WO2022133973A1 - Filtration aseptique de fibrinogène - Google Patents

Filtration aseptique de fibrinogène Download PDF

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Publication number
WO2022133973A1
WO2022133973A1 PCT/CN2020/139213 CN2020139213W WO2022133973A1 WO 2022133973 A1 WO2022133973 A1 WO 2022133973A1 CN 2020139213 W CN2020139213 W CN 2020139213W WO 2022133973 A1 WO2022133973 A1 WO 2022133973A1
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Prior art keywords
filter
fibrinogen
filtration
solution
glass fiber
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PCT/CN2020/139213
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English (en)
Inventor
Aibin Yu
Shuang Chen
Hai Li
Xuemei Zhang
Yufu Li
Original Assignee
Guangzhou Bioseal Biotech Co., Ltd.
Ethicon, Inc.
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Application filed by Guangzhou Bioseal Biotech Co., Ltd., Ethicon, Inc. filed Critical Guangzhou Bioseal Biotech Co., Ltd.
Priority to CN202080108137.2A priority Critical patent/CN116744986A/zh
Priority to PCT/CN2020/139213 priority patent/WO2022133973A1/fr
Publication of WO2022133973A1 publication Critical patent/WO2022133973A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/106—Fibrin; Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
    • A61L2/0017—Filtration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
    • A61L2/022—Filtration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3616—Blood, e.g. platelet-rich plasma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2202/00—Aspects relating to methods or apparatus for disinfecting or sterilising materials or objects
    • A61L2202/20—Targets to be treated
    • A61L2202/22—Blood or products thereof

Definitions

  • the present invention relates, inter alia, to methods of sterile filtration of a fibrinogen solution.
  • Fibrinogen is a glycoprotein complex that circulates in the blood of vertebrates.
  • Fibrinogen is an essential protein for blood coagulation. Its polymerization into insoluble fibrin formed at the end of the cascade of reactions governs coagulation, resulting in the formation of a clot closing the vascular breach responsible for the bleeding. Placement of the clot is therefore essential to ensure that the bleeding stops.
  • compositions comprising fibrinogen in particular for therapeutic purposes, requires purification techniques leading to a product that is sufficiently purified from contaminants of various nature.
  • U.S. Patent No. 5,259,971 discloses a method of purifying native, intact fibrinogen from a liquid sample containing contaminants having molecular weights higher and/or lower than that of the fibrinogen. The method comprises subjecting the sample to filtration using one or more filters having a molecular weight cut-off such that the native, intact fibrinogen is separated from the contaminants.
  • U.S. Patent No. 1,0493,133 discloses a method for preparing a highly concentrated fibrinogen solution includes adding amino acid or amino acid derivatives, and/or salts to a lowly concentrated fibrinogen solution, followed by a ultra-filtration concentration.
  • U.S. Patent No. 7,816,495 discloses processes for the purification of fibrinogen, and to readily solubilised fibrinogen preparations.
  • U.S. Patent No. 7,309,428 discloses a process and apparatus for separating blood plasma, having a mixing unit in the form of a first injection having a first connecting tube and a first piston to provide a compartment for a mixture composed of plasma to be separated and the protein-precipitating agent; a separating unit composed of a filtering tube for separating and preserving a solid material after separation.
  • U.S. Patent No. 9,561,478 discloses a separation membrane which includes a membrane comprising a polymer, characterized in that a functional layer is formed on the surface in one side of the membrane, the peak area percentage of carbon derived from ester group measured by the electron spectroscopy for chemical analysis (ESCA) on the surface of the preceding functional layer is 0.1% (by atomic number) or more but not more than 10 (% by atomic number) , and the peak area percentage of carbon derived from ester group measured by the electron spectroscopy for chemical analysis (ESCA) on the surface opposite to the functional layer is not more than 10 (%by atomic number) .
  • a separation membrane module suffering from little sticking of organic matters, proteins, platelets and so on is provided with the separation membrane as a built-in membrane.
  • JP Patent Application No. 2007215569 discloses a plasma component separator which is a hollow fiber membrane type plasma component separator in which a hollow fiber membrane is composed of a polysulfone polymer and polyvinylpyrrolidone, the permeability of bovine plasma immunoglobulin G is ⁇ 80%and ⁇ 100%, the permeability of bovine plasma fibrinogen is ⁇ 10%and ⁇ 30%, the liquid surface elevation value of the hollow fiber membrane measured by a capillary-rise method is ⁇ 80mm and ⁇ 120mm when converted to the hollow fiber membrane of an inner diameter 200 ⁇ m, and the circle conversion rate diameter of the hole diameter of the inner surface of the hollow fiber membrane is 13nm-64nm.
  • the present invention relates, inter alia, to methods of sterile filtration of a fibrinogen solution.
  • the Fibrinogen protein concentration in typical formulations reaches 60 to 120 mg/ml.
  • the fibrinogen molecule has a typical molecular weight of about 340 kDa, has a rod-like shape with dimensions of 9 ⁇ 47.5 ⁇ 6 nm, and easily forms multimeric particles in aqueous solutions. Such particles are not dissolved and may easily accumulate on the surface of filtration membranes used in sterilizing filtration operations, blocking the membranes and making the filtration difficult to perform.
  • PVDF polyvinylidene difluoride
  • filter membrane is used, with one square meter of membrane being capable of filtering only about 5 to 10 L fibrinogen solution, due to clogging of the filter membrane.
  • the filter needs to be replaced multiple times, the operation is complex, and the risk of microbial contamination is high.
  • An object of the present disclosure is to develop a method for an aseptic filtration of a fibrinogen solution which allows obtaining a sterile fibrinogen composition, and has an increased filtration capacity (more than about 90%) .
  • the inventors have developed a new filtration method which surprisingly provides extraordinary filtering capacity for fibrinogen formulation liquid e.g., 50 to 100 L/m 2 membrane, which is easy to implement in an industrial scale in an acceptable industrial cost price.
  • a method of sterile filtration of a protein-of-interest-comprising solution comprising: filtering the solution with at least one filter selected from a glass fiber pre-filter and an aseptic filter membrane comprising polysulfide or a derivative thereof.
  • the derivative of the polysulfone is selected from polyether sulfone (PES) , polyphenyl sulfone (PPSU) and mixtures thereof.
  • the polysulfone comprises PES.
  • the method comprises filtering the solution with the pre-filter and with polyvinylidene difluoride (PVDF) .
  • PVDF polyvinylidene difluoride
  • the method comprises filtering the solution with polysulfone.
  • the filter comprising polysulfone or a derivative thereof has a removal rating of 0.15 to 0.25 ⁇ m.
  • the glass fiber pre-filter has a removal rating of 0.35 to 0.55 ⁇ m.
  • the method comprises filtering the solution with the glass fiber pre-filter prior to the filtering with the polysulfone.
  • the filtration is carried out at a pressure of 2 to 30 psi.
  • the solution is obtained from a plasma fraction.
  • the plasma fraction is obtained from porcine.
  • the protein-of-interest comprises fibrinogen.
  • the method is characterized by a filtration capacity of the solution, e.g., a fibrinogen solution, of at least 5 kg/m 2 . In some embodiments of any aspect provided herein, the method is characterized by a filtration capacity of the solution, e.g., a fibrinogen solution, of at least 15 kg/m 2 . In some embodiments of any aspect provided herein, the method is characterized by a filtration capacity of the solution, e.g., a fibrinogen solution, of at least 40 kg/m 2 .
  • the method is characterized by a fibrinogen recovery of at least 85%.
  • the at least one filter has non-uniform pore size distribution.
  • a sterile filtration of a fibrinogen comprising solution comprising a first filtration carried out on a filter having a removal rating of 0.35 to 0.55 ⁇ m, and a second filtration carried out on a filter having a removal rating of 0.15 to 0.25 ⁇ m.
  • a sterile fibrinogen solution obtained by a method according to any aspect provided herein.
  • Fig. 1 presents a bar graph showing comparative filtration capacities (kg/m 2 ) of several batches of fibrinogen solution using in each triplet: polyvinylidene difluoride ( "PVDF” ; left bar) , glass fiber pre-filter ( “PRE” ; middle bar) , and polyethersulfone ( “PES” ; right bar) .
  • PVDF polyvinylidene difluoride
  • PRE glass fiber pre-filter
  • PES polyethersulfone
  • Fig. 2 presents a bar graph showing comparative filtration capacities (kg/m 2 ) of several batches of fibrinogen solution as presented in Fig. 1, with further showing the filtration capacity upon using PRE+PES filters (right bars added in each quartet) .
  • An object of the present invention is to provide an efficient method for aseptic filtration of fibrinogen from a solution.
  • a method of sterile filtration of a protein-of-interest-comprising mixture or solution comprising: filtering the mixture or solution with one or more filters selected from a glass fiber pre-filter and an aseptic filter comprising polysulfone.
  • a filter or membrane is employed to yield product (s) .
  • the method will either not yield the desired product having a desired purity, concentration, and the like, or an increased capacity.
  • the filter or membrane component is positioned within the fluid process stream and is capable of filtering the aforementioned mixture or solution as it passes therethrough.
  • filtration includes all of those separation processes as well as any other processes utilizing a filter that separates one.
  • the step of passing the fibrinogen-containing solution in the filter allows to remove therefrom micro floc which may be present in the solution.
  • the filtration of the disclosure is not an ultrafiltration step or process.
  • ultrafiltration refers to the process of separating a liquid into fractions by pressure-driven flow through semi-permeable membranes having molecular weight cutoffs in the range from 200 to about 330,000 and pore diameters from about 10 to 1000 Angstroms.
  • filter also referred to as "filter membrane” may refer to sieves, sheet metal screens or net-like structures for filtration.
  • the filter may for example be designed as a planar membrane or as a planar screen.
  • the filter may be permeselective and serve for the filtering out of particles from a fluid, in particular for the production of highly pure proteins of interest.
  • filtering the solution it is meant to refer to a step of passing the solution, e.g., source solution containing the protein of interest e.g., fibrinogen, in a filter to remove therefrom micro floc which may be present. That is, the fibrinogen may pass the filter with the contaminant e.g., bacteria, retaining trapped into or onto the filter.
  • the source solution is fresh, or, in some embodiments, the source solution had been frozen and was thawed prior to filtration, which typically may have more multimers that may reduce the filtration capacity as compared to the fresh solution (i.e. has not been frozen) .
  • mixture refers, but is not limited to, a combination of components in any physical form, e.g., blend, solution, suspension, dispersion, or the like.
  • sterile filtration is a functional description that a preparation is filtered through a sterile filter to remove contaminants such as bacterial and/or mycoplasma.
  • sterile refers to a substance that is free, or essentially free, of microbial and/or viral contamination.
  • contaminant means a material that is different from desired protein (s) of interest.
  • the step of aseptic filtration may achieve at least 1-log at least 2-log, at least 3-log, at least 4-log, or at least 5-log reduction of microorganisms in the source solution.
  • the glass fiber filter is made of resin-bonded glass fiber such as, without limitation, glass wool, however, rock wool, long glass fibers (filaments) or glass fiber cloth may be used.
  • resin-bonded glass fiber such as, without limitation, glass wool, however, rock wool, long glass fibers (filaments) or glass fiber cloth may be used.
  • acid resistant or alkali resistant glass fibers containing an alkaline oxide can be used as the glass fiber for the filter layer.
  • the density of the glass fiber filter may be in the range of 150 -250 kg/m 3 when pressure loss is considered, and the thickness of the glass fiber filter may be in the range of 40 -60 mm.
  • the mixture is a liquid sample comprising fibrinogen.
  • the fibrinogen is present at a concentration of 15 mg/ml or less.
  • the purified protein, e.g., fibrinogen may be precipitated using chemical agents followed by centrifugation or lyophilization. The sample may be concentrated by the filtration in the absence of further dilution.
  • pre-filter designates a filter that is likely to stop relatively large-sized solid particles, typically in excess of 100 micrometers. Typically, but not exclusively, the pre-filter is positioned upstream of the main, primary filter (s) .
  • the protein-of-interest-comprising mixture comprises a source solution.
  • Source solution broadly refers to a combination, mixture and/or admixture of ingredients having at least one liquid component and a protein of interest. Solutions typically include at least one solvent in greater quantity or volume than a solute. Typical solvents include water.
  • the source solution comprises a mixture of proteins.
  • the source solution comprises plasma, typically blood plasma or a fraction thereof.
  • the plasma comprises oxalated plasma.
  • the source solution comprises plasma harvested from a mammal.
  • the mammal is selected from a human, an equine, a bovine and a porcine.
  • the source solution comprises porcine plasma.
  • the plasma proteins may include one or more blood clotting factors (F) such as fibrinogen, prothrombin, thrombin, FX, FXa, FIX, FIXa, FVII, FVIIa, FVIII, FVIIIa, FXI, FXIa, FXII, FXIIa, FXIII, FXIIIa, von Willebrand factor etc., transport proteins such as albumin, transferrin, ceruloplasmin, haptoglobin, hemoglobin, hemopexin, etc., protease inhibitors such as ⁇ -antithrombin, ⁇ -antithrombin, ⁇ 2-macroglobulin, Cl-inhibitor, tissue factor pathway inhibitor (TFPI) , heparin cofactor II, protein C inhibitor (PAI-3) , Protein C, Protein S, etc., antiangionetic proteins such as latent-antithrombin, etc., highly glycosylated proteins including alfa-1-acid glycoprotein
  • proteins such as histidine-rich glycoprotein, mannan binding lectin, C4-binding protein, fibronectin, GC-globulin, plasminogen, blood factors such as erythropoeitin, interferon, tumor factors, tPA, gCSF and derivatives and muteins thereof.
  • the protein of interest comprises fibrinogen.
  • fibrinogen refers to a precursor protein of the blood clot matrix.
  • the fibrinogen has a molecular weight of about 340,000 Daltons and has three pairs of non-identical polypeptide chains, A ⁇ , B ⁇ and ⁇ , linked together by disulfide bonds.
  • fibrinogen has a trinodular structure: two identical D terminal globular domains and a central E globular domain connected by supercoiled ⁇ -helices.
  • the fibrinogen originates from a fibrinogen concentrate. In some embodiments, the fibrinogen is a blood derived fibrinogen concentrate.
  • the terms "originates from” or “derived from” are used interchangeably and refer to an origin or source of the relevant component, which may include naturally occurring, recombinant, processed, unpurified or purified molecules (e.g., the relevant protein) .
  • the source solution comprises plasma fraction obtained from prepurified blood plasma.
  • plasma fraction obtained from prepurified blood plasma it is meant to refer to any part or sub-part of human blood plasma, which has been the subject of one or more purification steps.
  • plasma fractions thus include the supernatant of cryoprecipitated plasma, the cryoprecipitate of plasma (resuspended) , fraction I obtained by ethanolic fractionation (according to the method of Cohn or Kistler &Nitschmann) , the eluates of chromatography, and the non-adsorbed fractions.
  • the fibrinogen composition subjected to the process of the invention undergoes an additional chromatography step.
  • the fibrinogen composition subjected to the method according to the invention is a chromatography eluate or a non-adsorbed fraction from a chromatography column, including multicolumn chromatography.
  • polysulfone is used generically to describe any polymer containing repeating or recurring units of one or more diamyl sulfone groups (e.g., monomers) of general formula - (Ar-SO 2 -Ar) -, where Ar is a substituted or unsubstituted aryl group such as a phenyl, biphenyl, bisphenol or any other aryl group containing an aromatic hydrocarbon or hetero-aromatic ring.
  • the polysulfone is selected from polysulfone (PSU) , polyethersulfone (PES) , polyphenylsulfone (PPSU) and mixtures or derivative thereof.
  • derivative refers to subject molecules which has been chemically modified but retain a major portion thereof unchanged, e.g., subject molecules which are substituted by additional or different substituents, subject molecules in which a portion thereof has been oxidized or hydrolyzed, and the like.
  • polymer describes an organic substance composed of a plurality of repeating structural units (monomeric units) covalently connected to one another.
  • the method comprises filtering the source solution with a glass fiber pre-filter, and polyvinylidene difluoride (PVDF) filter downstream or below thereof. In some embodiments, the method comprises filtering the source solution with a glass fiber pre-filter, and polysulfone filter downstream or below thereof.
  • PVDF polyvinylidene difluoride
  • the glass fiber pre-filter may have a removal rating of 0.3 to 0.6 ⁇ m, e.g., 0.4 ⁇ m, 0.5 ⁇ m, or 0.6 ⁇ m, including any value and range therebetween, which means that particles larger than the indicated size are effectively trapped by the filter.
  • the filter may have a pore size of about 1 ⁇ m or less, or about 0.5 ⁇ m or less. It will be appreciated that the removal rating and the pore size are different parameters, the removal rating reflecting the performance of the filter, and the pore size being a property of the filter film, although these parameters may influence one another and be closely related.
  • the glass fiber pre-filter is characterized by maximum operating pressure of 20 to 70 psi at around room temperature. In some embodiments, the glass fiber pre-filter is characterized by maximum operating pressure of 20, 30, 40, 50, 60, or 70 psi, including any value and range therebetween, at around room temperature.
  • maximum operating pressure refers to the highest pressure that does not damage the filter. Differential pressure is the difference in pressure between the filter inlet and the filter outlet.
  • the operating pressure is controlled by a pump e.g., to develop a differential pressure output side and the input side of the filters with the pressure at the output side being greater.
  • a pump refers to any device that causes the movement of fluids by applying suction or pressure e.g., by a compressed air.
  • around room temperature it is meant to refer to at least one temperature value within the range of 10 to 40 °C, or 15 to 37 °C. e.g., 10, 15, 20, 25, 30, 35, 37, or 40 °C, including any value and range therebetween.
  • the progress of the filtration process may be monitored using a variety of means e.g., by detecting contaminant concentration on the filter.
  • the filtration capacity of the fibrinogen solution is increased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, as compared to the old process using PVDF filter alone.
  • the filtration capacity is increased by 30%to 150%as compared to the traditional process using PVDF filter alone.
  • filtration capacity it is meant to refer to the protein of interest (e.g., fibrinogen) maximal load per membrane area, expressed in kg of the protein per m 2 of membrane of the relevant filter (or combination of filters) .
  • the filtration capacity is at least 5, at least 5.5 or at least 6 kg/m 2 . In some embodiments, upon using a glass fiber pre-filter followed by PVDF filter the filtration capacity is 4.5 to 15 kg/m 2 . In some embodiments, upon using a glass fiber pre-filter followed by PVDF filter the filtration capacity is 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5 or 15 kg/m 2 , including any value and range therebetween.
  • a method for increasing a filtration capacity of PVDF filtration by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, in sterile filtration of a protein-of-interest (e.g., fibrinogen) -comprising solution comprising filtering the solution with at least one pre-filter comprising a glass fiber prior to PVDF filtration.
  • a protein-of-interest e.g., fibrinogen
  • the solution comprising the protein-of-interest comprises fibrinogen which has not been previously frozen and/or thawed prior to the filtration process.
  • a material which has been frozen and thawed may have more multimers, insoluble, aggregated, or partially denatured material, thereby reducing the filtration capacity (when using a pre-filter) by about 50%as compared to a fresh solution, i.e. a solution which has not been previously frozen and/or thawed.
  • the fibrinogen recovery upon using a glass fiber pre-filter followed by PVDF filter the fibrinogen recovery is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, upon using a glass fiber pre-filter followed by PVDF filter the fibrinogen recovery is about 88%to approximately 100%.
  • the fibrinogen recovery is about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, including any value and range therebetween.
  • the fibrinogen recovery (from the fibrinogen source solution) is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, or at least 12%, compared to a process using PVDF filter and without the pre-filter.
  • the fibrinogen recovery from the fibrinogen solution is increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%, including any value and range therebetween, compared to a process using PVDF filter and without the pre-filter.
  • fibrinogen recovery also referred to as “fibrinogen activity recovery” and “filtration yield” ) describes the fibrinogen concentration (content) found after the filtration process divided by the fibrinogen concentration in the solution prior to the filtration process.
  • the method is characterized by a protein recovery of at least about 95%. In some of any embodiments of any aspect provided herein, the method is characterized by a protein recovery of about 95%to about 98%.
  • protein recovery also referred to as “protein yield”
  • protein recovery describes the protein content found after the filtration process divided by the protein content prior to the filtration process.
  • Protein content refers the amount of protein contained in a substance. Actually, aseptic filtration in the disclosed process of removing microorganisms from a fluid stream (without adversely affecting product quality) allows almost all proteins including fibrinogen pass through the filter.
  • the method comprises filtering the source solution with the polysulfone filter.
  • the filtration capacity is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 6-fold, as compared to the old process using PVDF filter.
  • the filtration capacity is increased by 2-fold to 6-fold as compared to the old process using PVDF filter.
  • the filtration capacity is at least 10, at least 12 or at least 15 kg/m 2 . In some embodiments, upon using a polysulfone (e.g., PES) filter the filtration capacity is 15 to 30 kg/m 2 . In some embodiments, upon using a polysulfone (e.g., PES) filter the filtration capacity is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 29, or 30 kg/m 2 , including any value and range therebetween.
  • a polysulfone e.g., PES
  • the filtration capacity is at least 10, at least 12 or at least 15 kg/m 2 . In some embodiments, upon using a polysulfone (e.g., PES) filter the filtration capacity is 15 to 30 kg/m 2 . In some embodiments, upon using a polysulfone (e.g., PES) filter the filtration capacity is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 29, or 30 kg/m 2 , including any value and range
  • the fibrinogen recovery from the fibrinogen solution is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.
  • a polysulfone e.g., PES
  • the method comprises filtering the source solution with a glass fiber pre-filter and thereafter polysulfone (e.g., PES) filter (i.e. downstream thereof) .
  • polysulfone e.g., PES
  • the use of a glass fiber pre-filter makes it possible to further increase the filtration capacity as compared to the sulfone filter alone.
  • the filtration capacity of the fibrinogen solution is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, or at least 7-fold, as compared to using sulfone filter alone, i.e. without using a glass fiber pre-filter.
  • the filtration capacity is increased by 30%to 150%as compared to the sulfone filter alone.
  • polysulfone e.g., PES
  • the fibrinogen recovery upon using a glass fiber pre-filter followed by the polysulfone (e.g., PES) filter the fibrinogen recovery is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 89%. In some embodiments, upon using a glass fiber pre-filter followed by PVDF filter the fibrinogen recovery is about 88%to approximately 100%.
  • PES polysulfone
  • the fibrinogen recovery is about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 89%, including any value and range therebetween.
  • the polysulfone e.g., PES
  • the hole-or pore shape of the filter may affect the filter's filtration capability. Assumingly, the asymmetric hole filter has a higher interception ability to the small particles than the filter with a uniform (symmetric) hole shape. The pore shapes and sizes of the old PVDF filter membrane appears to be much more uniform than that of glass fiber and PES. At the same time, the 0.45-micron glass fiber pre-filter may intercept large particles in the solution, to help increase the filtration flux of final filter.
  • a method of sterile filtration of a protein-of-interest (e.g., fibrinogen) -comprising mixture comprising: filtering the mixture with at least one filter, having there a gradient pore size from one planar surface to the other or a gradient pore size within the membrane.
  • the filter membrane, or, in some embodiments, a plurality of filter membranes form an overall porous member which is asymmetrically porous, having pores of progressively decreasing diameter in a progression from the upper (dosing) surface to the lower surface such that the overall membrane pores form a “V” shape, are non-uniform, and are not easily blocked by particles in the solution. Additionally, or alternatively, the overall membrane pores are linear and non-uniform.
  • asymmetric or “non-uniform” , it is meant to refer to a non-uniform or broad pore size distribution.
  • this term refers to a median pore size or a removal rating of the upper (dosing) surface being higher than a median pore size or the removal rating of a lower surface by 50%to 400%, or, in some embodiments, by 100 to 200%, e.g., 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, or 400%, including any value and range therebetween.
  • the pore sizes in at least one surface of the membrane vary by 50 to 400%, or, in some embodiments, by 100 to 200%, e.g., 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, or 400%, including any value and range therebetween.
  • size it is meant to refer to at least one dimension of the pore, typically micropore, e.g., diameter.
  • the method of sterile filtration of this aspect comprises forming a gradient pore size by using at least one filter selected from a glass fiber pre-filter and an aseptic filter comprising polysulfone.
  • filters selected from a glass fiber pre-filter and an aseptic filter comprising polysulfone.
  • a method of sterile filtration of a fibrinogen-comprising solution comprising a first filtration carried out on a filter having a removal rating of about 0.35 to about 0.55 ⁇ m (e.g., 0.35, 0.4, 0.45, 0.50, or 0.55 ⁇ m, including any value and range therebetween) , and a second (e.g., sequent) filtration carried out on a filter having a removal rating of about 0.15 to about 0.25 ⁇ m (e.g., 0.15, 0.20, or 0.25 ⁇ m, including any value and range therebetween) .
  • the filtration capacity of the solution is at least 15 kg/m 2 to at least 85 kg/m 2 , e.g., 15, 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 kg/m 2 , including any value and range therebetween.
  • a sterile solution of a fibrinogen obtained by the method disclosed herein according to any aspect or embodiments described above.
  • compositions comprising, “comprising” , “includes” , “including” , “contains” , “containing” , “has” , “having” , and their conjugates mean ” including but not limited to” .
  • the term “consisting of” means “including and limited to” .
  • the term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • 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.
  • method or “process” which may be used herein interchangeably refer to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, analytical, pharmacological, biological, biochemical and medical arts.
  • the terms “by weight” , “w/w” , “weight percent” , or “wt. %” which are used herein interchangeably describe the concentration of a particular substance out of the total weight of the corresponding mixture, solution, formulation or composition.
  • the purpose of this study is to explore a safe and effective new method to increase the filtration flux of the Fibrinogen formulation liquid, so as to achieve the goal of improving the final product yield and saving the filter consumables.
  • Fibrinogen liquid formulation is manufactured by Guangzhou Bioseal biotechnology Co, Ltd, containing 30-50 mg/ml of fibrinogen and is usually stored at -20 to about -30°C and thawed before performing filtration experiments. Various batches were used as described below.
  • Three parallel filtration processing lines were designed to filter the fibrinogen liquid formulation at the same time, and to weigh the filtered liquid in order to compare the filtration capabilities of the different filtration lines.
  • Filtration temperature room temperature, 20-26 degrees.
  • Filtration pressure 2-30 psi, compressed air was used to provide the desired pressure.
  • the experimental parameters of processing lines were as follows:
  • Line 1 Using one Millipore polyvinylidene difluoride (PVDF) filter. Filtration area: 500 cm 2 ; catalog number: MPGL10CL3. This is the same kind of filter used in the old processes.
  • PVDF Millipore polyvinylidene difluoride
  • Line 2 Adding a glass fiber pre-filter (also referred to as "PRE" ) before Millipore PVDF final filter, filter area: 900 cm 2 ; catalogs number: DFA3001UBC.
  • PRE glass fiber pre-filter
  • Line 3 Using a polyether sulfone (PES) final filter instead of Millipore PVDF filter for sterilizing filtration; filter area: 230 cm 2 ; Catalogs number: KA02ECV2FT.
  • PES polyether sulfone
  • the average filtration capacity of PVDF was about 4.7 kg/m 2 membrane; the average filtering capacity of PRE + PVDF was 8.7 kg/m 2 membrane, and the average filtering capacity of PES was much higher: 20.0 kg/m 2 membrane.
  • the combined filtration method of PRE + PES (as the final filter in the filtering process) can further improve the filtration capacity of the fibrinogen formulation liquid, and the average filtration capacity can reach an average of about 80 kg/m 2 membrane.
  • the reason for the difference in the results of '904 and '205 may be due to the fact that the material of '904 had been frozen and was then reconstituted, and upon freezing and then thawing more multimers could be formed which may affect the filtration capacity, while the '205 comprised fresh materials.
  • the hole (pore) shape of the filter membrane affects the filter's filtration capability.
  • the asymmetric hole filter has a higher interception ability to the small particles than the filter with a uniform hole shape.
  • the pore shape of the PVDF filter membrane is much more uniform than that of glass fiber and PES.
  • the uniform membrane might be completely blocked by particles in solution.
  • 0.45-micron glass fiber pre-filter can intercept large particles in the solution. In the glass fiber pre-filter the membrane pores are linear and non-uniform, and are not easily blocked by particles in the solution. And this may help increase the filtration flux of final filter.
  • PES Membrane has “V” shape pores and is also non-uniform, and therefore is not easily blocked by particles in the solution.
  • the average filtration capacity of PRE + PES can reach about 80 kg/m 2 and more. If the density of the fibrinogen formulation liquid is regarded as 1 g/ml, the average filtration capacity of PRE +PES can reach about 80 L/m 2 . The '904 fibrinogen formulation liquid had been frozen and was thereafter reconstituted, and in such a worse condition the filtration capacity of this method also reaches 54.26 L/m 2 .
  • the PALL 20-inch PES final filter has a membrane area of 2.08 m 2 , and the batch size of fibrinogen formulation liquid used is 70 L, so the use of 20-inch PALL PES filter is sufficient to filter a full batch of material.
  • the sterilizing filter to be selected is a PALL 20-inch PES filter (catalog number: AB2UECV7PH4) .
  • the pre-filter a 900-cm 2 the pre-filter was selected for the experiment, filtering 5 L of the liquid fibrinogen solution quickly and smoothly.
  • the 0.45-micron 20-inch glass fiber filter was selected as the pre-filter (catalog number: AB2UB7PH4) having filtration surface area of 1.30 m 2 , however the size of the pre-filter can be adjusted according to the actual production.
  • the experimental process was the same as the above-mentioned filter filtration capacity research experiment. After filtration, the fibrinogen liquid formulation was sampled before and after filtration to detect fibrinogen ( "FIB” ) activity and protein content was determined to evaluate the adsorption of the target protein by the new filter.
  • fibrinogen "FIB”
  • FIB activity was determined by Clauss method, which is widely used in the clinical laboratory.
  • the guideline for this procedure is approved by National Committee for Clinical Laboratory Standard (NCCLS) .
  • NCLS National Committee for Clinical Laboratory Standard
  • Stago STA-compact an automatic coagulation analyzer based on Clauss method was used for fibrinogen detection.
  • the assay uses diluted fibrinogen sample where clotting is initiated with a high concentration of reagent thrombin.
  • a calibration curve is plotted using serial dilutions of a reference fibrinogen standard.
  • the test plasma or fibrinogen sample is diluted, incubated, and FIB detection agent containing phospholipid is used. Thrombin and calcium are added, and timing begins with the addition of FIB detection agent.
  • the time taken for a clot to form is compared to the internal reference calibration curve to derive the fibrinogen concentration (e.g., mg/mL) of the test sample.
  • fibrinogen concentration e.g.,
  • Protein content refers the amount of protein contained in a substance.
  • aseptic filtration is the process of removing microorganisms from a fluid stream without adversely affecting product quality, which means the process will allow almost all proteins including fibrinogen pass through the filter.
  • BCA bicinchoninic acid
  • an amount of 200 ⁇ L of the BCA working solution was added to each well and the plate was mixed thoroughly on a plate shaker for 30 seconds. The plate was then covered and incubated at 37 °C for 30 minutes. Next, the plate was cooled to room temperature, and the absorbance at or near 562 nm on a plate reader was read.

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Abstract

Sont divulgués des procédés de filtration stérile d'une solution de fibrinogène, le procédé étant composé de la filtration de la solution avec un ou plusieurs filtres sélectionnés parmi un préfiltre de fibre de verre et un filtre aseptique constitué de polysulfone ou d'un dérivé de celle-ci. Sont en outre divulguées des solutions de fibrinogène aseptique obtenues par les procédés.
PCT/CN2020/139213 2020-12-25 2020-12-25 Filtration aseptique de fibrinogène WO2022133973A1 (fr)

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Citations (8)

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EP1457497A1 (fr) * 2003-03-06 2004-09-15 Probitas Pharma, S.A. Procédé d'élimination de virus dans une solution de fibrinogène et fibrinogène obtenu par ce procédé
JP2007215569A (ja) 2006-02-14 2007-08-30 Asahi Kasei Medical Co Ltd 血漿成分分離器及び二重濾過血液浄化装置
US7309428B2 (en) 2003-12-23 2007-12-18 Industrial Technology Research Institute Apparatus for separating components from blood plasma
US7816495B2 (en) 2002-07-10 2010-10-19 Nhs Blood And Transplant Processes for the preparation of fibrinogen
US20140116941A1 (en) * 2011-06-24 2014-05-01 Sartorius Stedim Biotech Gmbh Method for removing biopolymer aggregates and viruses from a fluid
US9561478B2 (en) 2008-03-31 2017-02-07 Toray Industries, Inc. Separation membrane, method of producing the same and separation membrane module using the separation membrane
US10493133B2 (en) 2011-10-27 2019-12-03 Green Cross Holdings Corporation Method for preparing highly concentrated fibrinogen solution and method for preparing fibrin sealant by using thereof

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US5259971A (en) 1992-03-02 1993-11-09 Cryolife, Inc. Method of preparing fibrinogen
US7816495B2 (en) 2002-07-10 2010-10-19 Nhs Blood And Transplant Processes for the preparation of fibrinogen
EP1457497A1 (fr) * 2003-03-06 2004-09-15 Probitas Pharma, S.A. Procédé d'élimination de virus dans une solution de fibrinogène et fibrinogène obtenu par ce procédé
US7309428B2 (en) 2003-12-23 2007-12-18 Industrial Technology Research Institute Apparatus for separating components from blood plasma
JP2007215569A (ja) 2006-02-14 2007-08-30 Asahi Kasei Medical Co Ltd 血漿成分分離器及び二重濾過血液浄化装置
US9561478B2 (en) 2008-03-31 2017-02-07 Toray Industries, Inc. Separation membrane, method of producing the same and separation membrane module using the separation membrane
US20140116941A1 (en) * 2011-06-24 2014-05-01 Sartorius Stedim Biotech Gmbh Method for removing biopolymer aggregates and viruses from a fluid
US10493133B2 (en) 2011-10-27 2019-12-03 Green Cross Holdings Corporation Method for preparing highly concentrated fibrinogen solution and method for preparing fibrin sealant by using thereof

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MAIK W. JORNITZ, THEODORE H. MELTZER: "Filtration and Purification in the Biopharmaceutical Industry vol 1", 1 January 2008, INFORMA HEALTHCARE USA, INC., New York, USA, ISBN: 0-8493-7953-9, article JORNITZ ET AL: "Filtration and Purification in the Biopharmaceutical Industry vol 1", pages: 1 - 21, XP055436615 *

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