WO2024119285A1 - Préparation de fibrinogène - Google Patents

Préparation de fibrinogène Download PDF

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Publication number
WO2024119285A1
WO2024119285A1 PCT/CN2022/136420 CN2022136420W WO2024119285A1 WO 2024119285 A1 WO2024119285 A1 WO 2024119285A1 CN 2022136420 W CN2022136420 W CN 2022136420W WO 2024119285 A1 WO2024119285 A1 WO 2024119285A1
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Prior art keywords
prothrombin
plasma
fibrinogen
depleted
blood
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PCT/CN2022/136420
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English (en)
Inventor
Hai Li
Caixia Jiang
Yufu Li
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Guangzhou Bioseal Biotech Co., Ltd.
Ethicon, Inc.
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Priority to PCT/CN2022/136420 priority Critical patent/WO2024119285A1/fr
Publication of WO2024119285A1 publication Critical patent/WO2024119285A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/32—Extraction; Separation; Purification by precipitation as complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6429—Thrombin (3.4.21.5)

Definitions

  • the present invention relates, inter alia, to a method for fibrinogen preparation from a plasma fraction depleted of prothrombin.
  • Fibrinogen and thrombin are two major biological components of fibrin sealant.
  • Thrombin is derived from prothrombin and is a serine protease that facilitates blood clotting by catalyzing the conversion of fibrinogen to fibrin.
  • Thrombin is also responsible for activating platelets and indirectly responsible for regulation of its own production and inhibition through multiple proteolytic feedback mechanisms.
  • Thrombin is also involved in activation of factor VIII, factor V, factor XI, factor XIII and protein C.
  • Thrombin is widely used in clinical applications as a coagulation factor to staunch bleeding of wounds by conversion of fibrinogen to fibrin, is a common component of surgical dressings, and has been used in combination with fibrinogen and other coagulation proteins in two-component hemostatic systems such as fibrin glues, adhesives, and sealants.
  • Thrombin is produced by proteolytic activation of the precursor (zymogen) prothrombin.
  • prothrombin For the production of thrombin, prothrombin must be cleaved at two sites generating intermediate products.
  • the conversion of prothrombin to thrombin in the body is catalyzed by the prothrombinase complex which includes activated Factor X and Factor V and assembles on negatively charged phospholipid membranes in the presence of calcium ions.
  • fibrinogen and prothrombin are separated from plasma respectively and while one of component is separated, the other component contained in the rest of raw material is then discarded.
  • US20180016567A is directed to methods for the production of thrombin from a source of prothrombin using a given BaSO 4 reagent as an adsorbent of prothrombin as well as methods for evaluating the suitability of a given BaSO 4 reagent for use in preparation of thrombin.
  • CN102286095B discloses preparation method for fibrinogen with the advantages of high dissolution rate in the production process, short production period, fast solubility in clinical use, good biological activity, high safety in clinical use and high yield.
  • CN112972755A discloses a preparation method of a biological hemostatic material based on porcine fibrinogen and thrombin.
  • CN105950576A discloses a method for extracting multiple proteins from bovine blood.
  • CN101214391B discloses a high-efficiency bio gel sealant which consists of main gel lyophilized powder, and catalyst lyophilized powder.
  • the present invention relates, inter alia, to a method for fibrinogen preparation from a plasma fraction depleted of prothrombin.
  • a method for preparation of fibrinogen comprising purifying fibrinogen from a plasma fraction being depleted of prothrombin.
  • the step of obtaining the plasma fraction depleted of prothrombin comprises: adding an anticoagulant to a source of blood; separating plasma from the source of blood, and extracting prothrombin from the plasma, thereby obtaining a prothrombin depleted plasma fraction and a prothrombin comprising plasma fraction.
  • the step of separating plasma from the source of blood further includes a sub step of viral inactivation comprising treating the plasma with solvent detergent (SD) .
  • SD solvent detergent
  • the anticoagulant comprises an oxalate ion.
  • the step of extracting prothrombin from the plasma is carried out by: adding to the plasma fraction barium sulfate (BaSO 4 ) under conditions allowing adsorption of prothrombin by barium sulfate and thereby obtaining barium sulfate comprising adsorbed prothrombin; and removing the barium sulfate comprising adsorbed prothrombin from the plasma.
  • barium sulfate BaSO 4
  • the anticoagulant comprises the oxalate ion that originates from potassium oxalate.
  • the step of purifying fibrinogen from a plasma fraction depleted of prothrombin comprises a step of removal of plasmin and/or plasminogen by an affinity chromatography. In some embodiments, purifying fibrinogen from a plasma fraction depleted of prothrombin comprises one or more steps of salting out.
  • the method further comprises a step of drying the purified fibrinogen.
  • the method further comprises formulating the dried fibrinogen to include dried thrombin.
  • the method comprises one or more viral inactivation sub-steps.
  • the one or more viral inactivation sub-steps comprise heat inactivation of the dried fibrinogen.
  • the method further comprises thrombin preparation from the prothrombin-comprising plasma fraction.
  • the prothrombin-comprising plasma fraction comprises prothrombin eluted from barium sulfate comprising adsorbed prothrombin.
  • the preparation of thrombin comprises activation of the prothrombin in the prothrombin-comprising plasma fraction.
  • the method is for preparation of fibrinogen and thrombin from plasma derived from a single blood source.
  • a prothrombin-depleted plasma fraction comprising fibrinogen obtainable by the method disclosed herein in any embodiment thereof.
  • prothrombin-depleted plasma fraction comprising fibrinogen, solvent detergent, and potassium oxalate.
  • the prothrombin depleted plasma the detergent comprises Polysorbate 80 at a final concentration of about 5 to about 12g/L, and the solvent comprises tri-n-butyl phosphate (TNBP) at a final concentration of about 1 to about 3.6 g/L, and potassium oxalate at a final concentration about 2g/L (or about 12 mmol/L) to about 3 g/L (or about 18 mmol/L) .
  • TNBP tri-n-butyl phosphate
  • FIG. 1A-1B shows a schematic purification process of embodiments of the invention (Figure 1A) compared to currently applied ( "old” ) methods of fibrinogen preparation starting from raw plasma ( Figure 1B) .
  • the invention relates, inter alia, to a method for preparation of fibrinogen.
  • the method comprises purification of fibrinogen from a plasma fraction being depleted of prothrombin.
  • Fibrinogen purification comprises one or more steps intended to isolate, separate and/or enrich fibrinogen from a mixture of proteins comprising fibrinogen, e.g., from plasma or a plasma fraction being depleted of prothrombin.
  • fibrinogen preparation starting from a plasma fraction depleted of prothrombin.
  • the pure product may be termed "a fibrinogen isolate” , "a fibrinogen preparation” , “a purified fibrinogen” or "a fibrinogen enriched fraction” .
  • the disclosed method of fibrinogen preparation starting from prothrombin depleted plasma may shorten the production cycle time, increase manufacture capacity by 50%or more and/or reduce costs compared to fibrinogen preparation starting from raw plasma.
  • the method of the invention may use 40%less blood to manufacture the same amount of product, and therefore it is more environmentally friendly as compared to other known methods of fibrinogen preparation (such as fibrinogen preparation methods employing raw plasma comprising prothrombin) .
  • Using a solution comprising prothrombin depleted plasma fraction to prepare fibrinogen may reduce the risk of fibrinogen clotting in the solution and/or prevent or reduce its degradation during the preparation process, thereby keeping the fibrinogen intact.
  • a "prothrombin depleted plasma fraction” includes plasma depleted of prothrombin and/or its complex.
  • prothrombin complex may include 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.
  • prothrombin complex comprises factors selected from Factor II, VII, IX and X.
  • a plasma preparation may be obtained by adding an anticoagulant to a source of blood; and separating plasma from the cells being present in the source of blood.
  • plasma is obtained by adding an anticoagulant to a source of blood at suitable conditions allowing separation of the plasma from a cell fraction.
  • plasma is obtained by adding an anticoagulant to a source of blood for a suitable period of time and at a suitable temperature to allow separation of plasma from a cell fraction in the source of blood and collecting the plasma.
  • a centrifugation step may be carried out, for example, to remove cellular debris.
  • raw plasma a collected a plasma preparation from a source of blood
  • a plasma fraction depleted of prothrombin may be obtained by extracting (or removing) prothrombin from the plasma, thereby obtaining a prothrombin depleted plasma fraction.
  • the prothrombin depleted plasma fraction typically comprises fibrinogen.
  • a "prothrombin depleted plasma fraction” relates to a plasma fraction being free of prothrombin and comprising fibrinogen.
  • a prothrombin depleted plasma fraction is used to produce fibrinogen.
  • free of prothrombin refers to being less than about 20%, less than about 15%, less than about 10%, less than about 5%of prothrombin, by weight, or even being absent of prothrombin left, as compared the initial amount of prothrombin prior to applying the prothrombin depletion step disclosed herein.
  • Prothrombin comprising plasma fraction relates to a fraction obtained by collecting prothrombin extracted from plasma. Typically, prothrombin comprising plasma fraction is free of fibrinogen. Prothrombin comprising plasma fraction may be used to prepare thrombin, if desired.
  • Prothrombin depleted plasma fraction and prothrombin-comprising plasma fraction may be obtained from the same source of blood and may be used in a method for preparing fibrinogen and thrombin, respectively.
  • Non-limiting examples of a source of blood for plasma preparations or fractions thereof include venous, arterial or capillary blood of a vertebrate.
  • the vertebrate may be e.g., human, pig, bovine, goat, sheep and horse.
  • a source of blood for plasma preparations or fractions thereof may include venous, arterial or capillary blood of a vertebrate being porcine.
  • Blood comprises a body fluid in humans or other vertebrates that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells.
  • blood is composed of blood cells suspended in blood plasma.
  • plasma constitutes about 55%of blood fluid, is mostly water (about 92%by volume) , and, inter alia, contains proteins, glucose, mineral ions, and hormones.
  • Albumin is the main protein in plasma.
  • isolated plasma e.g., obtained from collected blood with added anticoagulant present in a supernatant after centrifugation
  • anticoagulant relates to an additive that inhibits blood and/or plasma from clotting so that the constituents are non-significantly changed prior to the purification process.
  • anticoagulation relates to stabilization of the constituents in a blood sample for a certain period of time.
  • concentrations and properties of cellular and extra-cellular constituents, in the presence of an anticoagulant remain relatively unaltered when compared to their in-vivo state.
  • Anticoagulation may occur e.g., by binding calcium ions (e.g., chelation) and/or by inhibiting thrombin activity.
  • calcium ions e.g., chelation
  • Non-limiting examples of anticoagulants include EDTA, citrate, oxalate, heparinates, hirudin and salts thereof.
  • non-limiting examples of substances binding calcium ions include EDTA, citrate, and oxalate and salts thereof.
  • non-limiting examples of substances inhibiting thrombin activity include heparinates and/or hirudin and salts thereof.
  • a solid or liquid anticoagulant is mixed with blood, followed by incubation under refrigeration for a period of time to allow separation of blood into an upper layer comprising plasma and a lower layer comprising blood cells.
  • the upper layer comprising plasma is collected and centrifuged to obtain a supernatant comprising a cell free plasma.
  • the mixture of blood and anticoagulant may be incubated at 2-12 °C for 6-8 hours.
  • the mix separates into an upper layer comprising plasma and a lower layer comprising cells (e.g., red blood cells) .
  • the upper layer comprising plasma may be collected (e.g., using suction by a siphon method) , and the lower layer comprising cells is discarded.
  • the collected upper layer comprising plasma may be subjected to centrifugation to remove remaining cells, cell debris and/or particles. Centrifugation may be carried out at a speed of e.g., 3000 rev/mins to 10,000 rev/mins at low temperatures, such as about 10 °C, and for several minutes e.g., 30-60 minutes.
  • the collected plasma may be subjected to filter sterilization e.g., with a with filter membrane having a pore size ranging from about 0.2 to about 0.45 ⁇ m, such as, about 0.2, about 0.22, and about 0.45 ⁇ m pore size membrane.
  • Centrifugation facilitates rapid separation of blood cells from plasma, e.g., by using an increased relative centrifugal force (rcf) .
  • Anticoagulant oxalate may be prepared as a solution stock and the solution stock may be mixed with the blood ranging from about 1: 17 to about 1: 10, by volume, respectively.
  • the anticoagulant oxalate stock solution may contain potassium oxalate, for example at a concentration ranging from about 20 to about 30 g/L.
  • a suitable concentration of anticoagulant oxalate for use in the method for preparation of fibrinogen may range from about 20 g/L to about 30 g/L, such as, about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 g/L in a stock solution, including any value and range therebetween.
  • the anticoagulant oxalate stock solution contains 0.135 mol/L potassium oxalate.
  • This stock solution may be diluted with blood in a range of about 1: 7 to about 1: 10, by volume.
  • a solid or liquid anticoagulant comprises an oxalate salt, such as potassium oxalate.
  • Oxalate salt may be mixed with blood immediately after blood collection followed by incubation for a period of e.g., about 8 hours at about 12 °C to allow separation of blood into an upper layer comprising plasma and a lower layer comprising blood cells. Then, the upper layer comprising plasma may be collected by suction e.g., using a siphon, centrifuged at a speed of about 4000 rpm at about 10°C for about 50 minutes and a supernatant comprising cell free plasma may be filter sterilized.
  • a suitable concentration of anticoagulant in the blood or plasma is one that does not interfere with certain analytical tests used in the method of fibrinogen preparation.
  • a suitable amount of anticoagulant in the blood or plasma is one that does not change the concentration of the constituents to be measured (see Ref. World Health Organization Use of Anticoagulants in Diagnostic Laboratory Investigations) .
  • a suitable amount of anticoagulant for use in the method for preparation of fibrinogen is typically in the range of about 20 g/L to about 30 g/L, or of about 120 mmol/L to about 180 mmol/L, at times 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.
  • Non-limiting examples of oxalate salt include potassium oxalate, sodium oxalate, and ammonium oxalate.
  • the anticoagulant oxalate stock solution contains potassium oxalate.
  • a suitable concentration of potassium oxalate anticoagulant for use in the method for preparation of fibrinogen may range from about 120 mmol/L to about 180 mmol/L, such as, about 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 mmol/L in a stock solution, including any value and range therebetween.
  • a suitable final concentration of potassium oxalate anticoagulant in the blood for use in the method for preparation of fibrinogen may range from about 2 g/L to about 3 g/L such as about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 g/L, including any value and range therebetween.
  • a suitable final concentration of potassium oxalate anticoagulant in the blood for use in the method for preparation of fibrinogen may range from about 12.0 mmol/L to about 18.0 mmol/L, such as, about 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, or 18.0 mmol/L, including any value and range therebetween.
  • a suitable concentration of potassium oxalate in the anticoagulant for use in the method for preparation of fibrinogen ranges from about 23 to about 28g/L in a stock solution which is next diluted to the corresponding final concentration in blood of e.g., about 2.3 to about 2.8 g/L.
  • a suitable concentration of potassium oxalate in the anticoagulant stock solution for use in the method for preparation of fibrinogen ranges from about 20 g/L (120 mmol/L) to about 30 g/L (180 mmol/L) which is next diluted to the corresponding final concentration of potassium oxalate ranging from about 2 g/L (12 mmol/L) to about 3 g/L (18 mmol/L) in the blood.
  • Citrate anticoagulant may comprise trisodium citrate with 0.100 to 0.136 mol/L citric acid. Citrate may be buffered to pH 5.5 to 5.6 with e.g., 84 mmol/L Tris sodium citrate with 21 mmol/L citric acid.
  • Oxalated plasma comprises a cell-free fraction of blood containing oxalate anticoagulant e.g., potassium oxalate.
  • concentration of anticoagulant in plasma e.g., potassium oxalate may be at a final concentration range of about 2 g/L (12 mmol/L) to about 3 g/L (18 mmol/L) in blood, such as at a final concentration range of about 2.3-2.8 g/L potassium oxalate after stock solution dilution with blood.
  • Plasma may be obtained from anticoagulated blood selected from oxalated blood, citrated blood, and/or heparinized blood.
  • Plasma may be autologous, vertebrated including pooled plasma, such as pooled human plasma.
  • Vertebrated plasma may be of non-human source e.g., pig, including pig pooled plasma.
  • Vertebrate blood-derived products may carry a risk of transmitting infectious agents such as viruses.
  • Several measures are usually taken in order to minimize the risk of viral and/or unknown pathogens transmission, including routine testing of donated samples for the presence of certain viruses, and viral inactivation/removal steps during the manufacture process.
  • Effective reduction of viral transmission risk may be achieved by including at least two orthogonal viral inactivation steps that do not alter the beneficial properties of the product.
  • Lipid-enveloped viruses are quickly and efficiently inactivated by the Solvent Detergent (SD) treatment which destroys the lipid membrane of the viruses.
  • Additional viral inactivation treatments are e.g., a heat treatment (e.g., pasteurization) step and nanofiltration.
  • obtaining plasma e.g., oxalated plasma
  • obtaining plasma is carried out by separating cells from the source of blood, next plasma is filtered and subjected to a sub step of viral inactivation.
  • the plasma e.g., oxalated plasma
  • the plasma is subjected to viral inactivation (e.g., by SD treatment) .
  • SD treatment may be carried out by adding to the plasma a detergent (e.g., Tween 80, C 24 H 44 O 6 (C 2 H 4 O) n ) at a final concentration ranging from about 5 to 12 g/L, and a solvent (e.g. Tributyl phosphate; TNBP) at a final concentration ranging from about 1 to 3.6 g/L.
  • a detergent e.g., Tween 80, C 24 H 44 O 6 (C 2 H 4 O) n
  • TNBP Tributyl phosphate
  • the SD treatment includes 0.3%TNBP and 1%Tween at 24°C for 6h, or 0.3%TNBP and 1%Triton X-100 ( (C 14 H 22 O (C 2 H 4 O) n ) at 24°C for 4h.
  • SD treatment may be carried out by adding to the plasma a detergent (e.g., Tween 80 at a final concentration of about 5, 6, 7, 8, 9, 10, 11, or 12 g/L, including any value and range therebetween, and a solvent (e.g., TNBP) at a final concentration of about 1, 1.5, 2, 2.5, 3, or 3.6 g/L, including any value and range therebetween.
  • a detergent e.g., Tween 80 at a final concentration of about 5, 6, 7, 8, 9, 10, 11, or 12 g/L, including any value and range therebetween
  • a solvent e.g., TNBP
  • SD treatment may be carried out by adding to the plasma a detergent (e.g., Tween 80 at a final concentration ranging from about 5 to about 12 g/L, and a solvent (e.g., Tributyl phosphate or TNBP) at a final concentration ranging from about 1 to about 3.6 g/L, at pH ranging from about 7.0 to 9.0 upon incubating at about 24-26 °C e.g., for about 6 hours.
  • a detergent e.g., Tween 80 at a final concentration ranging from about 5 to about 12 g/L
  • a solvent e.g., Tributyl phosphate or TNBP
  • SD treatment may be carried out by adding to the plasma a detergent (e.g., Tween 80) at a final concentration ranging from about 0.5%to about 1.2% (w/v; i.e. 1.2 g/100ml) , and a solvent (e.g., TNBP) at a final concentration ranging from about 0.15 to about 0.36% (w/v) , at pH ranging from about 7.0 to about 9.0 and incubating at about 24-26 °C, e.g., for about 6 hours.
  • a detergent e.g., Tween 80
  • TNBP solvent
  • the plasma (e.g., oxalated plasma) is subjected to viral inactivation e.g., by an SD treatment.
  • the SD treatment may be carried out by adding to the plasma Tween 80 at a final concentration ranging from about 0.5 to about 1.2% (w/v) and TNBP at a final concentration ranging from about 0.15 to about 0.36% (w/v) , at pH ranging from about 7.0 to about 9.0 and incubating at about 24-26 °C, e.g., for about 6 hours.
  • SD-treated oxalated plasma is obtained.
  • SD-treated oxalated plasma may comprise plasma (e.g., filter sterilized plasma) comprising fibrinogen, prothrombin, Tween 80 ranging from about 0.5 to about 1.2% (w/v) , TNBP ranging from about 0.15 to about 0.36% (w/v) , and potassium oxalate at a concentration ranging from about 2.3 to about 2.8 g/L.
  • the SD-treated oxalated plasma may be obtained from e.g., human, pig, or bovine blood or a plasma fraction thereof comprising fibrinogen and prothrombin.
  • the SD treatment is carried out by adding to the plasma a detergent at a final concentration of 0.8 to 1.2% (w/v) or 8 to 12 g/L, and a solvent, the solvent being (e.g., TNBP) at a final concentration of 0.24%to 0.36% (w/v) or 2.4 to 3.6 g/L, at pH 7.0 to 9.0 upon incubating at about 24 to 26 °C e.g., for about 6 hours.
  • a detergent at a final concentration of 0.8 to 1.2% (w/v) or 8 to 12 g/L
  • a solvent e.g., TNBP
  • the plasma e.g., oxalated plasma
  • the SD treatment may be carried out by adding to the plasma Tween 80 at a final concentration ranging from about 0.8 to about 1.2% (w/v) and TNBP at a final concentration ranging from about 0.24 to about 0.36% (w/v) , at pH ranging from about 7.0 to about 9.0 upon incubating at about 24-26 °C e.g., for about 6 hours.
  • SD-treated oxalated plasma comprises plasma (e.g., filter sterilized plasma) comprising fibrinogen, prothrombin, Tween 80 at a concentration range of about 0.8 to about 1.2% (w/v) , TNBP at a concentration range of about 0.24%to about 0.36% (w/v) and potassium oxalate at a concentration range of about 2.3 to about 2.8 g/L.
  • plasma e.g., filter sterilized plasma
  • TNBP at a concentration range of about 0.24%to about 0.36%
  • potassium oxalate at a concentration range of about 2.3 to about 2.8 g/L.
  • SD-treated oxalated plasma may be obtained e.g., from human, pig, or bovine blood or a plasma fraction thereof comprising fibrinogen and prothrombin.
  • Preparation of both fibrinogen and thrombin may be carried out starting from one single "SD-treated oxalated plasma" .
  • SD-treated oxalated plasma is subjected to prothrombin extraction for obtaining prothrombin depleted plasma for fibrinogen preparation and for obtaining extracted prothrombin for thrombin preparation.
  • Non-limiting examples of depleting or extracting prothrombin from plasma include subjecting the plasma to chromatographic methods selected from barium sulfate, prothrombin adsorption, anion exchange prothrombin adsorption, affinity chromatography prothrombin adsorption, size exclusion and prothrombin precipitation.
  • a carrier may be added to plasma for a period of time to allow adsorption and/or binding of prothrombin to the carrier.
  • a carrier may be added to SD-treated oxalated plasma or a fraction thereof comprising fibrinogen and prothrombin for a period of time allowing adsorption and/or binding of prothrombin to the carrier.
  • the carrier may be selected from, without being limited thereto, a prothrombin adsorbent, an anion exchanger, and an affinity carrier capable of binding prothrombin.
  • the carrier may be selected from, without being limited thereto, barium sulfate, diethylaminoethanol (DEAE) , and a carrier comprising a ligand (e.g., antibodies) capable of binding prothrombin.
  • a ligand e.g., antibodies
  • the plasma fraction is separated from the carrier.
  • a prothrombin depleted plasma fraction may be obtained after the removal of the carrier comprising adsorbed (or bound) prothrombin.
  • barium sulfate in order to extract prothrombin, is added to SD-treated oxalated plasma or a fraction thereof comprising fibrinogen and prothrombin for a period of time allowing adsorption and/or binding of prothrombin to the barium sulfate.
  • the liquid plasma fraction may be separated from the barium sulfate.
  • a prothrombin depleted plasma fraction may be obtained after removal of the barium sulfate comprising adsorbed/bound prothrombin.
  • a liquid plasma solution remained after the removal (or extraction) of the barium sulfate with adsorbed/bound prothrombin may comprise a prothrombin depleted plasma fraction.
  • barium sulfate in order to extract prothrombin, is added to SD-treated potassium oxalated plasma or a fraction thereof comprising fibrinogen and prothrombin for a period of time allowing adsorption and/or binding of prothrombin to the barium sulfate.
  • the liquid plasma fraction is separated from the barium sulfate.
  • a prothrombin depleted plasma fraction may be obtained after removal of the barium sulfate comprising adsorbed/bound prothrombin.
  • a liquid plasma solution remaining after removal (or extraction) of the barium sulfate with adsorbed/bound prothrombin may comprise prothrombin depleted plasma fraction.
  • the specific barium sulfate reagent used may significantly affect the pro-coagulant activity of the barium sulfate prothrombin.
  • adsorption is carried out with a suitable barium sulfate product. It is disclosed that using certain barium sulfate products, at least some of the prothrombin is converted to thrombin and/or thrombin intermediates (see e.g., WO2016123804) .
  • Non suitable barium sulfate product may induce at least some of the prothrombin to convert to thrombin and/or thrombin intermediates before or during fibrinogen purification.
  • a pro-coagulation assay may be used to qualitatively determine whether a given BaSO 4 reagent is suitable in the fibrinogen preparation method. Using a suitable BaSO 4 may reduce the clotting risk of fibrinogen in the solution and/or prevent or reduce its degradation during the preparation process and thereby keep fibrinogen intact.
  • evaluating the suitability of a given BaSO 4 reagent for use in a method of fibrinogen preparation comprises: providing the given BaSO 4 reagent and plasma comprising prothrombin; contacting a sample of the given BaSO 4 reagent and the plasma under conditions allowing adsorption of prothrombin from the plasma to the given BaSO 4 reagent thereby obtaining BaSO 4 -adsorbed prothrombin; evaluating pro-coagulant activity of the BaSO 4 -adsorbed prothrombin, wherein when the evaluation is carried out by comparing the pro-coagulant activity of the BaSO 4 -adsorbed prothrombin to a pro-coagulant activity of a normal mammalian plasma.
  • the suitability of the given BaSO 4 reagent for use in preparing fibrinogen is indicated by the procoagulant activity of the BaSO 4 -adsorbed prothrombin being not greater than the pro-coagulant activity of normal mammalian plasma.
  • the conditions allowing prothrombin adsorption to BaSO 4 comprises pH ranging from about 7.4 to about 8.6 and/or BaSO 4 at a concentration range of about 1%to about 22% (w/v) .
  • the conditions allowing prothrombin adsorption to BaSO 4 comprises pH ranging from about 7.4 to about 8.6 and/or BaSO 4 at a 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 value and range therebetween.
  • the SD-treated oxalated plasma or a fraction thereof obtained e.g., from human, pig, or bovine blood or a plasma fraction thereof comprising fibrinogen and prothrombin is subjected to prothrombin extraction or depletion.
  • SD-treated oxalated plasma or a fraction thereof obtained e.g., from human, pig, or bovine blood or a plasma fraction thereof comprising fibrinogen and prothrombin
  • the carrier e.g., a suitable barium sulfate
  • the plasma fraction and the carrier may be separated.
  • the remaining plasma solution comprises a prothrombin depleted plasma fraction.
  • an SD-treated oxalated prothrombin depleted plasma is obtained.
  • SD-treated oxalated prothrombin depleted plasma comprises: prothrombin depleted plasma (e.g., sterile prothrombin depleted and sterile filtered plasma) , fibrinogen, Tween 80 at a final concentration of about 5 to about 12 g/L, TNBP at a final concentration of about 1 to about 3.6 g/L, and potassium oxalate at a final concentration about 2 g/L (12 mmol/L) to about 3 g/L (18 mmol/L) .
  • prothrombin is extracted from plasma (such as oxalated plasma comprising potassium oxalate and SD) by barium sulfate adsorption.
  • BaSO 4 may be mixed with the plasma (e.g., at a final concentration in a range of about 1 to about 3%, such as in a range of about 1 to about 2%w/v after being mixed with the plasma) for a period of time (e.g., about 2 hours) at room temperature (e.g., at about 25 °C) , and may be gently mixed (e.g., by using a stirrer) .
  • the BaSO 4 with adsorbed prothrombin may be separated from the plasma. Separation may be carried out e.g., by centrifugation (e.g., at a speed of about 5000 rpm for about 40 minutes at a temperature of about 10 °C) .
  • the supernatant comprising prothrombin depleted plasma may be kept cold (e.g., at about 4 °C) until use.
  • the separated BaSO 4 together with adsorbed prothrombin comprises a prothrombin plasma fraction may be kept cold (e.g., below-10 °C) until use.
  • the SD-treated oxalated plasma or a fraction thereof obtained e.g., from a pig plasma fraction comprising fibrinogen and prothrombin is subjected to prothrombin extraction or depletion.
  • the SD-treated oxalated porcine plasma or a fraction thereof is incubated with a suitable barium sulfate for a time period ranging from about 2 to about 3 hours at a temperature ranging from about 20 to about 30 °C.
  • the liquid plasma fraction and the barium sulfate are separated.
  • the remining plasma liquid solution comprises a prothrombin depleted plasma fraction.
  • SD-treated oxalated prothrombin depleted plasma may comprise prothrombin depleted plasma (e.g., sterile prothrombin depleted and sterile filtered plasma) comprising fibrinogen, Tween 80 at a final concentration ranging from about 5 to about 12 g/L, TNBP at a final concentration ranging from about 1 to about 3.6 g/L, and potassium oxalate at a final concentration ranging from about 2 g/L (12 mmol/L) to about 3 g/L (18 mmol/L) .
  • prothrombin depleted plasma e.g., sterile prothrombin depleted and sterile filtered plasma
  • fibrinogen e.g., fibrinogen, Tween 80 at a final concentration ranging from about 5 to about 12 g/L
  • TNBP at a final concentration ranging from about 1 to about 3.6 g/L
  • potassium oxalate at a final concentration ranging from about 2
  • prothrombin is extracted from plasma (e.g., oxalated plasma comprising potassium oxalate and SD) by barium sulfate adsorption.
  • plasma e.g., oxalated plasma comprising potassium oxalate and SD
  • BaSO 4 may be mixed with the plasma (e.g., at a final concentration ranging from about 1 to about 2%w/v after being mixed with the plasma) for a period of time (e.g., about 2 hours) at room temperature (e.g., at about 25°C) and is gently mixed (e.g., by using a stirrer) .
  • the BaSO 4 with adsorbed prothrombin may be separated from the plasma.
  • Separation may be carried out e.g., by centrifugation (e.g., at a speed of about 5000 rpm for about 40 minutes at a temperature of about 10°C) .
  • the supernatant comprising prothrombin depleted plasma is typically kept cold (e.g., at about 4 °C) until use.
  • the separated BaSO 4 with adsorbed prothrombin of a prothrombin plasma fraction may be kept cold (e.g., below-10 °C) until use.
  • Prothrombin depleted plasma fraction includes a plasma fraction free of prothrombin. Free of prothrombin relates to less than 10%, or, in some embodiment less than 5%of the amount (by weight) of the prothrombin being present prior of its extraction from the plasma.
  • an SD-treated oxalated plasma that is free of prothrombin is obtained.
  • the SD-treated oxalated plasma that is free of prothrombin comprises a plasma comprising fibrinogen, Tween 80 at a final concentration ranging from about 5 to about 12 g/L, TNBP at a final concentration ranging from about 1 to about 3.6 g/L, and potassium oxalate at a final concentration ranging from about 2 g/L (12 mmol/L) to about 3 g/L (18 mmol/L) , and contains less than about 5%by weight of the prothrombin being present prior of the prothrombin's extraction process from the plasma as disclosed herein.
  • the carrier with bound and/or adsorbed prothrombin may be removed or extracted from the plasma solution.
  • Adsorption and/or binding of prothrombin from plasma with a carrier may be carried out e.g., in a batch form or in a column packed with a carrier. Removal of prothrombin in a column may be by carried out by loading a plasma fraction to a column packed with a carrier under conditions allowing the carrier to bind to prothrombin in the plasma and then collecting the flow through solution. Removal of prothrombin in batch may be carried out by incubating a carrier with plasma comprising prothrombin and fibrinogen under conditions allowing the carrier to bind and/or adsorb prothrombin and then separating the carrier from the liquid.
  • the separation of prothrombin from plasma may be performed by a column setup, a batch setup or any combination thereof, such as a hybrid setup.
  • a carrier e.g., solid medium
  • plasma is allowed to move through the column to allow settling and a flow-through material comprising prothrombin depleted plasma is next collected.
  • a batch setup typically includes the following: plasma is added to the carrier (e.g., a solid phase) in a vessel and is then mixed, and next, the solid phase is separated and the liquid phase is collected e.g., by centrifugation. The supernatant comprising prothrombin depleted plasma is collected and the pellet comprising the carrier and carrier-bound prothrombin/adsorbed prothrombin is removed.
  • the carrier e.g., a solid phase
  • the supernatant comprising prothrombin depleted plasma is collected and the pellet comprising the carrier and carrier-bound prothrombin/adsorbed prothrombin is removed.
  • an SD-treated oxalated plasma that is free of prothrombin which comprises a sterilized filtered plasma comprising fibrinogen, Tween 80 at a final concentration ranging from about 5 to about 12 g/L, TNBP at a final concentration ranging from about 1 to about 3.6 g/L, and potassium oxalate at a final concentration ranging from about 2 g/L (12 mmol/L) to about 3 g/L (18 mmol/L) , and less than about 5%by weight of the prothrombin compared to that was present in the plasma prior of the prothrombin's extraction process from the plasma as disclosed herein.
  • Preparation of both fibrinogen and thrombin may be carried out from the same SD-treated oxalated plasma.
  • a prothrombin plasma fraction or plasma fraction enriched with prothrombin may include a carrier with the adsorbed and/or bound prothrombin and/or a prothrombin comprising solution extracted from the carrier e.g., by elution.
  • the term "elution” as disclosed herein is interchangeable with the term “desorption” .
  • the elution buffer during the preparation of thrombin comprises calcium.
  • Chelating salt such as sodium citrate may be present at a concentration of e.g., 3.0 to 4.4% (w/v) .
  • the elution buffer has pH of between 6.3 and 7.4.
  • a prothrombin plasma fraction or plasma fraction enriched with prothrombin may include a prothrombin comprising solution obtained by elution of prothrombin from barium sulfate adsorbed with plasma, or obtained by elution of prothrombin bound to DEAE after its contacting with plasma.
  • potassium oxalate is added to 50 L pig blood and 5 L anticoagulant or 0.138 mol/L is left standstill at a temperature of about 5°C for 8 hours to allow plasma separation from cells.
  • upper plasma liquid is then collected, and filtered with a sterile filtration membrane (having a pore size ranging from about 0.2 to about 0.45 ⁇ m, such as, about 0.2, about 0.22, or about 0.45 ⁇ m pore size membrane) , and is centrifuged for 50 minutes at a temperature of about 10°C using a rotating speed of 3500 rev/mins.
  • 24 L plasma obtained is filtered using a sterile filtration membrane (having a pore size of about 0.2 to about 0.45 ⁇ m such as about 002, about 0.22, or about 0.45 ⁇ m pore size membrane) .
  • the potassium oxalate comprising plasma is subjected to SD viral inactivation.
  • 243.1g Tween 80, and 73.0g tributyl phosphate are added, followed by gently stirring for 6 hours at a temperature of about 25°C.
  • the filter sterilized oxalated plasma comprising potassium oxalate and SD is subjected to prothrombin removal to produce prothrombin depleted plasma.
  • prothrombin removal to produce prothrombin depleted plasma.
  • 480g barium sulfate are added to the oxalated-and SD-treated plasma in a batch form followed by gently mixing for about 2 hours to allow absorption of prothrombin from the plasma.
  • prothrombin adsorbed in barium sulfate is removed from the plasma by 40 minutes centrifugation at a temperature of about 10 °C using rotating speed of 5000 rev/mins followed by collecting the supernatant and leaving the pellet-comprising prothrombin adsorbed to barium sulfate.
  • a method for fibrinogen preparation using a prothrombin depleted plasma fraction comprising subjecting a prothrombin depleted plasma fraction to one or more salting out steps or sub-steps.
  • Salting out relates to a purification technique that utilizes reduced solubility of certain molecules in a solution comprising high ionic strength. Salting out may be used to precipitate large biomolecules, such as proteins.
  • a specific salt concentration e.g., of sodium acetate, and/or of sodium chloride, may be used to precipitate a target protein (e.g., fibrinogen) . This process may also be used to concentrate dilute solutions of proteins. Dialysis may be used to remove the salt if needed.
  • three salting out (or precipitation) steps are carried out.
  • the potassium ion content in a batch of final products is still present at a very low level compared with the potassium ion content limit (78ppm) of the commercial human serum albumin allowed for injection, so the potassium ion content of the final products prepared by the new process is considered relatively low, that is, there is no significant impact on the safety of fibrinogen final product with such low level of potassium ion content.
  • fibrinogen preparation or purification also comprises removal of contaminant proteins such as plasmin and/or plasminogen e.g., by affinity chromatography.
  • fibrinogen preparation starting from oxalated SD-treated and barium sulfate prothrombin depleted plasma, followed by a salting out step, an affinity chromatography sub-step, and one or more (e.g., two) additional salting out steps.
  • purified fibrinogen is formulated into a solution comprising fibrinogen and stabilizers, typically upon sterilization by a filter.
  • the formulated filter sterilized fibrinogen solution is filled in vials (e.g., borosilicate vials) and is dried (e.g., by lyophilization) .
  • Lyophilization is a process in which water is removed from a liquid comprising e.g., a drug, creating a solid powder, or cake.
  • a liquid comprising e.g., a drug, creating a solid powder, or cake.
  • the lyophilized hemostatic product is stable for extended periods of time and may allow storage at room temperatures.
  • fibrinogen lyophilized powder or cake is prepared by using barium sulfate adsorbed plasma, or plasma starting material that is free of prothrombin, subjecting the prothrombin free plasma to one or more salting out steps (e.g., two or three salting out steps) to precipitate fibrinogen, dissolving the obtained fibrinogen and subjecting the dissolved fibrinogen to an affinity chromatography sub-step (e.g., carried out after the first precipitation step) to remove impurities (e.g., plasmin and plasminogen) , formulating the purified fibrinogen with a solution comprising one or more stabilizers, subjecting the formulated fibrinogen to filter sterilization and to lyophilization to obtain a cake or powder.
  • salting out steps e.g., two or three salting out steps
  • an affinity chromatography sub-step e.g., carried out after the first precipitation step
  • impurities e.g., plasmin and plasminogen
  • a first precipitation (or salting out) step may be as follows in exemplary embodiments: glycine (e.g., used for protecting fibrinogen during precipitation) is added to plasma (e.g., 3.5 mg per ml plasma for obtaining fibrinogen salting precipitate) and followed by mixing.
  • plasma e.g., 3.5 mg per ml plasma for obtaining fibrinogen salting precipitate
  • the plasma is cooled (e.g., to 4°C)
  • sodium acetate e.g., 130 mg per ml plasma
  • the plasma is then gently mixed for a period of time (e.g., 30 minutes) and is 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 a pellet (e.g., 40 minutes) .
  • This first precipitate pellet is collected and crushed.
  • a buffer solution comprising sodium citrate and Tris at pH 7.5 may be added to the crushed first precipitate and is gently mixed.
  • the solution may be sterilized using a filter (e.g., with 1.0 ⁇ m and 0.22 ⁇ m filter elements in sequence) and the protein content is measured.
  • glycine is added, upon gently mixing until the glycine is dissolved, then the solution may be sterilized using a filter (e.g., with 1.0 ⁇ m and 0.22 ⁇ m filter elements in sequence) , and the filtrate is collected.
  • a filter e.g., with 1.0 ⁇ m and 0.22 ⁇ m filter elements in sequence
  • a second precipitation (or salting out) step may be as follows in exemplary embodiments: to collected fibrinogen solution obtained after the precipitation (e.g. 380 L) glycine is added (e.g., 2709 g or e.g., about 7 mg glycine/ml fibrinogen solution) , followed by gently mixing to dissolve the glycine, then the solution is cooled (e.g., at 0 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 followed by gently mixing (e.g., for about 30 minutes) .
  • glycine e.g., 2709 g or e.g., about 7 mg glycine/ml fibrinogen solution
  • sodium acetate e.g. 87,020 g or e.g., about 150 g/l sodium acetate/m
  • the solution is centrifuged (e.g., at a speed of 4000 rpm) in a refrigerated centrifuge (e.g., at a temperature of 3°C) , and the precipitate is collected (e.g., to obtain 25,818 g of total protein) .
  • a refrigerated centrifuge e.g., at a temperature of 3°C
  • a third precipitation (or salting out) step may be as follows in exemplary embodiments: to the second precipitate containing fibrinogen (e.g., 25,818 g total protein) obtained in the preceding step a buffer solution of pH 10.0 made of sodium citrate and Tris is added (e.g., about 360 liters) followed by gently mixing, followed by a sterile filtration. Next, glycine (e.g., 1152 g or 3.2 g glycine/L) is added followed by gently mixing to dissolve the glycine. The solution is then cooled (e.g., to 3°C) , and sodium acetate is added (e.g., 52,200 g) followed by gently mixing (e.g., for 30 minutes) .
  • glycine e.g., 1152 g or 3.2 g glycine/L
  • the solution is centrifugated in a refrigerated centrifuge (e.g., at about 3°C at about 4000 rpm for about 40 min, ) and the precipitate is then collected.
  • This precipitate is washed 4 times under 4°C, then 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 buffer solution, as well as 1600 ml albumin solution which contained 40 g protein, with water for injection (WFI) being added to adjust the total volume of the solution to about 80L) .
  • WFI water for injection
  • the solution may be prepared with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier which is suitable for administration to a human or other vertebrates.
  • carrier denotes an ingredient with which the components are combined to facilitate the application of the composition in a manner such that the desired efficiency is substantially retained.
  • fibrinogen composition comprises a co-stabilizer and an excipient selected from histidine, sodium citrate, sucrose, sodium chloride, Tween 80, arginine, albumin, and combinations thereof.
  • Fibrinogen-containing powder may be obtained, in exemplary embodiments, by lyophilization of the formulated solution in Borosilicate vials (e.g., frozen under a temperature of about -40°C for 10 hours, drying under a temperature of about -30°C for 62 hours, and second drying under a temperature of about 40°C for 8 hours) .
  • Another viral inactivation sub-step may be applied, for example by heat inactivation of dried material. That is, beside SD, after lyophilization, another viral inactivation step may be carried out by heat inactivation using dry heat at e.g., 100 °C for 30 minutes.
  • composition is a process in which different chemical substances, including the active drug, e.g., fibrinogen, are combined to produce a final medicinal product.
  • active drug e.g., fibrinogen
  • stabilizers are added to preserve the structure of the molecule (s) and excipients.
  • a lyophilized drug is reconstituted as a liquid composition before being administered.
  • Reconstitution is carried out by combining a liquid diluent (e.g., aqueous solution such as water for injection) with the freeze-dried powder/cake, mixing, and then applying it in the desired site.
  • a liquid diluent e.g., aqueous solution such as water for injection
  • Fibrinogen liquid diluent may comprise sodium acetate and/or the sodium chloride solution.
  • the disclosed method using prothrombin depleted plasma/sample is cost effective as it allows preparation of both fibrinogen and thrombin from the same animal or vertebrate blood source or sample e.g., blood from same animal or vertebrate, and/or from same pool of blood.
  • the disclosed method using prothrombin depleted plasma/sample is cost effective as it allows preparation of both fibrinogen and thrombin from the same pig blood source or sample e.g., blood from same pig, and/or from same pool of blood.
  • both fibrinogen products have similar properties, meet the acceptance criteria, and are stable.
  • the obtained results show that the process of preparation of fibrinogen using prothrombin depleted plasma has no negative impact neither on the formulation, filtration, lyophilization process, nor on the yield of intermediate product or of the final product.
  • the critical quality of the final product meets all the 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 the fibrinogen was prepared using plasma in which prothrombin has been removed, compared to fibrinogen prepared using raw plasma. The disclosed process is therefore controllable.
  • ml/L refers to after lyophilization+reconstitution
  • the preparation of fibrinogen comprises not less than 55% (w/v) fibrinogen out of the total protein content.
  • fibrinogen manufacture is prepared using plasma in which prothrombin has been removed as a starting material (e.g., by treating plasma with barium sulfate) instead of using raw plasma.
  • This disclosed approach is advantageous from the economic point of view as it allows preparing both fibrinogen and thrombin from the same blood source as mentioned above. Also, the utilization rate of plasma produced using plasma in which prothrombin has been removed as a starting material (e.g., by treating plasma with barium sulfate) may be improved significantly compared to a process of producing fibrinogen and thrombin from raw plasma. As mentioned, the disclosed method using prothrombin depleted is cost effective as it allows preparation of both fibrinogen and thrombin from the same animal or vertebrate plasma source or sample e.g., blood from same animal or vertebrate, and/or from same pool of blood plasma.
  • Thrombin may be prepared from the prothrombin-comprising plasma fraction.
  • preparation of thrombin comprises the steps of elution of prothrombin from the carrier in the prothrombin-comprising plasma fraction and activation of the eluted prothrombin.
  • preparation of thrombin comprises elution of prothrombin from barium sulfate in the prothrombin-comprising plasma fraction and activation of the eluted prothrombin.
  • conditions which allow conversion of prothrombin into thrombin comprise subjecting the prothrombin to an activator such as calcium ions.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • Porcine plasma samples (A-167.0 L, B-218.0 L, and C-141L) were obtained by adding anticoagulant stock solution in a 1/10 dilution with blood (assume about 40%of plasma can be obtained from the whole blood; the calculated results for total blood in each case can be 1/0.4 i.e. A-417L, B-545L and C-352L) .
  • the anticoagulant stock solution contained 0.135 mol/L potassium oxalate.
  • the blood/anticoagulant mixture was placed in a 12°C cryogenic storage for 8 hours and was separated to an upper layer of plasma and a lower layer of red blood cell. Plasma suction was carried out by a siphon method, and the cells were discarded.
  • the upper layer of plasma was centrifuged at a speed of 4000 rpm at 10°C for 50 minutes, and the plasma was collected and filtered with a 0.22 ⁇ m filter.
  • the filtered plasma was subjected to SD treatment.
  • SD treatment was carried out by adding Tween 80 at a final concentration of 0.5%(v/v) and TNBP at a final concentration of 0.15% (v/v) , at pH 7.0-9.0 and then incubating at 25°C for 6 hours.
  • BaSO 4 adsorption was carried out. Briefly, BaSO 4 reagent (purchased from Shang Hai Merck Chemical Co., Ltd. ) was added to a unit of SD-treated plasma. A final concentration of 2%w/v BaSO 4 was gently mixed with the plasma for 2 hours at 25°C. Next, the BaSO 4 with adsorbed prothrombin was separated from the plasma. Separation was carried out by centrifugation at a speed of 5000 rpm for 40 minutes at a temperature of about 10°C. The supernatant or "prothrombin depleted and SD-treated plasma" was kept cold (4 °C) until use. The separated BaSO 4 with adsorbed prothrombin or prothrombin plasma fraction was kept cold ( ⁇ -10 °C) until use.
  • Fibrinogen lyophilized powder was prepared using prothrombin depleted and SD-treated plasma as follows:
  • a first precipitation step (by salting out) : an amount of 3.5 mg glycine was added per ml plasma and was dissolved. The plasma was cooled to 4°C, and 130 mg of sodium acetate per ml plasma was added, gently mixed for 30 minutes, followed by centrifugation at 10°C in a high-speed refrigerated centrifuge at 3500 rpm for 40 minutes. This first precipitate was collected and crushed.
  • Second precipitation (by salting out) : to collected 380 L fibrinogen solution, 2709 g of glycine were added, gently mixed until the glycine was dissolved, then the solution was cooled to 0-4°C, and 87,020 g of sodium acetate were added and gently mixed for 30 minutes. Next, the solution was centrifugated at a speed of 4000 rpm at a temperature of 3°C, and the precipitate was collected to obtain 25,818 g of the first purified product of the fibrinogen.
  • Third precipitate and lyophilization (by salting out) : to the 25,818 g (total precipitate weight obtained) in the preceding step 360 L of a buffer solution of pH 10.0 made of sodium citrate and Tris was added and gently mixed until dissolved and followed by filtration. Next, 1152 g glycine were added and gently mixed until dissolved. The solution was cooled to 3°C, and 52,200 g of sodium acetate were added and gently mixed for 30 minutes. Next, the solution was centrifugated at 4000 rpm 40 min at 3°C, and the precipitate was collected. This precipitate was washed and filtrated 4 times under 4°C to wash away unwanted residues.
  • Total protein was retained after filtration, then the protein was dissolved and formulated (560 g histidine, 720 g sodiumcitrate, 1200 g sucrose, 160 g sodium chloride, 40 g Tween 80 and 14.4 g arginine as a buffer solution were added, as well as 1600 ml albumin solution containing 40 g protein content, and water for injection (WFI) was added to adjust the total volume to 80 L) .
  • the final protein concentration was 50 g/L (albumin: 0.5 g/L) .
  • Fibrinogen-containing powder was obtained by lyophilization of the formulated solution in Borosilicate vials (frozen under-40°C for 10 hours; primary drying was caried out under-30°C for 62 hours, followed by a second drying under 40°C for 8 hours) .
  • another viral inactivation step i.e. in addition to SD in the oxalated plasma
  • heat inactivation was carried out by heat inactivation.
  • the lyophilized powder was subjected to dry heat at 100 °C for 30 minutes.
  • Thrombin was prepared from BaSO 4 adsorbed prothrombin separated from SD-treated plasma as in Example 1.
  • BaSO 4 adsorbed prothrombin was washed (added 9%sodium chloride solution 8L, gently mixed 30 min at room temperature, then centrifugated 30 min at a speed of 3200 rpm at a temperature of 4°C; this step was repeated two times) .
  • Prothrombin was thereafter eluted (added 10%sodium citrate solution 8L, gently mixed 30 min at room temperature, then centrifugated 30 min at a speed of 3500 rpm at a temperature of 4°C, this step was repeated four times) .
  • the prothrombin was thereafter activated (added 1.3L 36%calcium chloride solution and 1.3L 20%glycine solution, gently mixed and adjusted pH to 7.0, stored 8 hours at room temperature then turned to store at a temperature of 2°C for 2 days. An amount of 3.2L thrombin bulk solution was obtained by ultrafiltration and centrifugation) .
  • the produced thrombin was thereafter formulated (added 800 g glycine and 6.7L 6%w/v albumin solution, added WFI to adjust to a total volume of 27L) .
  • the formulated thrombin was lyophilized (frozen under-40°C for 10 hours, a primary drying step was performed at-25°C for 21 hours, a second drying step was performed at 40 °C for 8 hours) .
  • the lyophilized thrombin was then subjected to heat inactivation (dry heat at 100°C for 30 minutes) .
  • fibrinogen manufacturing is obtained using plasma in which prothrombin has been removed as a starting material (by treating the plasma with barium sulfate) instead of using raw plasma.
  • this approach is advantageous from the economic point of view as it allows preparing both fibrinogen and thrombin from the same blood source.
  • FIG. 1A shows a schematic purification process of the invention in embodiments thereof (Figure 1A) compared to fibrinogen preparation starting from raw plasma ( Figure 1B) .
  • the fibrinogen content obtained from raw plasma (the process is described in CN101214391B which is incorporated herein by reference)
  • that obtained from the prothrombin depleted plasma were both within the range of the upper and lower control limits and within internal acceptance criteria. Therefore, the fibrinogen content of each batch of lyophilized powder obtained either from raw plasma or from prothrombin depleted plasma meets the acceptance criteria.
  • the production process using prothrombin depleted plasma is controllable as there is no abnormal point out of the control limit.
  • the fibrinogen purity as obtained from raw plasma and from prothrombin depleted plasma were both within the range of the upper and lower control limits and within internal acceptance criteria. Therefore, the fibrinogen purity of each batch of lyophilized powder, obtained either from the raw plasma or from the prothrombin depleted plasma meets the acceptance criteria, there is no abnormal point out of the control limit, so the production process using prothrombin depleted plasma is controllable also in this regard.
  • the water content of the products obtained from raw plasma and from prothrombin depleted plasma were both within the range of the upper and lower control limits and within the internal acceptance criteria. Therefore, the water content of each batch of lyophilized powder obtained either from the raw plasma or from the prothrombin depleted plasma meets the acceptance criteria, there is no abnormal point out of the control limit, so the production process using prothrombin depleted plasma is controllable also in this regard.
  • sucrose content of each product obtained from either the raw plasma or from the prothrombin depleted plasma was within the range of the upper and lower control limits and met the internal acceptance criteria. Therefore, the sucrose content of each batch of lyophilized powder obtained either from the raw plasma or from the prothrombin depleted plasma meets the acceptance criteria, there is no abnormal point out of the control limit, so the production process using prothrombin depleted plasma is controllable also in this regard.
  • the sodium chloride content of each point of a product or an intermediate product obtained either from raw plasma or from prothrombin depleted plasma was within the range of the upper and lower control limits and met the internal acceptance criteria. Therefore, the sodium chloride content of each batch of either the lyophilized powder from raw plasma or the prothrombin depleted plasma met the acceptance criteria, there is no abnormal point out of the control limit, so the production process using prothrombin depleted plasma is controllable also in this regard.
  • the Tween 80 residue of each point of a product or an intermediate product obtained either from raw plasma or from prothrombin depleted plasma was within the range of the upper and lower control limits and met the internal acceptance criteria. Therefore, the Tween 80 residue of each batch of lyophilized powder obtained either from raw plasma or from prothrombin depleted plasma meets the acceptance criteria, there is no abnormal point out of the control limit, so the production process using prothrombin depleted plasma is controllable also in this regard.
  • the TNBP residue of each point of a product or an intermediate product obtained either from raw plasma or from prothrombin depleted plasma was within the range of the upper and lower control limits and met the internal acceptance criteria. Therefore, the TNBP residue of each batch of lyophilized powder obtained either from raw plasma or from prothrombin depleted plasma meets the acceptance criteria, there is no abnormal point out of the control limit, so the production process using prothrombin depleted plasma is controllable also in this regard.
  • the 1 st precipitation solution protein of each point of an intermediate product obtained from raw plasma or from prothrombin depleted plasma was within the range of the upper and lower control limits and met the internal acceptance criteria. Therefore, the 1 st precipitation solution protein of each batch of lyophilized powder obtained from raw plasma or from prothrombin depleted plasma meets the acceptance criteria, there is no abnormal point out of the control limit, so the production process using prothrombin depleted plasma is controllable also in this regard.
  • the fibrinogen yield of each point of the product obtained either from raw plasma or from prothrombin depleted plasma was within the range of the upper and lower control limits and met the internal acceptance criteria. Therefore, the fibrinogen yield of each batch of lyophilized powder obtained either from raw plasma or from prothrombin depleted plasma meets the acceptance criteria, there is no abnormal point out of the control limit, so the production process from using prothrombin depleted plasma is controllable also in this regard.
  • the potassium ion residue contained in the final fibrinogen product was explored in view of the potassium oxalate anticoagulant used in blood collection.
  • the level of potassium was compared with that in a fibrinogen product using sodium citrate as anticoagulant.
  • Table 2 presents a list of potassium ion content of fibrinogen prepared from raw plasma (in which sodium citrate was used as an anticoagulant) or from prothrombin depleted plasma (in which potassium oxalate was used as the anticoagulant, 25g/L; anticoagulation: blood ratio was 8-to-10: 1, respectively) .
  • the potassium ion content of the final product is within the range of the upper and lower control limits and meets the acceptance criteria for human albumin for injection no matter if sodium citrate or potassium oxalate are used as the anticoagulant. Therefore, there is no significant change in potassium ion content between using sodium citrate or potassium oxalate as anticoagulant, so the production process using potassium oxalate as anticoagulant is controllable.
  • Table 2 shows fibrinogen product from anticoagulant treated citrated plasma and potassium oxalate treated plasma.

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Abstract

L'invention concerne des procédés de préparation de fibrinogène par purification de fibrinogène à partir d'une fraction de plasma appauvrie en prothrombine. La fraction de plasma appauvrie en prothrombine est obtenue, par exemple, par ajout d'un anticoagulant à une source de sang ; séparation du plasma de la source de sang, et extraction de la prothrombine du plasma, ce qui permet d'obtenir une fraction de plasma appauvrie en prothrombine et une fraction de plasma constituée de prothrombine.
PCT/CN2022/136420 2022-12-05 2022-12-05 Préparation de fibrinogène WO2024119285A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102286095A (zh) * 2011-07-06 2011-12-21 大田华灿生物科技有限公司 纤维蛋白原的制备方法
CN105950576A (zh) * 2016-05-26 2016-09-21 成都远睿生物技术有限公司 一种从牛血中提取多种蛋白的方法
CN105985940A (zh) * 2015-02-06 2016-10-05 广州倍绣生物技术有限公司 用于制备凝血酶的方法
WO2017078947A1 (fr) * 2015-10-21 2017-05-11 Cambryn Biologics, Llc Procédés de purification de protéines du plasma
CN107485728A (zh) * 2016-06-12 2017-12-19 胡庆柳 一种胶原纤维蛋白复合止血贴及其制备方法
WO2021108976A1 (fr) * 2019-12-03 2021-06-10 Omrix Biopharmaceuticals Ltd. Purification de pro-thrombine
CN115109142A (zh) * 2022-08-08 2022-09-27 长春西诺生物科技有限公司 一种从犬血浆中同时分离犬纤维蛋白原和犬凝血酶原复合物的方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102286095A (zh) * 2011-07-06 2011-12-21 大田华灿生物科技有限公司 纤维蛋白原的制备方法
CN105985940A (zh) * 2015-02-06 2016-10-05 广州倍绣生物技术有限公司 用于制备凝血酶的方法
WO2017078947A1 (fr) * 2015-10-21 2017-05-11 Cambryn Biologics, Llc Procédés de purification de protéines du plasma
CN105950576A (zh) * 2016-05-26 2016-09-21 成都远睿生物技术有限公司 一种从牛血中提取多种蛋白的方法
CN107485728A (zh) * 2016-06-12 2017-12-19 胡庆柳 一种胶原纤维蛋白复合止血贴及其制备方法
WO2021108976A1 (fr) * 2019-12-03 2021-06-10 Omrix Biopharmaceuticals Ltd. Purification de pro-thrombine
CN115109142A (zh) * 2022-08-08 2022-09-27 长春西诺生物科技有限公司 一种从犬血浆中同时分离犬纤维蛋白原和犬凝血酶原复合物的方法

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