US20140154233A1 - Method of purifying therapeutic proteins - Google Patents

Method of purifying therapeutic proteins Download PDF

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Publication number
US20140154233A1
US20140154233A1 US13/803,740 US201313803740A US2014154233A1 US 20140154233 A1 US20140154233 A1 US 20140154233A1 US 201313803740 A US201313803740 A US 201313803740A US 2014154233 A1 US2014154233 A1 US 2014154233A1
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Prior art keywords
fibrinogen
solution
vwf
factor viii
resin
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US13/803,740
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Hung Pham
Jeffrey Michael HEY
Darren NGUY
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CSL BEHRING GmbH
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CSL Ltd
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Priority to US13/803,740 priority Critical patent/US20140154233A1/en
Assigned to CSL LIMITED reassignment CSL LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HEY, JEFFREY MICHAEL, NGUY, DARREN, PHAM, HUNG
Priority to EP18207150.6A priority patent/EP3483173A1/en
Priority to DK13861334.4T priority patent/DK2928905T3/en
Priority to CN201811091750.9A priority patent/CN109125714B/zh
Priority to PCT/AU2013/001414 priority patent/WO2014085861A1/en
Priority to JP2015545601A priority patent/JP6411360B2/ja
Priority to ES13861334T priority patent/ES2711455T3/es
Priority to CN201380063945.1A priority patent/CN104981476B/zh
Priority to SG10201704484QA priority patent/SG10201704484QA/en
Priority to KR1020157017824A priority patent/KR102240978B1/ko
Priority to PL13861334T priority patent/PL2928905T3/pl
Priority to CA2893373A priority patent/CA2893373A1/en
Priority to SG10201911709RA priority patent/SG10201911709RA/en
Priority to BR112015012854-8A priority patent/BR112015012854B1/pt
Priority to RU2015126551A priority patent/RU2685956C2/ru
Priority to CA3185085A priority patent/CA3185085A1/en
Priority to KR1020217010484A priority patent/KR102403847B1/ko
Priority to EP13861334.4A priority patent/EP2928905B1/en
Priority to US14/649,374 priority patent/US10188965B2/en
Priority to SG11201504372UA priority patent/SG11201504372UA/en
Priority to TR2019/02450T priority patent/TR201902450T4/tr
Priority to AU2013354899A priority patent/AU2013354899B2/en
Publication of US20140154233A1 publication Critical patent/US20140154233A1/en
Priority to US14/535,085 priority patent/US9598461B2/en
Assigned to CSL BEHRING GMBH reassignment CSL BEHRING GMBH CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: CSL LIMITED
Priority to HK16102793.7A priority patent/HK1214833A1/zh
Priority to JP2018178301A priority patent/JP6836562B2/ja
Priority to US16/214,519 priority patent/US11426680B2/en
Priority to JP2021016182A priority patent/JP2021073280A/ja
Abandoned legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
    • A61J1/00—Containers specially adapted for medical or pharmaceutical purposes
    • A61J1/05—Containers specially adapted for medical or pharmaceutical purposes for collecting, storing or administering blood, plasma or medical fluids ; Infusion or perfusion containers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/363—Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4833—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • 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
    • C07K1/18—Ion-exchange chromatography
    • 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
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • 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
    • C07K1/36—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
    • 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
    • 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
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to a method of reducing the level of impurities in a solution containing at least one therapeutic protein and to the resultant therapeutic-protein containing solutions. More specifically, to a method of reducing the level of plasminogen and/or tissue plasminogen activator and/or other protease(s) in a feedstock comprising fibrinogen and/or Factor VIII and/or Von Willebrand factor (VWF).
  • the present invention also relates generally to solutions and pharmaceutical formulations comprising the fibrinogen and/or Factor VIII and/or VWF recovered by such methods, and uses thereof.
  • Haemostasis is an important physiological process that prevents bleeding following damage (e.g. a rupture) to blood vessels.
  • tissue factor Factor III
  • Factor XII which is released by activated platelets, activates Factor XI.
  • Activated Factor VII and Factor XI promote a cascade of enzymatic reactions that lead to the activation of Factor X.
  • Active Factor X Factor X (Factor Xa), along with Factor III, Factor V, Ca 2+ , and platelet thromboplastic factor (PF 3 ), activate prothrombin activator.
  • Prothrombin activator converts prothrombin to thrombin, which converts fibrinogen (Factor I) to fibrin, which forms an initial mesh over the site of damage. The initial mesh is then converted to a dense fibrin clot by Factor XIII, sealing the rupture until the site is repaired.
  • thrombin will also activate Factor VIII, a glycoprotein pro-cofactor that in the circulation is mainly complexed to von Willebrand factor (VWF).
  • VWF von Willebrand factor
  • Factor VIII interacts with Factor IXa to activate Factor X in the presence of Ca +2 and phospholipids.
  • Current treatment options are limited to the administration of a pharmaceutical preparation of one or more therapeutic proteins, with a view to restoring endogenous levels of said proteins and maintaining haemostasis.
  • existing pharmaceutical preparations which are typically derived from donated blood plasma or a recombinant source, comprise zymogens and proteases (e.g., prothrombin, plasminogen, tissue plasminogen activator (tPA) and/or other proteases), which can destabilise the therapeutic proteins, such as fibrinogen, Factor VIII, or VWF during storage.
  • proteases e.g., prothrombin, plasminogen, tissue plasminogen activator (tPA) and/or other proteases
  • tPA tissue plasminogen activator
  • preparations are relatively unstable in aqueous solution, with long-term storage limited to lyophilized or frozen preparations.
  • fibrinogen is typically purified from human plasma, where it accounts for only about 2-5% (1.5-4.0 g/L) of total plasma proteins.
  • the purification of fibrinogen from plasma is carried out by classical plasma fractionation, where fibrinogen is cryo-precipitated from plasma followed by precipitation with either ethanol, ammonium sulphate, ⁇ alanine/glycine, polymers (e.g., polyethelene glycol) or low ionic strength solutions.
  • Such methods can achieve relative high yield and homogeneity. Where a greater level of purity is required, chromatographic techniques are often employed.
  • fibrinogen preparations that comprise contaminating proteins such as zymogens or proteases (e.g., prothrombin, tissue plasminogen activator (tPA) and plasminogen), which can destabilize fibrinogen in solution.
  • zymogens or proteases e.g., prothrombin, tissue plasminogen activator (tPA) and plasminogen
  • prothrombin when prothrombin is present, it can be activated to the serine protease thrombin which will in turn convert fibrinogen into fibrin.
  • tPA tissue plasminogen activator
  • tPA tissue plasminogen activator
  • plasminogen preparations are relatively unstable in aqueous solution, with long-term storage limited to lyophilized or frozen preparations.
  • EP1240200 (U.S. Pat. No. 6,960,463) is directed to methods of purifying fibrinogen from a fibrinogen-containing solution using ion exchange (IEX) chromatography.
  • IEX ion exchange
  • the method involves applying a fibrinogen-containing solution to an ion exchange matrix under conditions that allow the fibrinogen to bind to the matrix and then washing the ion exchange matrix with a solution comprising at least one omega amino acid. This is done to promote the differential removal of plasminogen from the resin. Fibrinogen that is bound to the matrix is then eluted from the matrix.
  • WO2012038410 provides a method of purifying fibrinogen using anion exchange resins which contain a hydroxylated polymer support grafted with tertiary or quaternary amines that bind fibrinogen.
  • EP1519944 teaches the use of an immobilized metal ion affinity chromatography matrix under conditions that fibrinogen and plasminogen bind to the matrix, and selectively eluting the fibrinogen and plasminogen separately, such that the main fibrinogen fraction contains about 600 ng of plasminogen per mg of protein.
  • the present invention provides a method of reducing the level of plasminogen and/or tissue plasminogen activator and/or other protease(s) in a solution comprising fibrinogen and/or Factor VIII and/or VWF.
  • the purified protein(s) are stable during storage as liquid preparations and can be used for clinical or veterinary applications, including treating or preventing conditions associated with a deficiency in the level of said protein(s).
  • a pharmaceutical formulation comprising a solution comprising fibrinogen and/or Factor VIII and/or VWF recovered by the method of the present invention, as herein described, and a pharmaceutically acceptable carrier.
  • a method of treating or preventing a condition associated with fibrinogen deficiency comprising administering to a subject in need thereof the solution or pharmaceutical formulation of the present invention, as herein described.
  • a fibrin glue comprising the solution of the present invention, as herein described.
  • a method of producing a stable liquid fibrinogen solution comprising:
  • a method of producing a stable liquid fibrinogen solution comprising:
  • FIG. 1 shows the percentage recovery of fibrinogen, plasminogen, t-PA and Factor II from a fibrinogen solution over a range of pH levels when the solution is passed through a HEA Hypercel in negative mode with respect to fibrinogen.
  • FIG. 2 shows the percentage recovery of fibrinogen, plasminogen, t-PA, and Factor II from a fibrinogen solution over a range of pH levels when the solution is passed through a PPA Hypercel in negative mode with respect to fibrinogen.
  • FIG. 3 shows the percentage recovery of fibrinogen, plasminogen, t-PA, and Factor II from a fibrinogen solution over a range of pH levels when the solution is passed through a MEP Hypercel in negative mode with respect to fibrinogen.
  • FIG. 4 a shows the percentage recovery of fibrinogen, plasminogen, t-PA and Factor II from a fibrinogen-containing solution over a range of pH levels when the solution is passed through a HEA Hypercel in negative mode with respect to fibrinogen.
  • FIG. 4 b shows the stability of the fibrinogen containing solution recovered from the drop through fraction of the HEA Hypercel column ( FIG. 4 a ) over 6 days at room temperature (approx. 20° C.), as measured by % of initial clottable protein.
  • FIG. 4 c shows the stability of the fibrinogen containing solution recovered from the drop through fraction of the HEA Hypercel column ( FIG. 4 a ) over 6 days at room temperature (approx. 20° C.), as measured by % clottable protein.
  • FIG. 5 shows the percentage process recovery for fibrinogen, t-PA, plasminogen and Factor II in fractions derived from a method according to an embodiment of the present invention, from the plasma cryoprecipitate through to the Macroprep-HQ eluate.
  • FIG. 6 shows the purity of monomeric fibrinogen that was recovered from the Macroprep-HQ chromatographic resin, as measured by analytical size exclusion HPLC chromatogram.
  • a solution comprising at least 80% total protein of fibrinogen is taken to mean a solution comprising fibrinogen at a concentration of at least 80% w/w of total protein. This can be calculated for example by dividing the amount of fibrinogen derived from the clottable protein assay by the total protein amount derived from a standard protein assay (e.g. Biuret) and multiplying by 100.
  • a standard protein assay e.g. Biuret
  • thrombin is added to a sample to form a clot, which is almost all fibrin.
  • the clot can be separated from the supernatant containing non-clottable proteins by centrifugation. Subsequently the clot is washed and dissolved by alkaline urea or other substances and the protein concentration is determined by spectrophotometry. Since the majority of the clot is fibrin, the protein concentration will be equivalent to the fibrinogen concentration. Hence the amount of clottable protein in a sample is equivalent to the difference between the total protein and the non-clottable protein component of the sample.
  • the purification of fibrinogen and/or Factor VIII and/or VWF from a feedstock is typically carried out by conventional fractionation, where the fibrinogen and/or Factor VIII and/or VWF is precipitated from the solution using, for example, ethanol, ammonium sulphate, 13 alanine/glycine, polymers (e.g., polyethylene glycol) and/or low ionic strength solutions.
  • a feedstock e.g., plasma or cell culture supernatant
  • ethanol e.g., ammonium sulphate
  • 13 alanine/glycine e.g., polyethylene glycol
  • polymers e.g., polyethylene glycol
  • Impurities such as prothrombin, tissue plasminogen activator (tPA) and plasminogen are particularly problematic, as destabilizing levels of these impurities can hydrolyse fibrinogen in aqueous solution, thus rendering the fibrinogen unstable, particularly during manufacture and/or long-term storage.
  • tPA tissue plasminogen activator
  • the present invention is predicated, at least in part, on the finding that passing a feedstock comprising fibrinogen and/or Factor VIII and/or VWF through a hydrophobic charge-induction chromatographic (HCIC) resin and recovering the solution comprising fibrinogen and/or Factor VIII and/or VWF that passes through the resin is an efficient alternative to existing purification processes for reducing the destabilizing level of plasminogen and/or tissue plasminogen activator and/or other protease(s) in the solution.
  • HCIC hydrophobic charge-induction chromatographic
  • the concentration of the plasminogen and/or tissue plasminogen activator and/or other protease(s) in the recovered solution comprising fibrinogen and/or Factor VIII and/or VWF is reduced by at least 60%, by at least 70%, by at least 80%, or by at least 90% or by at least 95% or by at least 98% compared to the feedstock.
  • a method of producing a stable liquid fibrinogen solution comprising:
  • the concentration of the plasminogen and/or tissue plasminogen activator and/or other protease(s) in the recovered solution comprising fibrinogen is reduced by at least 60%, by at least 70%, by at least 80%, or by at least 90% or by at least 95% or by at least 98% compared to the feedstock.
  • Chromatographic processes typically employ a solid support, also referred to interchangeably herein as a resin or matrix.
  • Suitable solid supports would be familiar to persons skilled in the art. Examples include inorganic carriers, such as glass and silica gel, organic, synthetic or naturally occurring carriers, such as agarose, cellulose, dextran, polyamide, polyacrylamides, vinyl copolymers of bifunctional acrylates, and various hydroxylated monomers, and the like.
  • Commercially available carriers are sold under the names of SephadexTM, SepharoseTM, HypercelTM, CaptoTM, FractogelTM, MacroPrepTM, UnosphereTM, GigaCapTM, TrisacrylTM, UltrogelTM, DynospheresTM, MacrosorbTM and XADTM resins.
  • the chromatography steps will generally be carried out under non-denaturing conditions and at convenient temperatures in the range of about +10° C. to +30° C., more usually at about ambient temperatures.
  • the chromatographic steps may be performed batch-wise or continuously, as convenient. Any convenient method of separation may be employed, such as column, centrifugation, filtration, decanting, or the like.
  • HCIC Hydrophobic Charge Induction Chromatography
  • HCIC uses binding moieties attached to a solid support, wherein the binding moieties may have specificity for one or more proteins that, in accordance with the methods of the present invention, represent impurities in the feedstock (e.g., zymogens and proteases such prothrombin, tPA and plasminogen).
  • the HCIC resin comprises a ligand selected from the group consisting of mercaptoethylpyridine (4-mercaptoethylpyridine, e.g., MEP Hypercel), n-hexylamine (e.g., HEA Hypercel) and phenylpropylamine (e.g., PPA Hypercel).
  • the HCIC resin comprises n-hexylamine.
  • HCIC ligands such as HEA, MEP and PPA have an advantage in that they permit separation based on the surface hydrophobicity of proteins, but do not require the addition of lyotropic salts often seen in other processes for the purification of fibrinogen using hydrophobic chromatography (e.g., hydrophobic interaction chromatography; HIC).
  • hydrophobic chromatography e.g., hydrophobic interaction chromatography; HIC
  • HIC hydrophobic interaction chromatography
  • HCIC is controlled on the basis of pH, rather than salt concentration.
  • HCIC resins also provide high binding capacity and high flow rates, ideal for both laboratory- and industrial-scale purification.
  • HCIC resins are often packed into columns with bed heights from about 2 cm to about 40 cm. At industrial scale the bed heights are usually at least 10 cm and are typically in the range from about 15 cm to 25 cm. The column diameters of industrial columns can range from 20 cm up to about 1.5 m. Such columns are operated at flow rates in accordance with HCIC resin manufacture instructions with flow rates in the 50-100 cm/hr range typical.
  • the upper flow rate limitation is in part due to HCIC resin pressure constraints. For HEA resin for example the upper operating pressure limit is ⁇ 3 bar ( ⁇ 300 kPa).
  • Typical dynamic binding capacities (10% breakthrough of a binding protein) for HCIC resins are about 20 to 30 mg of bound protein per mL of resin.
  • this enables relative large amounts of protein to be loaded onto the HCIC column as the abundant proteins like fibrinogen can pass through the column whilst less abundant proteins such as plasminogen and/or tissue plasminogen activator and/or other protease(s) bind to the HCIC resin, This is advantageous for industrial scale manufacture as either smaller sized columns and/or less column cycles are required to process a batch.
  • the pH of the solution or feedstock comprising fibrinogen and/or Factor VIII and/or VWF that is passed through the HCIC resin in accordance with the methods of the present invention can be adjusted to control the recovery of the fibrinogen and/or Factor VIII and/or VWF and removal of impurities.
  • the solution or feedstock comprising fibrinogen and/or Factor VIII and/or VWF that is passed through the HCIC resin has a pH from about 6.0 to about 9.5.
  • the solution or feedstock comprising fibrinogen and/or Factor VIII and/or VWF is passed through the HCIC resin preferably at a pH of about 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, or 9.5.
  • the solution or feedstock comprising fibrinogen and/or Factor VIII and/or VWF that is passed through the HCIC resin has a pH of about 7.0.
  • the HCIC resin is equilibrated prior to loading of the solution or feedstock at a pH of about 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, or 9.5.
  • the method of the present invention may also employ the use of more than one additional chromatography step to remove further impurities, if necessary, and thus improve the purity of the final preparation.
  • Additional chromatographic purification steps can be implemented either before or after the purification of fibrinogen and/or Factor VIII and/or VWF through the HCIC resin in accordance with the present invention.
  • the solution comprising fibrinogen and/or Factor VIII and/or VWF that is recovered from the HCIC resin in step (ii) can be passed through another chromatographic resin.
  • the additional chromatographic purification steps may employ another HCIC resin.
  • a method of reducing the level of plasminogen and/or tissue plasminogen activator and/or other protease(s) in a solution comprising fibrinogen and/or Factor VIII and/or VWF comprising:
  • the concentration of the plasminogen and/or tissue plasminogen activator and/or other protease(s) in the solution comprising fibrinogen and/or Factor VIII and/or VWF that is recovered in step (iv) is reduced by at least 60%, by at least 70%, by at least 80%, or by at least 90% or by at least 95% or by at least 98% compared to the feedstock.
  • a method of producing a stable liquid fibrinogen solution comprising:
  • the concentration of the plasminogen and/or tissue plasminogen activator and/or other protease(s) in the solution comprising fibrinogen that is recovered in step (iv) is reduced by at least 60%, by at least 70%, by at least 80%, or by at least 90% or by at least 95% or by at least 98% compared to the feedstock.
  • the second HCIC resin is different from the first HCIC resin.
  • the first and second hydrophobic charge-induction chromatographic resins are the same. Where the solution comprising fibrinogen and/or Factor VIII and/or VWF that is recovered from the HCIC resin in step (ii) is passed through the same HCIC resin, it may be desirable to wash the HCIC resin after step (ii) and prior to passing the recovered solution through the HCIC resin in step (iii) again in order to remove any impurities that may be bound to the resin.
  • the additional chromatographic resin may also be an anion exchange chromatographic resin.
  • anion exchange chromatography negatively charged molecules are attracted to a positively charged solid support.
  • a positively charged solid support can be prepared by any means known to persons skilled in the art and will usually involve the covalent attachment of a negatively charged functional ligand onto a solid support. Suitable negatively charged functional ligands will invariably depend on the molecule to be separated from solution.
  • suitable anion exchange resins are ones comprising a functional quaternary amine group (Q) and/or a tertiary amine group (DEAE), or a diethylaminopropyl group (ANX).
  • the anion exchange resin is a strong anion exchange resin.
  • the strong anion exchange resin comprises a quaternary amine functional ligand (e.g., —N+(CH3)3 as seen, for example, in Macroprep-HQTM; Bio-Rad Laboratories).
  • the anion exchange resin is trimethylamine groups grafted to a hydroxylated methacrylic polymer via a linking group such as GigaCap Q-650M®.
  • anion exchange chromatography is performed in positive mode with respect to the fibrinogen and/or Factor VIII and/or VWF. That is, the conditions used are such that, when the solution or feedstock comprising fibrinogen and/or Factor VIII and/or VWF is passed through the anion exchange chromatographic resin, the fibrinogen and/or Factor VIII and/or VWF bind(s) to the positively-charged functional groups attached to the resin, allowing impurities in the solution to pass through the resin in the flow-through (drop-through) fraction, where they can be discarded or recovered for other purposes.
  • the anion exchange chromatographic resin can be washed with a suitable wash buffer known to persons skilled in the art. The constituents of the wash buffer and the conditions of the wash step will typically be selected to retain the fibrinogen and/or Factor VIII and/or VWF bound to the resin during the wash step.
  • the resin prior to eluting the fibrinogen and/or Factor VIII and/or VWF from the anion exchange chromatographic resin, the resin is washed with a wash solution comprising epsilon-aminocaproic acid ( ⁇ -ACA).
  • ⁇ -ACA epsilon-aminocaproic acid
  • the addition of ⁇ -ACA to the wash buffer can promote the elution of proteases (such as plasminogen) that may be bound to the anion exchange chromatographic resin during the first pass.
  • proteases such as plasminogen
  • An example of a suitable wash step is described in U.S. Pat. No. 6,960,463.
  • any suitable elution buffer known to persons skilled in the art can be used.
  • an elution buffer comprising from about 150 mM to about 300 mM NaCl allows fibrinogen monomers to be eluted from the anion exchange resin while minimizing the elution of fibrinogen aggregates and/or other proteins (e.g., Factor VIII, VWF, fibronectin or proteases) that may be also bound to the resin.
  • the fibrinogen is eluted from the anion exchange resin with an elution buffer comprising from about 150 mM to about 300 mM NaCl.
  • an elution buffer comprising from about 150 mM to about 300 mM NaCl. This equates to an elution buffer having a conductivity range of about 14 mS/cm (150 mM NaCl) to about 25 mS/cm (300 mM NaCl).
  • the fibrinogen is eluted from the anion exchange resin with an elution buffer comprising from about 150 mM to about 270 mM NaCl.
  • an elution buffer comprising from about 150 mM to about 270 mM NaCl. This equates to an elution buffer having a conductivity range of about 14 mS/cm (150 mM NaCl) to about 23 mS/cm (270 mM NaCl).
  • the fibrinogen is eluted from the anion exchange resin with an elution buffer comprising from about 170 mM to about 230 mM NaCl. This equates to an elution buffer having a conductivity range of about 15 mS/cm (170 mM NaCl) to about 20 mS/cm (230 mM NaCl).
  • the fibrinogen is eluted from the anion exchange resin with an elution buffer comprising from about 200 mM to about 220 mM NaCl. This equates to an elution buffer having a conductivity range of about 18 mS/cm (200 mM NaCl) to about 19 mS/cm (220 mM NaCl).
  • the fibrinogen is eluted from the anion exchange resin with an elution buffer comprising from about 150 mM to about 190 mM NaCl.
  • the elution buffer comprises a free amino acid at a concentration that promotes the elution of fibrinogen monomer over aggregates thereof.
  • the elution buffer comprises a free amino acid at a concentration of about 1 to 3% (w/v). Any suitable free amino acid may be used in this capacity.
  • the free amino acid is arginine.
  • anion exchange chromatography is performed in negative mode with respect to the fibrinogen and positive mode in respect to Factor VIII and/or VWF. That is, the conditions used are such that, when the solution or feedstock comprising fibrinogen and Factor VIII and/or VWF is passed through the anion exchange chromatographic resin, the Factor VIII and/or VWF bind(s) to the positively-charged functional groups attached to the resin, allowing fibrinogen in the solution to pass through the resin in the flow-through (drop-through) fraction.
  • the anion exchange chromatographic resin can be washed with a suitable wash buffer known to persons skilled in the art. The constituents of the wash buffer and the conditions of the wash step will typically be selected to retain the Factor VIII and/or VWF bound to the resin during the wash step.
  • the solution or feedstock comprising fibrinogen and/or Factor VIII and/or VWF is passed through the anion exchange chromatographic resin in the presence of about 150 mM to about 270 mM NaCl. This equates to a conductivity range of about 14 mS/cm (150 mM NaCl) to about 23 mS/cm (270 mM NaCl). Under these conditions the fibrinogen particularly the monomeric form, passes through the anion exchange chromatographic resin whilst fibrinogen containing aggregates and other impurities such as IgG and fibronectin bind to the resin.
  • the solution or feedstock comprising fibrinogen and/or Factor VIII and/or VWF is passed through the anion exchange chromatographic resin in the presence of about 170 mM to about 230 mM NaCl (about 15 mS/cm to about 20 mS/cm) or about 200 mM to about 220 mM NaCl (about 18 mS/cm to about 19 mS/cm). Under these types of conditions it is expected that Factor VIII and/or vWF will bind to the anion exchange chromatographic resin.
  • Factor VIII and/or VWF can be eluted from the anion exchange resin with an elution buffer comprising at least 300 mM of a salt such as NaCl.
  • a salt such as NaCl.
  • Factor VIII and/or VWF are eluted from the anion exchange resin with about 500 mM NaCl.
  • the elution step can be conducted such that the fibrinogen is initially eluted (for example using conditions set out in the embodiments above) and then the Factor VIII and/or VWF can be eluted using a higher concentration of salt such as 500 mM NaCl.
  • an anion exchange chromatography step it can be performed either before and/or after passing the feedstock comprising fibrinogen and/or Factor VIII and/or VWF through the HCIC resin.
  • the method further comprises passing the solution comprising fibrinogen and/or Factor VIII and/or VWF that is recovered in step (ii) through an anion exchange chromatographic resin.
  • first and second HCIC chromatographic steps are employed, as herein described, the method further comprising passing the solution comprising fibrinogen and/or Factor VIII and/or VWF that is recovered in step (ii) and/or step (iv) through an anion exchange chromatographic resin.
  • the method further comprises passing the feedstock comprising fibrinogen and/or Factor VIII and/or VWF through an anion exchange chromatographic resin prior to step (i).
  • the number of additional chromatographic steps used in accordance with the present invention will depend on the level of purity required in the final preparation.
  • the method of the present invention may comprise 2, 3, 4 or 5 chromatography steps, as disclosed herein.
  • the sequence of steps will be HCIC/IEX or HCIC/HCIC or IEX/HCIC; where the method comprises 3 chromatography steps, the sequence of steps will be HCIC/IEX/HCIC or HCIC/HCIC/IEX or HCIC/HCIC/HCIC or HCIC/IEX/IEX or IEX/HCIC/HCIC or IEX/HCIC/IEX or IEX/IEX/HCIC; where the method comprises 4 chromatography steps, the sequence of steps will be HCIC/IEX/HCIC/HCIC or HCIC/HCIC/IEX/HCIC or HCIC/HCIC/HCIC/HCIC or HCIC/IEX/IEX or HCIC/HCIC/IEX/IEX or HCIC/HCIC/IEX/IEX or HCIC/IEX/HCIC/IEX or HCIC/IEX/HCIC/IEX or HCIC/IEX/HCIC/IEX or HCIC/IEX/HCIC/IE
  • the required level of purity may be dictated by the intended use of the solution (e.g., for treatment of patient with a fibrinogen and/or Factor VIII and/or VWF deficiency) and/or where a longer storage period is required as an aqueous preparation.
  • Chromatography can be performed using any means known to persons skilled in the art.
  • the chromatography steps according to the present invention can use axial flow columns, such as those available from GE Healthcare, Pall Corporation and Bio-Rad, or radial flow columns, such as those available from Proxcys.
  • the chromatography steps according to the present invention can also be conducted using expanded bed technologies.
  • the concentration of the plasminogen and/or tissue plasminogen activator and/or other protease(s) in the recovered solution comprising fibrinogen and/or Factor VIII and/or VWF is reduced by at least 60%, by at least 70%, by at least 80%, or by at least 90% or by at least 95% compared to the feedstock.
  • Methods that maximize the removal of impurities such as plasminogen and/or tissue plasminogen activator and/or other protease(s) are particularly advantageous, because the stability and efficacy of the fibrinogen and/or Factor VIII and/or VWF in solution is decisively improved, particularly during long-term storage.
  • Storage in liquid form is particularly advantageous for solutions comprising fibrinogen and/or Factor VIII and/or VWF because immediate use in a patient is possible. This is in contrast to the use of lyophilised preparations of purified fibrinogen and/or Factor VIII and/or VWF, which require reconstituting the lyophilized protein(s) in a suitable buffer and/or water for injection immediately prior to administration into a subject in need thereof.
  • An advantage of depleting proteases or their zymogens (such as plasminogen) from a solution comprising fibrinogen and/or Factor VIII and/or VWF is that it minimises the need to add anti-fibrinolytic agents to inhibit any residual protease and/or zymogen (e.g., plasmin or plasminogen).
  • anti-fibrinolytic agents include aprotinin, a bovine protein inhibitor of plasmin; ortranexamic acid, a synthetic plasmin inhibitor also associated with neurotoxic side-effects.
  • a further advantageous feature is that plasminogen, which has been separated from the solution comprising fibrinogen and/or Factor VIII and/or VWF by HCIC, may be further processed to yield a plasminogen-containing concentrate for, for example, clinical use.
  • HCIC may therefore be used to prepare both plasminogen and solutions comprising fibrinogen and/or Factor VIII and/or VWF from a single starting solution.
  • a further advantageous feature is that the production costs of the HCIC resin is far more economical than the cost of lysine-Sepharose or immobilised lysine resin which are used in affinity chromatography procedures.
  • HCIC could be used to replace aluminium hydroxide (e.g. Alhydrogel) steps for the removal of proteases (e.g Factor II).
  • Alhydrogel is currently widely used in the commercial production of Factor VIII and VWF.
  • the material is, however, relatively costly with 100 kg's typically used per batch.
  • alhydrogel often requires manual handling and the material is discarded after a single use.
  • HCIC steps can be fully automated and the resin can be used in the manufacture of multiple batches.
  • HCIC resin is compatible with 1M NaOH which can be used for inactivation and removal of pathogens including viruses and prions during column cleaning and resin sanitisation procedures.
  • Liquid preparations derived from the methods of the present invention also have advantages over the use of frozen preparations, which require expensive storage and transport means and must be thawed prior to immediate use. Even where the fibrinogen and/or Factor VIII and/or VWF is stored as a lyophilized or frozen preparation, it is advantageous for the reconstituted or thawed protein to be stable for longer. This is evident, for example, where material has been reconstituted as a precaution for a medical procedure, but its use was not required on the basis of medical considerations. This material is typically discarded, as the fibrinogen is only stable over a short-term period due to the presence of prothrombin and/or t-PA and/or other protease(s).
  • the solution comprising fibrinogen and/or Factor VIII and/or VWF recovered by the methods of the present invention are advantageous because they provide a preparation of fibrinogen and/or Factor VIII and/or VWF that has greater stability than existing lyophilised preparations, even at room temperature. This can be particularly advantageous on lengthy transport routes where low temperatures may, where appropriate, not be ensured throughout transport and/or storage. Stable storage of fibrinogen and/or Factor VIII and/or VWF in solution also facilitates, in many respects, the production, usage, transport and administration to a patient in need thereof.
  • stable means that there is little or no substantial loss of activity of the fibrinogen and/or Factor VIII and/or VWF after a period of time in storage as compared to the level of activity of the fibrinogen and/or Factor VIII and/or VWF before storage (e.g., as compared to the level of activity determined immediately after recovery of the solution comprising fibrinogen and/or Factor VIII and/or VWF in accordance with the present invention).
  • the solution comprising fibrinogen and/or Factor VIII and/or VWF retains at least 70% activity, preferably at least 80% activity, more preferably at least 90% activity, even more preferably at least 95% activity and most preferably 100% activity after a period of time in storage at a temperature of about 0° C. to about 30° C.
  • the fibrinogen recovered by the methods of the present invention retains from about 90% to 100% activity after at least 4 weeks in storage at a temperature of about 2° C. to about 8° C., preferably retains about 90% activity after 4 weeks in storage at a temperature of about 2° C. to about 8° C.
  • the fibrinogen retains from about 60% to about 80% activity after at least 4 weeks in storage at a temperature of about 30° C., preferably retains from about 60% to about 70% activity after 5 weeks in storage at a temperature of about 30° C.
  • the level of activity of the fibrinogen and/or Factor VIII and/or VWF can be determined by any means known to persons skilled in the art. Examples of suitable methods for determining the activity of fibrinogen, for example, are summarised by Mackie et al. (British J. Haematol. 121:396-404, 2003). Particular methods include Clauss (Clauss, 1957, Acta-Haematol. 17, 237-246) and/or clottable protein (Jacobsson K., Scand J Clin Lab Invest 1955; 7 (supp 14):1-54 or Fibrin sealant Ph. Eur. Monograph 903, 2012). Results can be reported as % clottable protein; % of initial clottable protein, and/or % of initial fibrinogen activity as determined using the Clauss method or similar.
  • the concentration of the plasminogen and/or tissue plasminogen activator and/or other protease(s) in the recovered solution comprising fibrinogen and/or Factor VIII and/or VWF is likely to dictate the length of storage and/or storage conditions (e.g., temperature).
  • a preparation in which the concentration of the plasminogen and/or tissue plasminogen activator and/or other protease(s) in the recovered solution is reduced by 80% compared to the feedstock may be stored for a longer period of time and/or at higher temperatures without significantly destabilising the activity of the fibrinogen and/or Factor VIII and/or VWF as compared to a preparation in which the concentration of the plasminogen and/or tissue plasminogen activator and/or other protease(s) in the recovered solution comprising fibrinogen and/or Factor VIII and/or VWF is reduced by only 50% compared to the feedstock.
  • the methods of the present invention can be performed at laboratory scale, they can be scalable up to industrial size without significant changes to conditions.
  • the methods of the present invention are performed on an industrial or commercial scale.
  • the methods of the invention are suitable for the commercial scale manufacture of fibrinogen and/or Factor VIII and/or VWF.
  • plasma fractions derived from at least about 500 kg of plasma. More preferably, the starting plasma fraction will be derived from at least about 5,000 kg, 7,500 kg, 10,000 kg and/or 15,000 kg of plasma per batch.
  • the solutions and pharmaceutical formulations comprising fibrinogen and/or Factor VIII and/or VWF of the present invention are manufactured at commercial scale from a plasma fraction or a recombinant feedstock.
  • a solution comprising fibrinogen and/or Factor VIII and/or VWF is to be used for clinical or veterinary applications (e.g., for administration to a subject with fibrinogen and/or Factor VIII and/or VWF deficiency or for use as a fibrin glue)
  • a solution comprising fibrinogen and/or Factor VIII and/or VWF is to be used for clinical or veterinary applications (e.g., for administration to a subject with fibrinogen and/or Factor VIII and/or VWF deficiency or for use as a fibrin glue)
  • the feedstock comprising fibrinogen and/or Factor VIII and/or VWF i.e., the starting material
  • Methods of reducing the virus titre in a solution will be known to persons skilled in the art.
  • Examples include pasteurization (for example, incubating the solution at 60° C. for 10 hours in the presence of high concentrations of stabilisers such as glycine (e.g. 2.75M) and sucrose (e.g. 50%) and/or other selected excipients or salts), dry heat treatment, virus filtration (passing the solution through a nano-filter; e.g., 20 nm cutoff) and/or subjecting the solution to treatment with a suitable organic solvent and detergent for a period of time and under conditions to inactivate virus in the solution.
  • Solvent detergent has been used for over 20 years to inactivate enveloped viruses particularly in plasma-derived products including fibrinogen and factor VIII and/or VWF.
  • Suitable solvents include tri-n-butyl phosphate (TnBP) and ether, preferably TnBP (typically at about 0.3%).
  • Suitable detergents include polysorbate (Tween) 80, polysorbate (Tween) 20 and Triton X-100 (typically at about 0.3%).
  • a preferred detergent is polysorbate 80 and a particularly preferred combination is polysorbate 80 and TnBP.
  • the feedstock may be stirred with solvent and detergent reagents at a temperature and for a time sufficient to inactivate any enveloped viruses that may be present.
  • the solvent detergent treatment may be carried out for about 4 hours at 25° C.
  • the solvent detergent chemicals are subsequently removed by for example adsorption on chromatographic media such as C-18 hydrophobic resins or eluting them in the drop-through fraction of ion exchange resins under conditions which adsorb the protein of interest.
  • the virus inactivation step can be performed at any suitable stage of the methods disclosed herein.
  • the feedstock comprising fibrinogen and/or Factor VIII and/or VWF is subject to a viral inactivation step prior to step (i).
  • the solution comprising fibrinogen and/or Factor VIII and/or VWF that is recovered from the hydrophobic charge-induction chromatographic resin i.e., from steps (ii) and/or (iv)
  • the viral inactivation step comprises pasteurisation or treatment with an organic solvent and detergent.
  • the virus inactivation step comprises virus filtration.
  • the feedstock or solution comprising fibrinogen and/or Factor VIII and/or VWF is subject to a viral inactivation step before it is passed through the anion exchange chromatographic resin.
  • a virus inactivation step such as solvent detergent treatment prior to passing the treated solution or feedstock through an anion exchange chromatographic resin is that the anion exchange resin allows for the removal of the organic solvent and detergent from the treated solution by utilizing conditions that promote binding of the fibrinogen and/or Factor VIII and/or VWF to the resin and removal of the organic solvent and detergent with the flow-through (drop-through) fraction.
  • Pasteurization can generate protein aggregates and polymers, particularly in a solution comprising fibrinogen (also referred to herein as a “fibrinogen solution”). Therefore, it may be desirable in some instances to reduce the level of aggregates/polymers in a pasteurized solution. This can be achieved by any means knows to persons skilled in the art, although conveniently can be achieved by further chromatographic purification.
  • the pasteurized solution or feedstock is passed through an anion exchange chromatographic resin in positive mode with respect to the fibrinogen and/or Factor VIII and/or VWF such that any aggregates or polymers are removed with the flow-through (drop-through) fraction.
  • feedstock is used herein to denote any solution comprising fibrinogen and/or Factor VIII and/or VWF.
  • the feedstock may also comprise other proteins (e.g., therapeutic proteins) known to persons skilled in the art. Examples include proteins involved in the blood coagulation cascade.
  • the feedstock comprises fibrinogen.
  • Suitable feedstock comprising fibrinogen and/or Factor VIII and/or VWF will be known to persons skilled in the art.
  • plasma or plasma fractions such as solubilised plasma cryoprecipitate or solubilised Fraction I paste derived from human or animal plasma or a plasma fraction, cell culture fractions from recombinant technology, fractions derived from milk from transgenic animals, etc.
  • Sources of recombinant fibrinogen and/or Factor VIII and/or VWF proteins are also suitable for use as a feedstock in accordance with the present invention.
  • the feedstock is plasma or a plasma fraction, it can be either pooled plasma donations or it can be from an individual donor.
  • the feedstock comprising fibrinogen and/or Factor VIII and/or VWF is a solubilised plasma cryoprecipitate.
  • This component either derived from whole blood or collected via apheresis, is prepared by controlled thawing fresh frozen plasma between 1-6° C. and recovering the precipitate. The cold-insoluble precipitate is refrozen.
  • One unit of cryoprecipitate apheresis is approximately equivalent to 2 units of cryoprecipitate derived from whole blood. It contains most of the fibrinogen, Factor VIII and VWF along with other proteins such as factor XIII and fibronectin from fresh frozen plasma.
  • Fraction I precipitate An alternate source of fibrinogen is Fraction I precipitate which can be prepared from frozen plasma by thawing and removing the cryoprecipitate by either centrifugation or filtration. The resultant cryosupernatant is then mixed with ethanol to precipitate Fraction I.
  • a Fraction I precipitate can be obtained by adding about 8% (v/v) ethanol at pH 7.2 and controlling the temperature to about ⁇ 3° C. (Cohn, et al. 1946, J. Am. Chem. Soc. 62: 459-475).
  • the feedstock comprising fibrinogen and/or Factor VIII and/or VWF is cryoprecipitate.
  • protease and/or its zymogen present in the feedstock or solution comprising fibrinogen and/or Factor VIII and/or VWF that, when exposed to a HCIC resin, is capable of binding to HCIC resin under conditions where the fibrinogen and/or Factor VIII and/or VWF passes through the resin.
  • Proteases may be any type, including serine proteases (e.g., plasmin, thrombin, trypsin), threonine proteases, cysteine proteases (e.g., cathepsin B and cathepsin H), aspartate proteases (e.g., pepsin), metallo-proteases (e.g., collagenases and gelatinases) and glutamic proteases.
  • serine proteases e.g., plasmin, thrombin, trypsin
  • cysteine proteases e.g., cathepsin B and cathepsin H
  • aspartate proteases e.g., pepsin
  • metallo-proteases e.g., collagenases and gelatinases
  • the proteases/zymogens may include plasminogen, tissue plasminogen activator (tPA), thrombin, elastase, Factor VIIa, Factor IXa, Factor Xa, Factor XIa, Factor XIIa, Factor XIIIa, plasma kallikreins and the like.
  • tissue plasminogen activator tPA
  • thrombin thrombin
  • elastase Factor VIIa
  • Factor IXa Factor Xa
  • Factor XIa Factor XIIa
  • Factor XIIIa Factor kallikreins and the like.
  • plasminogen tissue plasminogen activator
  • protease/zymogens to be removed from a solution comprising fibrinogen and/or Factor VIII and/or VWF are t-PA, pro- and/or active thrombin (Factor II/IIa).
  • the protease/zymogen can include any host cell protease, such as serine proteases (e.g., caseinases), metalloproteases (e.g., gelatinases, matrix metalloproteases (MMP) including MMP3, MMP10 or MMP12), aspartic proteases (cathepsin D), acid proteases amongst others.
  • step (i) it may be desirable to remove or reduce the level of impurities from the feedstock before passing the feedstock through an HCIC resin in step (i).
  • Removing or reducing the level of impurities from the feedstock can reduce the load on the HCIC resin during chromatographic purification and thus improve the efficiency of separation of plasminogen and/or tissue plasminogen activator and/or other protease(s) from the feedstock Impurities may be removed or reduced, for example, by precipitating fibrinogen and/or Factor VIII and/or VWF from the feedstock and recovering the precipitated protein(s).
  • Suitable methods of precipitating fibrinogen and/or Factor VIII and/or VWF from a feedstock comprising fibrinogen and/or Factor VIII and/or VWF will be known to persons skilled in the art.
  • An example includes adding aluminium hydroxide suspension to the feedstock, which is particularly useful for removing vitamin K-dependent proteins (for example, the clotting factors II, VII, IX, and X) and other proteins that have binding affinity for aluminium hydroxide, such as prothrombin (factor II) and t-PA, from plasma or plasma cryoprecipitate.
  • vitamin K-dependent proteins for example, the clotting factors II, VII, IX, and X
  • prothrombin factor II
  • t-PA t-PA
  • vitamin K-dependent proteins are removed or reduced from the feedstock.
  • vitamin K-dependent proteins are removed or reduced by addition of aluminium hydroxide to the feedstock.
  • Aluminium hydroxide may be in the form of Alhydrogel®, added to the feedstock to a final concentration of about 10% to about 80% w/w.
  • the aluminium hydroxide is added to the feedstock to a final concentration in the range from about 10% to about 50% (w/w). In preferred embodiments, the concentration is from about 15% to about 30% (w/w).
  • the aluminium hydroxide is added to the feedstock at about 20% to about 25% (w/w) for optimum fibrinogen recovery and removal of impurities such as prothrombin.
  • impurities such as prothrombin.
  • vitamin K-dependent proteins are removed from the feedstock by batch adsorption using aluminium hydroxide.
  • a solution comprising fibrinogen and/or Factor VIII and/or VWF that is recovered by the method of the present invention, as herein described.
  • the level of plasminogen and/or tissue plasminogen activator and/or other protease(s) in the solution will be less than 20% of total protein, preferably less than 10% of total protein, and more preferably less than 5% of total protein.
  • the skilled person will understand that the level of plasminogen and/or tissue plasminogen activator and/or other protease(s) that is present in the solution comprising fibrinogen and/or Factor VIII and/or VWF may depend on the intended use of the solution or length of storage.
  • the solution comprises more than about 10% plasminogen and/or tissue plasminogen activator and/or other protease(s) (of total protein).
  • the solution may be stored for at least 4 weeks at a temperature of about 30° C., it may be desirable that the solution comprises less than about 10% plasminogen and/or tissue plasminogen activator and/or other protease(s) (of total protein).
  • a solution comprising fibrinogen recovered by the method of the present invention.
  • the solution comprises at least 80% total protein of fibrinogen.
  • the solution further comprises:
  • the solution further comprises:
  • the concentration of the fibrinogen and/or Factor VIII and/or VWF in the solution recovered by the methods disclosed herein and the concentration of impurities can be measured by any means known to persons skilled in the art. Examples of suitable assays for measuring fibrinogen are described by Mackie et al. ( Br J Haematol. 2003 May; 121(3):396-404). Size exclusion HPLC may also be used to measure the concentration of fibrinogen and/or Factor VIII or an impurity in the solution comprising fibrinogen and/or Factor VIII (e.g., Cardinali et al. 2010, Arch. Biochem. Biphys.
  • impurities e.g., plasminogen and/or tissue plasminogen activator and/or other protease(s)
  • HPLC also allows the skilled person to discriminate between monomers and aggregates of fibrinogen.
  • concentration of fibrinogen and/or Factor VIII and/or VWF may differ depending on the sensitivity of the assay that is used. For instance, the concentration of fibrinogen in a solution as measured using the Clauss assay may be slightly lower than the concentration as measured in the same solution by HPLC.
  • the concentration of monomeric fibrinogen in the solution is at least 75%, at least 80%, at least 90% or at least 95% of total protein as measured by size exclusion HPLC.
  • a pharmaceutical formulation comprising a solution comprising fibrinogen and/or Factor VIII and/or VWF recovered by the methods disclosed herein, and a pharmaceutically acceptable carrier.
  • Suitable pharmaceutically acceptable carriers including pharmaceutically acceptable diluents and/or excipients, will be known to those skilled in the art. Examples include solvents, dispersion media, antifungal and antibacterial agents, surfactants, isotonic and absorption agents and the like.
  • the pharmaceutical formulation may also be formulated by the addition of a combination of suitable stabilisers, for example, an amino acid, a carbohydrate, a salt, and a detergent.
  • the stabiliser comprises a mixture of a sugar alcohol and an amino acid.
  • the stabilizer may comprise a mixture of a sugar (e.g. sucrose or trehalose), a sugar alcohol (e.g. mannitol or sorbitol), and an amino acid (e.g. proline, glycine and arginine).
  • the formulation comprises an amino acid such as arginine.
  • the formulation comprises divalent metal ions in a concentration up to 100 mM and a complexing agent as described in U.S. Pat. No.
  • the formulation is formulated without the addition of any anti-fibrinolytic agents or stabilising proteins such as albumin.
  • the pH is preferably about 6.5 to 7.5 and the osmolality is at least 240 mosmol/kg.
  • the pharmaceutical formulation may also be sterilised by filtration prior to dispensing and long term storage.
  • the formulation will retain substantially its original stability characteristics for at least 2, 4, 6, 8, 10, 12, 18, 24, 36 or more months.
  • formulations stored at 2-8° C. or 25° C. can typically retain substantially the same molecular size distribution as measured by HPLC-SEC when stored for 6 months or longer.
  • Particular embodiments of the pharmaceutical formulation can be stable and suitable for commercial pharmaceutical use for at least 6 months, 12 months, 18 months, 24 months, 36 months or even longer when stored at 2-8° C. and/or room temperature.
  • solutions and pharmaceutical formulations of the present invention may be formulated into any of many possible dosage forms, such as injectable formulations.
  • the formulations and their subsequent administration (dosing) are within the skill of those in the art. Dosing is dependent on the responsiveness of the subject to treatment, but will invariably last for as long as the desirable effect is required (e.g., a return to normal plasma levels of fibrinogen). Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.
  • the pharmaceutical formulation of the present invention has a volume of at least 5 mL and comprises at least 5 mg/mL fibrinogen. In another embodiment, the pharmaceutical formulation has a volume of at least 5 mL and comprises at least 20 mg/mL fibrinogen. In particular embodiments, the pharmaceutical formulation has a volume of at least 5 mL and comprises fibrinogen at a concentration of about 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL, 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 90 mg/mL or 100 mg/mL. In another aspect, there is provided a vessel containing at least 5 mL of a stable pharmaceutically acceptable fibrinogen solution, wherein the concentration of fibrinogen is at least 20 mg/mL.
  • a method of treating or preventing a condition associated with fibrinogen and/or Factor VIII and/or VWF deficiency comprising administering to a subject in need thereof a solution comprising fibrinogen and/or Factor VIII and/or VWF recovered by the method of the present invention, as herein disclosed, or the pharmaceutical formulation of the present invention, as herein disclosed.
  • a solution comprising fibrinogen and/or Factor VIII and/or VWF recovered by the method of the present invention, as herein disclosed, in the manufacture of a medicament for treating or preventing a condition associated with fibrinogen and/or Factor VIII and/or VWF deficiency.
  • the fibrinogen condition is selected from the group consisting of afibrinogenemia, hypofibrinogenemia and dysfibrinogenemia.
  • the Factor VIII and/or VWF condition is selected from the group consisting of hemophilia A, bleeding disorders (e.g., defective platelet function, thrombocytopenia or von Willebrand's disease), vascular injury, bleeding from trauma or surgery, bleeding due to anticoagulant therapy, bleeding due to liver disease.
  • bleeding disorders e.g., defective platelet function, thrombocytopenia or von Willebrand's disease
  • vascular injury bleeding from trauma or surgery, bleeding due to anticoagulant therapy, bleeding due to liver disease.
  • a solution comprising fibrinogen and/or Factor VIII and/or VWF recovered by the methods of the present invention include major wounds and severe haemorrhaging and burns.
  • a solution comprising fibrinogen prepared in accordance with the present invention can be injected intravenously into the patient in need thereof in order to compensate the state of fibrinogen deficiency and dosages can be determined by the skilled person based on the degree of deficiency.
  • a solution comprising fibrinogen recovered by the methods of the present invention also has advantages in the use of fibrin glues (also known as fibrin sealants) due to the lack of destabilizing levels of plasminogen and/or tissue plasminogen activator and/or other protease(s).
  • fibrin glues also known as fibrin sealants
  • tPA converts plasminogen to its active form plasmin which in turn digests the fibrin clot and therefore reduces clot formation upon topical application (e.g. for haemostasis).
  • Fibrin glues typically comprise two components: (i) fibrinogen (frequently together with factor XIII and a fibrinolysis inhibitor such as aprotinin), and (ii) thrombin (frequently together with calcium ions). The two components are reconstituted in order to prepare the glue ready for use. Fibrin glue is used in clinical and veterinary applications to simulate the last step of coagulation through the formation of cross-linked fibrin fibres using a combination of fibrinogen with thrombin in the presence of calcium and Factor XIII. Fibrin glue has diverse applications in clinical and veterinary medicine, including haemostasis, wound closure, adhesion prophylaxis and wound healing.
  • Fibrin glue can also be used to close skin wounds (including skin transplant), for sealing sutures and for bonding connective tissues, such as bone, cartilage and tendons.
  • a fibrin glue comprising the solution comprising fibrinogen recovered by the methods of the present invention, as herein disclosed.
  • Human pooled plasma cryoprecipitate was used as the starting material (i.e., fibrinogen-containing feedstock). Briefly, the pooled plasma cryoprecipitate was solubilised in an extraction buffer containing 20 mM Tri-sodium citrate, 200 mM epsilon-amino caproic acid ( ⁇ -ACA), 60 IU/mL heparin and 500 mM NaCl (pH 7.2 ⁇ 2) at 31 ⁇ 2° C. for 30 minutes (1 g cryoprecipitate per 4 g of buffer). Aluminium hydroxide 2% (w/w) was then added to the solubilised cryoprecipitate at a concentration of 25% (w/w). After which the aluminium hydroxide gel was removed by either centrifugation or depth filtration and the fibrinogen-containing supernatant was recovered for further chromatographic purification through a HCIC chromatographic resin.
  • ⁇ -ACA epsilon-amino caproic acid
  • the fibrinogen-containing supernatant was applied to chromatography columns that were packed with 1.8 mL of either HEA, PPA or MEP Hypercel resin.
  • the chromatography columns were pre-equilibrated in 25 mM Tris at a different pH, ranging from 6.5 to 8.5.
  • the fibrinogen-containing supernatant was loaded onto the chromatography column at a ratio of approximately 11 mL/mL resin.
  • HCIC purification was performed in negative mode with respect to fibrinogen, in which fibrinogen was allowed to drop-through in the unbound flow-through fraction, whilst the majority of t-PA, plasminogen and Factor II remained bound to the resin.
  • FIGS. 1 to 3 show step recovery of fibrinogen, plasminogen, t-PA and Factor II post-chromatographic purification using HEA Hypercel, PPA Hypercel and MEP Hypercel.
  • the results show that pH has little or no effect on plasminogen binding to these resins, whereas the binding of t-PA to the resins appears to be most effective at the lower pH range.
  • the HEA Hypercel column showed the highest fibrinogen recovery in the drop-through fraction and the operating pH range tested appeared to have little effect on fibrinogen recovery compared to that observed for both PPA and MEP Hypercel columns
  • Both PPA and MEP columns displayed highest fibrinogen recovery in the drop-through fraction at pH 8.5.
  • FIG. 4 a shows step recovery of fibrinogen, plasminogen, t-PA and Factor II during post-chromatographic purification using HEA Hypercel.
  • the fibrinogen remained stable in solution for at least 6 days at room temperature (approx. 20° C.).
  • the results are presented in FIGS. 4 b and 4 c.
  • the fibrinogen-containing supernatant post-alhydrogel adsorption step generated in accordance with Example 1 was subjected to a further precipitation step by adding a saline solution comprising 2.4M glycine, 2.7M NaCl, 2.1 mM CaCl 2 and 23 mM Tri-sodium citrate (pH 6.6-7.3).
  • the solubilised precipitate was warmed to 30° C. before being added to the glycine buffer, which was also incubated to 30° C., at a product to buffer ratio of 1:2.
  • the mixture was allowed to stir for 10 minutes and the resultant precipitate was recovered from the liquid phase by centrifugation.
  • the liquid phase which contained predominantly fibronectin and IgG, was discarded and the fibrinogen-containing precipitate was collected and resuspended in a solubilisation buffer containing 100 mM NaCl, 1.1 mM CaCl 2 , 10 mM Trisodium citrate, 10 mM Tris-(hydroxymethyl methylamine) and 4.5 mM sucrose (pH 7.0).
  • the solubilised fibrinogen intermediate 250 mL was clarified using a 1 ⁇ m filter before being passed through an XK 16/30 column packed with 36 mL of HEA Hypercel resin pre-equilibrated in 25 mM Tris pH 7.0.
  • the drop-through fraction was collected for fibrinogen, plasminogen, t-PA and Factor II testing and a summary of the results is provided in Table 2 below.
  • Process Step 2 Alhydrogel (aluminium hydroxide) adsorption (Alhydrogel concentration: target 20% w/w, ranging from 15 to 50% w/w) of solubilised plasma cryoprecipitate and recovery of fibrinogen-containing supernatant using methods such as centrifugation or depth filtration in the presence of a filter aid.
  • this step can be replaced with either a HCIC chromatographic step in negative mode (drop through) with respect to fibrinogen or a combination of HCIC and anion exchange chromatography both in negative mode with respect to the fibrinogen. If both HCIC and anion exchange chromatography are used in combination then Process Step 3 is optional;
  • Step 3 glycine precipitation of fibrinogen from the fibrinogen-containing supernanant of Step 2.
  • this step can be replaced with an anion exchange chromatography in negative mode with respect to the fibrinogen.
  • Process Step 4 passing the solubilised glycine precipitate from Step 3 through a HCIC chromatographic resin in negative mode with respect to fibrinogen;
  • Process Step 5 treating the purified fibrinogen solution recovered in Step 4 with solvent or detergent or pasteurisation to inactivate pathogens.
  • Process Step 6 passing the treated solution from Step 5 through an anion exchange chromatographic resin in positive mode with respect to fibrinogen, washing the weakly bound proteins from the resin and eluting the fibrinogen from the resin;
  • Step 7 substrateing the fibrinogen eluted from the anion exchange resin in Step 6 to nanofiltration (35 nm or 20 nm or a combination of 35/20 nm);
  • Process Step 8 subjecting the filtered fibrinogen from Step 7 to ultrafiltration (50, 100, 200 and 300 kDa membrane filters).
  • cryoprecipitate was prepared according to the steps described in Examples 1 to 3 through to the mixed mode chromatography step using a HEA Hypercel column.
  • the drop-through fraction from the HEA Hypercel column which contained predominantly fibrinogen, was subjected to an overnight solvent/detergent treatment for virus inactivation.
  • the virus inactivated solution was then diluted to ⁇ 10 mS/cm using 25 mM Tris (pH 8.0) prior to being loaded onto an anion exchange column (XK 50/30, GE Healthcare), packed with approximately 412 mL of Macroprep-HQ resin that was pre-equilibrated with 25 mM Tris (pH 8.0).
  • the flow-though fraction was discarded and the Macroprep-HQ column washed with 4 column volumes of a washing buffer containing 90 mM NaCl, 50 mM Tris, 20 mM EACA (pH 8.0).
  • the initial flow-through fraction and the wash fractions contain predominantly plasminogen and t-PA, whilst fibrinogen remained bound to the chromatographic resin.
  • the monomeric form of fibrinogen was selectively eluted from the Macroprep-HQ column using an elution buffer comprising 200 mM NaCl, 10 mM Tris, 10 mM Tri-sodium citrate, 46 mM sucrose and 1.1 mM CaCl 2 (pH 7.0), leaving fibrinogen aggregates and low molecular weight proteins bound to the Macroprep-HQ resin.
  • the purity of fibrinogen that was recovered from the Macroprep-HQ chromatographic resin was greater than 95%, as revealed by analytical size exclusion HPLC chromatography.
  • a representative of the HPLC profile of fibrinogen analysed on TSKGel G4000SWXL (Tosoh Corporation) is shown in FIG. 6 .
  • the fibrinogen monomer is eluted at a retention time of approximately 17.4 minutes and contributed to 96.6% of the total peak area, whilst fibrinogen dimer and/or other high molecular weight proteins are eluted at a retention time of approximately 14.8 minutes.
  • Purified fibrinogen solution recovered by the method described in Example 5 above was sterile filtered and subjected to a stability study over a 9/7 week period at 2°-8° C. or 30° C.
  • the liquid fibrinogen preparation placed at 2°-8° C. retained from about 90% of its original activity as measured by the Clauss method after the 9 week storage period.
  • the liquid fibrinogen preparation placed at 30° C. retained about 70% of its original activity after the 2 week storage period and did not lose further activity for at least 5 weeks. A further reduction in activity to below 60% was observed by week 7 of incubation at 30° C. The loss of fibrinogen activity at 30° C.

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