Fibrinogen preparation

Abstract

Provided herein are methods for preparing fibrinogen by purifying fibrinogen from a plasma fraction from which prothrombin has been rejected. The prothrombin-rejected plasma fraction is obtained, for example, by adding an anticoagulant to a blood source; plasma is separated from the blood source, and the prothrombin is extracted from the plasma, thereby obtaining a plasma fraction from which prothrombin has been removed and a plasma fraction consisting of prothrombin.

Description

Technical Field

[0001] The present invention particularly relates to a method for preparing fibrinogen from a plasma fraction depleted of prothrombin. Background Art

[0002] Fibrinogen and thrombin are two main biological components of fibrin sealants. Thrombin is derived from prothrombin and is a serine protease that promotes blood clotting by catalyzing the conversion of fibrinogen into fibrin. Thrombin is also responsible for activating platelets and indirectly for regulating its own production and inhibition through multiple proteolytic feedback mechanisms. Thrombin is also involved in the activation of factor VIII, factor V, factor XI, factor XIII, and protein C.

[0003] Thrombin is widely used in clinical applications as a clotting factor for wound hemostasis by converting fibrinogen into fibrin, is a common component of surgical dressings, and has been used in combination with fibrinogen and other clotting proteins in two-component hemostatic systems such as fibrin glue, adhesives, and sealants.

[0004] Thrombin is produced by proteolytic activation of the precursor (zymogen) prothrombin. To produce thrombin, prothrombin must be cleaved at two sites to produce an intermediate product. The conversion of prothrombin to thrombin in the body is catalyzed by the prothrombinase complex containing activated factor X and factor V and is assembled on negatively charged phospholipid membranes in the presence of calcium ions.

[0005] In currently applied methods, fibrinogen and prothrombin are each separated from plasma, and when one component is separated, the other component contained in the remaining raw material is subsequently discarded.

[0006] US20180016567A relates to a method for producing thrombin from a source of prothrombin using a given BaSO4 reagent as an adsorbent for prothrombin and a method for evaluating the suitability of a given BaSO4 reagent for preparing thrombin.

[0007] CN102286095B discloses a method for preparing fibrinogen, which has the advantages of high dissolution rate during production, short production cycle, rapid dissolution during clinical use, good biological activity, high safety during clinical use, and high yield.

[0008] CN112972755A discloses a method for preparing a biological hemostatic material based on porcine fibrinogen and thrombin.

[0009] CN105950576A discloses a method for extracting multiple proteins from bovine blood.

[0010] CN101214391B discloses a highly efficient biogel sealant composed of a main gelled dry powder and a catalyst dry powder. Summary of the Invention

[0011] The present invention particularly relates to a method for preparing fibrinogen from a plasma fraction from which prothrombin has been removed.

[0012] According to one aspect of the present invention, there is provided a method for preparing fibrinogen, the method comprising purifying fibrinogen from a plasma fraction from which prothrombin has been removed.

[0013] In some embodiments, the step of obtaining a plasma fraction from which prothrombin has been removed comprises: adding an anticoagulant to a blood source; separating plasma from the blood source, and extracting prothrombin from the plasma, thereby obtaining a plasma fraction from which prothrombin has been removed and a plasma fraction containing prothrombin.

[0014] In some embodiments, the step of separating plasma from the blood source further comprises a sub-step of virus inactivation, the sub-step of virus inactivation comprising treating the plasma with a solvent detergent (SD).

[0015] In some embodiments, the anticoagulant contains oxalate ions.

[0016] In some embodiments, the step of extracting prothrombin from the plasma is carried out by the following steps: adding barium sulfate (BaSO4) to the plasma fraction under conditions allowing the adsorption of prothrombin by barium sulfate, thereby obtaining barium sulfate containing adsorbed prothrombin; and removing the barium sulfate containing adsorbed prothrombin from the plasma.

[0017] In some embodiments, the anticoagulant contains oxalate ions derived from potassium oxalate.

[0018] In some embodiments, the step of purifying fibrinogen from a plasma fraction from which prothrombin has been removed comprises the step of removing plasmin and / or plasminogen by affinity chromatography. In some embodiments, purifying fibrinogen from a plasma fraction from which prothrombin has been removed includes one or more steps of salting out.

[0019] In some embodiments, the method further comprises a step of drying the purified fibrinogen.

[0020] In some embodiments, the method further comprises formulating the dried fibrinogen to contain dried thrombin.

[0021] In some embodiments, the method includes one or more sub-steps of virus inactivation. In some embodiments, one or more sub-steps of virus inactivation include heat inactivation of the dried fibrinogen.

[0022] In some embodiments, the method further comprises preparing thrombin from a plasma fraction containing prothrombin.

[0023] In some embodiments, the plasma fraction containing prothrombin comprises prothrombin eluted from barium sulfate containing adsorbed prothrombin. In some embodiments, there is provided

[0024] In some embodiments, the preparation of thrombin comprises activating prothrombin in the plasma fraction containing prothrombin.

[0025] In some embodiments, the method is used to prepare fibrinogen and thrombin from plasma derived from a single blood source.

[0026] According to one aspect of some embodiments, there is provided a plasma fraction depleted of prothrombin and containing fibrinogen obtainable by the methods disclosed herein in any of its embodiments.

[0027] According to another aspect of the present invention, there is provided a plasma fraction depleted of prothrombin and containing fibrinogen, a solvent detergent, and potassium oxalate. In a specific embodiment, in the plasma fraction depleted of prothrombin, the detergent comprises polysorbate 80 at a final concentration of about 5 g / L to about 12 g / L, and the solvent comprises tributyl phosphate (TNBP) at a final concentration of about 1 g / L to about 3.6 g / L and potassium oxalate at a final concentration of about 2 g / L (or about 12 mmol / L) to about 3 g / L (or about 18 mmol / L).

[0028] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the operation or testing of the embodiments of the present invention, the exemplary methods and materials are described below. In case of conflict, the patent specification and its definitions shall prevail. Additionally, the materials, methods, and examples are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figures 1A to 1B Shows a schematic purification method ([[]] Figure 1B ) of an embodiment of the present invention compared to the currently applied (“old”) method for preparing fibrinogen starting from raw plasma ([[]] Figure 1A ). DETAILED DESCRIPTION

[0030] The present invention particularly relates to a method for preparing fibrinogen. The method comprises purifying fibrinogen from a plasma fraction depleted of prothrombin.

[0031] Fibrinogen purification includes one or more steps aimed at separating, isolating, and / or enriching fibrinogen from a mixture of proteins containing fibrinogen, such as from plasma with prothrombin removed or from plasma fractionation, isolation, and / or enrichment.

[0032] Separating and / or purifying fibrinogen from all other proteins in plasma or plasma fractions is challenging. A method for preparing fibrinogen starting from a plasma fraction with prothrombin removed is disclosed herein. The pure product can be referred to as "fibrinogen isolate", "fibrinogen preparation", "purified fibrinogen", or "fibrinogen-enriched fraction".

[0033] Advantageously, compared to preparing fibrinogen starting from raw plasma, the disclosed method for preparing fibrinogen starting from a plasma fraction with prothrombin removed can shorten the production cycle time, increase the manufacturing capacity by 50% or more, and / or reduce costs. Compared to existing methods, the method of the present invention can use 40% less blood to produce the same amount of product, and thus is more environmentally friendly compared to other known fibrinogen preparation methods (such as fibrinogen preparation methods using raw plasma containing prothrombin).

[0034] Using a solution containing a plasma fraction with prothrombin removed to prepare fibrinogen can reduce the risk of fibrinogen coagulation in the solution and / or prevent or reduce fibrinogen degradation during the preparation process, thus maintaining fibrinogen integrity.

[0035] As used herein, "plasma fraction with prothrombin removed" includes plasma with prothrombin removed and / or its complexes.

[0036] As used herein, the prothrombin complex can contain vitamin K-dependent coagulation factors, such as factor II, and optionally one or more factors selected from VII, IX, X, protein C, and protein S.

[0037] In one embodiment, the prothrombin complex contains factors selected from factor II, VII, IX, and X.

[0038] A plasma preparation can be obtained by adding an anticoagulant to a blood source and separating the plasma from the cells present in the blood source.

[0039] Generally, plasma is obtained by adding an anticoagulant to a blood source under suitable conditions that allow separation of the plasma from the cell fraction. For example, plasma is obtained by adding an anticoagulant to a blood source at a suitable temperature for a suitable period of time to allow separation of the plasma from the cell fraction in the blood source and collecting the plasma.

[0040] After collecting the plasma, a centrifugation step can be carried out, for example, to remove cell debris.

[0041] Sometimes, a plasma preparation collected from a blood source is referred to as "crude plasma".

[0042] A prothrombin-depleted plasma fraction can be obtained by extracting (or removing) prothrombin from plasma, thereby obtaining a prothrombin-depleted plasma fraction. The prothrombin-depleted plasma fraction generally contains fibrinogen.

[0043] "Prothrombin-depleted plasma fraction" refers to a plasma fraction that is free of prothrombin and contains fibrinogen. In this method, the prothrombin-depleted plasma fraction is used to produce fibrinogen.

[0044] As used throughout this text, the term "free of prothrombin" means that, by weight, the amount of prothrombin is less than about 20%, less than about 15%, less than about 10%, less than about 5%, or even the remaining prothrombin is absent, compared to the initial amount of prothrombin before applying the prothrombin removal step disclosed herein.

[0045] "Plasma fraction containing prothrombin" refers to a fraction obtained by collecting prothrombin extracted from plasma. Generally, the plasma fraction containing prothrombin is free of fibrinogen. If desired, the plasma fraction containing prothrombin can be used to prepare thrombin.

[0046] Disclosed is a method for preparing fibrinogen and thrombin respectively from a prothrombin-depleted plasma fraction and a plasma fraction containing prothrombin obtained from the same blood source.

[0047] The prothrombin-depleted plasma fraction and the plasma fraction containing prothrombin can be obtained from the same blood source and can be used respectively in methods for preparing fibrinogen and thrombin.

[0048] Non-limiting examples of blood sources for plasma preparations or their fractions include venous blood, arterial blood, or capillary blood of vertebrates. Vertebrates can be, for example, humans, pigs, cows, goats, sheep, and horses.

[0049] In one embodiment, the blood source for plasma preparations or their fractions can include venous blood, arterial blood, or capillary blood of a vertebrate pig.

[0050] "Blood" includes the body fluid of a human or other vertebrate that delivers essential substances (such as nutrients and oxygen) to cells and transports metabolic waste from the same cells. In vertebrates, blood consists of blood cells suspended in plasma.

[0051] Typically, plasma constitutes approximately 55% of the blood fluid, and plasma is mainly water (about 92% by volume), and particularly contains proteins, glucose, mineral ions, and hormones.

[0052] Albumin is the main protein in plasma. Typically, isolated plasma (e.g., obtained from collected blood with an anticoagulant added (present in the supernatant after centrifugation)) contains a cell-free blood fraction containing the anticoagulant.

[0053] The term "anticoagulant" refers to an additive that inhibits blood and / or plasma clotting so that the components remain unchanged significantly before the purification process.

[0054] The term "anticoagulation" refers to stabilizing the components in a blood sample for a period of time. Typically, in blood, in the presence of an anticoagulant, the concentration and properties of the cells and extracellular components remain relatively unchanged compared to their in vivo state.

[0055] Anticoagulation can be carried out, for example, by binding calcium ions (e.g., chelation) and / or by inhibiting thrombin activity.

[0056] Non-limiting examples of anticoagulants include EDTA, citrate, oxalate, heparin, hirudin, and their salts.

[0057] Non-limiting examples of substances that bind calcium ions typically include EDTA, citrate, and oxalate, and their salts.

[0058] Non-limiting examples of substances that inhibit thrombin activity typically include heparin and / or hirudin, and their salts.

[0059] Typically, after blood collection, a solid anticoagulant or a liquid anticoagulant is mixed with the blood, and then incubated at a low temperature for a period of time to allow the blood to separate into an upper layer containing plasma and a lower layer containing blood cells. The upper layer containing plasma is collected and centrifuged to obtain a supernatant containing cell-free plasma.

[0060] For example, a mixture of blood and an anticoagulant can be incubated at 2°C to 12°C for 6 to 8 hours. Typically, the mixture is separated into an upper layer containing plasma and a lower layer containing cells (e.g., red blood cells). The upper layer containing plasma can be collected (e.g., using aspiration by a siphon method), and the lower layer containing cells can be discarded. The collected upper layer containing plasma can be centrifuged to remove remaining cells, cell debris, and / or particles. The centrifugation can be carried out at a low temperature (such as about 10°C) at a speed of, for example, 3000 revolutions per minute to 10,000 revolutions per minute for several minutes (e.g., 30 minutes to 60 minutes). Then, the supernatant containing plasma is collected. The collected plasma can be filter sterilized, for example, using a filtration membrane with a pore size of about 0.2 μm to about 0.45 μm, such as membranes with pore sizes of about 0.2 μm, about 0.22 μm, and about 0.45 μm.

[0061] Centrifugation promotes rapid separation of blood cells from plasma, for example, by using an increased relative centrifugal force (rcf). The rcf and revolutions per minute (rpm) are calculated using the radius of rotation r (the distance between the axis of rotation and the bottom of the container, in mm) by the following formula: rcf = 1,118 × r (rpm / 1000) 2 。

[0062] The anticoagulant oxalic acid can be prepared as a stock solution, and the stock solution can be mixed with blood at about 1:17 to about 1:10 by volume, respectively. The anticoagulant oxalic acid stock solution can contain potassium oxalate, for example, at a concentration in the range of about 20 g / L to about 30 g / L.

[0063] A suitable concentration of the anticoagulant oxalic acid for use in a method for preparing fibrinogen can be in the range of about 20 g / L to about 30 g / L in the stock solution, such as about 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, and 30 g / L, including any values and ranges therebetween.

[0064] In one embodiment, the anticoagulant oxalic acid stock solution contains 0.135 mol / L potassium oxalate. The stock solution can be diluted with blood at about 1:7 to about 1:10 by volume.

[0065] In one embodiment, the solid or liquid anticoagulant contains oxalate, such as potassium oxalate. The oxalate can be mixed with blood immediately after blood collection and then incubated at about 12°C for, for example, about 8 hours to allow the blood to separate into an upper layer containing plasma and a lower layer containing blood cells. Then, the upper layer containing plasma can be centrifuged at about 10°C at a speed of about 4000 rpm for about 50 minutes and collected by aspiration (e.g., using a siphon), and the supernatant containing cell-free plasma can be filter sterilized.

[0066] Typically, the appropriate concentration of an anticoagulant in blood or plasma is a concentration that does not interfere with the specific analytical tests used in the fibrinogen preparation method. Advantageously, the appropriate amount of an anticoagulant in blood or plasma is an amount that does not alter the concentration of the component to be measured (see reference World Health Organization Use of Anticoagulants in Diagnostic Laboratory Investigations).

[0067] In one embodiment, the appropriate amount of an anticoagulant for use in a method of preparing fibrinogen is generally in the range of about 20 g / L to about 30 g / L, or about 120 mmol / L to about 180 mmol / L, sometimes diluted 1:10 in blood, i.e., about 2 g / L to about 3 g / L or about 12 mmol / L to about 18 mmol / L in blood.

[0068] Non-limiting examples of oxalates include potassium oxalate, sodium oxalate, and ammonium oxalate. In one embodiment, the anticoagulant oxalate stock solution contains potassium oxalate. The appropriate concentration of potassium oxalate anticoagulant for use in a method of preparing fibrinogen can be in the range of about 120 mmol / L to about 180 mmol / L in the stock solution, such as about 120 mmol / L, 125 mmol / L, 130 mmol / L, 135 mmol / L, 140 mmol / L, 145 mmol / L, 150 mmol / L, 155 mmol / L, 160 mmol / L, 165 mmol / L, 170 mmol / L, 175 mmol / L, or 180 mmol / L, including any values and ranges therebetween.

[0069] The appropriate final concentration of potassium oxalate anticoagulant in blood for use in a method of preparing fibrinogen can be in the range of about 2 g / L to about 3 g / L, such as about 2.0 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L, or 3.0 g / L, including any values and ranges therebetween.

[0070] The suitable final concentration of potassium oxalate anticoagulant in blood for use in a method of preparing fibrinogen can be in the range of about 12.0 mmol / L to about 18.0 mmol / L, such as, about 12.0 mmol / L, 12.5 mmol / L, 13.0 mmol / L, 13.5 mmol / L, 14.0 mmol / L, 14.5 mmol / L, 15.0 mmol / L, 15.5 mmol / L, 16.0 mmol / L, 16.5 mmol / L, 17.0 mmol / L, 17.5 mmol / L or 18.0 mmol / L, including any values and ranges therebetween.

[0071] In one embodiment, the suitable concentration of potassium oxalate in the anticoagulant for use in a method of preparing fibrinogen is in the range of about 23 g / L to about 28 g / L in the stock solution, which is then diluted to a corresponding final concentration in blood, such as about 2.3 g / L to about 2.8 g / L.

[0072] In one embodiment, the suitable concentration of potassium oxalate in the anticoagulant stock solution for use in a method of preparing fibrinogen is in the range of about 20 g / L (120 mmol / L) to about 30 g / L (180 mmol / L), which is then diluted to a corresponding final concentration of potassium oxalate in blood in the range of about 2 g / L (12 mmol / L) to about 3 g / L (18 mmol / L).

[0073] The citrate anticoagulant can comprise trisodium citrate and 0.100 mol / L to 0.136 mol / L citric acid. The citric acid can be buffered to pH 5.5 to 5.6 with, for example, 84 mmol / L trisodium citrate and 21 mmol / L citric acid.

[0074] Oxalated plasma comprises the cell-free fraction of blood containing an oxalate anticoagulant (e.g., potassium oxalate). The concentration of the anticoagulant (e.g., potassium oxalate) in the plasma can be in the range of a final concentration in blood of about 2 g / L (12 mmol / L) to about 3 g / L (18 mmol / L), such as a final concentration range of about 2.3 g / L to 2.8 g / L potassium oxalate after dilution of the stock solution with blood.

[0075] Plasma can be obtained from anticoagulated blood selected from oxalated blood, citrated blood, and / or heparinized blood.

[0076] Plasma can be autologous, vertebrate, including pooled plasma, such as pooled human plasma. Vertebrate plasma can be non-human, e.g., porcine, including pooled porcine plasma.

[0077] Vertebrate blood-derived products may have a risk of transmitting infectious agents such as viruses. Usually, several measures are taken to minimize the risk of virus and / or unknown pathogen transmission, including routine testing of donor samples for the presence of certain viruses and virus inactivation / removal steps during the manufacturing process. Effective reduction of the virus transmission risk can be achieved by including at least two orthogonal virus inactivation steps that do not alter the beneficial properties of the product.

[0078] Enveloped viruses are rapidly and effectively inactivated by solvent-detergent (SD) treatment, which disrupts the lipid membrane of the virus. Other virus inactivation treatments are, for example, heat treatment (e.g., pasteurization) steps and nanofiltration. Usually, plasma (e.g., oxalated plasma) is obtained by separating cells from a blood source, and then the plasma is filtered and subjected to sub-steps of virus inactivation.

[0079] In one embodiment, plasma (e.g., oxalated plasma) is subjected to virus inactivation (e.g., by SD treatment).

[0080] SD treatment can be carried out by adding a detergent (e.g., Tween 80, C 24 H 44 O6(C2H4O) n )(final concentration in the range of about 5 g / L to 12 g / L) and a solvent (e.g., tributyl phosphate; TNBP) (final concentration in the range of about 1 g / L to 3.6 g / L) to the plasma. Usually, the SD treatment includes treating with 0.3% TNBP and 1% Tween at 24 °C for 6 hours, or treating with 0.3% TNBP and 1% Triton X-100 (C 14 H 22 O(C2H4O) n ) at 24 °C for 4 hours.

[0081] SD treatment can be carried out by adding a detergent (e.g., Tween 80, final concentration of about 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L or 12 g / L, including any values and ranges therebetween) and a solvent (e.g., TNBP) (final concentration of about 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L or 3.6 g / L, including any values and ranges therebetween) to the plasma.

[0082] SD treatment can be carried out by adding a detergent (e.g., Tween 80, final concentration in the range of about 5 g / L to about 12 g / L) and a solvent (e.g., tributyl phosphate or TNBP) (final concentration in the range of about 1 g / L to about 3.6 g / L) to plasma at a pH in the range of about 7.0 to 9.0 after incubation at about 24°C to 26°C for about 6 hours, for example.

[0083] SD treatment can be carried out by adding a detergent (e.g., Tween 80) (final concentration in the range of about 0.5% to about 1.2% (w / v; i.e., 1.2 g / 100 ml)) and a solvent (e.g., TNBP) (final concentration in the range of about 0.15% to about 0.36% (w / v)) to plasma at a pH in the range of about 7.0 to about 9.0 and incubating at about 24°C to 26°C for about 6 hours.

[0084] In one embodiment, plasma (e.g., oxalated plasma) is subjected to virus inactivation (e.g., by SD treatment). SD treatment can be carried out by adding Tween 80 (final concentration in the range of about 0.5% to about 1.2% (w / v)) and TNBP (final concentration in the range of about 0.15% to about 0.36% (w / v)) to plasma at a pH in the range of about 7.0 to about 9.0 and incubating at about 24°C to 26°C for about 6 hours, for example.

[0085] In one embodiment, SD-treated oxalated plasma is obtained. The SD-treated oxalated plasma can contain plasma (e.g., filter-sterilized plasma) (containing fibrinogen, prothrombin), Tween 80 (concentration in the range of about 0.5% to about 1.2% (w / v)), TNBP (concentration in the range of about 0.15% to about 0.36% (w / v)), and potassium oxalate (concentration in the range of about 2.3 to about 2.8 g / L). The SD-treated oxalated plasma can be obtained from, for example, human, porcine, or bovine blood or their plasma fractions containing fibrinogen and prothrombin.

[0086] In one embodiment, SD treatment is carried out by adding a detergent (final concentration 0.8% to 1.2% (w / v) or 8 g / L to 12 g / L) and a solvent (the solvent is e.g., TNBP) (final concentration 0.24% to 0.36% (w / v) or 2.4 g / L to 3.6 g / L) to plasma at pH 7.0 to 9.0 after incubation at about 24°C to 26°C for about 6 hours, for example.

[0087] In one embodiment, plasma (e.g., oxalated plasma) is subjected to viral inactivation (e.g., by SD treatment). The SD treatment can be carried out by adding Tween 80 (final concentration in the range of about 0.8% to about 1.2% (w / v)) and TNBP (final concentration in the range of about 0.24% to about 0.36% (w / v)) to the plasma at a pH in the range of about 7.0 to about 9.0 after incubation at about 24°C to 26°C for, for example, about 6 hours.

[0088] In one embodiment, the SD-treated oxalated plasma comprises plasma (e.g., filter-sterilized plasma) (comprising fibrinogen, prothrombin), Tween 80 (concentration in the range of about 0.8% to about 1.2% (w / v)), TNBP (concentration in the range of about 0.24% to about 0.36% (w / v)), and potassium oxalate (concentration in the range of about 2.3 g / L to about 2.8 g / L). The SD-treated oxalated plasma can be obtained, for example, from human, porcine, or bovine blood or their plasma fractions comprising fibrinogen and prothrombin.

[0089] The preparation of both fibrinogen and thrombin can be started from a single "SD-treated oxalated plasma". For example, prothrombin extraction is performed on the SD-treated oxalated plasma to obtain prothrombin-depleted plasma for fibrinogen preparation and the extracted prothrombin for thrombin preparation.

[0090] Non-limiting examples of removing or extracting prothrombin from plasma (e.g., SD-treated oxalated plasma) include subjecting the plasma to a chromatographic method selected from barium sulfate, prothrombin adsorption, anion exchange prothrombin adsorption, affinity chromatography prothrombin adsorption, size exclusion, and prothrombin precipitation.

[0091] To extract prothrombin, a carrier can be added to the plasma for a period of time to allow prothrombin to adsorb and / or bind to the carrier. For example, to extract prothrombin, a carrier can be added to the SD-treated oxalated plasma or its fraction comprising fibrinogen and prothrombin for a period of time to allow prothrombin to adsorb and / or bind to the carrier. The carrier can be selected from, but is not limited to, prothrombin adsorbents, anion exchangers, and affinity carriers capable of binding prothrombin. The carrier can be selected from, but is not limited to, barium sulfate, diethylaminoethanol (DEAE), and carriers comprising a ligand (e.g., an antibody) capable of binding prothrombin. Subsequently, the plasma fraction is separated from the carrier. The prothrombin-depleted plasma fraction can be obtained after removing the carrier containing the adsorbed (or bound) prothrombin.

[0092] In one embodiment, to extract prothrombin, barium sulfate is added to SD-treated oxalated plasma or a fraction thereof containing fibrinogen and prothrombin for a period of time to allow prothrombin to adsorb and / or bind to the barium sulfate. Subsequently, the liquid plasma fraction can be separated from the barium sulfate. The prothrombin-depleted plasma fraction can be obtained after removing the barium sulfate containing the adsorbed / bound prothrombin. The remaining liquid plasma solution can contain the prothrombin-depleted plasma fraction after removing (or extracting) the barium sulfate with the adsorbed / bound prothrombin.

[0093] In one embodiment, to extract prothrombin, barium sulfate is added to SD-treated potassium oxalated plasma or a fraction thereof containing fibrinogen and prothrombin for a period of time to allow prothrombin to adsorb and / or bind to the barium sulfate. Subsequently, the liquid plasma fraction is separated from the barium sulfate. The prothrombin-depleted plasma fraction can be obtained after removing the barium sulfate containing the adsorbed / bound prothrombin. The remaining liquid plasma solution can contain the prothrombin-depleted plasma fraction after removing (or extracting) the barium sulfate with the adsorbed / bound prothrombin.

[0094] The specific barium sulfate reagent used can significantly affect the procoagulant activity of barium sulfate prothrombin. Preferably, adsorption is carried out with a suitable barium sulfate product. It has been disclosed that, using a specific barium sulfate product, at least some prothrombin can be converted to thrombin and / or thrombin intermediates (see, for example, WO2016123804). An inappropriate barium sulfate product can induce the conversion of at least some prothrombin to thrombin and / or thrombin intermediates before or during fibrinogen purification. When using barium sulfate for the preparation of fibrinogen from prothrombin-depleted plasma, it is advantageous to avoid using a BaSO4 product that promotes the conversion of prothrombin to thrombin in the presence of fibrinogen. It is advantageous to avoid premature conversion of prothrombin to thrombin by the barium sulfate product. It is advantageous to use a suitable barium sulfate product without procoagulant activity as a starting material for the preparation of fibrinogen to obtain the prothrombin-depleted plasma fraction.

[0095] A procoagulation assay can be used to qualitatively determine whether a given BaSO4 reagent is suitable for the fibrinogen preparation method. Using a suitable BaSO4 can reduce the risk of fibrinogen clotting in the solution and / or prevent or reduce the degradation of fibrinogen during the preparation process, thus keeping the fibrinogen intact.

[0096] In one embodiment, evaluating the suitability of a given BaSO4 reagent for use in a fibrinogen preparation method includes: providing the given BaSO4 reagent and plasma containing prothrombin; contacting a sample of the given BaSO4 reagent with the plasma under conditions that permit adsorption of prothrombin from the plasma to the given BaSO4 reagent, thereby obtaining BaSO4-adsorbed prothrombin; evaluating the procoagulant activity of the BaSO4-adsorbed prothrombin, wherein the evaluation is performed by comparing the procoagulant activity of the BaSO4-adsorbed prothrombin with the procoagulant activity of normal mammalian plasma. The suitability of the given BaSO4 reagent for use in preparing fibrinogen is indicated by the procoagulant activity of the BaSO4-adsorbed prothrombin being no greater than the procoagulant activity of normal mammalian plasma.

[0097] In one embodiment, the conditions that permit adsorption of prothrombin to BaSO4 include a pH range of about 7.4 to about 8.6 and / or a BaSO4 concentration range of about 1% to about 22% (w / v).

[0098] In one embodiment, the conditions that permit adsorption of prothrombin to BaSO4 include a pH range of about 7.4 to about 8.6 and / or a BaSO4 concentration range of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21% or 22% (w / v) (including any values and ranges therebetween).

[0099] In one embodiment, prothrombin extraction or depletion is performed on SD-treated oxalated plasma or a fraction thereof (e.g., obtained from human, porcine or bovine blood or plasma fractions thereof containing fibrinogen and prothrombin). For this purpose, the SD-treated oxalated plasma or a fraction thereof (e.g., obtained from human, porcine or bovine blood or plasma fractions thereof containing fibrinogen and prothrombin) can be incubated with a carrier (e.g., a suitable barium sulfate) at room temperature (about 20°C to 30°C) for a period of time (such as about 2 hours to 3 hours) to permit binding, adsorption or complexation of prothrombin with the carrier. Subsequently, the plasma fraction and the carrier can be separated. After separation, the remaining plasma solution contains the plasma fraction depleted of prothrombin. After extraction (or depletion) of prothrombin, SD-treated oxalated plasma depleted of prothrombin is obtained.

[0100] In one embodiment, the SD-treated oxalated prothrombin-depleted plasma comprises: prothrombin-depleted plasma (e.g., sterile prothrombin-depleted plasma and sterile filtered plasma), fibrinogen, Tween 80 at a final concentration of about 5 g / L to about 12 g / L, TNBP at a final concentration of about 1 g / L to about 3.6 g / L, and potassium oxalate at a final concentration of about 2 g / L (12 mmol / L) to about 3 g / L (18 mmol / L).

[0101] In one embodiment of the method, prothrombin is extracted from plasma (such as oxalated plasma containing potassium oxalate and SD) by barium sulfate adsorption. BaSO4 can be mixed with plasma (e.g., after mixing with plasma, at a final concentration in the range of about 1% to about 3%, such as in the range of about 1% to about 2% w / v) at room temperature (e.g., at about 25 °C) for a period of time (e.g., about 2 hours), and can be gently mixed (e.g., by using a stirrer).

[0102] Next, the BaSO4 adsorbed with prothrombin can be separated from the plasma. The separation can be carried out, for example, by centrifugation (e.g., at a speed of about 5000 rpm for about 40 minutes at a temperature of about 10 °C). The supernatant containing prothrombin-depleted plasma can be kept cold (e.g., stored at about 4 °C) until use. The separated BaSO4 can be kept cold (e.g., below -10 °C) together with the adsorbed prothrombin (containing the prothrombin plasma fraction) until use.

[0103] In one embodiment of the method, the SD-treated oxalated plasma or a fraction thereof obtained, for example, from a porcine plasma fraction containing fibrinogen and prothrombin, is subjected to prothrombin extraction or depletion. For example, the SD-treated oxalated porcine plasma or a fraction thereof is incubated with a suitable barium sulfate at a temperature in the range of about 20 °C to about 30 °C for a period of about 2 hours to about 3 hours. Then, the liquid plasma fraction and barium sulfate are separated. After separation, the remaining plasma liquid solution contains the prothrombin-depleted plasma fraction. After extracting (or depleting) prothrombin, SD-treated oxalated prothrombin-depleted plasma is obtained. The SD-treated oxalated prothrombin-depleted plasma can comprise prothrombin-depleted plasma (e.g., sterile prothrombin-depleted plasma and sterile filtered plasma), which contains fibrinogen, Tween 80 at a final concentration in the range of about 5 g / L to about 12 g / L, TNBP at a final concentration in the range of about 1 g / L to about 3.6 g / L, and potassium oxalate at a final concentration in the range of about 2 g / L (12 mmol / L) to about 3 g / L (18 mmol / L).

[0104] In one embodiment of the method, prothrombin is extracted from plasma (e.g., oxalated plasma containing potassium oxalate and SD) by adsorption onto barium sulfate. BaSO4 can be mixed with plasma (e.g., at a final concentration in the range of about 1% to about 2% w / v after mixing with plasma) at room temperature (e.g., at about 25°C) for a period of time (e.g., about 2 hours), and gently mixed (e.g., by using a stirrer). Next, the BaSO4 adsorbed with prothrombin can be separated from the plasma. The separation can be carried out, for example, by centrifugation (e.g., at a speed of about 5000 rpm for about 40 minutes at a temperature of about 10°C). The supernatant, which generally contains plasma depleted of prothrombin, is kept cold (e.g., stored at about 4°C) until use. The separated BaSO4 together with the adsorbed prothrombin of the prothrombin plasma fraction can be kept cold (e.g., below -10°C) until use.

[0105] The plasma fraction depleted of prothrombin includes a plasma fraction free of prothrombin. Free of prothrombin means that the amount (by weight) of prothrombin present before extraction of prothrombin from plasma is less than 10%, or in one embodiment less than 5%.

[0106] In one embodiment, SD-treated oxalated plasma free of prothrombin is obtained. In one embodiment, the SD-treated oxalated plasma free of prothrombin includes plasma that contains fibrinogen, Tween 80 at a final concentration in the range of about 5 g / L to about 12 g / L, TNBP at a final concentration in the range of about 1 g / L to about 3.6 g / L, and potassium oxalate at a final concentration in the range of about 2 g / L (12 mmol / L) to about 3 g / L (18 mmol / L), and contains less than about 5 wt% of the prothrombin that was present prior to the process of extracting prothrombin from plasma as disclosed herein.

[0107] After prothrombin binding and / or adsorption, the carrier with bound and / or adsorbed prothrombin can be removed or extracted from the plasma solution.

[0108] The adsorption and / or binding of prothrombin from plasma to the carrier can be carried out, for example, in batch form or in a column packed with the carrier. The removal of prothrombin from the column can be carried out by loading a plasma fraction onto a column packed with the carrier under conditions that allow the carrier to bind to prothrombin in the plasma, and then collecting the flow-through solution. The batch removal of prothrombin can be carried out by incubating the carrier with plasma containing prothrombin and fibrinogen under conditions that allow the carrier to bind and / or adsorb prothrombin, and then separating the carrier from the liquid.

[0109] The separation of prothrombin from plasma can be carried out by column set-up, batch set-up, or any combination thereof (such as a mixing set-up).

[0110] Typically, in a column set-up, a carrier (e.g., a solid medium) is packed onto a column, and plasma is allowed to move through the column to allow sedimentation, followed by collection of the flow-through material containing the plasma from which prothrombin has been removed.

[0111] A batch set-up generally comprises the following: plasma is added to a carrier (e.g., a solid phase) in a container, then mixed, and then the solid phase is separated, and the liquid phase is collected, for example, by centrifugation. The supernatant containing the plasma from which prothrombin has been removed is collected, and the particles containing the carrier and the carrier-bound prothrombin / carrier-adsorbed prothrombin are removed.

[0112] In another embodiment, SD-treated oxalated plasma free of prothrombin is obtained, the SD-treated oxalated plasma comprising sterile-filtered plasma containing fibrinogen, Tween 80 at a final concentration in the range of about 5 g / L to about 12 g / L, TNBP at a final concentration in the range of about 1 g / L to about 3.6 g / L, and potassium oxalate at a final concentration in the range of about 2 g / L (12 mmol / L) to about 3 g / L (18 mmol / L), and less than about 5% by weight of prothrombin compared to the prothrombin present prior to the process of extracting prothrombin from plasma as disclosed herein.

[0113] The preparation of both fibrinogen and thrombin can be carried out from the same SD-treated oxalated plasma.

[0114] A prothrombin plasma fraction or a prothrombin-rich plasma fraction can comprise a carrier having adsorbed prothrombin and / or bound prothrombin and / or a solution containing prothrombin extracted from the carrier, for example, by elution. Generally, the term "elution" as disclosed herein can be interchanged with the term "desorption". In one embodiment, the elution buffer during the preparation of thrombin contains calcium. A chelating salt (such as sodium citrate) can be present at a concentration of, for example, 3.0% to 4.4% (w / v). In some embodiments, the elution buffer has a pH between about 6.3 and 7.4.

[0115] A prothrombin plasma fraction or a prothrombin-rich plasma fraction can include a solution containing prothrombin obtained by eluting prothrombin from barium sulfate adsorbed with plasma or by eluting prothrombin bound to DEAE after contact with plasma.

[0116] In an exemplary embodiment, potassium oxalate is added to 50 L of porcine blood, and 5 L of anticoagulant or 0.138 mol / L is allowed to stand at a temperature of about 5 °C for 8 hours to allow plasma to separate from cells. In an exemplary embodiment, the upper plasma liquid is then collected and filtered through a sterile filtration membrane (having a pore size range of about 0.2 μm to about 0.45 μm, such as a membrane with a pore size of about 0.2 μm, about 0.22 μm, or about 0.45 μm), and centrifuged at a speed of 3500 revolutions per minute at a temperature of about 10 °C for 50 minutes. In an exemplary embodiment, a sterile filtration membrane (having a pore size of about 0.2 μm to about 0.45 μm, such as a membrane with a pore size of about 0.2 μm, about 0.22 μm, or about 0.45 μm) is used to filter the obtained 24 L of plasma. Generally, the plasma containing potassium oxalate is subjected to SD virus inactivation. In an exemplary embodiment, 243.1 g of Tween 80 and 73.0 g of tributyl phosphate are added to the filtered plasma containing potassium oxalate, and then gently stirred at a temperature of about 25 °C for 6 hours.

[0117] In one embodiment, the filtered and sterilized oxalated plasma containing potassium oxalate and SD is subjected to prothrombin removal to produce prothrombin-depleted plasma. For this purpose, in an exemplary embodiment, 480 g of barium sulfate is added in batches to the oxalated plasma and SD-treated plasma, and then gently mixed for about 2 hours to allow prothrombin to be absorbed from the plasma. In an exemplary embodiment, prothrombin adsorbed in barium sulfate is removed from the plasma by centrifuging at a speed of 5000 revolutions per minute at a temperature of about 10 °C for 40 minutes, and then collecting the supernatant and leaving the particles containing prothrombin adsorbed to barium sulfate.

[0118] In some aspects, a method for preparing fibrinogen using a prothrombin-depleted plasma fraction is provided, where generally the method includes performing one or more salting-out steps or sub-steps on the prothrombin-depleted plasma fraction. Salting out involves a purification technique that utilizes the reduced solubility of specific molecules in a solution containing a high ionic strength. Salting out can be used to precipitate large biomolecules such as proteins. The concentration of a specific salt (such as sodium acetate and / or sodium chloride) can be used to precipitate the target protein (such as fibrinogen). The method can also be used to concentrate dilute solutions of proteins. If desired, dialysis can be used to remove the salt.

[0119] In one embodiment of the disclosed method for fibrinogen preparation, three salting-out (or precipitation) steps are performed.

[0120] In an exemplary embodiment, compared with the potassium ion content limit (78 ppm) of commercial human serum albumin that allows injection, the potassium ion content in the final product batch still exists at a very low level. Therefore, the potassium ion content of the final product prepared by the new method is considered to be relatively low, that is to say, it has no significant impact on the safety of the fibrinogen final product with such a low level of potassium ion content.

[0121] Typically, fibrinogen preparation or purification also includes, for example, removing contaminating proteins (such as plasmin and / or plasminogen) by affinity chromatography.

[0122] The present invention also provides a method for preparing fibrinogen, which starts from oxalated SD-treated plasma and barium sulfate-depleted prothrombin plasma, and then undergoes a salting-out step, an affinity chromatography sub-step, and one or more (e.g., two) additional salting-out steps. Typically, the purified fibrinogen is formulated into a solution containing fibrinogen and a stabilizer, usually by filtration sterilization. Typically, the formulated and filtration-sterilized fibrinogen solution is filled into vials (e.g., borosilicate vials) and dried (e.g., by lyophilization).

[0123] Lyophilization or freeze-drying is a process of removing water from a liquid containing, for example, a drug to produce a solid powder or a lyophilized cake. Typically, lyophilized hemostatic products are stable over an extended period of time and allow storage at room temperature.

[0124] In an exemplary embodiment of the method, the fibrinogen lyophilized powder or lyophilized cake is prepared by using plasma adsorbed with barium sulfate or prothrombin-free plasma starting material, performing one or more salting-out steps (e.g., two or three salting-out steps) on the prothrombin-free plasma to precipitate fibrinogen, dissolving the obtained fibrinogen and performing an affinity chromatography sub-step (e.g., after the first precipitation step) on the dissolved fibrinogen to remove impurities (e.g., plasmin and plasminogen), formulating the purified fibrinogen with a solution containing one or more stabilizers, filtering and sterilizing the formulated fibrinogen, and lyophilizing to obtain a lyophilized cake or lyophilized powder.

[0125] In an exemplary embodiment, the first precipitation (or salting out) step may be carried out as follows: Glycine (e.g., for protecting fibrinogen during precipitation) is added to plasma (e.g., 3.5 mg / ml plasma for obtaining a fibrinogen salting out precipitate), and then mixed. The plasma is cooled (e.g., cooled to 4 °C), and sodium acetate (e.g., 130 mg / ml plasma) is added to the plasma. Then the plasma is gently mixed for a period of time (e.g., 30 minutes) and centrifuged in a refrigerated centrifuge (e.g., at 10 °C) at a high speed (e.g., at 3500 rpm) for a period of time sufficient to collect the precipitate in particulate form (e.g., 40 minutes). The first precipitate particles are collected and crushed. To dissolve the precipitate, a buffer solution containing sodium citrate and Tris at pH 7.5 is added to the crushed first precipitate and gently mixed. Then, the solution can be sterilized using filters (e.g., using 1.0 μm and 0.22 μm filter elements in sequence), and the protein content is measured. Then, glycine is added and gently mixed until dissolved, and then the solution can be sterilized using filters (e.g., using 1.0 μm and 0.22 μm filter elements in sequence), and the filtrate is collected.

[0126] In an exemplary embodiment, the second precipitation (or salting out) step may be carried out as follows: Glycine (e.g., 2709 g, or e.g., about 7 mg glycine / ml fibrinogen solution) is added to the collected fibrinogen solution obtained after precipitation (e.g., 380 L), then gently mixed to dissolve the glycine, then the solution is cooled (e.g., at 0 °C to 4 °C), and sodium acetate (e.g., 87,020 g, or e.g., about 150 g / L sodium acetate / ml fibrinogen glycine solution) is added, and then gently mixed (e.g., for about 30 minutes). Then, the solution is centrifuged in a refrigerated centrifuge (e.g., at a temperature of 3 °C) at a speed (e.g., at 4000 rpm), and the precipitate is collected (e.g., to obtain 25,818 g of total protein).

[0127] In an exemplary embodiment, the third precipitation (or salting out) step may be carried out as follows: A buffer solution with a pH of 10.0 prepared from sodium citrate and Tris (e.g., approximately 360 liters) is added to the second precipitate containing fibrinogen obtained in the foregoing step (e.g., 25,818 g total protein), then gently mixed, and then sterile filtered. Next, glycine is added (e.g., 1152 g or 3.2 g glycine / L), and then gently mixed to dissolve the glycine. Then the solution is cooled (e.g., cooled to 3°C), and sodium acetate is added (e.g., 52,200 g), and then gently mixed (e.g., for 30 minutes). Next, the solution is centrifuged in a refrigerated centrifuge (e.g., centrifuged at approximately 4000 rpm at approximately 3°C for approximately 40 minutes), and then the precipitate is collected. The precipitate is washed 4 times at 4°C, and then the precipitate is dissolved and formulated (e.g., by adding 560 g histidine, 720 g sodium citrate, 1200 g sucrose, 160 g sodium chloride, 40 g Tween 80, and 14.4 g arginine as a buffer solution, and 1600 ml albumin solution containing 40 g protein, and adding water for injection (WFI) to adjust the total volume of the solution to approximately 80 L).

[0128] Solutions can be prepared with pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to a carrier suitable for administration to humans or other vertebrates. The term "carrier" refers to an ingredient that is combined with the components to facilitate the administration of the composition in a manner such that the desired efficiency is substantially maintained.

[0129] In one embodiment of the present invention, the fibrinogen composition comprises a co-stabilizer and an excipient selected from histidine, sodium citrate, sucrose, sodium chloride, Tween 80, arginine, albumin, and combinations thereof. In an exemplary embodiment, a solution formulated in a borosilicate vial is lyophilized (e.g., frozen at a temperature of approximately -40°C for 10 hours, dried at a temperature of approximately -30°C for 62 hours, and secondarily dried at a temperature of approximately 40°C for 8 hours) to obtain a fibrinogen-containing powder.

[0130] Another viral inactivation sub-step can be applied, for example, by heat inactivation of the dried material. That is, in addition to SD, after lyophilization, another viral inactivation step can be carried out by using dry heat treatment at, for example, 100°C for 30 minutes.

[0131] Drug formulation is the process of combining different chemicals (including active drugs, e.g., fibrinogen) to produce a final pharmaceutical product. Generally, in protein formulations, stabilizers are added to maintain the structure of the molecules and excipients.

[0132] Before administration, the lyophilized drug is reconstituted into a liquid composition prior to administration. Reconstitution is carried out by combining and mixing a liquid diluent (e.g., an aqueous solution such as water for injection) with the lyophilized powder / cake, and then the reconstituted product is administered to the desired site. Reconstitution generally also requires a delivery system to ensure proper mixing and administration of the drug. The fibrinogen liquid diluent may contain a sodium acetate and / or sodium chloride solution.

[0133] Advantageously, the disclosed method using prothrombin-depleted plasma / samples is cost-effective because it allows for the preparation of both fibrinogen and thrombin from the same animal or vertebrate blood source or sample (e.g., blood from the same animal or vertebrate) and / or from the same blood pool. In one embodiment, the disclosed method using prothrombin-depleted plasma / samples is cost-effective because it allows for the preparation of both fibrinogen and thrombin from the same porcine blood source or sample (e.g., blood from the same pig) and / or from the same blood pool.

[0134] Based on a comparative analysis of the test results of other methods according to fibrinogen batches prepared using native plasma and fibrinogen batches prepared using the method disclosed herein that includes prothrombin-depleted plasma, the two fibrinogen products have similar characteristics, meet the acceptance criteria, and are stable. The results obtained indicate that the method of using prothrombin-depleted plasma to prepare fibrinogen has no negative impact on the formulation, filtration, lyophilization process, or the yield of the intermediate or final product. The critical quality of the final product meets all acceptance criteria (see Table 1 below). It has been found according to the present invention that there is no significant difference in fibrinogen content and purity when fibrinogen is prepared using plasma from which prothrombin has been removed compared to fibrinogen prepared using native plasma. Thus, the disclosed method is controllable.

[0135] Table 1

[0136]

[0137] *NLT = not less than

[0138] ml / L refers to after lyophilization + reconstitution

[0139] Thus, in some embodiments, after completion of the disclosed method, the preparation of fibrinogen contains not less than 55% (w / v) of the total protein content as fibrinogen.

[0140] In some embodiments of the present application, fibrinogen products are prepared using plasma from which prothrombin has been removed (e.g., by treating plasma with barium sulfate) as the starting material instead of using native plasma.

[0141] From an economic perspective, this disclosed method is advantageous because it allows for the preparation of both fibrinogen and thrombin from the same blood source as described above. Additionally, compared to the process of generating fibrinogen and thrombin from raw plasma, the utilization rate of plasma produced using plasma that has been depleted of prothrombin as the starting material (e.g., by treating plasma with barium sulfate) can be significantly increased. As described above, the disclosed method using prothrombin-depleted plasma is cost-effective because it allows for the preparation of both fibrinogen and thrombin from the same animal or vertebrate plasma source or sample (e.g., blood from the same animal or vertebrate) and / or from the same plasma pool.

[0142] Thrombin can be prepared from a plasma fraction containing prothrombin. For example, the preparation of thrombin includes the steps of eluting prothrombin from a carrier in a plasma fraction containing prothrombin and activating the eluted prothrombin.

[0143] In one embodiment, the preparation of thrombin includes eluting prothrombin from barium sulfate in a plasma fraction containing prothrombin and activating the eluted prothrombin. In one embodiment, the conditions that allow for the conversion of prothrombin to thrombin include subjecting prothrombin to an activator (such as calcium ions).

[0144] As used herein, the term "about" is also intended to include values that are 10% higher or lower than the indicated value. Unless otherwise specified, all numbers such as those representing, for example, ratios, weights, moles / mole, amounts, viscosities, temperatures, etc. should in all cases be understood to be modified by the term "about". Thus, unless otherwise indicated, the numerical parameters set forth in this specification and the appended claims are approximations that may vary by up to ±10% depending on the desired properties sought to be obtained by the present invention.

[0145] Whenever a numerical range is indicated herein, it is meant to include any recited number (fraction or integer) within the indicated range. The phrases "range is between" a first indicated number and a second indicated number and "range is from" a first indicated number "to" a second indicated number are used interchangeably herein and are meant to include the first and second indicated numbers and all fractions and integers therebetween.

[0146] The various embodiments and aspects of the present invention as described above and claimed in the claims section below are experimentally supported in the following examples.

[0147] Examples

[0148] Materials and Methods

[0149] Example 1 - Preparation of Plasma Treated with SD and Depleted of Prothrombin

[0150] Porcine plasma samples (A - 167.0 L, B - 218.0 L, and C - 141 L) were obtained by adding an anticoagulant stock solution diluted 1 / 10 with blood (assuming that approximately 40% of plasma can be obtained from whole blood; in each case, the calculated total blood can be 1 / 0.4, i.e., A - 417 L, B - 545 L, and C - 352 L). The anticoagulant stock solution contained 0.135 mol / L potassium oxalate. The blood / anticoagulant mixture was placed in a cryogenic storage at 12 °C for 8 hours, and the mixture was separated into the upper plasma and the lower red blood cells. Plasma aspiration was performed by the siphon method, and the cells were discarded. The upper plasma was centrifuged at 4000 rpm for 50 minutes at 10 °C, the plasma was collected and filtered through a 0.22 μm filter. The filtered plasma was subjected to SD treatment. The SD treatment was carried out by the following steps: adding Tween 80 with a final concentration of 0.5% (v / v) and TNBP with a final concentration of 0.15% (v / v) at pH 7.0 to 9.0, and then incubating at 25 °C for 6 hours.

[0151] Next, BaSO4 adsorption was carried out. Briefly, BaSO4 reagent (purchased from Shang Hai Merck Chemical Co., Ltd.) was added to the unit of SD - treated plasma. BaSO4 with a final concentration of 2% w / v was gently mixed with the plasma at 25 °C for 2 hours. Next, the BaSO4 adsorbed with prothrombin was separated from the plasma. The separation was carried out by centrifuging at 5000 rpm for 40 minutes at a temperature of about 10 °C. The supernatant or "prothrombin - depleted and SD - treated plasma" was kept at low temperature (4 °C) until use. The separated BaSO4 could be kept at low temperature (< - 10 °C) together with the adsorbed prothrombin or prothrombin plasma fraction until use.

[0152] Example 2 - Purification of Fibrinogen from Plasma Treated with SD and Depleted of Prothrombin

[0153] The prothrombin - depleted and SD - treated plasma was used to prepare freeze - dried fibrinogen as described below:

[0154] The first precipitation step (by salting - out): Glycine was added in an amount of 3.5 mg per ml of plasma and dissolved. The plasma was cooled to 4 °C, 130 mg of sodium acetate was added per ml of plasma, gently mixed for 30 minutes, and then centrifuged at 3500 rpm for 40 minutes in a high - speed refrigerated centrifuge at 10 °C. The first precipitate was collected and crushed.

[0155] A 360 L buffer solution containing sodium citrate and Tris at pH 7.5 was added to the ground first precipitate (an amount of precipitated protein of about 7.1 kg), and gently mixed until dissolved. Then, the solution was filtered, and the protein content was measured (about 18.6 g / L in a total volume of 380 L). An amount of 1653 g of glycine was added, gently mixed until the glycine was dissolved, and then the solution was filtered sequentially using 1.0 μm and 0.22 μm filter elements, and the filtrate was collected thereafter.

[0156] Second precipitation (by salting out): 2709 g of glycine was added to the collected 380 L fibrinogen solution, gently mixed until the glycine was dissolved, then the solution was cooled to 0 °C to 4 °C, 87,020 g of sodium acetate was added, and gently mixed for 30 minutes. Then, the solution was centrifuged at a speed of 4000 rpm at a temperature of 3 °C, and the precipitate was collected to obtain a first purified product of 25,818 g of fibrinogen.

[0157] Third precipitation and lyophilization (by salting out): A 360 L buffer solution at pH 10.0 prepared from sodium citrate and Tris was added to 25,818 g of the total precipitate obtained in the previous step, and gently mixed until dissolved, then filtered. Then, 1152 g of glycine was added and gently mixed until dissolved. The solution was cooled to 3 °C, 52,200 g of sodium acetate was added and gently mixed for 30 minutes. Then, the solution was centrifuged at 4000 rpm at 3 °C for 40 minutes, and the precipitate was collected. The precipitate was washed and filtered 4 times at 4 °C to wash away unwanted residues. The total protein was retained after filtration, and then the protein was dissolved and formulated (560 g of histidine, 720 g of sodium citrate, 1200 g of sucrose, 160 g of sodium chloride, 40 g of Tween 80 and 14.4 g of arginine were added as buffer solutions, and a 1600 ml albumin solution containing 40 g of protein content, and water for injection (WFI) was added to adjust the total volume to about 80 L). The final protein concentration was 50 g / L (albumin: 0.5 g / L). A fibrinogen-containing powder was obtained by lyophilizing the solution formulated in borosilicate vials (frozen at -40 °C for 10 hours; dried once at -30 °C for 62 hours, and then dried twice at 40 °C for 8 hours). After lyophilization, another virus inactivation step was carried out by heat inactivation (i.e., in addition to SD carried out in oxalated plasma). For this purpose, the lyophilized powder was subjected to dry heat treatment at 100 °C for 30 minutes.

[0158] The results showed that the method for preparing fibrinogen from prothrombin-depleted plasma had no negative impact on the formulation, filtration, lyophilization processes, and the yields of intermediate and final products. The critical quality of the final product met all acceptance criteria.

[0159] Example 3 - Thrombin Preparation

[0160] As described in Example 1, thrombin was prepared from prothrombin adsorbed on BaSO4 (isolated from SD-treated plasma). The BaSO4-adsorbed prothrombin was washed (8 L of 9% sodium chloride solution was added, gently mixed at room temperature for 30 minutes, and then centrifuged at 3200 rpm for 30 minutes at 4 °C; this step was repeated twice).

[0161] Subsequently, the prothrombin was eluted (8 L of 10% sodium citrate solution was added, gently mixed at room temperature for 30 minutes, and then centrifuged at 3500 rpm for 30 minutes at 4 °C, and this step was repeated four times).

[0162] Subsequently, the prothrombin was activated (1.3 L of 36% calcium chloride solution and 1.3 L of 20% glycine solution were added, gently mixed and the pH was adjusted to 7.0, stored at room temperature for 8 hours, and then transferred to storage at 2 °C for 2 days. A 3.2 L volume of the bulk thrombin solution was obtained by ultrafiltration and centrifugation).

[0163] Subsequently, the produced thrombin was formulated (800 g of glycine and 6.7 L of 6% w / v albumin solution were added, and WFI was added to adjust to a total volume of 27 L).

[0164] The formulated thrombin was lyophilized (frozen at -40 °C for 10 hours, subjected to a primary drying step at -25 °C for 21 hours, and a secondary drying step at 40 °C for 8 hours). Then the lyophilized thrombin was heat-inactivated (dry heat treatment at 100 °C for 30 minutes).

[0165] It is noteworthy that both the lyophilized fibrinogen and thrombin powders were prepared from the same plasma, so the plasma utilization rate can be significantly improved. In this application, plasma from which prothrombin has been removed (by treating plasma with barium sulfate) was used as the starting material instead of using the original plasma to obtain the fibrinogen product. As described above, from an economic perspective, this method is advantageous because it allows both fibrinogen and thrombin to be prepared from the same blood source.

[0166] Example 4 - Comparison of Fibrinogen Compositions Prepared from Native Plasma and Plasma Depleted of Prothrombin Comparison

[0167] The pH of each batch of the lyophilized powder prepared from the original plasma or from the plasma from which prothrombin has been removed is within the acceptance criteria because there are no abnormal points exceeding the control limits, and the production process using the plasma from which prothrombin has been removed is controllable.

[0168] The drawings show the fibrinogen preparation starting from the original plasma ( Figure 1B) In contrast, the exemplary purification method in the embodiments of the present invention ( Figure 1A ).

[0169] According to the comparative analysis of the results, the fibrinogen content obtained from the original plasma (the method is described in CN101214391B, which is incorporated herein by reference) and the fibrinogen content obtained from the plasma from which prothrombin has been removed are both within the upper control limit and the lower control limit and within the internal acceptance criteria. Therefore, the fibrinogen content of each batch of the lyophilized powder obtained from the original plasma or from the plasma from which prothrombin has been removed meets the acceptance criteria. Therefore, the production process using the plasma from which prothrombin has been removed is controllable because there are no abnormal points beyond the control limits.

[0170] According to the comparative analysis of the results, the fibrinogen purity obtained from the original plasma and from the plasma from which prothrombin has been removed are both within the upper control limit and the lower control limit and within the internal acceptance criteria. Therefore, the fibrinogen purity of each batch of the lyophilized powder obtained from the original plasma or from the plasma from which prothrombin has been removed meets the acceptance criteria, and there are no abnormal points beyond the control limits. Therefore, the production process using the plasma from which prothrombin has been removed is also controllable in this regard.

[0171] According to the comparative analysis of the results, the water content of the products obtained from the original plasma and from the plasma from which prothrombin has been removed are both within the upper control limit and the lower control limit and within the internal acceptance criteria. Therefore, the water content of each batch of the lyophilized powder obtained from the original plasma or from the plasma from which prothrombin has been removed meets the acceptance criteria, and there are no abnormal points beyond the control limits. Therefore, the production process using the plasma from which prothrombin has been removed is also controllable in this regard.

[0172] According to the comparative analysis of the results, the sucrose content of each product obtained from the original plasma or from the plasma from which prothrombin has been removed is within the upper control limit and the lower control limit and meets the internal acceptance criteria. Therefore, the sucrose content of each batch of the lyophilized powder obtained from the original plasma or from the plasma from which prothrombin has been removed meets the acceptance criteria, and there are no abnormal points beyond the control limits. Therefore, the production process using the plasma from which prothrombin has been removed is also controllable in this regard.

[0173] According to the comparative analysis of the results, the sodium chloride content of each point of the product or intermediate product obtained from the original plasma or from the plasma from which prothrombin has been removed is within the upper control limit and the lower control limit and meets the internal acceptance criteria. Therefore, the sodium chloride content of each batch of the lyophilized powder from the original plasma or the plasma from which prothrombin has been removed meets the acceptance criteria, and there are no abnormal points beyond the control limits. Therefore, the production process using the plasma from which prothrombin has been removed is also controllable in this regard.

[0174] Based on the comparative analysis of the results, the Tween 80 residue at each point of the product or intermediate obtained from the original plasma or the plasma with prothrombin removed is within the upper control limit and the lower control limit and meets the internal acceptance criteria. Therefore, the Tween 80 residue of each batch of the lyophilized powder obtained from the raw material plasma or the plasma with prothrombin removed meets the acceptance criteria, and there are no abnormal points beyond the control limits. Thus, the production process using the plasma with prothrombin removed is also controllable in this regard.

[0175] Based on the comparative analysis of the results, the TNBP residue at each point of the product or intermediate obtained from the original plasma or the plasma with prothrombin removed is within the upper control limit and the lower control limit and meets the internal acceptance criteria. Therefore, the TNBP residue of each batch of the lyophilized powder obtained from the raw material plasma or the plasma with prothrombin removed meets the acceptance criteria, and there are no abnormal points beyond the control limits. Thus, the production process using the plasma with prothrombin removed is also controllable in this regard.

[0176] Based on the comparative analysis of the results, the protein of the first precipitation solution at each point of the intermediate obtained from the original plasma or the plasma with prothrombin removed is within the upper control limit and the lower control limit and meets the internal acceptance criteria. Therefore, the protein of the first precipitation solution of each batch of the lyophilized powder obtained from the original plasma or the plasma with prothrombin removed meets the acceptance criteria, and there are no abnormal points beyond the control limits. Thus, the production process using the plasma with prothrombin removed is also controllable in this regard.

[0177] Based on the comparative analysis of the results, the fibrinogen yield at each point of the product obtained from the original plasma or the plasma with prothrombin removed is within the upper control limit and the lower control limit and meets the internal acceptance criteria. Therefore, the fibrinogen yield of each batch of the lyophilized powder obtained from the raw material plasma or the plasma with prothrombin removed meets the acceptance criteria, and there are no abnormal points beyond the control limits. Thus, the production process using the plasma with prothrombin removed is also controllable in this regard.

[0178] Considering the potassium oxalate anticoagulant used in blood collection, the potassium ion residues contained in the final fibrinogen product were studied. The potassium levels were compared with those of fibrinogen products using sodium citrate as the anticoagulant. Table 2 provides a list of the potassium ion contents of fibrinogen prepared from either native plasma (where sodium citrate was used as the anticoagulant) or prothrombin-depleted plasma (where potassium oxalate was used as the anticoagulant, 25 g / L; anticoagulant:blood ratios were 8:1 to 10:1). The results show that, compared with the potassium ion content limit (78 ppm) of, for example, commercial injectable human serum albumin, the potassium ion content of the final product prepared using potassium oxalate as the anticoagulant remained at a very low level, and thus the potassium ion content of the final product prepared using prothrombin-depleted plasma and potassium oxalate as the anticoagulant was very low.

[0179] Based on the comparative analysis of the results, regardless of whether sodium citrate or potassium oxalate was used as the anticoagulant, the potassium ion content of the final product was within the upper and lower control limits and met the acceptance criteria for injectable human albumin. Therefore, there was no significant change in the potassium ion content between using sodium citrate or potassium oxalate as the anticoagulant, and thus the production process using potassium oxalate as the anticoagulant was controllable.

[0180] Table 2 shows fibrinogen products from anticoagulant-treated citrated plasma and potassium oxalate-treated plasma.

[0181] Table 2

[0182]

[0183] Although the present invention has been described in connection with its specific embodiments, it is apparent that many alternatives, modifications, and variations will be obvious to those skilled in the art. Accordingly, it is intended to cover all such alternative forms, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. A method for preparing fibrinogen, the method comprising purifying fibrinogen from a prothrombin-depleted plasma fraction.

2. The method according to claim 1, wherein obtaining the prothrombin-depleted plasma fraction comprises the steps of: i) adding an anticoagulant to a blood source; ii) separating plasma from the blood source, and iii) extracting prothrombin from the plasma to obtain a prothrombin-depleted plasma fraction and a plasma fraction containing prothrombin.

3. The method according to claim 2, wherein step ii) further comprises a sub-step of virus inactivation, the sub-step of virus inactivation comprising treating the plasma with a solvent detergent (SD).

4. The method according to claim 2 or 3, wherein the anticoagulant contains oxalate ions.

5. The method according to any one of claims 2 to 4, wherein extracting prothrombin from the plasma is carried out by the steps of: (a) adding the barium sulfate to the plasma fraction under conditions allowing the adsorption of prothrombin by the barium sulfate to obtain barium sulfate adsorbed with prothrombin; and (b) removing the barium sulfate adsorbed with the prothrombin from the plasma fraction.

6. The method according to claims 4 and 5, wherein the anticoagulant comprises potassium oxalate.

7. The method according to any one of claims 1 to 6, wherein purifying fibrinogen from the prothrombin-depleted plasma fraction comprises one or more salting-out steps.

8. The method according to any one of claims 1 to 7, wherein purifying fibrinogen from the prothrombin-depleted plasma fraction comprises the step of removing plasmin and / or plasminogen by affinity chromatography.

9. The method according to any one of claims 1 to 8, the method further comprising drying the purified fibrinogen.

10. The method according to claim 9, wherein the drying is carried out by freeze-drying to obtain dried purified fibrinogen.

11. The method according to claim 9 or 10, the method further comprising formulating the dried fibrinogen to contain dried thrombin.

12. The method according to any one of claims 1 to 11, the method comprising one or more virus inactivation sub-steps.

13. The method according to claim 12, wherein the one or more virus inactivation sub-steps comprise heat inactivation of the dried fibrinogen.

14. The method according to any one of claims 2 to 13, wherein the method further comprises a thrombin preparation obtained from the plasma fraction containing the prothrombin.

15. The method according to claim 14, wherein the plasma fraction containing the prothrombin contains prothrombin eluted from the barium sulfate adsorbed with the prothrombin.

16. The method according to claim 15, wherein the preparation of thrombin comprises the activation of the prothrombin in the plasma fraction.

17. The method according to any one of claims 14 to 16, the method further comprising preparing fibrinogen, wherein the plasma is derived from a single blood source.

18. A prothrombin-depleted plasma fraction containing fibrinogen, said prothrombin-depleted plasma fraction containing fibrinogen being obtainable by the method according to any one of claims 1 to 17.

19. A prothrombin-depleted plasma fraction, said prothrombin-depleted plasma fraction containing fibrinogen, a solvent detergent (SD) and potassium oxalate.

20. The prothrombin-depleted plasma according to claim 18 or 19, said prothrombin-depleted plasma comprising: SD, i.e. a detergent containing polysorbate 80 at a final concentration of about 5 g / L to about 12 g / L and a solvent containing tributyl phosphate (TNBP) at a final concentration of about 1 g / L to about 3.6 g / L; and potassium oxalate at a final concentration of about 2 g / L (or about 12 mmol / L) to about 3 g / L (or about 18 mmol / L).

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