Fibrinogen Compositions and Methods of Preparation

Abstract

The present invention relates to the field of blood products, in particular fibrinogen and fibrinogen formulations. The invention provides fibrinogen preparations in a dry, e.g. freeze-dried, state with a residual water content of 2-5% (w / w). The inventors have found that said residual water content is advantageous for viral inactivation by dry heat. This advantageous inactivation results in a particularly virus-free and stable formulation. The invention further provides fibrinogen preparations in a dry, e.g. freeze-dried state, with a particularly low number of subvisible particles (SVPs), and a group of such preparations. The preparations are suitable for reconstituting 1 g of fibrinogen in an injection solution to obtain a fibrinogen solution with a content of 6000 or less SVPs between 10-100 μm and 600 or less SVPs between 25-100 μm. A method for the preparation of the preparations of the invention and these preparations for use in the treatment of fibrinogen deficiency are also disclosed.

Description

[Technical field]

[0001] The present invention relates to the field of blood products, in particular fibrinogen and fibrinogen formulations. The present invention provides fibrinogen in a dry, e.g. freeze-dried, state with a residual water content of 2-5% (w / w). The inventors have discovered that said residual water content is favorable for the inactivation of viruses by dry heat treatment, resulting in a stable formulation and a container containing said formulation with a high safety margin against infectious viruses. The present invention further provides a fibrinogen formulation in a dry, e.g. freeze-dried state, containing a particularly low number of sub-visible particles (SVPs), and a container containing said formulation. In addition, the present invention also provides a group of containers or formulations as described above. The formulation is suitable for reconstitution, e.g. one container containing 1 g of fibrinogen of said formulation, in an aqueous solution, e.g. water for injection, to obtain a fibrinogen solution with a content of not more than 6000 SVPs of 10-100 μm and not more than 600 SVPs of 25-100 μm. Methods for preparing the formulations of the invention and for use in treating fibrinogen deficiency are also disclosed. [Background technology]

[0002] Fibrinogen is the major structural protein in blood and is responsible for blood clot formation. During tissue and blood vessel injury, fibrinogen is enzymatically converted by thrombin into fibrin, which then becomes a fibrin-based meshwork that forms the basis of blood clots. The primary role of fibrin clots is to seal injured blood vessels and stop bleeding. Fibrin also binds to thrombin and reduces its activity. This action provides a feedback mechanism to prevent excessive clotting. In addition, fibrin contributes to the expansion of platelets and endothelial cells, differentiation of tissue fibroblasts, formation of capillaries, and angiogenesis, thereby facilitating revascularization and wound repair. It can be used to treat hemostatic disorders.

[0003] The fibrinogen molecule circulates in the blood as a soluble plasma glycoprotein composed of two trimers of three different polypeptide chains: fibrinogen α-chain, fibrinogen β-chain, and fibrinogen γ-chain. The typical molecular weight of fibrinogen is ~340 kDa. The normal fibrinogen concentration in human plasma is 150-400 mg / dL, and can be significantly decreased or increased beyond this range with pathological bleeding and / or thrombosis (Wikipedia).

[0004] In fibrinogen deficiency, the ability to form blood clots is impaired, resulting in a greatly increased risk of fatal bleeding and delayed hemostasis.

[0005] In severe congenital fibrinogen deficiency, the patient's ability to synthesize sufficient amounts of functional fibrinogen is impaired or completely lost. These patients are required to receive frequent injections of concentrated fibrinogen. In acquired fibrinogen deficiency, the patient loses endogenous fibrinogen, a condition that can lead to uncontrollable bleeding. A common cause of acquired deficiency is massive blood loss during complex surgery, but it can also occur as a result of severe trauma. In this case, it is necessary to stop the bleeding by administering fibrinogen intravenously to raise the fibrinogen concentration above the fatal value.

[0006] Thus, fibrinogen preparations and methods for preparing them are well known in the art. For example, Haemocomplettan® is manufactured by CSL Behring (Marburg, Germany). WO00 / 47621A1 discloses a method for preparing a composition comprising fibrinogen and fibronectin. WO2018 / 115800A1, WO2008 / 117746A1, EP0085923A1, EP0804933A2, WO95 / 26749A1, WO98 / 55105A1 and EP0345246A2 relate to fibrinogen compositions with various stabilizers.

[0007] WO01 / 48016A1, WO2004 / 007533A1, WO2012 / 038410A1, WO2009 / 155626A2 disclose methods for producing fibrinogen compositions. For example, since fibrinogen is a very sensitive protein and prone to forming aggregates, WO2006 / 015704 relates to heat treatment of fibrinogen preparations, whereby the formation of aggregates is minimized.

[0008] In general, formulations containing proteins may contain particulate matter, which may be the result of self-aggregation of proteins in the formulation to form particles (Carpenter et al., J Pharm Sci. 2009 Apr: 98(4): 1201 - 1205). Particles may be visible or invisible to the naked eye. Subvisible particles (SVPs) generally have diameters up to 100 μm. Their maximum diameter represents the limit of detection by the human naked eye. Thus, particles smaller than 100 μm are termed "subvisible".

[0009] One aspect of the stability and quality of a formulation is the presence or absence of SVPs (Abraham et al., 2011. BioPharm International 24(4)). Such particles may consist of aggregated proteins and / or components shed from processing materials or container closure systems and can directly affect the efficacy and immunogenicity of the formulation. SVPs also often act as nuclei for further protein aggregation and / or lead to the development of larger particles by accumulation. Measuring the particle size and concentration of SVPs in formulations is a necessary precursor to their effective control and is becoming increasingly important as the industry strives for 'defect-free' and 'essentially particle-free' products (Carpenter et al., 2015, www.europeanpharmaceuticalreview.com / article / 35952 / meeting-biopharmaceutical-analytical-requirements-for-subvisible-particle-sizing-and-counting / ). Light Obscuration Methods <788> Current United States Pharmacopeia (USP) requirements for standard tests for SVP analysis specify that particulates larger than 10 μm should be controlled to no more than 6000 per container, and particles larger than 25 μm should be limited to no more than 600 per container. These limits are related to concerns that particles may block capillaries (average diameter about 7 μm) upon injection. Other health issues, such as increased immunogenicity, may also be present with SVPs of all particle sizes (Carpenter et al. 2009. Journal of Pharmaceutical Sciences 98(4):1201-1205).

[0010] Currently, there are exceptions for plasma proteins and proteins for intramuscular and subcutaneous injection, since these proteins may contain relatively large amounts of SVPs, but it is clear that this is not due to the absence of concerns, but rather to the fact that it has not been possible to reproducibly and routinely produce plasma preparations with sufficiently low SVP content.

[0011] WO2013 / 106772A2 discloses a method for determining the characteristics of particle populations using a particle analyzer and explains that the generation of SVPs may be caused by the conditions of the preparation and / or the conditions of the packaging process.

[0012] WO2016 / 057739A1, WO2014 / 100143A2, and WO2019 / 060062A1 disclose antibody compositions having a low SVP content and comprising fatty acid esters such as polysorbates, and / or surfactants.

[0013] Nevertheless, there remains a great demand for plasma products, such as fibrinogen products, with a low SVP content.

[0014] Fibrinogen is typically purified from plasma, but despite the demands of stringent screening and donation testing of donors, it cannot be guaranteed that the plasma is free of infectious viruses. Another common problem is viral contamination. This forces the incorporation of a viral removal or viral inactivation step into the manufacturing process. Removal can be done, for example, by nanofiltration (as in the case of Fibclot® from LFB SA (Courtaboeuf Cedex, France)), but this method has the disadvantage that the filters are expensive and often clog. Chromatography can also be used. Viral inactivation can be based on solvent / detergent (S / D) treatment, pasteurization, or heat treatment (as described, for example, in WO 97 / 42980 A1), inactivation by acidic pH, or by irradiation, for example by UV light. Thermal inactivation in the presence of sucrose is performed for the CSL Behring formulation Haemocomplettan®, but may be considered inappropriate due to problems that arise in diabetic patients. A combination of effective methods for viral inactivation / removal is also frequently used in the art for lipid-enveloped viruses, as mandated under the European Medicines Agency (EMA) jurisdiction.

[0015] Viruses in factor VIII preparations are inactivated by heat treatment of the freeze-dried composition with a residual moisture content of 0.8% in, for example, EP0844005A1. JPS6289628A describes drying fibrinogen to a residual moisture content of 0.05-3% and heat treatment at 60°C for 65-90 hours in the presence of disaccharides. WO93 / 05067A1 presents polysorbate 80 as an antiviral substance and as a solubilizer of topical fibrinogen complexes. Successful virus inactivation depends on several factors in the composition and preparation preparation and needs to be confirmed experimentally, e.g., by virus addition experiments. Summary of the Invention [Problem to be solved by the invention]

[0016] Considering the current state of the art, the inventors have addressed the problem of providing an advantageous fibrinogen formulation that addresses one or more of these problems. In particular, the inventors have addressed the problem of providing a fibrinogen formulation that is active, stable for a long period of time, and free of viral risks, in a particularly safe formulation, e.g. for intravenous administration. Preferably, the fibrinogen preparation should be characterized by good solubility, a very low SVP content, and a substantial absence of accessory proteins for stabilization or other uses. Furthermore, the blood product should be producible in a well-standardized process in a reproducible manner. [Means for solving the problem]

[0017] Fibrinogen preparation of the present invention This problem is solved by the subject matter of the present invention, e.g. as claimed in the claims. Advantageously, the present invention provides fibrinogen preparations, which are particularly safe to use in terms of viral safety and / or the number of SVPs, or both, and containers containing such preparations.

[0018] In one embodiment, the invention provides a container containing a fibrinogen preparation in a dry, preferably lyophilized state, with a residual moisture content of 2-5% (w / w), preferably 2.5-4% (w / w). A residual moisture content of 2.5-3.5%, e.g. about 3% (w / w), has been shown to be particularly preferred with respect to virus inactivation and stability optimization. Thus, more preferably, the fibrinogen preparation of the invention is in a dry, preferably lyophilized state, and has a residual moisture content of 2.5-3.5% (w / w), preferably about 3% (w / w). The residual moisture content is preferably measured according to NIR spectroscopy. Alternatively, the residual moisture content can be determined by the Karl Fischer method. Unless otherwise stated, the residual moisture content % refers to w / w (weight of water / weight of preparation).

[0019] As known to those skilled in the art, a formulation is a pharmaceutical composition, i.e., a final dosage form, ready to be administered to a patient as a medicine, after further reconstitution with a solvent if necessary, and / or ready for sale and / or supply to a patient or physician. Generally, a formulation is prepared from a bulk drug substance. In this disclosure, the formulation of the invention, or a container containing the formulation of the invention, is also referred to as an article of the invention.

[0020] According to the present invention, the formulations of the invention are dry, which is not intended to mean that there is no water present, but rather that the formulations are solid and typically dry. Compared to other fibrinogen formulations of the state of the art, the formulations of the invention have a relatively high residual water content of 2-5% (w / w) as mentioned above. Freeze drying is the preferred option for drying, but as an alternative the formulations can be spray dried or spray freeze dried. Moisture refers to water. The formulations do not contain any significant amount of other solvents, and in particular do not contain any amount of solvents (or other components) that is inappropriate for pharmaceutical use, such as intravenous administration. Effect of the Invention

[0021] The inventors have surprisingly found that the effectiveness of virus inactivation by dry heat treatment, especially for non-enveloped viruses, is significantly improved at said residual moisture content, and that the formulations produced by adding said viruses with said residual moisture content still have high stability and good solubility. Such particularly efficient virus inactivation and high stability and solubility are illustrated by the following examples and figures. Thus, the formulations of the present invention are particularly safe for viruses, highly stable, and have good solubility upon reconstitution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] As used herein, "fibrinogen" refers to the major structural protein responsible for the formation of blood clots and present in plasma, and preferably refers to the entire glycoprotein form of fibrinogen. Preferably, "fibrinogen" refers to plasma fibrinogen, i.e., plasma-derived fibrinogen. Alternatively, fibrinogen for use in the present invention may be recombinantly produced.

[0023] Preferably, the formulation of the present invention is packaged in a dosage unit form. Typically, for fibrinogen, this means that about 1 g of fibrinogen is packaged. Before administration to a patient, the dried, e.g. lyophilized, fibrinogen formulation is typically reconstituted in a solvent, particularly an aqueous solvent, to obtain a solution. 1 g of fibrinogen may be reconstituted, for example, in water for injection, buffer, or plasma, typically in 50 mL thereof, to provide a solution with a total protein content of about 20 g / L, mainly fibrinogen.

[0024] In another embodiment, the present invention provides a container containing a fibrinogen preparation in a dry state, the fibrinogen preparation being suitable for reconstitution in an aqueous solution, preferably in water for injection, and the fibrinogen solution reconstituted from 1 g of fibrinogen of the preparation has an SVP content of 6000 or less at 10-100 μm and an SVP content of 600 or less at 25-100 μm. "Suitable for reconstitution" means that when the contents of one container are reconstituted in a solvent, in particular in water for injection, a fibrinogen solution is obtained having an SVP content of 6000 or less at 10-100 μm and an SVP content of 600 or less at 25-100 μm. In this specification, the amount of SVP refers to the absolute amount of SVP in the respective container in the respective dosage unit (e.g. 50 mL or 50-100 mL).

[0025] Typically, a fibrinogen preparation contains about 50-98% (w / w), preferably 70-95%, for example 80-90% active fibrinogen. Thus, if it is desired to package 1 g (active) fibrinogen, more than 1 g of preparation is often contained in the container, for example about 1.5-2 g of fibrinogen preparation. The preparation typically further contains additives, for example as described below. Preferably, said maximum concentration of SVP applies when reconstituting a single container of fibrinogen preparation containing, for example, 1 g of fibrinogen. Said maximum concentration of SVP may also apply when reconstituting a single container of fibrinogen preparation containing, for example, more than 2 g of fibrinogen, possibly containing 3 g or 5 g of fibrinogen.

[0026] In one embodiment, the present invention provides a dried, preferably freeze-dried, fibrinogen formulation having a residual moisture content of 2-5% (w / w), optionally 2.5-3.5% (w / w), for example about 3% (w / w), which formulation is suitable for reconstitution into an aqueous solution, preferably an injection solution, and which has an SVP content of 10-100 μm or less and an SVP content of 25-100 μm or less for 1 g of fibrinogen from said formulation.

[0027] Official requirements for SVP according to European Pharmacopoeia 2.9.19 and United States Pharmacopoeia 788 (documents providing standards for particulate matter for injections) are met for the first time with the formulation of the present invention for fibrinogen formulations. European Pharmacopoeia 2.9.19 and United States Pharmacopoeia 788 generally require no more than 6000 particles per 50 mL container for particles 10 μm or larger and no more than 600 particles per 50 mL container for particles 25 μm or larger, which corresponds to 120 particles per mL for particles 10 μm or larger and 12 particles per mL for particles 25 μm or larger.

[0028] The number of fibrinogen molecules with a diameter of 0.05 μm (Hall et al., 1959) (fibrinogen concentration in reconstituted formulation (DP): 20 g / L, molecular weight approximately 340 kDa, concentration approximately 60 μmol / L) is, for example, approximately 17.7·10 per mL. 18 There can be one.

[0029] Reconstitution is performed in a pharma- ceutically acceptable solvent, typically an aqueous solvent. Preferably, water for injection is used, but it can also be reconstituted in a buffer, such as, for example, saline or PBS. During reconstitution, the fibrinogen preparation is dissolved in the solvent. Reconstitution can be performed, for example, by adding the solvent to the container, and can optionally include mixing, for example, by vortexing, shaking, drawing up into a syringe, and can optionally be repeated. Reconstitution performed for the purpose of analyzing the number of SVPs does not include filtration.

[0030] According to European Pharmacopoeia 2.9.19 and US Pharmacopoeia 788, the amount of SVPs is measured by the light obscuration method, e.g., by a particle counter Hiac Model 9703+ (Beckman, Krefeld, Germany) equipped with a Hiac Model HRLD-400 sensor for particle sizes ranging from 2 to 400 μm. For each measurement, 5 mL of protein solution is aspirated through a fine tube and passed next to the sensor. The first measurement is used to wash the system and the measurement result is discarded. The average of the following four measurements is used to determine the number of SVPs in the solution. It is important to note that the protein solution is not filtered before the measurement.

[0031] In the final fibrinogen preparation (DP), the number of SVPs ≥ 10 μm and ≥ 25 μm is preferably determined. For this determination, the lyophilized DP is preferably reconstituted in 50 mL of water for injection (WFI). The reconstituted solution is transferred to the particle counter without any further filtration. All samples are preferably counted in triplicate. The average value of the total amount of particles per container is reported. The number of SVPs ≥ 10 μm is always greater than the number of SVPs ≥ 25 μm, since SVPs ≥ 25 μm are also included in the group of SVPs ≥ 10 μm.

[0032] Due to the low number of SVPs contained in the formulations of the invention, it is possible to safely administer said formulations without prior filtration, which is particularly advantageous in time-sensitive situations. However, filtration may also be carried out, for example to obtain a solution of the dry formulation in such a way as to avoid foam formation.

[0033] The present invention provides for the first time in a reproducible manner such a fibrinogen preparation and a container containing the fibrinogen preparation. The present invention therefore also provides a batch of containers containing a fibrinogen preparation in a dry state, in which for at least 10 containers of said batch, the fibrinogen preparation is suitable for reconstitution in an aqueous solution, preferably in water for injection, and in which the fibrinogen solution reconstituted from 1 g of fibrinogen of said preparation has an SVP content of 10-100 μm of less than 6000 and an SVP content of 25-100 μm of less than 600. Preferably, these conditions apply for at least 100 containers of the batch of containers of the invention, and optionally for at least 200 containers. This limitation may also apply for at least 500 containers of the batch of containers of the invention, and optionally for substantially all containers of said batch. The containers of the present invention may be selected randomly from the group, or preferably, may be selected evenly or approximately evenly from the first half of the group in which the containers are filled first and the second half of the group in which the containers are filled next.

[0034] The invention also provides a group of containers comprising a fibrinogen preparation in a dry state, wherein for at least the last 10% of the containers of the group, preferably for at least the last 20% of the containers of said group, and optionally for substantially all the containers of said group, the fibrinogen preparation is suitable for reconstitution in an aqueous solution, preferably in water for injection, and wherein the reconstituted fibrinogen solution from 1 g of fibrinogen of said preparation has an SVP content of 10-100 μm of not more than 6000 and an SVP content of 25-100 μm of not more than 600. "Last" in this context means the last filled.

[0035] Preferably, the fibrinogen preparation used in the present invention has an even lower SVP content. For example, the preparation is suitable for reconstitution into an aqueous solution, preferably water for injection, and the fibrinogen solution reconstituted from 1 g of fibrinogen of the preparation has an SVP content of 3500 or less at 10-100 μm and an SVP content of 60 or less at 25-100 μm. Optionally, the fibrinogen preparation used in the present invention is suitable for reconstitution into an aqueous solution, preferably water for injection, and the fibrinogen solution reconstituted from 1 g of fibrinogen of the preparation has an SVP content of 3500 or less at 10-100 μm and an SVP content of 35 or less at 25-100 μm. The inventors can demonstrate that the present invention also provides a fibrinogen preparation of the present invention which is suitable for reconstitution into an aqueous solution, preferably water for injection, and which, for each gram of fibrinogen in said preparation, has a fibrinogen solution containing less than 2500 10-100 μm SVPs and less than 30 25-100 μm SVPs.

[0036] Insofar as the invention relates to a group of containers containing a fibrinogen preparation of the invention, said fibrinogen preparation has a residual moisture content of 2-5% (w / w), preferably 2.5-3.5% (w / w), which means that each preparation in the group has said residual moisture content.

[0037] The fibrinogen preparation of the invention is packaged in a container such as a vial, e.g. a glass or plastic (e.g. polyethylene) vial. Preferably, the container is a glass container. The container may have a capacity to allow for the addition of, e.g., at least 50 mL of solution. Preferably, the container has a nominal fill volume of 100 mL. The container may be a syringe, e.g. a single chamber syringe. The container may be a double chamber syringe, one of which contains the fibrinogen preparation of the invention.

[0038] The present invention provides a kit comprising the formulation of the present invention in a suitable container, e.g., a vial, and / or a suitable transfer device. More preferably, the transfer device is a device that facilitates the clean or aseptic transfer of a suitable solvent, e.g., water for injection, from a container containing the solvent to a container containing the formulation. Even more preferably, the transfer device is a device that also facilitates the transfer of the dissolved formulation to a device for administration, e.g., a syringe. Such transfer devices are commercially available (e.g., Mix2Vial® (West), Nextaro® (SFM)) and are well known to those skilled in the art. The transfer device optionally comprises one or more filters, e.g., filters capable of removing bacteria and / or particles in the solution and / or formulation. Such filters may be used to ensure the removal of particles that enter the formulation or solution, e.g., by penetrating the lid of the container (e.g., material from the rubber stopper entering by penetrating the rubber stopper with a needle).

[0039] Mesh or membrane filters may be used where deemed appropriate. The pore size of such filters may range from 3 μm to 25 μm, preferably from 4 to 10 μm. The term "pore size" refers to the nominal pore size.

[0040] As regards the filter through which the dissolved formulation passes, preferably a membrane filter is used. Preferably, said filter has a pore size of 3 to 10 μm, more preferably 4 to 9 μm, for example 5 or 8 μm. The inventors have found that particularly good results are achieved by using a membrane filter made of an acrylic copolymer.

[0041] Therefore, the present invention also relates to a kit comprising the formulation of the present invention in a suitable container (e.g., a vial) and a suitable transfer device as described above. Preferably, the kit also comprises a suitable container comprising an aqueous solvent (e.g., water for injection) for dissolving the formulation. For example, the present invention also relates to a kit comprising (i) the formulation of the present invention in a suitable container (e.g., a vial), (ii) a suitable transfer device comprising a filter with a pore size of 3 to 10 μm for filtering the formulation, and (iii) a suitable container comprising an aqueous solvent (e.g., water for injection).

[0042] A container of the invention typically contains a fibrinogen preparation comprising 1 g of fibrinogen, but may contain other amounts of fibrinogen, for example 0.5-10 g, or 1-5 g, 2 g or 5 g.

[0043] Preferably, the containers, e.g. vials, of the invention are suitable for reconstitution in 50 mL of water for injection at a fibrinogen concentration of 20 g / L. Reconstitution for counting the number of SVPs as defined herein is also typically carried out at said concentrations.

[0044] The fibrinogen formulations of the invention are in a dry state, preferably a freeze-dried state. The formulations may be spray-dried or spray-freeze-dried.

[0045] The fibrinogen preparation used in the present invention contains, in addition to fibrinogen and the residual water content, a) polysorbates, b) optionally, a filler; c) optionally, amino acids and / or d) Optionally, salt may include:

[0046] For example, the fibrinogen formulations used in the present invention preferably contain a polysorbate, such as polysorbate 80, since the inventors have been able to show that this polysorbate contributes to the reduction of viral load upon dry heat treatment at the preferred residual moisture content and may further aid in the reduction of SVPs. Polysorbate 20 is also an option.

[0047] The fibrinogen preparation or group of fibrinogen preparations of the present invention may also contain a bulking agent, optionally trehalose.Preferably, the bulking agent also acts as a cryoprotectant.Mannose can be used as another option, but the inventors have found that trehalose provides a better cake-like structure and better dissolution properties.

[0048] The fibrinogen preparation or group of fibrinogen preparations of the invention may further comprise an amino acid, optionally arginine, which preferably has a stabilizing effect. Lysine may be used as an alternative, but the inventors have found that arginine results in a better cake-like structure and better dissolution properties.

[0049] Furthermore, the fibrinogen preparation or group of fibrinogen preparations of the present invention may contain a salt, preferably sodium chloride, sodium citrate, or a combination thereof, and optionally sodium chloride and sodium citrate. The salt may function, for example, as a buffer and / or to provide an isotonic solution. Preferably, due to the toxicity of potassium, the compositions of the present invention do not contain significant amounts of potassium.

[0050] Therefore, the fibrinogen preparation used in the present invention may be, for example a) Polysorbate 80, b) trehalose, c) Arginine and d) Salts, preferably sodium chloride, sodium citrate, and combinations thereof. may include:

[0051] Preferably, the fibrinogen preparation used in the present invention comprises polysorbate 80, trehalose, arginine, sodium, chloride, citrate and residual water. The preparation may further comprise calcium, preferably at a concentration of less than 1 mM. Higher calcium concentrations may contribute to undesired complex formation. The fibrinogen preparation or group of fibrinogen preparations of the present invention may further comprise additional plasma proteins selected from the group comprising albumin, fibronectin, A2 macroglobulin, immunoglobulins such as IgG, IgA or IgM, von Willebrand factor (vWF), fibrinopeptide A and D-dimer, for example in small amounts thereof, preferably in the approximate concentrations provided in the examples below.

[0052] A preferred fibrinogen formulation for use in the present invention consists essentially of the above mentioned components: fibrinogen, a polysorbate such as polysorbate 80, a bulking agent such as trehalose, an amino acid such as arginine, a salt such as sodium chloride and / or sodium citrate, or a plasma protein selected from the group including albumin, fibronectin, A2 macroglobulin, an immunoglobulin such as IgG, IgA or IgM, von Willebrand factor, fibrinopeptide A and D-dimer, and residual water.

[0053] For example, in a preferred embodiment, the fibrinogen formulation used in the present invention may contain 95-135 mmol / L sodium, less than 1 mmol / L calcium, 100-160 mmol chloride, 3-7 mmol / L citrate, 25-55 mmol / L arginine, 22-38 mmol / L trehalose and 0.03-0.07% polysorbate 80 when 1 g of fibrinogen of said formulation is reconstituted in water for injection at 20 g / L, as described herein.

[0054] In particular, preferably, the formulation of the present invention does not contain significantly more albumin than the above. For example, the formulation of the present invention contains less than 0.5 g / L albumin, preferably less than 0.15 g / L albumin when reconstituted with a fibrinogen concentration of 20 g / L. Thus, appropriate administration of fibrinogen and albumin to the patient can be provided separately, and furthermore, there is no need to carry out quality control processes for both fibrinogen and albumin. Preferably, when reconstituted with a fibrinogen concentration of 20 g / L, each of the non-fibrinogen plasma proteins contains less than 0.5 g / L, allowing them to be administered separately.

[0055] Additionally, the fibrinogen preparation or group of fibrinogen preparations of the invention preferably does not contain saccharose and / or glutamic acid, preferably does not contain either saccharose or glutamic acid. Saccharose is particularly problematic in the context of diabetes. Concerns regarding glutamic acid have been discussed in the context of "Chinese restaurant syndrome".

[0056] The inventors have found that the fibrinogen preparations used in the present invention are very stable. The inventors have advantageously found that the formulations of the present invention are stable when stored dry at 2-25° C. for at least 6 months, preferably at least 1 year, and in some cases at least 5 years. Furthermore, as shown in the examples below, after reconstitution, the formulations may be stored at 2-8° C. for at least 8 weeks without significant degradation. By stable, it is preferably meant that the formulation maintains at least 80%, and in some cases at least 90%, of the specific fibrinogen activity originally contained therein.

[0057] The fibrinogen preparation or group of fibrinogen preparations of the present invention preferably contains less than 20%, preferably less than 10% aggregates. Aggregates are typically polymers of fibrinogen, such as dimers, trimers, etc. The percentage of aggregates can be determined by HP-SEC analysis. The percentage is described as the detected amount of fibrinogen present in the form of aggregates compared to the total amount of fibrinogen.

[0058] In one embodiment, the present invention also provides a fibrinogen solution of the present invention, which is obtained by reconstituting the dry formulation of the present invention in an aqueous solvent, in particular water for injection. This solution has a low SVP content, as defined herein. Also provided is a method for reconstituting said fibrinogen solution, which comprises adding water for injection or another aqueous solvent to the dry formulation of the present invention.

[0059] Method for preparing a container containing the fibrinogen preparation of the present invention In another embodiment, the present invention provides a method for producing a container comprising a fibrinogen formulation of the present invention, comprising the steps of: a) filter-sterilizing a bulk solution of fibrinogen drug substance; b) receiving the filter-sterilized bulk solution in a receiving tank, optionally with an agitator equipped with stirring means; c) optionally agitating the bulk solution in the receiving tank while the agitation means is submerged in said bulk solution; d) filling a container with a predetermined amount of said solution, for example by using a pump; e) freeze-drying the solution in the container to obtain a freeze-dried formulation; and f) subjecting the lyophilized formulation to a dry heat treatment; and g) optionally packaging the container containing the formulation. The present invention provides a method comprising:

[0060] In one embodiment, the method of the invention comprises a) filter-sterilizing the bulk solution of fibrinogen drug substance; b) receiving the sterilized filtered bulk solution in a receiving tank; c) Do not agitate the bulk solution in the receiving tank; d) filling a container with a predetermined amount of said solution, for example by using a pump; e) freeze-drying the solution in the container to obtain a freeze-dried formulation; and f) dry heat treatment of the lyophilized formulation; and g) optionally packaging the container containing the drug product; Includes.

[0061] The inventors have surprisingly discovered that it is not necessary to stir the bulk solution before and / or during the filling process. The solution remains homogeneous throughout the filling process, e.g., for 2-3 hours, without stirring. Samples taken during filling showed consistent concentrations of fibrinogen and other components throughout the filling process, even without any stirring. The inventors have also discovered that reducing or avoiding the stirring process reduces the amount of SVP.

[0062] Nevertheless, to avoid the risk of non-homogeneity, stirring for a short period of time and / or under gentle conditions may be included in the method of the invention. Thus, typically, the method of the invention comprises a) Sterilizing the bulk solution of fibrinogen drug substance by filtration b) receiving the filtered sterilized bulk solution in a receiving tank, the receiving tank having an agitator equipped with an agitation means; c) agitating the bulk solution in the receiving tank while the agitation means is submerged in said bulk solution; d) filling a container with a predetermined amount of said solution, for example by using a pump; e) freeze-drying the solution in the container to obtain a freeze-dried formulation; and f) subjecting the lyophilized formulation to a dry heat treatment; and g) optionally packaging the container containing the formulation. Includes.

[0063] Agitation has the advantage of ensuring that non-homogeneity of the solution and the resulting non-homogeneous packaging is avoided, however, the inventors have found that the solution is sufficiently homogeneous even without agitation.

[0064] A representative filling line that may be used with the present invention is shown in FIG.

[0065] The filter sterilization in step a) is preferably performed with a filter having a mesh size of 0.2 μm, which makes the solution filter-sterilized. The solution of fibrinogen drug substance passes through the filter from a delivery tank, which may typically be a stainless steel tank or a plastic container (such as a bag) and can hold, for example, 80 L. The solution is pressurized through the filter by a pressure line. The transfer pressure is up to 600 mbar (600 hPa). Optionally, after filter sterilization, the solution is directly transferred into a receiving tank (step b).

[0066] In step b), the filter-sterilized bulk solution is received in a receiving tank, for example a stainless steel tank. The volume may be, for example, 50 L. The receiving tank optionally has a stirring means, preferably a stirrer with a stirring bar or a stirring rod. In the context of the present invention, "a" is understood to mean "at least one". Thus, the stirred tank may have two or more stirrers. The stirrer may also have at least one stirring means, for example one stirring blade. Preferably, the stirrer has two or more stirring means, for example two or more stirring blades. In a stirrer with two blades, the blades are typically arranged opposite each other. The stirring means, for example the stirring blade, preferably rotates horizontally to stir the solution. The stirring blade may be fixed to a central drive shaft that typically rotates and moves the stirring blade. The stirring means may be a stir bar, for example a magnetic stir bar. The stirring means may be a swinging plate, or multiple swinging plates.

[0067] In optional step c), the bulk solution is stirred in the receiving tank when the stirring means is submerged in the bulk solution. Thus, the solution is not stirred before the stirring blades or other stirring means (if there are several, all stirring means or blades) are submerged. That is, in contrast to the prior art methods, the solution is not stirred before a sufficient amount of solution is received in the receiving tank for the stirring means to be submerged. This has the effect that the surface of the bulk solution is not disturbed by the stirring means during stirring. Thus, foam formation is prevented and shear forces on the solution are minimized. If the solution is stirred during filling, when the solution is filled from the receiving tank to the container and the level of the solution in the receiving tank drops again, stirring is stopped before the level of the solution drops below the stirring means, and the surface of the bulk solution is not disturbed by the stirring means during stirring.

[0068] For a 50 L receiving tank, to further minimize exposure to shear stress, the duration of agitation is preferably limited to a maximum of 1 hour, more preferably a maximum of 10 minutes, and most preferably a maximum of 5 minutes. This time can vary depending on the volume of the solution and tank, e.g., for larger tanks and / or more solution, the duration of agitation can be longer.

[0069] Small-scale experiments showed that the speed and velocity of stirring, the duration of stirring and the geometry of the stirrer can affect the number of SVPs formed.

[0070] Preferably, the agitator, e.g. as described above, mounted on a vertical drive shaft with two rotating parallel agitator blades, rotates at a maximum speed of 150 rpm, e.g. 30-100 rpm or 50-80 rpm. Preferably, the shear forces to which the solution is exposed are less than those experienced by a solution exposed to a preferred agitator with two agitator blades as described above rotating at a maximum speed of 150 rpm, e.g. 80 rpm, in a 50 L cylindrical stainless steel receiving tank.

[0071] In a particularly preferred embodiment, for example in a 50 L cylindrical stainless steel tank with filling line KS1025, e.g. model AS18.3 from Bausch & Stroebel, Ilshofen, Germany, agitation at a speed of 150 rpm or less, e.g. 80 rpm, for 0-5 minutes has been shown to minimize the formation of SVP.

[0072] It has further been shown that stirring during filling is not required. If additional bulk formulation solution is added to the receiving tank, stirring can be resumed for a limited time as described above. In this case, the maximum stirring time applies throughout the stirring to ensure that no formulation solution is stirred for an excessively long time. If additional solution must be added, it is advantageous to add the additional solution while the bulk of the solution still remains in the receiving tank, if a stirring blade is provided, so that the surface of the liquid is not disturbed as the stirring blade rotates.

[0073] The inventors have surprisingly found that such smooth or gentle stirring, and thus minimizing the shear forces to which the fibrinogen solution is exposed, results in an advantageously low number of SVPs in the formulation used in the present invention. Tests conducted by the inventors have shown that the shear forces to which the fibrinogen solution is exposed after sterilization by filtration can result in a significant increase in the number of SVPs in the formulation after reconstitution. In particular, stirring in the receiving tank is a critical step. Although stirring may be important to obtain a homogeneous formulation, it has been shown that excessive stirring or stirring under incorrect conditions, such as stirring when the surface of the solution is disturbed or bubbles are generated, results in a higher number of SVPs.

[0074] In step d) the container is filled with a predetermined amount of solution using a pump or other means. The pump may be a peristaltic pump used at a speed of up to 300 rpm, for example 200-290 rpm, for example up to 270 rpm. The predetermined amount of fibrinogen in the container is preferably 1-5 g, optionally 1 g, 2 g or 3 g, for example 1 g.

[0075] In step e), the drug substance in the filled container, e.g. vial, is freeze-dried. Preferably, the container is placed in a pre-chilled freeze-dryer. This allows rapid freezing of the protein solution and improves the sublimation of water, resulting in a preferred porous cake-like structure. In the method of the present invention, the freeze-drying conditions are preferably selected to obtain a residual moisture content after freeze-drying of 2-5% (w / w), optionally 2.5-4%, or 2.5-3.5% (w / w), e.g. about 3% (w / w).

[0076] The residual moisture content in the lyophilized formulations used in the present invention depends mainly on the lyophilization conditions. The components of the formulation, such as polysorbate 80, have an effect on the residual moisture content insofar as it may be necessary to improve the lyophilization conditions to obtain the desired residual moisture content.

[0077] A particular advantage of the relatively high residual moisture content of the preferred formulations of the present invention is that, as the inventors have discovered, this high residual moisture content allows for particularly effective viral inactivation during the dry heat treatment that follows lyophilization. Generally, high residual moisture content is detrimental to the integrity of the formulation, but the inventors have surprisingly found that formulations of the present invention having said residual moisture content are very stable. Formulations with a residual moisture content of 2.5-3.5% have been shown to be optimal with respect to both parameters.

[0078] Improved virus inactivation by dry heat treatment can be shown for non-enveloped viruses, such as porcine parvovirus (PPV). In tests against enveloped viruses, such as HIV (human immunodeficiency virus) or BVDV (bovine viral diarrhea virus), this effect was not very clearly shown. This may be due to other virus inactivation effects, such as the fact that polysorbates, which are preferably contained in the formulation of the invention, already bring about very good virus inactivation against enveloped viruses during lyophilization, i.e. before dry heat treatment, at all residual moisture contents analyzed, i.e. even at lower residual moisture contents. Therefore, in the preparation of fibrinogen formulations, non-enveloped viruses are preferably inactivated by dry heat treatment of a fibrinogen solution lyophilized to a residual moisture content of 2-5%, optionally in the presence of a polysorbate, such as polysorbate 80. Enveloped viruses are preferably inactivated as described herein by freeze-drying and / or UV-C treatment of the fibrinogen solution in the presence of a polysorbate, such as polysorbate 80. All these measures are preferably performed and contribute to providing a virus-free formulation.

[0079] In a preferred embodiment, the freeze-drying method of the present invention comprises the steps of: a) freezing (preferably quick freezing) at -29°C or below for at least 4 hours, preferably at -50°C or below for about 8 hours; b) a first drying step of stepwise drying at below -10°C and above 40 μbar (4 Pa), preferably starting from below -25°C and above 200 μbar (20 Pa), preferably 280 μbar (29 Pa), and increasing the temperature stepwise; c) A second drying step in which the mixture is dried at 17°C to 23°C for at least 2 hours. Includes.

[0080] As stated above, preferably the residual moisture content after said lyophilization is 2-5% (w / w).

[0081] The freeze-drying method is more specifically a) freezing at -52°C or lower for at least 8 hours; b) a first drying step comprising a first step at about -36°C and about 280 μbar (28 Pa) for about 5-48 hours, a second step at about -23°C and about 70 μbar (7 Pa) for about 25-45 hours, and a third step at about -10°C and about 40 μbar (4 Pa) for about 45-84 hours, e.g. a first step at about -36°C and about 280 μbar (28 Pa) for about 45 hours, a second step at about -23°C and about 70 μbar (7 Pa) for about 45 hours, and a third step at about -10°C and about 40 μbar (4 Pa) for about 45 hours, c) a second drying step at about 20° C. and about 10 μbar (1 Pa) for about 3-4 hours may include:

[0082] Alternatively, the first drying step of step b) may involve, for example, a first step at about -36°C for about 5 hours and about 280 μbar, a second step at about -23°C and about 70 μbar for about 25 hours, and a third step at -10°C and about 40 μbar for about 78 hours.

[0083] In the method of the present invention, in step f) a dry heat treatment is performed. The dry heat treatment may for example involve heating the formulation to 100°C ± 1.5°C, e.g. 99-100°C. The dry heat treatment may be performed for 20-60 minutes, preferably 20-40 minutes. Good results have been obtained with 30 ± 3 minutes. The dry heat treatment is preferably performed in a steam autoclave, of course after closing the container, e.g. with a pierceable rubber stopper.

[0084] The formulation is optionally packaged in step g). A package insert may be added, for example stating that the formulation is for dissolving in water for injection at 20 g / L fibrinogen and / or that the formulation is for intravenous administration. A filter may be packaged with the formulation container.

[0085] A preferred method for preparing a container containing a formulation of the present invention may comprise the following steps: After the drug substance, i.e. the prepared formulation, is filter-sterilized (0.2 μm), it is filled into a container, e.g. a vial. In filling, the drug substance is preferably received in a receiving tank and, optionally, carefully stirred, e.g. for up to 5 minutes, while the stirring blade of the stirrer or other stirring means is covered with the drug substance solution, or not stirred at all, while avoiding the formation of bubbles. The stirrer operates at 150 rpm or less, preferably 80 rpm or less. The drug substance is then filled into the container by a peristaltic pump, preferably operating at 290 rpm or less, e.g. 270 rpm or less. Typically, 32 mL, e.g. an amount corresponding to about 1 g of fibrinogen, is filled into the container. It is then lyophilized to a residual moisture content of 2-5%, and a dry heat treatment at about 99-100°C for about 30 minutes is performed for further virus inactivation. The container containing the formulation may then be packaged.

[0086] Optionally, the method of the invention further comprises the step of preparing a fibrinogen drug substance prior to step a). For example, the fibrinogen may be prepared according to the methods described below or in the Examples. The fibrinogen may also be prepared according to other methods, e.g. methods known in the art.

[0087] A typical starting material for the production of fibrinogen is human plasma. A cryoprecipitate of human plasma may be obtained by thawing frozen plasma at 0-4° C. and separating the precipitate by centrifugation or other separation means.

[0088] For each kg of cryoprecipitate, a mixture of 2.91 kg water (WFI), 114 g 25% (v / v) ethanol, and 9000 IU heparin may be prepared. Cryoprecipitate may be added to the stirred WFI / ethanol / heparin solution. The pH value may be adjusted to 7.0.

[0089] 108 g of a 2% aluminum hydroxide suspension may be added per kg of cryoprecipitate used and the mixture may be stirred at 22.5° C. The pH value may be adjusted to 6.55 and then centrifuged by continuously operating the centrifuge.

[0090] During stirring, 1% polysorbate 80 and 0.3% tri-n-butyl phosphate may be added. The protein solution may be stirred at 25° C. for at least 8 hours.

[0091] Anion exchange gel Toyopearl® TSK DEAE-650 (beads of hydroxylated methacrylic acid polymer as matrix material with diethylaminoethyl groups) may be used for further purification by column chromatography. Protein loading may be about 50±10 mg protein per mL of anion exchange gel.

[0092] The chloride content in the protein solution may be adjusted to 120 mmol / L by adding sodium chloride solution. The protein solution may be applied to the column. The flow-through fraction contains fibrinogen, which may be collected for further processing.

[0093] The resulting fibrinogen solution (flow through) may be subjected to glycine precipitation. Glycine may be added to a final concentration of 1.2 M to precipitate the fibrinogen. Sodium chloride may be added to a final concentration of 2 M. The fibrinogen-containing precipitate may then be separated by centrifugation. The fibrinogen paste may be stored at a temperature below -70°C.

[0094] The precipitate may be resuspended in a buffer (15 mM trisodium citrate dihydrate, pH value: 6.9±0.1, conductivity: 3.3±0.5 mS / cm). The composition contains TnBP, polysorbate 80, glycine and sodium chloride in addition to other proteins (e.g., 0.7-0.9 U / mg vWF). The composition may be filtered and may be subjected to UV-C treatment using a device such as a UVivatec® device (Sartorius Stedim Biotech) for virus inactivation. UV irradiation is preferably at 254 nm±1 nm and 125-200 J / m 2 This is done using

[0095] For the subsequent cation exchange chromatography step, a column (POROS™ 50 HS) may be equilibrated with equilibration buffer (15 mM trisodium citrate dihydrate, 65 mM sodium chloride, pH value: 6.5±0.1, conductivity: 9.0±1.5 mS / cm, 2-5 column volumes).

[0096] A liquid phase containing fibrinogen that has been subjected to a UV-C irradiation step may be prepared by adjusting the composition to 15 mM trisodium citrate, pH value: 6.5±0.1, and conductivity 9.0±1.5 mS / cm. The column may be loaded with 10 g-20 g of protein per L of resin volume.

[0097] The column may be rinsed with wash buffer (15 mM trisodium citrate dihydrate, 65 mM sodium chloride, pH value: 6.5±0.1, conductivity: 9.0±1.0 mS / cm, 2-5 column volumes).

[0098] Then, fibrinogen may be eluted with an elution buffer (7.5 mM trisodium citrate dihydrate, 150 mM sodium chloride, 75 mM L-arginine hydrochloride, pH value: 7.0±0.1, conductivity 19.5±1.5 mS / cm). In this step, fibrinogen is eluted from the column. Most of the vWF is still bound to the column.

[0099] The column may then be rinsed with a higher salt buffer (15 mM trisodium citrate dihydrate, 1.5 M sodium chloride, pH: 6.5±0.1, conductivity 113.5±5.0 mS / cm), which elutes vWF from the column, and the column may then be washed with 1 M sodium hydroxide.

[0100] In this method, most of the albumin and IgG have already been separated from fibrinogen in the precipitation step, but they do not bind to the cation exchange material, and more than 50% of the vWF present in the liquid phase containing fibrinogen can be removed using this method.

[0101] In the production of drug substance, the eluted fractions may be concentrated by ultrafiltration and the protein concentration adjusted to 33 g of fibrinogen per liter with citrate buffer. Further components may be added to form the final drug substance, for example as described herein.

[0102] As explained in step a) and subsequent steps, the drug substance is then preferably filtered (0.2 μm) into different containers, such as vials, under the conditions described above, and dried, preferably lyophilized, to a residual moisture content of 2-5%. A final heat treatment, preferably by steam autoclaving (e.g. 100° C., 30 min), is then performed as a further viral inactivation step. The resulting formulation is surprisingly stable.

[0103] The method of the invention may be advantageously used to reduce the number of SVPs, in particular to the limits described herein. Mild loading conditions are particularly relevant for achieving this goal. The method of the invention may also be advantageously used to reduce the number of viruses, such as non-enveloped viruses. Dry heat treatment at a residual moisture content of 2-5%, preferably in the presence of a polysorbate, such as polysorbate 80, is particularly relevant for achieving this goal. In combination, the method of the invention results in a formulation that is particularly safe for patients.

[0104] The present invention also provides a container comprising a fibrinogen preparation of the invention, or a group of containers comprising a fibrinogen preparation, obtainable by the method of the invention as described herein.

[0105] Uses, especially pharmaceutical uses In another embodiment, the present invention provides a container comprising a fibrinogen preparation for use in treating fibrinogen deficiency. For example, the fibrinogen preparation may be for use in treating a hereditary fibrinogen-related disease. Preferably, the preparation is for use in treating a congenital fibrinogen deficiency. The fibrinogen deficiency may be, for example, congenital afibrinogenemia, congenital hypofibrinogenemia, fibrinogen storage disease, congenital dysfibrinogen plasma, hereditary fibrinogen Aα chain amyloidosis, congenital hypo / dysfibrinogenemia, cryofibrinogenemia, etc.

[0106] The fibrinogen preparation of the present invention may be for use in the treatment of acquired fibrinogen-related diseases such as acquired dysfibrinogenemia and acquired hypofibrinogenemia.

[0107] If the fibrinogen deficiency is acquired, the fibrinogen preparations of the invention are preferably used to treat blood loss or bleeding, optionally during (major) surgical procedures. Blood loss or bleeding may be due to trauma.

[0108] The preparation of the present invention may be administered instead of plasma preparations such as fresh frozen plasma. Administering the fibrinogen preparation of the present invention instead of such plasma preparations, or even instead of fibrinogen preparations that contain a large amount of other plasma proteins such as albumin, is advantageous in that the preparation allows the plasma proteins to be administered separately according to the patient's needs. In particular, administration of fibrinogen is required to prepare for bleeding, i.e. to treat bleeding, or to prevent bleeding in cases where there is a fibrinogen deficiency. The preparation of the present invention is also free of viral risks.

[0109] In another embodiment, the present invention provides a method for treating fibrinogen deficiency comprising administering a fibrinogen formulation of the present invention to a patient in need thereof, such as a patient suffering from any of the aforementioned diseases. The patient to whom the formulation is administered is preferably a human patient.

[0110] Typically, the fibrinogen preparation or group of fibrinogen preparations of the invention is prepared for intravenous administration to a patient after resuspension. Due to the small number of SVPs as described herein, the fibrinogen preparation or group of fibrinogen preparations of the invention is advantageously suitable for administration after resuspension without prior filtration. This saves time, especially in emergency cases. However, the fibrinogen preparation of the invention may also be used after resuspension and filtration. A filter may be included as part of the kit of the invention, including a container containing the fibrinogen preparation of the invention and a filter, preferably a transfer device or a syringe port filter. Filtration may be useful when resuspending the preparation of the invention, in particular to avoid or reduce foam formation. Alternatively, resuspension may be performed by gentle shaking to avoid foam formation.

[0111] The fibrinogen formulation or fibrinogen formulations of the invention may be used as fibrin glue, for example in bandages etc. Alternative applications include as part of cell culture medium or as part of organ printing, for example 3D organ printing.

[0112] EMBODIMENTS OF THE PRESENT DISCLOSURE The present invention provides, for example, the following embodiments:

[0113] In embodiment 1, the container contains a fibrinogen preparation in a dry state with a residual moisture content of 2-5% (w / w). In embodiment 2, the container contains the fibrinogen preparation of embodiment 1 with a residual moisture content of 2.5-3.5% (w / w), for example about 3% (w / w).

[0114] In embodiment 3, the formulation in the container of any of embodiments 1 or 2 is suitable for reconstitution in an aqueous solution, preferably in water for injection, and the fibrinogen solution reconstituted from 1 g of fibrinogen of said formulation has an SVP content of 10-100 μm of 6000 or less and an SVP content of 25-100 μm of 600 or less. In embodiment 4, the formulation in the container of any of embodiments 1-3, when one container is reconstituted in a solvent, in particular in water for injection, a fibrinogen solution is obtained having an SVP content of 10-100 μm of 6000 or less and an SVP content of 25-100 μm of 600 or less.

[0115] In embodiment 5, the present invention provides a container containing a fibrinogen preparation in a dry state, the fibrinogen preparation being suitable for reconstitution in an aqueous solution, preferably in water for injection, and the fibrinogen solution reconstituted from 1 g of fibrinogen of said preparation has an SVP content of 10-100 μm of not more than 6000 and an SVP content of 25-100 μm of not more than 600. In embodiment 6, for the preparation in the container of embodiment 5, when one container is reconstituted in a solvent, in particular in water for injection, a fibrinogen solution is obtained having an SVP content of 10-100 μm of not more than 6000 and an SVP content of 25-100 μm of not more than 600.

[0116] In embodiment 7, the present invention provides a group of containers containing a fibrinogen preparation in a dry state, wherein for at least 10 containers of said group, the fibrinogen preparation is suitable for reconstitution into an aqueous solution, preferably into an injection solution, and the fibrinogen solution reconstituted from 1 g of fibrinogen of said preparation has an SVP content of 10-100 μm of 6000 or less and an SVP content of 25-100 μm of 600 or less. In embodiment 8, these conditions apply for at least 100 containers of the group of embodiment 7, and optionally for at least 200 containers. In embodiment 9, these conditions apply for at least 500 containers of the group of embodiment 7, and optionally for substantially all containers of said group. In embodiment 10, these conditions apply for at least the last 10% of any group of embodiments 7-9, preferably for at least the last 20% of said group, and optionally for substantially all containers of said group.

[0117] In embodiment 11, the container comprising the fibrinogen preparation of any of embodiments 5-10 or the group of containers comprising the fibrinogen preparation has a residual moisture content of 2-5% (w / w).In embodiment 12, the container comprising the fibrinogen preparation of any of embodiments 5-11 or the group of containers comprising the fibrinogen preparation has a residual moisture content of 2.5-3.5% (w / w), for example about 3% (w / w).

[0118] In embodiment 13, the fibrinogen preparation of any of embodiments 1-12 is packaged in a vial, i.e., the container is a vial. In embodiment 14, the fibrinogen preparation in the container of any of embodiments 1-13 is in a lyophilized state. In embodiment 15, the container of any of embodiments 1-14 contains 1 to 3 g of fibrinogen, optionally containing 1 g of fibrinogen. In embodiment 16, the reconstitution of any of embodiments 1-11 is performed at a fibrinogen concentration of 20 g / L.

[0119] In embodiment 16, in a container or a group of containers containing a fibrinogen preparation according to embodiments 1-15, the fibrinogen preparation is suitable for reconstitution in an aqueous solution, preferably in water for injection, and the fibrinogen solution reconstituted from 1 g of fibrinogen of said preparation has an SVP content of 10-100 μm of 3500 or less and an SVP content of 25-100 μm of 60 or less. Thus, in embodiment 17, for a container containing a fibrinogen preparation according to any of embodiments 1-16, when one container is reconstituted in a solvent, in particular in water for injection, a fibrinogen solution is obtained having an SVP content of 10-100 μm of 3500 or less and an SVP content of 25-100 μm of 60 or less.

[0120] In embodiment 18, the fibrinogen preparation or group of fibrinogen preparations in any of the containers of embodiments 1-17 are suitable for reconstitution in an aqueous solution, preferably in water for injection, and for the fibrinogen solution reconstituted from 1 g of fibrinogen of said preparation, the SVP content of 10-100 μm is less than 3500 and the SVP content of 25-100 μm is less than 35. Thus, in embodiment 19, for the preparation in any of the containers of embodiments 1-18, when one container is reconstituted in water for injection, a fibrinogen solution is obtained having an SVP content of 10-100 μm less than 3500 and an SVP content of 25-100 μm less than 35.

[0121] In embodiment 20, the fibrinogen preparation or group of fibrinogen preparations in the containers of embodiments 1-19 are suitable for reconstitution in an aqueous solution, preferably in water for injection, and for the fibrinogen solution reconstituted from 1 g of fibrinogen of said preparation, the SVP content of 10-100 μm is less than 2500 and the SVP content of 25-100 μm is less than 30. Thus, in embodiment 21, for the preparation in any of the containers of embodiments 1-20, when one container is reconstituted in a solvent, in particular in water for injection, a fibrinogen solution is obtained that has an SVP content of 10-100 μm less than 2500 and an SVP content of 25-100 μm less than 30.

[0122] In embodiment 22, the fibrinogen preparation in the container of embodiments 1-21 or in the group of containers containing a fibrinogen preparation is a) polysorbates, b) optionally, a filler; c) optionally, amino acids and / or d) optionally a salt selected from the group including sodium chloride, sodium citrate, and combinations thereof; Includes.

[0123] In embodiment 23, the fibrinogen formulation contained in the container of embodiments 1-22 comprises a polysorbate, optionally polysorbate 80. In embodiment 24, the fibrinogen formulation or fibrinogen formulations contained in the container of embodiments 1-23 comprise a bulking agent, optionally trehalose. In embodiment 25, the fibrinogen formulation contained in the container of embodiments 1-24 comprises an amino acid, optionally arginine. In embodiment 26, the fibrinogen formulation contained in the container of embodiments 1-25 comprises a salt selected from the group comprising sodium chloride, sodium citrate, and combinations thereof, optionally sodium chloride and sodium citrate.

[0124] In embodiment 27, the fibrinogen preparation contained in the container of embodiments 1-26 is a) a polysorbate, preferably polysorbate 80; b) a bulking agent, preferably trehalose; c) amino acids, preferably arginine and d) a salt selected from the group including sodium chloride, sodium citrate, and combinations thereof; Includes.

[0125] In embodiment 28, the fibrinogen formulation contained in the container of embodiment 1-27 comprises polysorbate 80, trehalose, arginine, sodium, chloride, citrate and residual moisture. In embodiment 29, the fibrinogen formulation contained in the container of embodiment 1-28 comprises less than 1 mM calcium. In embodiment 30, the fibrinogen formulation contained in the container of embodiment 1-29 further comprises an additional plasma protein selected from the group comprising albumin, fibronectin, A2 macroglobulin, an immunoglobulin such as IgG, IgA or IgM, von Willebrand factor, fibrinopeptide A and D-dimer. In embodiment 31, the fibrinogen preparation contained in the container of embodiments 1-30 consists of the above-mentioned components, i.e. fibrinogen, a polysorbate such as polysorbate 80, a bulking agent such as trehalose, an amino acid such as arginine, a salt such as sodium chloride and / or sodium citrate, or a plasma protein selected from the group including albumin, fibronectin, A2 macroglobulin, an immunoglobulin such as IgG, IgA or IgM, von Willebrand factor, fibrinopeptide A and D-dimer, or a plasma protein consisting of said group.

[0126] In embodiment 32, the fibrinogen preparation contained in the container of embodiment 1-31 contains less than 0.5 g / L albumin, preferably less than 0.15 g / L albumin, when the fibrinogen concentration upon reconstitution is 20 g / L. In embodiment 33, the fibrinogen preparation contained in the container of embodiment 1-32 does not contain saccharose and / or glutamic acid, preferably does not contain either saccharose or glutamic acid. Preferably, none of the non-fibrinogen plasma proteins is present in a concentration greater than 0.5 g / L.

[0127] In embodiment 34, the fibrinogen formulation contained in the container of embodiments 1-33 is stable at 2-25° C. for at least 6 months, preferably at least 1 year, and optionally at least 5 years.

[0128] In embodiment 35, the fibrinogen preparation contained in the container of embodiments 1-34 contains less than 20% aggregates, preferably less than 10% aggregates.

[0129] In embodiment 36, the present invention relates to a method for producing a container comprising a fibrinogen formulation of any of embodiments 1-35, comprising the steps of: a) filter-sterilizing a bulk solution of fibrinogen drug substance; b) receiving the filter-sterilized bulk solution in a receiving tank, optionally with an agitator equipped with stirring means; c) optionally agitating the bulk solution in the receiving tank while the agitation means is submerged in said bulk solution; d) filling the container with a predetermined amount of solution, for example by using a pump; e) lyophilizing the solution in the container to obtain a lyophilized formulation; and f) Dry heat treatment of the freeze-dried formulation If the solution is stirred in step c), the receiving tank is equipped with an agitator having stirring means.

[0130] In embodiment 37, the method of embodiment 36 further comprises: g) packaging the containers containing the preparations; Includes.

[0131] In embodiment 38, the method of either embodiment 36 or 37, wherein the residual moisture content after lyophilization is 2-5% (w / w), optionally 2.5-3.5% (w / w), for example about 3% (w / w).

[0132] In embodiment 39, the freeze-drying method of any of embodiments 36-38, further comprising: a) freezing at -29°C or below for at least 4 hours, preferably at -50°C or below; b) a first drying step of stepwise drying at below -10°C and above 40 μbar (4 Pa), preferably starting from below -25°C and above 200 μbar (20 Pa) and increasing the temperature stepwise; c) The second drying step is performed at 17-23°C for at least 2 hours. and has a residual moisture content of 2-5% after lyophilization.

[0133] In embodiment 40, the freeze-drying method of embodiment 39 is a) freezing at -52°C or lower for at least 8 hours; b) a first drying step comprising a first step at about -36°C and about 280 μbar (28 Pa) for about 48 hours, a second step at about -23°C and about 70 μbar (7 Pa) for about 40 hours, and a third step at about -10°C and about 40 μbar (4 Pa) for about 78 hours; c) a second drying step at about 20° C. and about 10 μbar (1 Pa) for about 3-4 hours Includes.

[0134] In embodiment 41, in the method of any of embodiments 36-40, the dry heat treatment (step f) comprises heating the formulation to 100° C.±1.5° C. In embodiment 42, in the method of any of embodiments 36-41, the dry heat treatment is carried out for 30±3 minutes. In embodiment 43, in the method of any of embodiments 36-42, the dry heat treatment is carried out in a steam autoclave.

[0135] In embodiment 44, in any of the methods of embodiments 36-43, the solution is not stirred in step d), and is stirred in step c), for example for up to 1 hour, preferably for up to 1 hour. In embodiment 45, in any of the methods of embodiments 36-44, the stirring in step c) is performed for up to 10 minutes, for example for up to 5 minutes. In embodiment 46, in any of the methods of embodiments 36-45, the stirring is performed at up to 150 rpm, preferably where the receiving tank has a volume of 50-150 L and is cylindrical, and the stirring means is a stirring blade (for example in the form of a blade or rod) fixed to a central drive shaft rotating at up to 150 rpm, for example at 80 rpm. In embodiment 47, in any of the methods of embodiments 36-46, the solution is not stirred when the stirring rod may disturb the surface of the solution and / or bubbles may form.

[0136] In embodiment 48, the method of any of embodiments 36-43, wherein the solution is not stirred in either step c) or step d).

[0137] In embodiment 49, the present invention provides a container comprising a fibrinogen formulation or a group of containers comprising a fibrinogen formulation of any of embodiments 1-35, obtained from the method of any of embodiments 36-48.

[0138] In embodiment 50, the present invention provides a container or a group of containers comprising a fibrinogen preparation of any of embodiments 1-35 or 49, for use in treating fibrinogen deficiency. In embodiment 51, the fibrinogen preparation in the container of embodiment 49 is a preparation for use in treating inherited fibrinogen-related disorders. In embodiment 52, the fibrinogen preparation in the container of any of embodiments 49-50 is a preparation for use in treating congenital fibrinogen deficiency. In embodiment 53, the fibrinogen preparation in the container of embodiment 49 is a preparation for use in treating acquired fibrinogen-related disorders. In embodiment 54, the fibrinogen preparation in the container of any of embodiments 50 or 53 is a preparation for use in treating blood loss / bleeding, optionally during (major) surgical operations. In embodiment 55, the fibrinogen preparation in the container of any of embodiments 50 or 53 is a preparation for use in treating blood loss / bleeding due to trauma.

[0139] In embodiment 56, the fibrinogen preparation in the container of any of embodiments 50-55 is a preparation for intravenous administration to a patient after reconstitution. In embodiment 57, the fibrinogen preparation in the container of any of embodiments 50-56 is a preparation for administration after reconstitution and without prior filtration.

[0140] In embodiment 58, the method of any of embodiments 36-48 is used to reduce the number of SVPs in the formulation, in particular to the limits described herein. In embodiment 59, the method of any of embodiments 36-48 is used to reduce the number of viruses, such as non-enveloped viruses, in the formulation. In embodiment 60, the method of any of embodiments 36-48 is used to reduce the number of SVPs in the formulation, in particular to the limits described herein, and to reduce the number of viruses, such as non-enveloped viruses, in the formulation.

[0141] The present invention is further illustrated but not limited by the following examples and figures, all of which are hereby incorporated in their entirety herein. [Brief description of the drawings]

[0142] [Figure 1] Illustrative diagram of a preferred filling plant used in the present invention: (1): air inlet, (2): pressure line, (3): delivery tank, e.g., capacity 80 L, (4): final filter, (5): formulation outlet, (6): receiving tank, (7): stirrer, preferably up to 150 rpm, (8): stirring blade, (9): peristaltic pump, e.g., 290 rpm or less, (10): container, e.g., vial, (11): freeze dryer [Diagram 2] Residual moisture content significantly affects viral inactivation kinetics (I). Fibrinogen samples spiked with PPV were lyophilized to a residual moisture content of less than 1% (w / w) in the presence and absence of polysorbate 80 (PS80) (sample without polysorbate-H3, sample with 0.05% polysorbate in the reconstituted formulation-H5) and partially adjusted to other defined residual moisture contents as described in Example 4. Dry heat (DH) treatment was performed for all samples at 99°C for 0, 30, 45, or 60 min. [Diagram 3] Residual moisture content has a significant effect on virus inactivation kinetics (II). Fibrinogen samples spiked with PPV were lyophilized to a residual moisture content of less than 1% (w / w) in the presence and absence of polysorbate 80 (PS80) (sample without polysorbate-H3 (as shown in Figure 3A), sample with 0.05% polysorbate in the reconstituted formulation-H5 (as shown in Figure 3B)) and partially adjusted to other defined residual moisture contents as described in Example 4. Dry heat (DH) treatment was performed for all samples at 99°C for 0, 30, 45, or 60 min. [Figure 4]Stability of reconstituted fibrinogen formulations of the invention at 2-8° C. and 23-27° C. At 2-8° C., the fibrinogen formulation was stable for at least 8 weeks. At 23-27° C., the fibrinogen formulation was stable for at least 4 weeks (Test B). [Diagram 5] Stability of the fibrinogen preparation of the invention after 6 years of storage (Study C). [Figure 6] Homogeneity of stirred and unstirred API. The API in the receiving tank was briefly stirred and samples were taken from the surface, middle, and bottom of the solution (t=0, left column group). After 5 hours of incubation, an additional sample was taken from the same location (t=5, middle column group). The tank was then stirred again and an additional sample was taken (t=5+S, right column group). There were no significant differences in protein concentration (A), fibrinogen specific activity (B), SVPs ≥ 10 μm (C), SVPs ≥ 25 μm (D), or aggregates (E) depending on where the sample was taken or whether after incubation or stirring. In A and B, the dashed line indicates the lower specification limit and the dashed-dotted line in A indicates the upper specification limit. In CE, the dashed line indicates the upper specification limit. EXAMPLES

[0143] method Protein measurement Protein measurements were performed by UV absorption spectroscopy (Spectralphotometer Genesys™ 6, Spectralphotometer Genesys™ 10). Proteins in solution absorb UV light at a wavelength of 280 nm, mainly due to the presence of aromatic amino acids such as tyrosine and tryptophan. This property is the basis for protein measurement at 280 nm. The accuracy of protein measurement by UV spectroscopy can be reduced by light scattering by the test sample. To correct for this effect, the absorbance at 360 nm was subtracted from the absorbance at 280 nm.

[0144] Fibrinogen measurement (immunoturbidimetric method) Immunoturbidimetric concentrations were measured by nephelometry using a BN Prospec (Siemens) nephelometer. Fibrinogen forms a complex with a specific antibody. This complex causes scattering of the irradiated light. The increase in this scattering correlates with the fibrinogen concentration.

[0145] Measurement of fibrinogen activity by clottable proteins For the measurement of fibrinogen activity (=clotable protein), the prepared samples were mixed with an appropriate buffer solution containing sufficient thrombin and incubated at 37° C. The remaining protein was measured in the supernatant by UV spectroscopy at 280 / 360 nm, and the result was then subtracted from the total protein (above) to calculate the clottable protein.

[0146] Measurement of fibrinogen specific activity Specific fibrinogen activity was determined from the activity of clottable proteins relative to the total protein measured by UV absorbance (280 nm).

[0147] Addition of PPV At 22±4°C, the specimens were spiked with virus stock and 34 mL aliquots were filled into containers, e.g. vials. Samples were taken and titrated from the stock and from the specimens spiked with virus. All vials underwent lyophilization. After lyophilization, the residual water content of each container was measured by NIR (near infrared spectroscopy). For virus titration, the lyophilisates were resuspended in 50 mL of WFI (solution for injection).

[0148] Preparation of viral inoculum PPV virus stocks were prepared from virus-infected cells. For the release of virus stocks for virus confirmation testing, titers were confirmed by at least three independent titrations, with serial 3-fold dilutions for each titration, and eight replicates for each dilution.

[0149] Virus titration To determine the viral content of the samples, they were quantitatively analyzed by a virus-specific cell-based infectivity assay (virus titration). After resuspension (if applicable), the samples were diluted and immediately titrated onto a sensitive cell line. After a period of incubation, the cytopathic effect caused by the virus was evaluated.

[0150] Virus titer calculation method Preferably, the virus titer was calculated according to Spearman and Kaerber (Spearman C, Kaerber Gl In: Mayr A, Bachmann PA, Bibrack B, Wittmann G; eds. Virologische Arbeitsmethoden, Vol. I, p. 37-39 Fischer Verlag Stuttgart, 1974.) or by applying a Poisson distribution (e.g. when no infectivity was detected).

[0151] Calculation of viral reduction factor The virus reduction factor for each experiment was determined as the common logarithm of the ratio of the virus load (total virus) in the specimens in the virus spike test to the virus load in the specimens after heat treatment. The virus load was calculated as the product of the concentration of the virus titer and the volume.

[0152] Residual moisture measurement according to Karl Fischer and NIR (near infrared spectroscopy) These measurements were performed as described in EP0844005A1.

[0153] Measurement of aggregate content According to European Pharmacopoeia 2.2.30 and US Pharmacopoeia 621, aggregate content is measured by size-exclusion chromatography combined with UV detection. Thus, a protein solution is applied onto a column resin where the proteins interact with the porous resin in a size-dependent manner. Small proteins or protein fragments have a stronger interaction with the column resin and therefore a longer retention time, whereas larger proteins and protein aggregates (dimers, trimers, etc.) have a shorter retention time and therefore elute first. Protein fractions are detected by a UV detector at the end of the column.

[0154] SVP Measurement According to European Pharmacopoeia 2.9.19 and US Pharmacopoeia 788, the amount of SVPs is measured by the light obscuration method, e.g. with a particle counter Hiac Model 9703+ (Beckman, Krefeld, Germany) equipped with a Hiac Model HRLD-400 sensor for particle sizes ranging from 2 to 400 μm. For each measurement, the protein solution is aspirated through a fine tube and passed next to the sensor. The first measurement is used to wash the system and the measurement result is discarded. The average of the following four measurements is used to determine the number of SVPs in the solution. It is important to note that the protein solution is not filtered before the measurement.

[0155] Example 1 - Preparation of fibrinogen 1A) Comprehensive process information The following process is a preferred process for preparing fibrinogen that can be used to prepare the fibrinogen formulations of the present invention. Other processes, such as those known in the art, may also be used.

[0156] Concentrated fibrinogen drug substance (DS) may be produced, for example, from a collateral fraction of a process for producing factor VIII. The manufacturing process was carried out under GMP conditions. The process included an anion exchange chromatography step, in which factor VIII was bound to the chromatographic material and subjected to further processing. The anion exchange chromatography effluent of the factor VIII process was used for the fibrinogen preparation, and was also collected and stabilized with trisodium citrate. Fibrinogen was then precipitated by adding glycine, sodium chloride and calcium chloride. The precipitate was separated by flow-through centrifugation. The resulting intermediate "glycine paste" could be stored at or below -70°C until further use.

[0157] For the preparation of concentrated fibrinogen DS, the frozen intermediate "glycine paste" was dissolved in sodium citrate buffer (solution F01) and filtered. It was then subjected to UV-C irradiation for viral inactivation. For further purification, the protein was chromatographically purified by cation exchange chromatography using POROS 50 HS. The collected column elution fractions were concentrated by ultrafiltration (UF). After adding polysorbate 80 (PS80) and trehalose, the pH and protein concentration were adjusted. The final bulk DS was further processed into pharmaceutical formulation (DP).

[0158] Concentrated fibrinogen DP was produced from concentrated fibrinogen DS. The process was carried out under GMP conditions. The prepared final bulk DS was filled into the final container, where 100 mL glass vials were filled with 32.0 mL of protein solution. The cap was loosely attached to the vial and placed on the pre-cooled freeze-dryer shelf, and the freeze-drying process was started. After freeze-drying, the freeze-dried formulation was heat treated at 100°C (temperature of the formulation) for 30 minutes in a steam autoclave for inactivation of enveloped and especially non-enveloped viruses.

[0159] 1B) Detailed manufacturing process of concentrated fibrinogen drug substance and concentrated fibrinogen drug product The following list outlines the process steps for preparing Fibrinogen DS (Drug Substance) and Fibrinogen DP (Drug Product) in 1 g fill size.

[0160] Process DS1 Starting material (human plasma)

[0161] Step DS2 Plasma storage and thawing

[0162] Step DS3: Isolation of cryoprecipitate Centrifugation Temperature=2±2℃ Storage of Cryoprecipitate at -25°C or below

[0163] Step DS4: Storage of cryoprecipitate pH value = 7.05 ± 0.05 Temperature=22.5±2.5℃ Mixing time ≥ 30 min

[0164] Process DS5 Aluminum hydroxide treatment Addition of 2% aluminum hydroxide suspension for 15-30 minutes Mixing time = 5 min Temperature=22.5±2.5℃ pH value = 6.55 ± 0.05 Temperature=15℃±1.0℃

[0165] Process DS6 Polysorbate 80 / TnBP treatment (virus inactivation) Protein ≤ 10g / L Calcium chloride = 0.001M pH value = 7.1 ± 0.1 Temperature=25.0±1.0℃ Polysorbate 80 = 10 ± 3 g / kg Tri-n-butyl phosphate = 3±0.9g / kg Mixing time = 8-14 hours

[0166] Step DS7 Anion exchange column chromatography DEAE Toyopearl Temperature=22±4℃ Flow rate=192L / h

[0167] Step DS8 Effluent collection

[0168] Step DS9 Glycine Precipitation Temperature=22±4℃ 0.0059 kg trisodium citrate per kg protein solution, mixing time ≥ 5 min 0.091 g glycine per kg protein solution, mixing time ≥ 15 min 0.117 kg sodium chloride per kg protein solution and 0.000035 kg calcium chloride dihydrate per kg protein solution, mixing time ≥ 5 min Buffer A (equilibration and washing buffer for the anion exchange chromatography): 10 mM trisodium citrate, 120 mM sodium chloride, 120 mM glycine, 1 mM calcium chloride, pH 7.0-7.1 The flow-through was mixed with glycine and sodium chloride to precipitate fibrinogen (eg, glycine concentration: 1.2 M, sodium chloride concentration: 2.0 M at room temperature). A glycine paste was produced by flow-through centrifugation. The solid glycine paste is frozen, particularly quick frozen at or below -50°C (eg, -50°C to -70°C), to reduce aggregate formation.

[0169] Process DS10 Virus inactivation by UV-C light irradiation (virus inactivation) Temperature=18±2℃ UV-C irradiation amount=125-200J / m 2 For example, wavelength 254 nm Conductivity=9±1mS / cm The glycine paste was resuspended in sodium citrate buffer (buffer F01) and filtered. For irradiation, an irradiator with a circular tube was used according to EP 0 840 624 B1. 125-200 J / m at 254 nm 2 Irradiation was a particularly homogeneous and gentle treatment for the formulation.

[0170] Step DS11 Cation exchange column chromatography (CEX) POROS 50 HS Protein loading = 10-20g per L of resin per cycle Conductivity=9±1mS / cm Temperature=22±4℃ Flow rate=200±100cm / h CEX reduces the concentrations of vWF and further proteins as well as TnBP and polysorbate 80. The column was equilibrated with washing buffer F02 (15 mM citrate buffer (trisodium citrate), 65 mM sodium chloride, pH 6.5). The fibrinogen-containing fraction was added and fibrinogen was bound to the column. Elution was performed with elution buffer (F03: 7.5 mM citrate buffer (trisodium citrate), 150 mM sodium chloride, 75 mM arginine, pH 7.0) with arginine / citric acid / sodium chloride.

[0171] Process DS12 Ultrafiltration Protein concentration = 55g / L ± 10g / L

[0172] Process DS13: Preparation of protein solution, 0.2μm filter filtration and quality control of drug substance Trehalose dihydrate = 0.0170 ± 0.0017 per kg of protein solution Polysorbate 80 = 0.075 ± 0.0075% pH=7.0±0.5 Fibrinogen concentration = 30-36g / L The protein concentration was adjusted to 33 g / L and the pH was adjusted to 7.0 with citrate buffer. 0.017 kg trehalose was added per kg protein. 0.075% (w / v) polysorbate 80 (for stabilization, improved solubility, and enhanced reduction of non-enveloped viruses, e.g., parvoviruses). Prior to lyophilization, the polysorbate concentration was adjusted to 0.075% (w / v). This typically results in a polysorbate concentration of 0.05% after reconstitution. → API (final bulk)

[0173] Process DP1+2 Final sterilization by filtration and filling into final containers / sales units 0.2μm filter filtration Filling of approx. 32mL

[0174] Process DP3 Freeze drying Freezing below -29°C for more than 4 hours Staged primary drying below -10°C Secondary drying at 20°C ± 3°C for up to 4 hours Total drying time: approx. 150-200 hours

[0175] Process DP4 Heat treatment (virus inactivation) Formulation temperature: 100±1.5℃ for 30±3 minutes Residual moisture 2.0-5.0% Cooling to below 80℃

[0176] Process DP5+6 DP quality control and packaging Storage at 5℃±3℃

[0177] A preferred process for manufacturing the formulation is described in more detail in EP192149193 (application number, unpublished) and in the critical steps of the present invention below.

[0178] Example 2 - Filling Process In a preferred process of the present invention for the final filling of concentrated fibrinogen drug substance (DS), a standardized / mechanical filling line for filling drug proteins was employed (Bausch & Stroebel, Ilshofen, Germany, KS 1025, model: AS 18.2). The equipment is shown in FIG.

[0179] a) For filling, the final DS is filtered through filter 4 (e.g., filter type: Sartorius 5235307H9--SS, 0.2 m 2, cellulose acetate (CA) membrane) from a delivery tank 3 (typically a stainless steel tank, e.g. with a nominal filling volume of 80 L) to a receiving tank 6 (typically a stainless steel tank, e.g. with a nominal filling volume of 50 L) along with a formulation outlet 5 by superposition of pressure (e.g. below 600 mbar (600 hPa)) at the pressurized line 2 and air inlet 1. When the stirrer bar 8 (or stirrer rod) of the stirrer 7 is covered with fibrinogen solution, e.g. after about 15 L of the receiving tank 6 is filled, the stirrer 7 is started to run at a low speed, in particular below 150 rpm, preferably at 80 rpm ± 20 rpm, with the stirrer bar 8 submerged. This keeps the surface of the protein solution above the stirring means and prevents foam formation. Then, e.g. after the drug substance transfer to the receiving tank is completed, filling is started using a peristaltic pump 9 with a pump speed below 290 rpm, preferably below 270 rpm. Glass vials 10 (nominal fill volume: 100 mL, diameter: 5.2 cm) were each filled with 32 mL of drug substance. The stirrer 7 was stopped when or before the surface of the solution reached the stir bar / stirring means 8.

[0180] Some of the vials 10, in particular 50 of them, are collected in a frame, in particular a metal frame, and transferred without delay to a freeze-dryer 11, which has been pre-cooled (to below -50°C).

[0181] The resulting formulation could be reconstituted as described herein with low numbers of SVPs formed, which is attributable to gentle mixing conditions.

[0182] b) Alternatively, for filling, the final DS can be adjusted to, for example, filter type: Sartorius 5235307H9--SS, 0.2m 2, cellulose acetate (CA) membrane) from a delivery tank 3 (typically a stainless steel tank, e.g. with a nominal fill volume of 80 L) to a receiving tank 6 (typically a stainless steel tank, e.g. with a nominal fill volume of 50 L) by superposition of pressure (e.g. up to 600 mbar (600 hPa)) at the pressurized line 2 and air inlet 1 along with a formulation outlet 5. After transfer of the drug substance to the receiving tank was completed, the stirrer 7 was started at a low speed, in particular up to 150 rpm, preferably 80 rpm ± 20 rpm. The solution was stirred for 5 minutes and stirring was stopped. Filling was started with a peristaltic pump 9 with a pump speed of up to 290 rpm, preferably up to 270 rpm. Glass vials 10 (nominal fill volume: 100 mL, diameter 5.2 cm) were filled with 32 mL of drug substance each.

[0183] Before the receiving tank 6 was emptied, the remaining drug substance was filled into the receiving tank so that the agitator 7 could be used while it was submerged with the remaining and added drug substance solution. The solution was stirred for 5 minutes and then the agitator was stopped and the filling process was resumed. Alternatively, filling may begin during stirring if it is ensured that the stirring means is always submerged when stirring.

[0184] Some of the vials 10, in particular 50, were collected in a frame, in particular a metal frame, and transferred without delay to a pre-cooled (below -50°C) freeze-dryer 11.

[0185] By taking samples from different stages during the filling process and comparing the samples for e.g. fibrinogen concentration, the inventors surprisingly found that gentle and short agitation was sufficient for a homogenous formulation. Even without agitation, no inhomogeneity was found.

[0186] The number of SVPs was reproducibly lower than in a).

[0187] c) To compare homogeneity with and without stirring, the drug substance in receiving tank 6 was briefly stirred and samples were taken from the surface, middle and bottom of the solution. After 5 hours of incubation (t=5), an additional sample was taken from the same location. The tank was then stirred again and an additional sample was taken (t=5+S). There was no significant difference depending on where the sample was taken or whether it was after incubation or stirring (Figure 6).

[0188] Example 3 - Freeze-Drying It is particularly advantageous to place the filled container 10 into a pre-cooled freeze-dryer. This allows rapid freezing of the protein solution and improves water sublimation, resulting in a favorable porous cake-like structure. Freeze-drying was performed in such a way that a residual moisture content of 2.5-5% was achieved.

[0189] In the preferred process after filling of the final containers, the drying program was started. The first step consisted of a freezing step at -52°C for 8 hours, followed by primary drying. Primary drying was subdivided into three steps: drying at 280 μbar (28 Pa) and -36°C for 6 hours (step 1), drying at 70 μbar (7 Pa) and -23°C for 25-45 hours (step 2), and drying at 40 μbar (4 Pa) and -10°C for 45-78 hours (step 3) (preferably step 1 for 45 hours, step 2 for 45 hours and step 3 for 45 hours, or step 1 for 5 hours, step 2 for 25 hours and step 3 for 78 hours). This procedure improved the solubility of the final product and reduced the amount of SVP. To move to secondary drying, a pressure rise test (0.044 μbar (0.44 Pa) for 3 minutes) had to be passed. Secondary drying at 10 μbar (1 Pa) and 20° C. for 3 hours reduces the variability of the residual moisture and leads to a more homogenous lyophilisate behaviour after heat treatment (100° C. for 30 min). In addition, secondary drying at 20° C. reduced the amount of SVP in the final product.

[0190] Residual moisture was tested for each group using NIR or Karl Fischer method. NIR results can be correlated to Karl Fischer method using a calibrated table for the samples of the present invention. For analysis, water was extracted from the lyophilisates and chemically quantified by subsequent reactions.

[0191] The residual moisture content in the formulations prepared by the method of the present invention, as detected by Karl Fischer method, was typically between 2.5-3.5% (w / w), as shown in Table 1. In comparison with the commercially available formulations, the commercially available formulations showed significantly lower residual moisture content of less than 1% (w / w). [Table 1]

[0192] Example 4 - Comparison of virus inactivation depending on residual moisture content Enveloped viruses are reliably removed in the solvent / detergent step performed during the preparation of the drug substance as described above. Non-enveloped viruses must be removed in addition. The optimal conditions were analyzed in subsequent experiments.

[0193] For experiments with non-enveloped viruses, fibrinogen samples spiked with PPV were first lyophilized to a residual moisture content of less than 1% (w / w) in the presence or absence of polysorbate 80 (PS80) (sample without polysorbate - H3, sample with 0.05% polysorbate in the reconstituted formulation - H5). The moisture content of the samples after lyophilization was measured by NIR (near infrared spectroscopy).

[0194] Some samples were then opened and a defined amount of water for injection (WFI) was added into the vial without touching the lyophilisate. The vials were recapped and incubated until the added water for injection had evaporated. To obtain equal pressure in all vials, the vials were opened and placed in the chamber of the lyophiliser. The vials were then closed under vacuum. The water content was again measured by NIR or, in parallel control samples, after extraction into organic solvent, according to the Karl Fischer method as described herein.

[0195] Dry heat (DH) treatment was performed for all samples at 99°C for 0, 30, 45 or 60 min. 0 min represents lyophilized samples that were not subjected to dry heat treatment. Reduction in viral titer was measured using logarithmic (log 10 ) using the pre-lyophilized material as the starting value for the calculation of reduction. There was no significant reduction in virus at 0 min. At 4% residual moisture, increasing virus inactivation was observed with increasing heating time (30, 45, 60 min) (log 10 Values ​​of about 2.5-4, virus reduction factor log 10 (Some outliers were below the value of 2.) Virus inactivation in samples with less than 1% residual moisture was log 10 The values ​​ranged from 1-1.5. At such low moistures, no effect of polysorbate was seen. At higher residual moistures, there was a trend towards a beneficial effect of polysorbate 80. At all time points, the greatest reductions were obtained in samples with approximately 4% residual moisture and polysorbate 80 (Figure 2).

[0196] In a further series of experiments, the virus spiking experiment was repeated on different fibrinogen samples with different residual moisture contents, from 0 to 5%, measured and adjusted after lyophilization as described above. Samples without polysorbate 80 (H3) and with polysorbate 80 (H5 - 0.05% in reconstituted formulation) were tested.

[0197] The relationship between residual moisture and viral inactivation shown for PPV could be confirmed both for samples without and with PS80.

[0198] In Figures 3A and 3B, samples were grouped according to their residual moisture and virus reduction was shown for dry heat (DH) treatments performed at 99°C for different times. For samples subjected to 45 or 60 min of DH, significantly higher virus inactivation could be shown for the sample group with the highest residual moisture. In the presence of PS80 (Figure 3B), or higher than 3.2 and up to 4.3%, or preferably at least 4%, residual moisture appeared optimal for a significant effect of virus inactivation, whereas for samples without polysorbate (Figure 3A), 5% residual moisture was optimal.

[0199] Example 5 - Formulation of the formulation One container, e.g. one vial, of the fibrinogen preparation of the invention may, when reconstituted in 50 mL of water for injection at 20 g / L, conditions suitable for intravenous administration, have, for example, the following properties:

[0200] pH 7.0 Osmolality ≥ 240 mosmol / kg Protein 20g / L Specific activity ≥ 80%, e.g. 98% ± 0.8% (n = 7) Sodium 95-135 mmol / L, e.g., about 115 mmol / L Calcium < 1 mmol / L Chloride 100-160 mmol / L, e.g., about 130 mmol / L Citrate 3-7mmol / L, e.g., about 5mmol / L Arginine 25-55mmol / L, e.g., about 40mmol / L Trehalose 22-38mmol / L, e.g., about 30mmol / L Polysorbate 80 0.03-0.07%, e.g. about 0.05% Aggregate content ≤ 20%, e.g. 11 ± 1% (n = 11) (measured by HP-SEC analysis) [Table 2] [Table 3]

[0201] Example 6: Measurement of SVP According to European Pharmacopoeia 2.9.19 and United States Pharmacopoeia 788, the amount of SVP was determined as described above.

[0202] The final concentrated fibrinogen preparation (fibrinogen preparation, DP) was counted for particles ≥10 μm and ≥25 μm. Lyophilized DP was reconstituted in 50 mL water for injection (WFI). The reconstituted solution was transferred to the particle counter without further filtration. All samples were counted in triplicate. The average total particle amount per vial was reported. The count of particles ≥10 μm was always higher than the count of particles ≥25 μm, since particles ≥25 μm were also included in the group of particles ≥10 μm.

[0203] The results of the last five batches produced by the method of the present invention showed that the number of SVPs in the final product was always within specification (Table 4), so no additional filtration was required before administration. [Table 4]

[0204] For comparison purposes, Table 5 below shows the number of SVPs analyzed in several competitor formulations. [Table 5]

[0205] The low amount of SVP in the formulation of the present invention is preferably due to the final formulation, in particular the presence of polysorbates, the gentle filling process and the lyophilization process, appropriately applied to the fibrinogen.

[0206] Example 7 - Stability of formulations 7.1 Test A - Long-term stability of lyophilized formulations To investigate the long-term stability of the fibrinogen preparations, four groups of fibrinogen preparations were stored for stability studies: group 1 for 36 months and group 3 for 60 months at 5°C (±3°C) and 25°C (±2°C), respectively. Based on the data from this study, a shelf life of at least 60 months was demonstrated for the fibrinogen preparations at 5°C (±3°C) and 25°C (±2°C).

[0207] For stability testing, the groups were tested for activity after reconstitution at predefined time points for each storage condition (5° C., 25° C., 55-65% relative humidity (RH)): fibrinogen activity was tested in the reconstituted aqueous solution immediately after reconstitution and after incubation at room temperature (approximately 25° C.) for 6 and 24 hours after reconstitution. This study simulated the routine clinical use of fibrinogen concentrate.

[0208] Storage at 5°C (±3°C): All results for stability parameters remained essentially unchanged after 60 months (groups B524071, B524084 and B524032) and 36 months (group B524031), respectively. That is, activity, pH value, osmolality, color and proteinescence of the reconstituted fibrinogen remained unchanged. The dissolution time of the lyophilisates varied between 5 and 30 minutes, which was acceptable. No increase in aggregates was detected. At all time points, the stability of the reconstituted solutions was shown to extend to 24 hours at room temperature. The content of additives such as citrate, trehalose and arginine remained almost unchanged. All solutions tested were sterile and pyrogen-free. The residual moisture content measured in the groups was 2.1 to 3.0% after 36 and 60 months, respectively. This value was approximately 0.2 to 0.3 percentage points lower than before storage.

[0209] Storage at 25°C (±2°C):All results for stability parameters remained essentially unchanged after 60 months (groups B524071, B524084 and B524032) and 36 months (group B524031), respectively: activity, pH value, osmolality, color and proteinescence of the reconstituted fibrinogen remained unchanged. Dissolution times varied between 5 and 30 minutes, which was acceptable.

[0210] All samples tested in the stability study were sterile and pyrogen-free under all storage conditions. There was no aluminum concentration during storage for 60 months at 5°C (± 3°C) and 25°C (± 2°C).

[0211] In summary, comparison of results obtained immediately after reconstitution and after 6 and 24 hours at predefined time points over a 60 month shelf life at storage temperatures of 5°C and 25°C demonstrated that fibrinogen activity remained stable for at least the first 24 hours after reconstitution.

[0212] 7.2 Test B - Stability of reconstituted formulations A stability study was conducted for group number B524071, where after reconstitution the formulation was stored at 2-8° C. or 23-27° C. for up to 8 weeks (FIG. 4). The residual moisture of this group was measured to be 2.4%.

[0213] Storage at 2-8° C. for up to 8 weeks did not result in an increase in proteolytic activity or aggregate content, nor did it result in a significant decrease in specific activity. Samples were also stable for at least 4 weeks at 23-27° C. Only after 8 weeks did specific activity slowly decrease and proteolytic activity increase.

[0214] 7.3 Test C - Stability of reconstituted formulations (storage period of 6 years) A third stability study was conducted in which fibrinogen formulations stored for 6 years (group no. B524032, stored at 5° C. or 25° C. for 6 years, respectively) were tested for activity at defined times, i.e., 0, 6, 24 and 48 hours, after reconstitution in water for injection at 5° C.±3° C. and 25° C.±2° C. (60% RH±5% RH). The results are shown in FIG. 5.

[0215] Post-sampling microbial testing was performed at pre-determined time points to reveal microbial contamination during reconstitution. None of the samples showed microbial contamination. The pH of all samples was 6.9. Protein content and color were identical in all samples.

[0216] In conclusion, even after 6 years (72 months) of storage of the dry formulation, the reconstituted formulation showed no loss of fibrinogen activity. Moreover, the reconstituted formulation was still stable after 48 hours at 5°C and 25°C. All parameters tested were shown to be within the target ranges of the specifications both immediately after reconstitution and at all subsequent time points tested. Only minor variations were observed for the parameters "total protein", "aggregates" and "subvisible particles". This high stability is surprising, especially in light of the absence of stabilizing factors, such as significant amounts of albumin, as described herein.

[0217] Example 8 - Reconstitution characteristics It was shown that relatively high residual moisture did not have a significant detrimental effect on the solubility of the formulation. In comparison with other formulations, the formulation of the present invention had similar solubility.

[0218] The reconstitution or dissolution process is typically completed in 3-8 minutes for the formulations of the invention when dissolved in water for injection. FibCLOT® from LFB SA (Courtaboeuf Cedex, France) dissolves in about 4-5 minutes, where strong foam formation is observed. Fibryga® from Octapharma (Langenfeld, Switzerland) dissolves after 8 minutes. Haemocomplettan® from CSL Behring (Marburg, Germany) dissolves after about 10 minutes. The dissolution method itself (addition of water directly to the vial, or Mix2Vial® (addition of water through a filter and filtration of the dissolved formulation), shaking by hand or using a shaker) does not seem to have a significant effect on the results. Here, in general, foam formation is lower with the Mix2Vial® method.

[0219] In summary, lyophilization and dry heat processing according to the methods of the present invention, in combination with the filling process and compounding, results in a virus-free, active and highly soluble formulation.

Claims

1. A container (10) containing a fibrinogen preparation in a dry state with a residual moisture content of 2 - 5% (w / w), optionally 2.5 - 3.5% (w / w), preferably in a lyophilized state.

2. The fibrinogen preparation is suitable for reconstitution in an aqueous solution, preferably water for injection, and for the fibrinogen solution obtained by reconstituting 1 g of fibrinogen of the preparation, the SVP content of 10 - 100 μm is 6000 or less, and the SVP content of 25 - 100 μm is 600 or less. The container (10) according to claim 1.

3. A container (10) containing a fibrinogen preparation in a dry state, preferably in a lyophilized state, the fibrinogen preparation is suitable for reconstitution in an aqueous solution, preferably water for injection, and for the fibrinogen solution obtained by reconstituting 1 g of fibrinogen of the preparation, the SVP content of 10 - 100 μm is 6000 or less, and the SVP content of 25 - 1 hundred μm is 600 or less.

4. A group of containers (10) containing a fibrinogen preparation in a dry state, preferably in a lyophilized state, for at least 10 containers (10) in the group, the fibrinogen preparation is suitable for reconstitution in an aqueous solution, preferably water for injection, and for the fibrinogen solution obtained by reconstituting 1 g of fibrinogen of the preparation, the SVP content of 10 - 1 hundred μm is 6000 or less, and the SVP content of 25 - 1 hundred μm is 600 or less. A group of containers (10).

5. The fibrinogen preparation is a fibrinogen preparation with a residual moisture content of 2 - 5% (w / w), preferably 2.5 - 3.5% (w / w). The container (10) or group of containers (10) according to claim 3 or 4.

6. A container (10) containing the fibrinogen preparation according to any one of claims 1 - 4 or a group of containers (10) containing the fibrinogen preparation, a) Polysorbate, preferably polysorbate 80, b) Optionally, a filler, preferably trehalose, c) Optionally, an amino acid, preferably arginine, and / or, d) Optionally, a salt, such as sodium chloride, sodium citrate, or a combination thereof is included. The preparation preferably comprises polysorbate 80, trehalose, arginine, sodium, chloride, citrate and residual moisture, and is in a container (10) or a group of containers (10).

7. A container (10) or a group of containers (10) containing a fibrinogen preparation according to any one of claims 2 - 4, wherein the concentration upon reconstitution is 20 g / L.

8. A container (10) or a group of containers (10) containing a fibrinogen preparation according to any one of claims 2 - 4, wherein when the fibrinogen concentration upon reconstitution is 20 g / L, the albumin concentration is 0.5 g / L and it does not contain saccharose or glutamic acid.

9. A container (10) or a group of containers (10) containing a fibrinogen preparation according to any one of claims 1 - 4, wherein the fibrinogen preparation is suitable for reconstitution in an aqueous solution, preferably water for injection, and for the fibrinogen solution after reconstitution per 1 g of fibrinogen in the preparation, the SVP content of 10 - 100 μm is 3500 or less and the SVP content of 25 - 100 μm is 35 or less. Optionally, a container (10) or a group of containers (10), wherein the fibrinogen preparation is suitable for reconstitution in an aqueous solution, preferably water for injection, and for the fibrinogen solution after reconstitution per 1 g of fibrinogen in the preparation, the SVP content of 10 - 100 μm is 2500 or less and the SVP content of 25 - 100 μm is 30 or less.

10. A container (10) or a group of containers (10) containing a fibrinogen preparation according to any one of claims 1 - 4, which is stable at 2 - 25°C for at least 6 months, preferably at least 5 years.

11. A container (10) or a group of containers (10) containing a fibrinogen preparation according to any one of claims 1 - 4, wherein the proportion of aggregates contained is less than 20%.

12. A method for manufacturing a container (10) containing a fibrinogen preparation according to any one of claims 1 - 4, comprising: a) a step of filter - sterilizing a bulk solution of fibrinogen raw drug; b) a step of receiving the solution after filter - sterilization in a receiving tank (6), optionally in a receiving tank (6) having a stirrer (7) equipped with stirring means (8). c) Optionally, a step of stirring the bulk solution in the receiving tank (6) when the stirring means (8) is submerged in the bulk solution; d) A step of filling the container (10) with a predetermined amount of solution; e) A step of lyophilizing the solution in the container (10) to obtain a lyophilized formulation; f) A step of heat-treating the lyophilized formulation; g) Optionally, a step of packaging the container (10) containing the formulation A method comprising.

13. The receiving tank (6) has a capacity of 50 - 150 L and is cylindrical, and the stirring means (8) is a plurality of blades (or rods) fixed to a central drive shaft that rotates at a maximum of 150 rpm, the stirring time is a maximum of 1 hour, optionally about 5 minutes, preferably the stirring in step c) is at least 10 minutes, and the bulk solution is not stirred in step d). The method according to claim 12.

14. The method according to claim 12, wherein Lyophilization is a) A step of freezing at -29 °C or lower, preferably -50 °C or lower for 4 hours or more; b) A first drying step of drying stepwise at -10 °C or lower and 40 μbar (4 Pa) or more, where preferably it starts from conditions of less than -25 °C and 200 μbar (20 Pa) or more and the temperature is increased stepwise; c) A second drying step of drying at 17 - 23 °C for 2 hours or more Including, the residual moisture content after lyophilization is 2 - 5% (w / w), Lyophilization optionally is a) A step of freezing at -52 °C or lower for 8 hours or more; b) A first drying step including a first step carried out at about -36 °C and about 280 μbar (28 Pa) for about 48 hours, a second step carried out at about -23 °C and about 70 μbar (7 Pa) for about 40 hours, and a third step carried out at about -10 °C and about 40 μbar (4 Pa) for about 78 hours; c) A second drying step of drying at about 20 °C and about 10 μbar (1 Pa) for about 3 - 4 hours Including, a method.

15. The heat treatment includes a step of heating the formulation at 100 ± 1.5 °C for 30 ± 3 minutes, and optionally this is carried out in a steam autoclave. The method according to claim 12.

16. A container (10) containing a fibrinogen formulation or a group of containers (10) containing a fibrinogen formulation obtainable by the method according to claim 12.

17. (i) The container (10) according to claim 1; (ii) A transfer device for facilitating the clean or aseptic transfer of a suitable solvent from a container containing the solvent to a container containing the formulation; and A suitable container containing an aqueous solvent for dissolving the formulation comprising a kit. **Claim 18**: The kit according to claim 17, wherein the transfer device comprises one or more filters, and optionally, the filter is a filter with a pore size of 3 to 10 μm for filtering the formulation. **Claim 19** A container (10) containing a fibrinogen formulation according to any of claims 1 - 4 for use in the treatment of fibrinogen deficiency, or a group of containers (10) containing a fibrinogen formulation, or the kit according to claim 17.

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