vWF (U / mg total protein) by a factor of at least 2, preferably at least 3, and more preferably up to 4 or more, where the factor is directly dependent on the vWF content of the loading material. In a preferred embodiment, the amount of vWF in the eluted fibrinogen fraction is reduced to less than 0.5 U / mg total protein, more preferably less than 0.4 U / mg total protein, more preferably less than 0.3 U / mg total protein, and even more preferably less than 0.2 U / mg. Typically, the eluted fibrinogen fraction from the cation exchange step may contain vWF in an amount of about 0.1-0.3 U / mg total protein. The ability of the cation exchange chromatography step to reduce the amount of vWF in the fibrinogen fraction was also tested with test material spiked with vWF. The inventors were able to show that the cation exchange chromatography step with spiked test material can achieve a vWF removal factor of 10 or even more.

[0080] The eluted fraction containing fibrinogen is preferably further concentrated and / or diluted to obtain a fibrinogen preparation with a defined fibrinogen content.

[0081] In a preferred embodiment, the elution fraction containing fibrinogen contains less than 0.01 mg / ml polysorbate 80 and less than 0.8 μg / ml TnBP.

[0082] In a preferred embodiment, the method of the present invention further comprises the step e) of formulating the fibrinogen into a pharmaceutical composition.

[0083] The use of the cation exchange chromatography step of the present invention in a purification protocol for producing fibrinogen from plasma has several advantages. First, the cation exchange step can effectively reduce the amount of vWF in the fibrinogen product. Second, additional pharmaceutical product components such as arginine and / or other formulation buffer components can be already included in the elution buffer for cation exchange chromatography, thereby avoiding additional buffer exchange steps such as costly and labor-intensive diafiltration. Third, the cation exchange chromatography step is highly effective in reducing or removing prions, thereby avoiding additional extra steps for prion reduction or removal. A fourth advantage of the cation exchange chromatography step is that it can very efficiently remove solvents / detergents such as polysorbate 80 and TnBP. In summary, the production method proposed by the present inventors, including a cation exchange chromatography step, is a highly effective, economical, and superior method for producing fibrinogen. The production method involves relatively few process steps that are relatively easy to implement.

[0084] As mentioned above, the liquid phase loaded onto the cation exchange material may already be a partially purified fibrinogen preparation. Thus, the method of the present invention for the production of a fibrinogen preparation may comprise preceding and / or subsequent purification and production steps, preferably including one or more viral inactivation steps. In a particularly preferred embodiment, the production method further comprises at least one of the following steps: Use of cryoprecipitate of human plasma as starting material; Al(OH)3 adsorption; S / D processing; anion exchange chromatography and use of the flow-through for further fibrinogen purification; Glycine precipitation; UV-C treatment; Ultrafiltration; freeze-drying; Dry heat treatment.

[0085] In a preferred embodiment, the complete manufacturing process starting from plasma includes the steps of anion exchange chromatography, glycine precipitation, and cation exchange chromatography as described herein. Preferably, additional steps of viral inactivation or viral reduction, such as S / D treatment and / or UV-C treatment and / or dry heat treatment, are performed. The anion exchange chromatography step can achieve a vWF removal factor of about 1-2. The glycine precipitation step can achieve a vWF removal factor of about 4-5. The cation exchange chromatography step can achieve a vWF removal factor of at least 3. The complete manufacturing process as a whole can achieve a vWF removal factor of about 20 or more relative to dissolved cryoprecipitate.

[0086] Preferably, the specific activity of the final product purified fibrinogen is at least 95% clottable protein activity relative to total protein, preferably 98%±0.8 (activity measured by clottable protein and OD280).

[0087] In a particularly preferred embodiment, the method of preparation comprises all of the above steps, wherein a cation exchange chromatography step as described herein is performed between the UV-C treatment and the ultrafiltration, preferably with the other steps being performed in the order listed above.

[0088] Preferred step embodiments are described in more detail below:

[0089] Cryoprecipitate of human plasma is the preferred source of fibrinogen in the methods of the present invention.

[0090] The cryoprecipitate is reconstituted or solubilized under appropriate buffer conditions, particularly at neutral pH, preferably in a solution buffer, and subjected to adsorption, particularly by Al(OH)3, and the resulting gel is removed, preferably by centrifugation. If necessary, the supernatant can be filtered. As a next step, the supernatant can be subjected to virus inactivation, preferably by solvent / detergent (S / D) treatment. S / D compounds such as polysorbate 80 and TnBP (tri-n-butyl phosphate) are preferred.

[0091] The resulting solution can then be further purified using chromatographic methods. This can typically be done by contacting the S / D-treated protein solution with an anion exchange material. A preferred material for this purpose is one having diethylaminoethyl (EDAE) groups as anion exchange groups grafted onto the matrix material. One example of such a material is Toyopeal DEAE, which uses hydroxylated methacrylic polymer beads as the matrix material. The solution is contacted with the anion exchange material under conditions in which fibrinogen does not bind to the weak anion exchange material. Fibrinogen is present in the flow-through.

[0092] The resulting fibrinogen solution can then be subjected to glycine precipitation, preferably with 1-1.5 M glycine, more preferably about 1.2 M glycine. Preferably, NaCl, preferably 1-3 M NaCl, more preferably about 2 M NaCl, is additionally used for glycine precipitation. The above concentrations can be achieved by adding glycine and / or NaCl directly to the solution. The solution is buffered to a pH range of 6.7-7.2 with 20-40 mM citrate. The fibrinogen-containing precipitate can then be separated by centrifugation, preferably flow-through centrifugation. A single fibrinogen precipitation is usually sufficient. The fibrinogen paste can be stored at temperatures below -70°C.

[0093] As a next step, the precipitate is resuspended and the solution can be subjected to UV-C treatment for further virus inactivation, especially parvovirus inactivation. In a preferred embodiment, a UVivatec system (Sartorius Stedim Biotech GmbH, Germany) is used. As a buffer, a sodium citrate buffer is preferably used. Preferably, the resulting solution is also suitable for cation exchange chromatography according to the present invention.

[0094] As a next step, the fibrinogen solution is subjected to cation exchange chromatography according to the method described above.

[0095] The resulting fibrinogen solution is then further concentrated by ultrafiltration, preferably to a concentration of 20-70 g / L, preferably 55±10 g / L. The resulting concentrate may be diluted with the corresponding buffer to the desired final concentration, preferably 25-40 g / L, preferably 33±3 g / L. During these steps, further ingredients for the final pharmaceutical product may be added. The pH of the solution may be adjusted, for example, to pH 7.0±0.5.

[0096] The resulting solution can be filtered (0.2 μm) into different vials and lyophilized, followed by a final dry heat treatment (e.g., in an autoclave at 100° C. for 30 minutes) as an additional viral inactivation step. In the final solution or lyophilizate, the total protein consists essentially of fibrinogen.

[0097] The resulting lyophilized product can be reconstituted to give a solution containing preferably 12 to 25 g / L fibrinogen, more preferably 18 to 24 g / L fibrinogen, 20 to 65 mmol / L arginine, more preferably 25 to 55 mmol / L arginine, and 2 to 10 mmol / L citrate, more preferably 3 to 7 mmol / L citrate, and having a pH of 6.5 to 7.5.

[0098] The present invention also provides a fibrinogen preparation obtained according to the production method of the present invention. The fibrinogen preparation is characterized by an advantageous purity and activity profile. In particular, the fibrinogen preparation is characterized by a very low factor XIII content. Preferably, the fibrinogen preparation has an FXIII concentration of 0.5-2.0 FXIII:Ag (% of norm) and / or an FXIII activity of less than 16 FXIII:Ac (% of norm), thus demonstrating improved purity compared to conventional fibrinogen products. This corresponds to an FXIII:Ac value of <0.008 IU / mg fibrinogen (at 20 mg / ml fibrinogen). 5-2.0 FXIII:Ag (% of norm) approximately corresponds to 0.0003-0.0010 IU / mg fibrinogen (at 20 mg / ml fibrinogen).

[0099] This allows fewer coexisting factors to be administered when the fibrinogen preparation of the present invention is used for medical purposes. Furthermore, the fibrinogen preparation of the present invention exhibits better or improved clot firmness compared to conventional fibrinogen products. Furthermore, the fibrinogen preparation of the present invention does not exhibit detectable amounts of D-dimer (<0.17 mg / L), which further demonstrates that the fibrinogen preparation of the present invention has particularly advantageous physiological activity and improved purity.

[0100] The present invention also relates to a fibrinogen product having an FXIII concentration of 0.5-2.0 FXIII:Ag (% of norm) and / or an FXIII activity of less than 16 FXIII:Ac (% of norm) and / or not containing detectable amounts of D-dimer (<0.17 mg / l) and / or a maximum clot firmness of 20-30 mm (Fib-tem assay at 2.5 g / l fibrinogen).

[0101] Pharmaceutical compositions obtained from the fibrinogen preparation are also subject of the present invention. Preferably, the pharmaceutical composition of the present invention comprises a fibrinogen preparation obtained by the method of the present invention as described, and preferably at least one pharmaceutical carrier.

[0102] The pharmaceutical composition can be filled into a suitable vial.

[0103] The resulting pharmaceutical composition exhibits good physiological properties and high stability as indicated by clot hardness assay. It has a low content of polymeric substances and excellent purity. The pharmaceutical composition is preferably provided as a lyophilizate.

[0104] When the pharmaceutical composition is provided as a solution, preferably from a lyophilized fibrinogen product, the vWF content is preferably less than 0.5 U / mg total protein, more preferably less than 0.4 U / mg total protein, for example, in a preferred embodiment the vWF content is less than 7 U / ml in a fibrinogen amount of 20 g / l, which corresponds to 0.35 U / mg total protein.

[0105] In a preferred embodiment, the pharmaceutical composition, when prepared as a solution, preferably from a lyophilisate, comprises the following components: fibrinogen at a concentration of 12-25 g / l, pH 6.5-7.5, arginine at 20-65 mmol / l, citrate at 2-10 mmol / l.

[0106] The pharmaceutical compositions obtained by the methods of the present invention are preferably suitable for intravenous use, preferably in humans.

[0107] The pharmaceutical composition is essentially useful for treating hemostatic disorders and / or preventing or treating bleeding. In a preferred embodiment, the hemostatic disorders are congenital fibrinogen deficiency, acquired fibrinogen deficiency, traumatic injury, and preventing or treating bleeding. The prevention or treatment of bleeding by administering the pharmaceutical composition of the present invention can be performed during or after surgery, particularly during spinal surgery or gynecological surgery. Therefore, the present invention also relates to a method for treating hemostatic disorders by administering the pharmaceutical composition of the present invention. [Example]

[0108] Example 1 Cryoprecipitate of human plasma was used as a fibrinogen source. Cryoprecipitate was obtained by thawing frozen plasma at 0-4°C and separating the precipitate.

[0109] For each kg of cryoprecipitate, prepare a mixture of 2.91 kg of water (WFI), 114 g of 25% ethanol (v / v), and 9000 IU of heparin. Add the cryoprecipitate to the WFI / ethanol / heparin solution while stirring. Adjust the pH to 7.0.

[0110] For each kg of cryoprecipitate used, 108 g of a 2% aluminum hydroxide suspension are added and the mixture is stirred at 22.5° C. The pH value is adjusted to 6.55 and then centrifuged in a continuously operating centrifuge.

[0111] 1% Polysorbate 80 and 0.3% Tri-n-butyl phosphate are added with stirring. The protein solution is stirred at 25.0° C. for at least 8 hours.

[0112] For further purification by column chromatography, anion exchange gel Toyopearl TSK DEAE-650 (hydroxylated methacrylic polymer beads with diethylaminoethyl groups as the matrix material) is used. The protein loading is approximately 50 ± 10 mg of protein per ml of anion exchange gel.

[0113] The chloride content of the protein solution is adjusted to 120 mmol / l by adding NaCl solution. The protein solution is applied to the column and the flow-through fraction is collected.

[0114] The resulting fibrinogen solution, containing 10 mM trisodium citrate, 120 mM NaCl, 120 mM glycine, 1 mM CaCl2, 0.1% polysorbate 80, and 0.3% TnBP at pH 7.0-7.1, is subjected to glycine precipitation. To precipitate fibrinogen, glycine is added to a final concentration of 1.2 M. NaCl is added to a final concentration of 2 M. The fibrinogen-containing precipitate is then separated by centrifugation. The fibrinogen paste can be stored at -70°C or below.

[0115] The precipitate is resuspended in a buffer (15 mM trisodium citrate dihydrate, pH 6.9±0.1, conductivity 3.3±0.5 mS / cm). The composition contains TnBP, polysorbate 80, glycine, and NaCl, as well as other proteins (e.g., 0.7-0.9 U / mg vWF). The composition is filtered and subjected to UV-C treatment for virus inactivation using a device such as a UVivatec device (Sartorius Stedim Biotech). Irradiation is preferably 125-200 J / m at 254 nm±1 nm. 2 This is done.

[0116] For the next cation exchange chromatography step, equilibrate the column (POROS™ 50 HS) with equilibration buffer (15 mM anhydrous trisodium citrate, 65 mM sodium chloride, pH 6.5 ± 0.1, conductivity 9.0 ± 1.5 mS / cm, 2-5 column volumes).

[0117] The fibrinogen-containing liquid phase from the UV irradiation step is prepared by adjusting the composition to 15 mM trisodium citrate, pH 6.5 ± 0.1, and conductivity 9.0 ± 1.5 mS / cm. The column is loaded with 10-20 g / L of protein per liter of gel volume.

[0118] Wash the column with wash buffer (15 mM anhydrous trisodium citrate, 65 mM sodium chloride, pH value 6.5 ± 0.1, conductivity 9.0 ± 1.0 mS / cm, 2–5 column volumes).

[0119] Then, fibrinogen is eluted using an elution buffer (7.5 mM anhydrous trisodium citrate, 150 mM sodium chloride, 75 mM L-arginine monohydrochloride, pH 7.0±0.1, conductivity 19.5±1.5 mS / cm). In this step, fibrinogen is eluted from the column. Most of the vWF remains bound to the column.

[0120] The column is then washed with a buffer with a higher salt concentration (15 mM anhydrous trisodium citrate, 1.5 mM sodium chloride, pH 6.5±0.1, conductivity 113.5±5.0 mS / cm). The vWF is eluted from the column. The column is then washed with 1 M sodium hydroxide.

[0121] In this method, albumin and IgG do not bind to the CEX material. More than 50% of the vWF present in the liquid phase containing fibrinogen can be removed using this method.

[0122] For preparation of the drug substance, the eluted fraction is concentrated by ultrafiltration and the protein concentration adjusted to 33 g of fibrinogen per liter with citrate buffer. Additional ingredients may be added to form the final drug substance.

[0123] The drug substance was filtered (0.2 μm) into different vials and lyophilized. An additional viral inactivation step was then performed by a final heat treatment in a steam autoclave (100°C, 30 min). The product is remarkably stable.

[0124] The final product exhibits good clot stability measured as maximum clot firmness (MCF) (Fib-tem assay at 2.5 g / l fibrinogen, 25 mm) compared to a standard human plasma control (27 mm) and a plasma pool (22 mm), providing strong evidence of good physiological activity.

[0125] Furthermore, the fibrinogen preparation had an FXIII concentration of less than 1.5% FXIII:Ag (% of norm) and an FXIII activity of less than 16% FXIII:Ac (% of norm). Furthermore, the fibrinogen preparation did not exhibit detectable amounts of D-dimer. Both parameters indicated that the fibrinogen product of the present invention had very good purity. The data are shown in Table 1 (Samples 1, 2, and 3: Preparations according to the method of Example 1 of the present invention - solubilized final product, protein 20 mg / ml).

[0126] [Table 1]

[0127] The commercial fibrinogen product sample exhibits much higher FXIII activity and higher D-dimer content compared to the product of the present invention (data not shown).

[0128] Example 2 25 g of glycine precipitate, prepared as described in Example 1, was UV-C treated and purified using cation exchange chromatography (POROS™ 50 HS) as described in Example 1. Table 2 shows the compositions of the loading solution, flow-through, and eluent.

[0129] [Table 2]

[0130] If the total protein corresponds substantially to fibrinogen, the protein yield in the eluate is 95.5% based on the protein concentration measured by OD280. 37.9% of the total vWF is present in the fibrinogen eluate. 100.3% of the fibrinogen, measured as Fib:Ag, is present in the eluate.

[0131] Because the vWF content in the load of this example is relatively low, the vWF removal factor in this example is 2.4 (from 0.45 to 0.19 U / mg). In other examples, the inventors have shown that vWF removal factors range from 3 to 6 when the vWF content in the load material ranges from 0.6 to 0.9 U / mg total protein.

[0132] Example 3 The cation exchange chromatography step was carried out in a similar manner to that described in Example 1, except that Macro-Prep® High S was used as the strong cation exchange column material instead of POROS™ 50 HS. For improved binding of fibrinogen to the column during loading and washing, the conductivity was set to 4.5 mS / cm, and the pH was set to 6.0, which is higher than the pI of fibrinogen. Under elution conditions of pH 7 and a conductivity of 19.5 mS / cm, 94.8% of fibrinogen was eluted from the column. The amount of vWF in the elution fraction was less than 0.4 U / mg protein. Only approximately 18.3% of the vWF in the protein load onto the CEX column could be detected in the eluate. No polysorbate 80 or TnBP could be detected in the elution fraction.

[0133] Example 4 To examine the ability of the cation exchange chromatography step in reducing the amount of vWF in the eluted fibrinogen fraction, cation exchange chromatography using a loading material enriched in vWF (spiked) was performed using a POROS™ 50 HS column as described in Example 1. The vWF content of the load ranged from 1.09 U / mg total protein (unspiked material) to 4.49 U / mg total protein in several spiked samples (see Table 3). After selective elution of fibrinogen (150 mM NaCl, pH 7.0), vWF was eluted in the high-salt fraction (1.5 M NaCl, pH 6.5). As can be seen from Table 3, the amount of vWF in the eluted fibrinogen fraction (fibrinogen eluate) remained essentially constant with increasing vWF content of the load, ranging from 0.21 U / mg total protein (unspiked material) to 0.35 U / mg total protein (load spiked with 4.49 U / mg total protein). The removal factor of vWF increased with spiking of the load material, from 5 for unspiked load material to 12 for load material spiked with 4.49 U / mg total protein.

[0134] On a larger scale, the removal factors compared to dissolved cryoprecipitate for typical samples are shown in Table 4. Over the entire process, a removal factor compared to dissolved cryoprecipitate of 24.2 is achieved.

[0135] [Table 3]

[0136] [Table 4]

[0137] Example 5 To demonstrate the robustness of the cation exchange chromatography step with respect to pH value and conductivity during column loading (POROS™ 50 HS), a pH range of 6.3 to 6.7 was tested during column loading according to Example 1. The conductivity during column loading was 7 to 11 mS / cm. The protein load was 20 g to 22 g protein per liter of column material. In all conditions tested, vWF was removed to less than 0.5 U / mg total protein, as can be seen in Figure 1. (Figure 1 shows the relationship between vWF amount and different values ​​of loading solution conductivity and pH for different protein loads (top row: pH 6.4-6.6 and 8-10 mS / cm; bottom row: pH 6.3-6.7 and 7-11 mS / cm). The results show that a conductivity range of 6.4-6.6 is particularly advantageous for reducing vWF in the fibrinogen eluate (where vWF is removed to less than 0.4 U / mg). The results further show that a conductivity range of 8-10 mS / cm is particularly advantageous for reducing vWF in the fibrinogen eluate (resulting in vWF removal to less than 0.4 U / mg). Furthermore, for effective removal of vWF, a total protein load of 21 g / L or less of column material, especially 20 g / L or less, is advantageous.)

[0138] Example 6 To test the ability of the cation exchange chromatography step to reduce the amount of polysorbate 80 and TnBP in the eluted fibrinogen fraction, cation exchange chromatography using a loading material enriched in both polysorbate 80 and TnBP (spiked) was performed using a POROS™ 50 HS column as described in Example 1, except that the UV-C irradiation step was omitted. The polysorbate 80 and TnBP contents of the load ranged from 1.11 mg / ml and approximately 20 μg / ml (unspiked material) to 10.74 mg / ml and 2590 μg / ml in several spiked samples (see Table 5). After selective elution of fibrinogen (150 mM NaCl, pH 7.0), vWF was eluted in the high-salt fraction (1.5 M NaCl, pH 6.5). The fibrinogen yield in the column eluate was estimated from the chromatograms to be constant across all experiments (data not shown). As can be seen from Table 5, the polysorbate 80 and TnBP contents in the eluted fibrinogen fraction (fibrinogen eluate) were below the detection limit of the analytical method for all polysorbate 80 / TnBP spikes applied, except for the highest TnBP spike, where approximately 30 μg / ml of TnBP was detected in the eluate. Thus, using POROS™ 50 HS chromatography of fibrinogen-containing samples, polysorbate 80 and TnBP could be removed by a factor of 1000. At the same time, the vWF content of the fibrinogen eluate remained virtually unaffected, with only a slight increase observed for the highest polysorbate 80 / TnBP spike.

[0139] [Table 5]

[0140] Example 7 The inventors were able to demonstrate that the cation exchange chromatography step effectively removes prions below the detection limit. To demonstrate this effect, hamster prions (strain 263K), a well-established test model for variant Creutzfeldt-Jakob disease, were tested. Prion-spiked test material was analyzed for removal capacity. Prion titers were measured by Western blot. In the eluate of the cation exchange chromatography step, prions were removed from fibrinogen by a log ≥ 3.27. 10 The prion was removed to below the detection limit, demonstrating reliable prion removal by the cation exchange chromatography step. Prions were stripped from the column with 1.5 M NaCl (high salt concentration).

[0141] The experimental results are summarized in Table 6. The test material was spiked with prion (hamster-adapted scrapie isolate, strain 263K, supplied by ViruSure GmbH, Austria), a sample was taken from the prion stock and spiked into the test material, and the prion titer was measured by Western blot. The spiked test material was loaded onto the column in a volume of approximately 94 ml, and the column was washed. The flow-through and wash were collected and the volume was measured. Fibrinogen was eluted from the column in a volume of 50 ml, and the column was washed with 1.5 M NaCl. A sample of each fraction was taken and analyzed for prion titer. The results of this study are shown in Table 6 below. Fibrinogen to prion was detected at a log conversion of ≧3.27. 10 It is shown that the ions are removed to below the detection limit.

[0142] [Table 6] a) After calculation, round to one decimal place. b) A 10-fold concentrated sample (-1.0 log) was non-reactive. c) The sample was diluted.

[0143] Analysis method Protein measurement Protein measurements were performed by UV absorption (Spektralphotometer Genesys™ 6, Spektralphotometer Genesys™ 10). In solution, proteins absorb UV light at a wavelength of 280 nm due to the presence of aromatic amino acids, primarily tyrosine and tryptophan. This property is the basis for measuring proteins at 280 nm. The accuracy of UV spectroscopic measurements of proteins can be reduced by light scattering by the test sample. To correct for this effect, the absorbance at 360 nm is subtracted from the absorbance at 280 nm.

[0144] Fibrinogen (Fib:Ag) measurement Fib:Ag concentration is measured by nephelometry using a BN Prospec (Siemens) nephelometer. Fibrinogen forms a complex with a specific antibody. This complex causes scattering of irradiated light. The increase in scattering correlates with fibrinogen concentration.

[0145] Measurement of fibrinogen activity by clottable proteins For the assay of fibrinogen activity (= clottable protein), the sample preparation is mixed with a suitable buffer solution containing sufficient thrombin and incubated at 37° C. The remaining protein in the supernatant is measured by UV spectrophotometry at 280 / 360 nm and the result is subtracted from the total protein amount (see above) to calculate the clottable protein.

[0146] Determination of fibrinogen specific activity The specific activity of fibrinogen is determined by the clottable protein activity relative to the total protein measured by UV absorption (280 nm).

[0147] Clot stability measurement Clot stability is measured by the Fib-tem assay using a ROTEM whole blood analyzer (Tem Innovations GmbH, Munich), which is an established viscoelastic method for hemostatic testing of whole blood.

[0148] The test is performed according to the manufacturer's instructions. The validity of the method is tested using control formulations (Rotrol N and P).

[0149] For the determination of Fib-tem, fibrinogen samples are dissolved according to the manufacturer's instructions and further dilutions are made with fibrinogen-deficient plasma to give fibrinogen concentrations of 1.5 g / l, 2.0 g / l and 2.5 g / l.

[0150] vWF activity (vWF:Ag) measurement The test kit "vWF Ag" contains reagents for the immunoturbidimetric determination of von Willebrand factor antigen (vWF:Ag) in human plasma or plasma products, measured with the Behring coagulation system (BCS XP).

[0151] Perform the test according to the manufacturer's instructions, including the provided reagents and the defined test definitions and measurement instructions for the coagulation system BCS XP. Sample results are evaluated using the standard / reference curve.

[0152] For the preparation of the standard / reference curve, standard human plasma (Siemens) is used in duplicate at different dilution steps. The coagulation system (BCS XP) automatically dilutes the calibrator in the range of 10-200% of the standard. The validity of the reference curve is tested using a control preparation (Control Plasma N).

[0153] For the measurement, prepare a dilution series of the fibrinogen concentrate sample at 11:1 and 1:10 dilutions in Owren's Veronal Buffer. All other dilutions, incubations, and use of the different reagents provided in the reagent kit are prepared automatically by the test system (BCS XP).

[0154] FXIII activity (FXIII:Ac) measurement FXIII activity is measured by a photometric test (Berichrom FXIII, Siemens Healthcare Diagnostics GmbH) measured with a Behring coagulation system (BCS XP, Siemens Healthcare).

[0155] Perform the test according to the manufacturer's instructions, including the provided reagents and the defined test definitions and measurement instructions for the coagulation system BCS XP. Sample results are evaluated using the standard / reference curve.

[0156] To generate the standard / reference curve, standard human plasma (Siemens) is used in duplicate at different dilution steps. The coagulation system (BCS XP) automatically dilutes the calibrator in the range of 15 to 130% of the standard. The validity of the reference curve is tested using a control preparation (Control Plasma N). Here, 100% of the standard (Siemens standard human plasma (CoA)) corresponds to 1 IU / ml according to the WHO standard.

[0157] For the measurement, a dilution series of the fibrinogen concentrate sample is prepared at 1:1, 1:3 and 1:5 dilutions in NaCl solution (0.9%, w / v). All other dilutions, incubations and use of the different reagents provided in the reagent kit are prepared automatically by the coagulation system (BCS XP).

[0158] FXIII:Ag concentration measurement The assay is based on a sandwich ELISA assay using a Matched-Pair Antibody Set and VisuLize Buffer Pak (both from Affinity Biologicals). The test is performed according to the manufacturer's instructions, including the provided reagents and the defined test definition and measurement instructions. For calibration purposes, standard human plasma (Siemens) is used at seven 1:2 dilutions starting from 1:100.

[0159] Affinity-purified polyclonal antibodies against the FXIII A subunit are coated onto microtiter plates. Remaining binding sites are blocked with bovine serum albumin. After washing, standards and samples are applied. Bound FXIII is detected with peroxidase-conjugated FXIII antibodies. Peroxidase activity is detected with OPD (o-phenylenediamine) and stopped with H2SO4. The OD at 490 nm is measured. 100% of the standard corresponds approximately to 1 IU / ml according to the WHO standard.

[0160] D-dimer measurement INNOVANCE D-Dimer is a particle-enhanced immunoturbidimetric assay for the quantitative measurement of cross-linked fibrin degradation products (D-dimers) in human plasma or plasma products, measured on a Behring coagulation system (BCS XP).

[0161] Tests are performed according to the manufacturer's instructions, including the provided reagents and the defined test definitions and measurement instructions for the coagulation system BCS XP. Samples are evaluated using a standard / reference curve.

[0162] For the preparation of the standard / reference curve, INNOVANCE D-dimer calibrator is used in duplicate at different dilution steps. The coagulation system (BCS XP) automatically dilutes the calibrator in the range of 0.17 to 4.4 mg / L. The validity of the reference curve is tested using a control preparation (Control Plasma N).

[0163] For the measurement, a dilution series of the fibrinogen concentrate sample is prepared at 1:1 and 1:5 dilutions in the diluent (provided in the test kit). All other dilutions, incubations, and use of different reagents provided in the test kit are automatically prepared by the test system (BCS XP).

[0164] TnBP concentration measurement N-Hexane was used to extract TNBP from the sample solution. A reference solution containing the same amount as the standard limit was prepared and subjected to the same extraction procedure as the other samples. The hexane phases from the reference and sample solutions were analyzed by gas chromatography.

[0165] Compliance with the specification is assessed by comparison of the TNBP peak heights in the chromatograms from the samples and the corresponding reference solutions, which are prepared by dilution from the validated standard solution from the TNBP-Assay-Test.

[0166] Polysorbate 80 concentration measurement The determination of polysorbate 80 is carried out by photometric method according to the latest edition of the European Pharmacopoeia 2.2.25.

[0167] Polysorbate 80 in protein solutions is measured by a photometric assay. Polyoxylated compounds such as polysorbate 80 form a blue complex with ammonium cobalt thiocyanate.

[0168] Interference due to high protein content is avoided by deproteinization with ethanol. After protein precipitation with ethanol, the supernatant is evaporated to near dryness. The complex is extracted with dichloromethane and measured photometrically at 620 nm. A calibration function is performed without the deproteinization step.

[0169] Ristocetin cofactor activity (RCoF, vWF:RiCo) Cofactor activity of von Willebrand factor is measured by the BC von Willebrand reagent, an in vitro test for the determination of ristocetin cofactor activity of von Willebrand factor in human plasma or plasma products by platelet aggregation measured in the Behring coagulation system (BCS XP).

[0170] The test is carried out according to the manufacturer's instructions, including the reagents provided. The reference curve is changed from 20-150% of the standard to 5-100%. The test definition and measurement instructions are adapted to the coagulation system BCS XP. The samples are evaluated using the standard / reference curve.

[0171] For the preparation of the calibration / reference curve, standard human plasma is used in duplicate at different dilution steps. The coagulation system (BCS XP) automatically dilutes the calibrator in the range of 5-100% of the standard. The validity of the reference curve is tested using a control preparation (Control Plasma N).

[0172] For the measurement, a dilution series of the fibrinogen concentrate sample is prepared at 1:1 and 1:5 dilutions in NaCl solution (0.9%, w / v). All other dilutions, incubations, and use of the different reagents provided in the test kit are prepared automatically by the test system (BCS XP). Aspects of the present invention Aspects of the present invention are further described in the following sections: [Section 1] A method for producing a fibrinogen preparation from a plasma-derived fibrinogen-containing source, comprising the steps of: a) providing a liquid phase comprising plasma fibrinogen; b) contacting the liquid phase with a cation exchange chromatography (CEX) material under conditions that result in binding of fibrinogen; wherein the liquid phase has a pH in the range of 5.6 to 7.0; c) optionally washing unbound compounds from the cation exchange chromatography material; d) Eluting the fibrinogen from the cation exchange material with an elution buffer. [Section 2] Item 2. The method according to item 1, wherein the process is used to reduce the amount of von Willebrand factor (vWF) when the source contains von Willebrand factor. [Section 3] 3. The method according to item 1 or 2 above, wherein the process is used to reduce the amount of prions. [Section 4] The method according to any one of items 1 to 3 above, wherein the pH of the liquid phase in step b) is in the range of 6.3 to 6.9, more preferably 6.4 to 6.8. [Section 5] 5. The method according to any one of items 1 to 4 above, wherein the liquid phase in step b) has an ionic strength of 5 to 15 mS / cm, preferably 7 to 11 mS / cm, more preferably 8 to 10 mS / cm. [Section 6] 6. The method according to any one of items 1 to 5 above, wherein the cation exchange chromatography material is a strong cation exchange chromatography material. [Section 7] 7. The method according to any one of items 1 to 6 above, wherein the cation exchange chromatography material is a macroporous material. [Section 8] 8. The method according to any one of items 1 to 7 above, wherein the cation exchange chromatography material is a material containing a sulfonate functional group, preferably a sulfopropyl group. [Section 9] 9. The method of claim 8, wherein the cation exchange chromatography material comprises a resin backbone consisting of cross-linked polystyrene divinylbenzene to which sulfonate functional groups are attached as sulfopropyl groups via a polyhydroxyl surface. [Section 10] 10. The method according to any one of items 1 to 9 above, wherein the washing is carried out using a wash buffer having a pH in the range of 5.6 to 7.0 and an ionic strength of 5 to 15 mS / cm. [Section 11] 11. The method according to any one of items 1 to 10 above, wherein elution is carried out using an elution buffer having a pH at least 0.2 units higher than the conditions that result in binding of fibrinogen. [Section 12] 12. The method according to any one of items 1 to 11, particularly item 11, wherein elution is carried out using an elution buffer having an ionic strength at least 2 mS / cm higher than the conditions that result in fibrinogen binding. [Section 13] 13. The method according to any one of items 1 to 12 above, wherein the elution buffer contains one or more pharmaceutical compounds. [Section 14] Item 14. The method according to item 13, wherein the pharmaceutical compound is at least one amino acid, preferably arginine. [Section 15] Item 15. The method according to item 14, wherein the method comprises the step e) of formulating fibrinogen into a pharmaceutical composition. [Section 16] 16. The method according to any one of items 1 to 15, further comprising at least one of the following steps: Use of cryoprecipitate of human plasma as starting material; Al(OH)3 adsorption; S / D processing; anion exchange chromatography, and the use of flow-through; Glycine precipitation; UV-C treatment; Ultrafiltration; freeze-drying; Dry heat treatment. [Section 17] Item 17. The method according to item 16, wherein the steps listed in item 16 are carried out, and a cation exchange chromatography step described in any one of items 1 to 14 is carried out between the UV-C treatment and the ultrafiltration. [Section 18] A fibrinogen preparation obtained by the method according to any one of items 1 to 17 above, preferably the method according to item 17 above. [Section 19] Item 19. The fibrinogen preparation according to item 18, having an FXIII concentration of 0.5 to 2.0 FXIII:Ag (% of norm) and / or an FXIII activity of less than 16 FXIII:Ac (% of norm). [Section 20] 20. The fibrinogen preparation according to item 18 or 19, wherein the fibrinogen preparation does not exhibit a detectable amount of D-dimer. [Section 21] 21. A pharmaceutical composition obtained from the fibrinogen preparation according to any one of items 18 to 20 above. [Section 22] Item 22. The pharmaceutical composition according to item 21, which is a lyophilized product. [Section 23] 23. The pharmaceutical composition according to item 21 or 22, which is for treating hemostatic disorders or for preventing or treating bleeding. [Brief explanation of the drawings]

[0173] (Not stated in the original text)

Claims

1. A method for producing a fibrinogen preparation from a human plasma-derived fibrinogen-containing source, comprising the steps of: a) providing a liquid phase comprising plasma fibrinogen; b) contacting the liquid phase with a cation exchange chromatography (CEX) material under conditions that result in binding of fibrinogen; wherein the liquid phase has a pH in the range of 5.6 to 7.0, the liquid phase has an ionic strength of 5 to 15 mS / cm, and the cation exchange chromatography material is a macroporous material comprising particles with pore diameters in the range of 100 to 1000 nm; c) optionally washing unbound compounds from the cation exchange chromatography material; d) eluting the fibrinogen from the cation exchange material with an elution buffer; wherein the elution buffer has a pH 0.2 to 1 unit higher than the conditions conducive to binding of fibrinogen and / or an ionic strength 5 to 15 mS / cm higher than the conditions conducive to binding of fibrinogen.

2. 10. The method of claim 1, wherein the method is used to reduce the amount of von Willebrand factor (vWF) when the source contains von Willebrand factor.

3. 3. The method of claim 1 or 2, wherein the method is used to reduce the amount of prions.

4. The method according to any one of claims 1 to 3, wherein the pH of the liquid phase in step b) is in the range of 6.3 to 6.9, more preferably pH 6.4 to 6.

8.

5. The method according to any of claims 1 to 4, wherein the ionic strength of the liquid phase in step b) is between 7 and 11 mS / cm, preferably between 8 and 10 mS / cm.

6. The method according to any one of claims 1 to 5, wherein the cation exchange chromatography material is a strong cation exchange chromatography material.

7. The method according to any one of claims 1 to 6, wherein cryoprecipitate of human plasma is used as starting material.

8. The method according to any one of claims 1 to 7, wherein the cation exchange chromatography material is a material containing sulfonate functional groups, preferably sulfopropyl groups.

9. 9. The method of claim 8, wherein the cation exchange chromatography material comprises a resin backbone consisting of cross-linked polystyrene divinylbenzene to which sulfonate functional groups are attached as sulfopropyl groups through a polyhydroxyl surface.

10. The method according to any one of claims 1 to 9, wherein the washing is carried out using a washing buffer having a pH in the range of 5.6 to 7.0 and an ionic strength of 5 to 15 mS / cm.

11. The method according to any one of claims 1 to 10, wherein the elution buffer contains one or more pharmaceutical formulation compounds.

12. The method according to claim 11, wherein the pharmaceutical formulation compound is at least one amino acid, preferably arginine.

13. The method according to claim 12, wherein the method includes step e) of formulating fibrinogen into a pharmaceutical composition.

14. The method according to any one of claims 1 to 13, further comprising at least one of the following steps: Use of cryoprecipitate of human plasma as a starting material; Al(OH) ３ suck; suck; S / D treatment; Use of anion exchange chromatography and flow-through; Glycine precipitation; UV-C treatment; Ultrafiltration; Lyophilization; Dry heat treatment.

15. The method according to claim 14, wherein all of the steps listed in claim 14 are carried out, and the cation exchange chromatography step described in any one of claims 1 to 12 is carried out between the UV-C treatment and the ultrafiltration.

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