WO2010014708A2 - Factor viii polymer conjugates - Google Patents

Factor viii polymer conjugates Download PDF

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Publication number
WO2010014708A2
WO2010014708A2 PCT/US2009/052103 US2009052103W WO2010014708A2 WO 2010014708 A2 WO2010014708 A2 WO 2010014708A2 US 2009052103 W US2009052103 W US 2009052103W WO 2010014708 A2 WO2010014708 A2 WO 2010014708A2
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WO
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Prior art keywords
fviii
rfviii
factor viii
peg
soluble polymer
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PCT/US2009/052103
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French (fr)
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WO2010014708A3 (en
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Peter Turecek
Juergen Siekmann
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Baxter International Inc.
Baxter Healthcare S.A.
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Priority to SI200931077T priority Critical patent/SI2318050T1/en
Priority to NZ590569A priority patent/NZ590569A/en
Priority to CN200980130950.3A priority patent/CN102112156B/en
Priority to PL09790935T priority patent/PL2318050T3/en
Priority to AU2009276625A priority patent/AU2009276625B2/en
Priority to CA2730714A priority patent/CA2730714C/en
Priority to EP14178828.1A priority patent/EP2810662B1/en
Priority to JP2011521284A priority patent/JP2013500238A/en
Priority to MX2011001238A priority patent/MX2011001238A/en
Priority to EP09790935.2A priority patent/EP2318050B1/en
Priority to EP14178827.3A priority patent/EP2799088B1/en
Priority to BRPI0916675-0A priority patent/BRPI0916675B1/en
Priority to KR1020167033032A priority patent/KR101879838B1/en
Priority to PL14178828T priority patent/PL2810662T3/en
Application filed by Baxter International Inc., Baxter Healthcare S.A. filed Critical Baxter International Inc.
Priority to KR1020117004739A priority patent/KR101681574B1/en
Priority to DK09790935.2T priority patent/DK2318050T3/en
Priority to ES09790935.2T priority patent/ES2524598T3/en
Priority to KR1020187020030A priority patent/KR102049190B1/en
Publication of WO2010014708A2 publication Critical patent/WO2010014708A2/en
Publication of WO2010014708A3 publication Critical patent/WO2010014708A3/en
Priority to HK11109099.8A priority patent/HK1155939A1/en
Priority to HRP20141083AT priority patent/HRP20141083T1/en
Priority to AU2014268202A priority patent/AU2014268202B2/en
Priority to SM201500014T priority patent/SMT201500014B/en
Priority to AU2017208311A priority patent/AU2017208311A1/en
Priority to AU2019222949A priority patent/AU2019222949B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • 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
    • 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
    • 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
    • C07K14/755—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 to a proteinaceous construct comprising coagulation factor VIII (FVIII) being bound to at least one water soluble polymer, including a poly(alkylene oxide) such as polyethylene glycol. Further the present invention relates to methods for prolonging the in vivo-half-life of FVIII in the blood of a mammal having a bleeding disorder associated with functional defects or deficiencies of FVIII.
  • FVIII coagulation factor VIII
  • Coagulation factor VIII (FVIII) circulates in plasma at a very low concentration and is bound non-covalently to von Willebrand factor (VWF). During hemostasis, FVIII is separated from VWF and acts as a cofactor for activated factor IX (F ⁇ Xa)-mediated factor X (FX) activation by enhancing the rate of activation in the presence of calcium and phospholipids or cellular membranes.
  • FVIII is synthesized as a single-chain precursor of approximately 270-330 kD with the domain structure A1-A2-B-A3-C1-C2.
  • FVIII is composed of a heavy chain (A1-A2-B) and a light chain (A3-C1-C2).
  • the molecular mass of the light chain is 80 kD whereas, due to proteolysis within the B domain, the heavy chain is in the range of 90-220 kD.
  • FVIII is also synthesized as a recombinant protein for therapeutic use in bleeding disorders.
  • Various in vitro assays have been devised to determine the potential efficacy of recombinant FVIII (rFVIII) as a therapeutic medicine. These assays mimic the in vivo effects of endogenous FVIII.
  • In vitro thrombin treatment of FVIII results in a rapid increase and subsequent decrease in its procoagulant activity, as measured by in vitro assay. This activation and inactivation coincides with specific limited proteolysis both in the heavy and the light chains, which alter the availability of different binding epitopes in FVIII, e.g.
  • FVIII FVIII to dissociate from VWF and bind to a phospholipid surface or altering the binding ability to certain monoclonal antibodies.
  • the lack or dysfunction of FVIII is associated with the most frequent bleeding disorder, hemophilia A.
  • the treatment of choice for the management of hemophilia A is replacement therapy with plasma derived or rFVIII concentrates. Patients with severe haemophilia A with FVIII levels below 1%, are generally on prophylactic therapy with the aim of keeping FVIII above 1% between doses. Taking into account the average half- lives of the various FVIII products in the circulation, this can usually be achieved by giving FVIII two to three times a week.
  • Therapeutic polypeptide drugs are rapidly degraded by proteolytic enzymes and neutralized by antibodies. This reduces their half- life and circulation time, thereby limiting their therapeutic effectiveness.
  • the addition of a soluble polymer or carbohydrate to a polypeptide has been shown to prevent degradation and increase the polypeptides half-life.
  • PEGylation of polypeptide drugs protects them and improves their pharmacodynamic and pharmacokinetic profiles (Harris J M et Chess R B, Nat Rev Drug Discov 2003;2:214-21).
  • the PEGylation process attaches repeating units of polyethylene glycol (PEG) to a polypeptide drug.
  • PEGylation of molecules can lead to increased resistance of drugs to enzymatic degradation, increased half-life in vivo, reduced dosing frequency, decreased immunogenicity, increased physical and thermal stability, increased solubility, increased liquid stability, and reduced aggregation.
  • U.S. Pat. No. 6,037,452 describes a poly(alkylene oxide)-FVIII or FIX conjugate, where the protein is covalently bound to a poly(alkylene oxide) through carbonyl- groups of said FVIII.
  • EP1258497B1 describes a method to prepare conjugates of FVIII and a biocompatible polymer. This patent was supplemented by a publication of R ⁇ stin et al. (Bioconj Chem 2000; 11:387-96). The conjugates comprise a B-domain deleted recombinant FVIII modified with monomethoxy polyethylene glycol. The conjugate had reduced FVIII function and the coagulant activity decreased rapidly with the degree of modification.
  • WO04075923A3 describes polymer-FVIII molecular conjugate comprising a plurality of conjugates wherein each conjugate has one to three water soluble polymers covalently attached to an FVIII molecule.
  • the FVIII molecule is B-domain-deleted.
  • U.S. Pat. No. 4,970,300 describes a modified FVIII, wherein an infusible conjugate comprising a protein having FVIII activity was covalently linked to a nonantigenic ligand.
  • U.S. Pat. No. 6,048,720 describes conjugates of a polypeptide and a biocompatible polymer.
  • WO94/15625 describes FVIII bound to polyethylene glycol having a preferred molecular weight of no greater than 5,000 Daltons.
  • an FVIII having an attached soluble polymer to extend the half-life of the FVIII in vivo, for example, a PEGylated FVIII, such as full-length FVIII having PEG greater than 10,000 Daltons conjugated thereto, which retains functional activity while providing an extended half-life in vivo, as compared to non-PEGylated FVIII.
  • a PEGylated FVIII such as full-length FVIII having PEG greater than 10,000 Daltons conjugated thereto, which retains functional activity while providing an extended half-life in vivo, as compared to non-PEGylated FVIII.
  • the present invention relates to a proteinaceous construct comprising a Factor VIII molecule which is conjugated to a water-soluble polymer via carbohydrate moieties of Factor VIII, and methods of preparing same.
  • a method of conjugating a water soluble polymer to an oxidized carbohydrate moiety of FVIII comprising contacting the oxidized carbohydrate moiety with an activated water soluble polymer under conditions that allow conjugation.
  • the water soluble polymer is selected from the group consisting of PEG, PSA and dextran.
  • the activated water soluble polymer is selected from the group consisting of PEG-hydrazide, PSA- hydrazine and aldehyde- activated dextran.
  • the carbohydrate moiety is oxidized by incubation in a buffer comprising NaIO 4 .
  • the oxidized carbohydrate moiety of FVIII is located in the B domain of FVIII.
  • a modified FVIII produced by the method according to any of the aforementioned methods is provided.
  • a proteinaceous construct comprising (a) a Factor VIII molecule; and (b) at least one water soluble polymer bound to said Factor VIII molecule, wherein the water soluble polymer is attached to the Factor VIII via one or more carbohydrate moieties located in the B domain Factor VIII.
  • the water soluble polymer is selected from the group consisting of PEG, PSA and dextran.
  • FIG. 1 shows the broadening and mass increase of rFVIII after conjugation with PEG measured by SDS-PAGE with subsequent immunoblotting.
  • FIG. 2 shows the pharmacokinetics of PEG-rFVIII conjugate compared to non-conjugated FVIII in hemophilic mice. Open squares: PEGrFVIII, dose 200 IU FVIII/kg. Closed diamonds: native rFVIII, dose 200 IU FVIII/kg.
  • FIG. 3 shows the detailed analysis of PEGylation sites by SDS-PAGE using various anti FVIII antibodies.
  • FIG. 4 shows the thrombin-induced activation and inactivation of native and PEGylated rFVIII.
  • FIG. 5 shows the bands demonstrating the domains of native and PEGylated rFVIII.
  • FIG. 6 shows the extent of PEGylation of various domains of native and PEGylated rFVIII.
  • FIG. 7 shows the thrombin inactivation rate of native and PEGylated rFVIII.
  • the invention is a proteinaceous construct comprising an FVIII molecule having at least a portion of the B domain intact, bound to a water-soluble polymer which include, a polyalkylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, polyoxazoline, a poly acryloylmorpholine or a carbohydrate, such as polysialic acid (PSA) or dextran.
  • a water-soluble polymer which include, a polyalkylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, polyoxazoline, a poly acryloylmorpholine or a carbohydrate, such as polysialic acid (PSA) or dextran.
  • the water soluble polymer is a polyethylene glycol molecule having a molecular weight of greater than 10,000 Daltons.
  • the water soluble polymer has a molecular weight of greater than 10,000 Da to about 125,000 Da, about 15,000 Da to 20,000 Da, or about 18,000 Da to about 25,000 Da.
  • the construct retains the full functional activity of standard therapeutic FVIII products, and provides an extended half- life in vivo, as compared to standard therapeutic FVIII products.
  • the construct retains at least 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, or 150 percent (%) biological activity relative to native Factor VIII.
  • the biological activities of the construct and native Factor VIII are determined by the ratios of chromo genie activity to FVIII antigen value (FVIII:Chr:FVIII:Ag).
  • the half-life of the construct is decreased or increased 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold relative to the in vivo half-life of native Factor VIII.
  • the starting material of the present invention is FVIII, which can be derived from human plasma, or produced by recombinant engineering techniques, as described in patents U.S. Pat. No. 4,757,006; U.S. Pat. No. 5,733,873; U.S. Pat. No. 5,198,349; U.S. Pat. No. 5,250,421; U.S. Pat. No. 5,919,766; EP 306 968.
  • the term "Factor VIII” or “FVIII” refers to any FVIII molecule which has at least a portion of the B domain intact, and which exhibits biological activity that is associated with native FVIII.
  • the FVIII molecule is full-length Factor VIII.
  • the FVIII molecule is a protein which is encoded for by DNA sequences capable of hybridizing to DNA encoding Factor VIILC. Such a protein may contain amino acid deletions at various sites between or within the domains A1-A2-B-A3- C1-C2 (U.S. Pat. No. 4,868,112).
  • the FVIII molecule may also be an analog of native FVIII wherein one or more amino acid residues have been replaced by site-directed mutagenesis.
  • the FVIII molecules useful for the present invention include the full-length protein, precursors of the protein, biologically active or functional subunits or fragments of the protein, and functional derivatives thereof, as well as variants thereof as described herein below.
  • Reference to FVIII is meant to include all potential forms of such proteins and wherein each of the forms of FVIII has at least a portion or all of the native B domain sequence intact.
  • the term "recombinant Factor VIII” may include any rFVIII, heterologous or naturally occurring, obtained via recombinant DNA technology, or a biologically active derivative thereof.
  • the term encompasses proteins as described above and nucleic acids, encoding a rFVIII of the invention.
  • nucleic acids include, for example and without limitation, genes, pre-mRNAs, mRNAs, polymorphic variants, alleles, synthetic and naturally-occurring mutants.
  • Proteins embraced by the term rFVIII include, for example and without limitation, those proteins and polypeptides described hereinabove, proteins encoded by a nucleic acid described above, interspecies homologs and other polypeptides that: (1) have an amino acid sequence that has greater than about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or greater amino acid sequence identity, over a region of at least about 25, about 50, about 100, about 200, about 300, about 400, or more amino acids (up to the full length sequence of 406 amino acids for the mature native protein), to a polypeptide encoded by a referenced nucleic acid or an amino acid sequence described herein; and/or (2) specifically bind to antibodies, e.g., polyclonal or monoclonal antibodies, generated against an immunogen comprising a referenced amino acid sequence as described herein
  • Polynucleotides encoding a rFVIII of the invention include, without limitation, those that (1) specifically hybridize under stringent hybridization conditions to a nucleic acid encoding a referenced amino acid sequence as described herein, and conservatively modified variants thereof; (2) have a nucleic acid sequence that has greater than about 95%, about 96%, about 97%, about 98%, about 99%, or higher nucleotide sequence identity, over a region of at least about 25, about 50, about 100, about 150, about 200, about 250, about 500, about 1000, or more nucleotides (up to the full length sequence of 1218 nucleotides of the mature protein), to a reference nucleic acid sequence as described herein.
  • endogenous FVIII includes FVIII which originates from the mammal intended to receive treatment.
  • the term also includes FVIII transcribed from a transgene or any other foreign DNA present in said mammal.
  • exogenous FVIII includes FVIII which does not originate from said mammal.
  • Variant (or analog) polypeptides include insertion variants, wherein one or more amino acid residues are added to an FVIII amino acid sequence of the invention. Insertions may be located at either or both termini of the protein, and/or may be positioned within internal regions of the FVIII amino acid sequence.
  • Insertion variants include for example, fusion proteins and proteins including amino acid tags or other animo acid labels.
  • the FVIII molecule may optionally contain an N-terminal Met, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli.
  • deletion variants one or more amino acid residues in a FVIII polypeptide as described herein are removed.
  • Deletions can be effected at one or both termini of the FVIII polypeptide, and/or with removal of one or more residues within the FVIII amino acid sequence.
  • Deletion variants therefore, include all fragments of a FVIII polypeptide sequence.
  • substitution variants one or more amino acid residues of a FVIII polypeptide are removed and replaced with alternative residues.
  • the substitutions are conservative in nature and conservative substitutions of this type are well known in the art.
  • the invention embraces substitutions that are also non- conservative. Exemplary conservative substitutions are described in Lehninger, [Biochemistry, 2nd Edition; Worth Publishers, Inc., New York (1975), pp.71-77] and set out immediately below.
  • a "naturally-occurring" polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or any mammal.
  • the nucleic acids and proteins of the invention can be recombinant molecules (e.g., heterologous and encoding the wild type sequence or a variant thereof, or non-naturally occurring).
  • Reference polynucleotide and polypeptide sequences include, e.g., UniProtKB/Swiss-Prot P00451 (FA8_HUMAN); Gitschier J et al., Characterization of the human Factor VIII gene, Nature, 312(5992): 326-30 (1984); Vehar GH et al., Structure of human Factor VIII, Nature, 312(5992):337-42 (1984); and Thompson AR. Structure and Function of the Factor VIII gene and protein, Semin Thromb Hemost, 2003:29;ll-29 (2002), (references incorporated herein in their entireties).
  • biologically active derivative or “biologically active variant” includes any derivative or variant of a molecule having substantially the same functional and/or biological properties of said molecule, such as binding properties, and/or the same structural basis, such as a peptidic backbone or a basic polymeric unit.
  • plasma-derived FVIII or "plasmatic” includes all forms of the protein found in blood obtained from a mammal having the property of activating the coagulation pathway.
  • production of rFVIII includes any method known in the art for (i) the production of recombinant DNA by genetic engineering, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by, for example and without limitation, transfection, electroporation or microinjection, (iii) cultivating said transformed cells, (iv) expressing rFVIII, e.g. constitutively or upon induction, and (v) isolating said rFVIII, e.g. from the culture medium or by harvesting the transformed cells, in order to (vi) obtain purified rFVIII.
  • the rFVIII is produced by expression in a suitable prokaryotic or eukaryotic host system characterized by producing a pharmacologically acceptable rFVIII molecule.
  • suitable prokaryotic or eukaryotic host system characterized by producing a pharmacologically acceptable rFVIII molecule.
  • eukaryotic cells are mammalian cells, such as CHO, COS, HEK 293, BHK, SK-Hep, and HepG2.
  • vectors are used for the preparation of the rFVIII and are selected from eukaryotic and prokaryotic expression vectors.
  • vectors for prokaryotic expression include plasmids such as, and without limitation, pRSET, pET, and pBAD, wherein the promoters used in prokaryotic expression vectors include one or more of, and without limitation, lac, trc, trp, recA, or araBAD.
  • vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as, and without limitation, pAO, pPIC, pYES, or pMET, using promoters such as, and without limitation, AOXl, GAP, GALl, or AUGl; (ii) for expression in insect cells, vectors such as and without limitation, pMT, pAc5, pIB, pMIB, or pBAC, using promoters such as and without limitation PH, plO, MT, Ac5, OpIE2, gp64, or polh, and (iii) for expression in mammalian cells, vectors such as and without limitation pSVL, pCMV, pRc/RSV, pcDNA3, or pBPV, and vectors derived from, in one aspect, viral systems such as and without limitation vaccinia virus, adeno-associated viruses, herpes viruses, or retroviruses, using promoters such as and without limitation
  • FVIII molecules are conjugated to a water soluble polymer by any of a variety of chemical methods (Roberts JM et al., Advan Drug Delivery Rev 2002;54:459-76).
  • FVIII is PEGylated by the conjugation of PEG to free amino groups of the protein using N-hydroxysuccinimide (NHS) esters.
  • NHS N-hydroxysuccinimide
  • the water soluble polymer, for example PEG is coupled to free SH groups using maleimide chemistry or the coupling of PEG hydrazides or PEG amines to carbohydrate moieties of the FVIII after prior oxidation.
  • FVIII is conjugated to other water soluble polymers, where the water soluble polymers are, for example, polyalkylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, polyoxazoline, a poly acryloylmorpholine, carbohydrate or a polysaccharide such as polysialic acid (PSA) or dextran.
  • the coupling of the water soluble polymer can be carried out by direct coupling to the protein or via linker molecules.
  • MBPH 4-[4-N-Maleimidophenyl]butyric acid hydrazide
  • carbohydrate-selective hydrazide and a sulfhydryl-reactive maleimide group
  • the conjugation can be performed by direct coupling (or coupling via linker systems) of the water soluble polymer to Factor VIII under formation of stable bonds.
  • degradable, releasable or hydrolysable linker systems can be used in the present invention (Tsubery et al. J Biol Chem 2004;279:38118-24 / Greenwald et al., J Med Chem 1999;42:3657-67 / Zhao et al., Bioconj Chem 2006;17:341-51 / WO2006/138572A2 / US7259224B2 / US7060259B2).
  • an embodiment of the invention is the coupling of the activated soluble polymer to the oxidized carbohydrate moiety of FVIII.
  • activated water soluble polymer is used herein to refer to water soluble polymers used for coupling to FVIII having an active functional group, which allows chemical conjugation of the water soluble polymer to a linker or directly to FVIII (which contains an active aldehyde group).
  • oxidized carbohydrate moiety refers to FVIII containing free aldehyde groups, which are generated by an oxidative agent such as NaIO 4 .
  • aldehyde- activated dextran (containing an active aldehyde groups) is coupled to the aldehyde groups of FVIII via a dihydrazide linker.
  • FVIII glycosylation pattern of FVIII (Lenting et al; Blood, 92:3983-96(1998))
  • conjugation of FVII via carbohydrate moieties should likely take place in the B domain of FVIII.
  • Targeting the B domain for such conjugation reactions is desired since the B domain does not play a role in the activity of FVIII.
  • Enzymatic glycoconjugation is described in US 2008/00700275.
  • FVIII was modified via lysine residues by use of polyethylene glycol derivatives containing an active N-hydroxysuccinimide ester (NHS) such as succinimidyl succinate, succinimidyl glutarate or succinimidyl propionate.
  • NPS N-hydroxysuccinimide ester
  • these derivatives react with the lysine residues of FVIII under mild conditions by forming a stable amide bond.
  • the chain length of the PEG derivative is 5,000 Da.
  • PEG derivatives with chain lengths of 500 to 2,000 Da, 2,000 to 5,000 Da, greater than 5,000 up to 10,000 Da or greater than 10,000 up to 20,000 Da, or greater than 20,000 up to 150,000 Da are used in various embodiments, including linear and branched structures.
  • an FVIII molecule is chemically modified using PEG derivatives that are commercially available. These PEG derivatives can have a linear or branched structures. Examples of PEG-derivatives containing NHS groups are listed below.
  • PEG derivatives are examples of those commercially available from Nektar Therapeutics (Huntsville, Ala.; see www.nektar.com/PEG reagent catalog; Nektar Advanced PEGylation, price list 2005-2006): mPEG- Succinimidyl propionate (mPEG-SPA)
  • PEG derivatives are commercially available from NOF Corporation (Tokyo, Japan; see www.nof.co.jp/english: Catalogue 2005)
  • propane derivatives show a glycerol backbone with a 1,2 substitution pattern.
  • branched PEG derivatives based on glycerol structures with 1,3 substitution or other branched structures described in US2003/0143596A1 can also be used.
  • PEG derivatives with degradable as described by Tsubery et al. (J Biol Chem 2004;279:38118-24) and Shechter et al. (WO04089280A3) can also be used in the present invention.
  • degradable linkers for example, hydrolysable linkers
  • the PEGylated FVIII of this invention exhibits full functional activity, combined with an extended FVIII half- life in vivo.
  • the PEGylated rFVIII seems to be more resistant against thrombin inactivation. This was shown by a variety of in vitro and in vivo methods, and is illustrated by the following examples.
  • sialic acid moieties includes sialic acid monomers or polymers ("polysaccharides") which are soluble in an aqueous solution or suspension and have little or no negative impact, such as side effects, to mammals upon administration of the PSA- FVIII -conjugate in a pharmaceutically effective amount.
  • the polymers are characterized, in one aspect, as having from 1 to 4 units. In certain aspects, different sialic acid units are combined in a chain.
  • sialic acid moieties are bound to FVIII for example by the method described in US Patent No. 4,356,170, which is herein incorporated by reference.
  • the polysaccharide compound is a naturally occurring polysaccharide, a derivative of a naturally occurring polysaccharide, or a naturally occurring polysaccharide derivative.
  • all of the saccharide residues in the compound are sialic acid residues.
  • the polysialic acid portion of the polysaccharide compound is highly hydrophilic, and in another embodiment the entire compound is highly hydrophilic. Hydrophilicity is conferred primarily by the pendant carboxyl groups of the sialic acid units, as well as the hydroxyl groups.
  • the saccharide unit may contain other functional groups, such as, amine, hydroxyl or sulphate groups, or combinations thereof. These groups may be present on naturally occurring saccharide compounds, or introduced into derivative polysaccharide compounds.
  • Polysaccharide compounds of particular use for the invention are, in one aspect, those produced by bacteria. Some of these naturally occurring polysaccharides are known as glycolipids. In one embodiment, the polysaccharide compounds are substantially free of terminal galactose units.
  • the in vivo half- life of the proteinaceous construct is prolonged.
  • the in vivo half- life of the proteinaceous construct is prolonged by at least a factor of two, while in another embodiment the in vivo half-life is prolonged by at least a factor of three, as compared to FVIII which is not bound to a water soluble polymer.
  • the proteinaceous construct of the present invention may be administered by injection, such as intravenous, intramuscular, or intraperitoneal injection.
  • compositions comprising a proteinaceous construct of the present invention to human or test animals
  • the compositions comprise one or more pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carriers include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like, including those agents disclosed above.
  • compositions may be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and or surgical implantation at a particular site is contemplated as well.
  • compositions are essentially free of pyrogens, as well as other impurities that could be harmful to the recipient.
  • compositions can be carried out with the dose levels and pattern being selected by the treating physician.
  • the appropriate dosage will depend on the type of disease to be treated, as described above, the severity and course of the disease, whether drug is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the drug, and the discretion of the attending physician.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of a proteinaceous construct as defined above.
  • the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, salt, buffer, or excipient.
  • the pharmaceutical composition can be used for treating the above-defined bleeding disorders.
  • the pharmaceutical composition of the invention may be a solution or a lyophilized product. Solutions of the pharmaceutical composition may be subjected to any suitable lyophylization process.
  • kits which comprise a composition of the invention packaged in a manner which facilitates its use for administration to subjects.
  • a kit includes a compound or composition described herein (e.g., a composition comprising a proteinaceous construct), packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition in practicing the method.
  • the kit contains a first container having a composition comprising a proteinaceous construct and a second container having a physiologically acceptable reconstitution solution for the composition in the first container.
  • the compound or composition is packaged in a unit dosage form.
  • the kit may further include a device suitable for administering the composition according to a specific route of administration.
  • the kit contains a label that describes use of the therapeutic protein or peptide composition.
  • reaction mixture was applied onto an equilibrated ion- exchange chromatography resin (Fractogel EMD TMAE 650M/Pharmacia XK-10 column, bed height: 15.0 cm) in 20 mM Hepes buffer, 150 mM NaCl, pH 7.4, containing 0.5% sucrose and 0.1% Polysorbate 80. Then the column was washed with 20 CV equilibration buffer to remove excess reagent and the PEGylated rFVIII was eluted with elution buffer (20 mM Hepes, 1.0 M NaCl, 0.5% sucrose, 0.1% Polysorbate 80, pH 7.4).
  • equilibrated ion- exchange chromatography resin Frazier EMD TMAE 650M/Pharmacia XK-10 column, bed height: 15.0 cm
  • the eluate was concentrated by ultrafiltration/diafiltration with a membrane consisting of regenerated cellulose and with a molecular weight cut-off of 30 kD using a buffer system consisting of 20 mM Hepes, 150 mM NaCl, 0.5% sucrose, pH 7.4.
  • RFVIII derived from the Advate manufacturing process was PEGylated according to Example 1 and the PEGylated FVIII product was biochemically characterized.
  • the functional activity of the PEG-rFVIII was determined by use of the FVIII chromogenic assay (Rosen S, Scand J Haematol 1984;33 (Suppl 40): 139-45). The method is based on Ph. Eur. 5th edition (5.05) 2.7.4 Assay of Blood Coagulation Factor VIII.
  • a sample, containing factor VIII is mixed with thrombin, activated factor IX (FIXa), phospholipids and factor X (FX) in a buffer containing calcium.
  • FVIII is activated by thrombin and subsequently forms a complex with phospholipids, FIXa and calcium ions.
  • This complex activates factor X to factor Xa, which in turn cleaves the chromogenic substrate FXa-I (AcOH*CH3OCO-D-CHA-Gly-Arg-pNA).
  • the time course of para-nitroaniline (pNA) released is measured with a micro plate reader at 405 nm. The slope of the reaction is proportional to the factor VIII concentration in the sample.
  • the FVIII antigen value was measured by use of an ELISA system commercially available (Cedarlane, Hornby, Ontario, Canada) with minor modifications. From these values the ratios FVIII chromogen/FVIII antigen were calculated.
  • the protein content in the preparations was determined by measuring the optical density at 280 nm. From these data the protein content was calculated (Hoyer LW in: Human Protein Data. Installments 1-6; Heberli Ed.; Wiley V C H, Weinheim, Germany, 1998) and expressed in mg/ml.
  • Native rFVIII was characterized by SDS PAGE under reducing conditions by using a 4-12% polyacrylamide gradient gel obtained from Invitrogen (Carlsbad, Calif., USA) according to the instructions of the manufacturer. As molecular weight markers (MW) Precision Plus markers (10 kD-250 kD) obtained from Bio-Rad (Hercules, Calif., USA) were used. Then the proteins were transferred on a PVDF membrane obtained from Bio-Rad (Hercules, Calif., USA) by electroblotting and subsequently incubated with a polyclonal sheep anti human FVIILC antibody obtained from Cedarlane (Hornby, Ontario, Canada).
  • MW molecular weight markers
  • Precision Plus markers 10 kD-250 kD obtained from Bio-Rad (Hercules, Calif., USA) were used. Then the proteins were transferred on a PVDF membrane obtained from Bio-Rad (Hercules, Calif., USA) by electroblotting and subsequently incubated with a poly
  • the last steps of the immuno staining procedure were the incubation with an alkaline phosphatase (ALP) conjugated anti-sheep antibody obtained from Accurate (Westbury, N. Y., USA) followed by the final visualization by use of an ALP substrate kit (Bio-Rad, Hercules, Calif., USA).
  • ALP alkaline phosphatase
  • FIG. 1 The blot demonstrates the domain structure of native and PEGylated rFVIII. It is shown that the PEGylated rFVIII has broader bands and high molecular masses than the native recombinant protein.
  • FVIII deficient mice described in detail by Bi et al. were used as a model of severe human hemophilia A.
  • Groups of 5 mice received a bolus injection (10 ml/kg) via the tail vein with either PEG-rFVIII (PEG-SS, 5K) prepared according to Example 1 or native rFVIII in a dose of 200 IU FVIII/kg bodyweight.
  • Citrate plasma by heart puncture after anesthesia was prepared from the respective groups, 5 minutes, 3, 6, 9 and 24 hours after injection. FVIII activity levels were measured in plasma samples. The results of this experiment are summarized in FIG. 2.
  • FVIII was incubated at 37 T! with 0.5 or 1 nM thrombin. Subsamples were withdrawn at time intervals between 0.5 to 40 minutes and added to a mixture of FIXa, FX, PL-vesicles and CaCl 2 also containing a specific thrombin inhibitor to stop the further thrombin-mediated reactions and incubated for 3 minutes. A subsample was added to a chromogenic substrate, which is selectively cleaved by FXa and contained EDTA to stop further Xa activation. After a 15 min incubation, the reaction was terminated by acetic acid.
  • the absorbance (A405) values which are proportional to the FXa concentrations, were measured in an ELISA reader and converted to FXa concentrations using a purified FXa reference curve. The generated FXa concentrations were plotted against the incubation time with thrombin.
  • Pseudo-first order inactivation rate of FVIII was determined by fitting the declining part of the curves with a single exponential fit.
  • a solution of rFVIII in 20 mM Hepes buffer pH 7.4 containing 150 mM NaCl, 0.5% sucrose and 0.1% Polysorbate 80 was prepared from bulk material derived from the Advate manufacturing process containing 489 IU FVIII/ml.
  • PEG-SG branched PEG succinimidyl glutarate
  • reaction mixture was applied onto an equilibrated ion- exchange chromatography resin (Fractogel EMD TMAE 650M/Pharmacia XK-50 column, bed height: 14.5 cm) in 20 mM Hepes buffer, 150 mM NaCl, pH 7.4, containing 0.5% sucrose and 0.1% Polysorbate 80 using a linear flow rate of 1 cm/min.
  • an equilibrated ion- exchange chromatography resin Frazier EMD TMAE 650M/Pharmacia XK-50 column, bed height: 14.5 cm
  • the column was washed with 25 CV equilibration buffer to remove excess reagent (linear flow rate: 2 cm/min) and the PEGylated rFVIII was eluted with elution buffer (20 mM Hepes, 1.0 M NaCl, 0.5% sucrose, 0.1% Polysorbate 80, pH 7.4) at a linear flow rate of 0.5 cm/min. Then the eluate was concentrated by ultrafiltration/diafiltration with a membrane consisting of regenerated cellulose and with a molecular weight cut-off of 30 kD using a buffer system consisting of 20 mM Hepes, 150 mM NaCl, 0.5% sucrose, pH 7.4.
  • RFVIII derived from the Advate manufacturing process was PEGylated via lysine residues using a branched PEG-SG reagent according to Example 7 and the PEGylated rFVIII product was biochemically characterized as described in Example 2.
  • the PEGylated rFVIII was characterized by SDS-PAGE and immunoblotting techniques under reducing conditions using a 4-12% polyacrylamide gradient gel as described in Example 3. The results are summarized in FIG. 5. The blot demonstrates the domain structure of native and PEGylated rFVIII. It is shown that the PEGylated rFVIII has broader bands and high molecular masses than the native recombinant protein.
  • a solution of rFVIII (final concentration: 1.2 mg/ml) is prepared in 25 mM phosphate buffer, pH 6.7. NaIO 4 is added (final concentration 0.3 mM) for the oxidation of carbohydrate residues (Roberts et al.; Advanced Drug Del Rev.; 54:459-76 (2002); Meir and Wilchek; Meth Enzymol;l38: 429-42(1987)).
  • the reaction was quenched by addition of glycerol in a final concentration of 10%, and the excess reagents were separated by repeated centrifugation using Amicon Micron-10 devices (Amicon, Billerica, MA). PEG-hydrazide (MW 3300 Da / Nektar, Huntsville, Alabama) was added to give a final concentration of 1.5 mM reagent. The PEGylation was then performed for 2h at room temperature. Subsequently, the conjugate obtained and the excess reagent was separated by repeated centrifugation on Amicon Micron-10 devices using 25 mM phosphate buffer, pH 6.7.
  • a solution of rFVIII (final concentration: 1 mg/ml) is prepared in 20 mM sodium acetate buffer, pH 6.0.
  • NaIO 4 is added (final concentration 0.25 mM) for the oxidation of carbohydrate residues. The oxidation is carried out for 60 min at 4 0 C in the dark.
  • Sodium bisulfite (final concentration 25 rnM) is added to stop the reaction. The excess sodium periodate is separated by gelfiltration on DG-10 columns (Bio-Rad).
  • PSA-hydrazine with a chain length of 20 kD (prepared according to WO2006/016168) is added (final concentration 10 mM).
  • the polysialylation procedure is carried out for 2 h at room temperature.
  • the polysialylated rFVIII is purified by HIC on Butyl-Sepharose (GE- Healthcare).
  • a 5 M NaCl solution is added to the mixture to give a final concentration of 3M NaCl.
  • Polysialic Acid was purified by anion-exchange chromatography on Q- Sepharose FF as described in WO06016161A1.
  • CV column volumes
  • the fraction eluted with 350 mM NaCl showed a molecular weight of 20 kDa as indicated by SDS gel electrophoresis.
  • This fraction was concentrated by ultrafiltration using a 5 kD membrane made of regenerated cellulose (Millipore, Billerica, MA) and subsequently diafiltrated against 50 mM phosphate buffer, pH 7.2.
  • the PSA was oxidized with NaIO 4 and a terminal primary amino group was introduced by reductive amination as described in WO05016973A1.
  • the mixture was then dialyzed against a (NH 4 ) 2 C ⁇ 3 solution (50 mg/L) containing 10 mM NaCl and subsequently against 50 mM phosphate buffer, pH 8.0, containing 5 mM EDTA.
  • a sulfhydryl group was next introduced by reaction of the terminal primary amino group with 2-iminothiolane (Traut's reagent / Pierce, Rockford, IL).
  • the reaction was carried out in 50 mM phosphate buffer, pH 8.0, containing 5 mM EDTA with 20 fold molar excess of reagent for Ih at room temperature.
  • the PSA solution containing a terminal free SH - group was subjected to ultrafiltration/diafiltration using a membrane with a cut-off of 5 kD and made of regenerated cellulose (Millipore, Billerica, MA).
  • the MPBH-PSA conjugate was added to the oxidized rFVIII (0.105 mg/ml protein) and the reaction mixture was incubated for 2h at RT under gentle shaking.
  • the rFVIII-PSA conjugate was purified by HIC using a prepacked Butyl Sepharose column (GE Healthcare, Butyl HiTrap FF 5 ml). To allow hydrophobic interactions of the conjugate with Butyl Sepharose the sample was cooled to 2-8 0 C and the ionic strength of the reaction mixture was increased to a conductivity of approx.
  • the conjugate was eluted with a buffer of low ionic strength, pH 7.4 (50 niM Hepes, 6.7 niM CaCl 2 ) with a flow rate of 1.2 cm /min. During the chromatography process, samples and buffers were cooled using an ice bath. Finally, the pH of the eluate was adjusted to 6.9.
  • rFVIII conjugation of rFVIII with dextran
  • 2 ml rFVIII (638 mg, 3.4 mg/ml protein) were transferred to oxidation buffer (50 mM sodium acetate, pH 6) using desalting columns (Bio-Rad Econopac 10 DG) according to the manufacturer's instruction.
  • the protein was then oxidated with 0.25mM NaIO 4 (Ih at 4 0 C in the dark).
  • the oxidated protein was first concentrated using vivaspin ultrafiltration spin columns (Sartorius Stedim Biotech GmbH) with a MWCO of 30 kDa according to the manufacturer's instructions.
  • the sample was next dialyzed against reaction buffer (50 mM sodium phosphate pH 7) over night at 4 0 C.
  • ADH adipic acid dihydrazide

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Abstract

The invention is a proteinaceous construct comprising a Factor VIII molecule which is conjugated to a water-soluble polymer via carbohydrate moieties of Factor VIII, and methods of preparing same. In one embodiment of the invention, the water-soluble polymer is selected from the group consisting of polyethylene glycol (PEG), polysialic acid (PSA) or dextran.

Description

FACTOR VIII POLYMER CONJUGATES FIELD OF THE INVENTION
[0001] This application is a continuation-in-part of U.S. Application No. 11/729,625, filed on March 29, 2007, which claims priority to U.S. Provisional Application Nos. 60/790,239 and 60/787,968, which were filed on June 6, 2006 and March 31, 2006, respectively.
[0002] The present invention relates to a proteinaceous construct comprising coagulation factor VIII (FVIII) being bound to at least one water soluble polymer, including a poly(alkylene oxide) such as polyethylene glycol. Further the present invention relates to methods for prolonging the in vivo-half-life of FVIII in the blood of a mammal having a bleeding disorder associated with functional defects or deficiencies of FVIII.
BACKGROUND OF THE INVENTION
[0003] Coagulation factor VIII (FVIII) circulates in plasma at a very low concentration and is bound non-covalently to von Willebrand factor (VWF). During hemostasis, FVIII is separated from VWF and acts as a cofactor for activated factor IX (FΙXa)-mediated factor X (FX) activation by enhancing the rate of activation in the presence of calcium and phospholipids or cellular membranes.
[0004] FVIII is synthesized as a single-chain precursor of approximately 270-330 kD with the domain structure A1-A2-B-A3-C1-C2. When purified from plasma (e.g., "plasma-derived" or "plasmatic"), FVIII is composed of a heavy chain (A1-A2-B) and a light chain (A3-C1-C2). The molecular mass of the light chain is 80 kD whereas, due to proteolysis within the B domain, the heavy chain is in the range of 90-220 kD.
[0005] FVIII is also synthesized as a recombinant protein for therapeutic use in bleeding disorders. Various in vitro assays have been devised to determine the potential efficacy of recombinant FVIII (rFVIII) as a therapeutic medicine. These assays mimic the in vivo effects of endogenous FVIII. In vitro thrombin treatment of FVIII results in a rapid increase and subsequent decrease in its procoagulant activity, as measured by in vitro assay. This activation and inactivation coincides with specific limited proteolysis both in the heavy and the light chains, which alter the availability of different binding epitopes in FVIII, e.g. allowing FVIII to dissociate from VWF and bind to a phospholipid surface or altering the binding ability to certain monoclonal antibodies. [0006] The lack or dysfunction of FVIII is associated with the most frequent bleeding disorder, hemophilia A. The treatment of choice for the management of hemophilia A is replacement therapy with plasma derived or rFVIII concentrates. Patients with severe haemophilia A with FVIII levels below 1%, are generally on prophylactic therapy with the aim of keeping FVIII above 1% between doses. Taking into account the average half- lives of the various FVIII products in the circulation, this can usually be achieved by giving FVIII two to three times a week.
[0007] There are many concentrates on the market for the treatment of hemophilia A. One of these concentrates is the recombinant product Advate®, which is produced in CHO-cells and manufactured by Baxter Healthcare Corporation. No human or animal plasma proteins or albumin are added in the cell culture process, purification, or final formulation of this product.
[0008] The aim of many manufacturers of FVIII concentrates and therapeutic polypeptide drugs is to develop a next generation product with enhanced pharmacodynamic and pharmacokinetic properties, while maintaining all other product characteristics.
[0009] Therapeutic polypeptide drugs are rapidly degraded by proteolytic enzymes and neutralized by antibodies. This reduces their half- life and circulation time, thereby limiting their therapeutic effectiveness. The addition of a soluble polymer or carbohydrate to a polypeptide has been shown to prevent degradation and increase the polypeptides half-life. For instance, PEGylation of polypeptide drugs protects them and improves their pharmacodynamic and pharmacokinetic profiles (Harris J M et Chess R B, Nat Rev Drug Discov 2003;2:214-21). The PEGylation process attaches repeating units of polyethylene glycol (PEG) to a polypeptide drug. PEGylation of molecules can lead to increased resistance of drugs to enzymatic degradation, increased half-life in vivo, reduced dosing frequency, decreased immunogenicity, increased physical and thermal stability, increased solubility, increased liquid stability, and reduced aggregation.
[0010] Thus, the addition of a soluble polymer, such as through PEGylation is one approach to improve the properties of a FVIII product. The state of the art is documented by different patents and patent applications:
[0011] U.S. Pat. No. 6,037,452 describes a poly(alkylene oxide)-FVIII or FIX conjugate, where the protein is covalently bound to a poly(alkylene oxide) through carbonyl- groups of said FVIII. [0012] EP1258497B1 describes a method to prepare conjugates of FVIII and a biocompatible polymer. This patent was supplemented by a publication of Rδstin et al. (Bioconj Chem 2000; 11:387-96). The conjugates comprise a B-domain deleted recombinant FVIII modified with monomethoxy polyethylene glycol. The conjugate had reduced FVIII function and the coagulant activity decreased rapidly with the degree of modification.
[0013] WO04075923A3 describes polymer-FVIII molecular conjugate comprising a plurality of conjugates wherein each conjugate has one to three water soluble polymers covalently attached to an FVIII molecule. The FVIII molecule is B-domain-deleted.
[0014] U.S. Pat. No. 4,970,300 describes a modified FVIII, wherein an infusible conjugate comprising a protein having FVIII activity was covalently linked to a nonantigenic ligand.
[0015] U.S. Pat. No. 6,048,720 describes conjugates of a polypeptide and a biocompatible polymer.
[0016] WO94/15625 describes FVIII bound to polyethylene glycol having a preferred molecular weight of no greater than 5,000 Daltons.
[0017] There remains a need for an FVIII having an attached soluble polymer to extend the half-life of the FVIII in vivo, for example, a PEGylated FVIII, such as full-length FVIII having PEG greater than 10,000 Daltons conjugated thereto, which retains functional activity while providing an extended half-life in vivo, as compared to non-PEGylated FVIII.
SUMMARY OF THE INVENTION
[0018] The present invention relates to a proteinaceous construct comprising a Factor VIII molecule which is conjugated to a water-soluble polymer via carbohydrate moieties of Factor VIII, and methods of preparing same.
[0019] In one embodiment of the invention, a method of conjugating a water soluble polymer to an oxidized carbohydrate moiety of FVIII is provided comprising contacting the oxidized carbohydrate moiety with an activated water soluble polymer under conditions that allow conjugation. In a related aspect, the water soluble polymer is selected from the group consisting of PEG, PSA and dextran. In still another aspect, the activated water soluble polymer is selected from the group consisting of PEG-hydrazide, PSA- hydrazine and aldehyde- activated dextran. In another aspect of the invention, the carbohydrate moiety is oxidized by incubation in a buffer comprising NaIO4. In still another aspect of the invention, the oxidized carbohydrate moiety of FVIII is located in the B domain of FVIII.
[0020] In another embodiment of the invention, a modified FVIII produced by the method according to any of the aforementioned methods is provided. In still another embodiment, a proteinaceous construct is provided comprising (a) a Factor VIII molecule; and (b) at least one water soluble polymer bound to said Factor VIII molecule, wherein the water soluble polymer is attached to the Factor VIII via one or more carbohydrate moieties located in the B domain Factor VIII. In a related aspect of the invention, the water soluble polymer is selected from the group consisting of PEG, PSA and dextran.
FIGURES
[0021] FIG. 1 shows the broadening and mass increase of rFVIII after conjugation with PEG measured by SDS-PAGE with subsequent immunoblotting.
[0022] FIG. 2 shows the pharmacokinetics of PEG-rFVIII conjugate compared to non-conjugated FVIII in hemophilic mice. Open squares: PEGrFVIII, dose 200 IU FVIII/kg. Closed diamonds: native rFVIII, dose 200 IU FVIII/kg.
[0023] FIG. 3 shows the detailed analysis of PEGylation sites by SDS-PAGE using various anti FVIII antibodies.
[0024] FIG. 4 shows the thrombin-induced activation and inactivation of native and PEGylated rFVIII.
[0025] FIG. 5 shows the bands demonstrating the domains of native and PEGylated rFVIII.
[0026] FIG. 6 shows the extent of PEGylation of various domains of native and PEGylated rFVIII.
[0027] FIG. 7 shows the thrombin inactivation rate of native and PEGylated rFVIII.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The invention is a proteinaceous construct comprising an FVIII molecule having at least a portion of the B domain intact, bound to a water-soluble polymer which include, a polyalkylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, polyoxazoline, a poly acryloylmorpholine or a carbohydrate, such as polysialic acid (PSA) or dextran. In one embodiment of the invention, the water soluble polymer is a polyethylene glycol molecule having a molecular weight of greater than 10,000 Daltons. In another embodiment, the water soluble polymer has a molecular weight of greater than 10,000 Da to about 125,000 Da, about 15,000 Da to 20,000 Da, or about 18,000 Da to about 25,000 Da. In one embodiment, the construct retains the full functional activity of standard therapeutic FVIII products, and provides an extended half- life in vivo, as compared to standard therapeutic FVIII products. In another embodiment, the construct retains at least 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, or 150 percent (%) biological activity relative to native Factor VIII. In a related aspect, the biological activities of the construct and native Factor VIII are determined by the ratios of chromo genie activity to FVIII antigen value (FVIII:Chr:FVIII:Ag). In still another embodiment of the invention, the half-life of the construct is decreased or increased 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold relative to the in vivo half-life of native Factor VIII.
[0029] The starting material of the present invention is FVIII, which can be derived from human plasma, or produced by recombinant engineering techniques, as described in patents U.S. Pat. No. 4,757,006; U.S. Pat. No. 5,733,873; U.S. Pat. No. 5,198,349; U.S. Pat. No. 5,250,421; U.S. Pat. No. 5,919,766; EP 306 968.
[0030] Herein, the term "Factor VIII" or "FVIII" refers to any FVIII molecule which has at least a portion of the B domain intact, and which exhibits biological activity that is associated with native FVIII. In one embodiment of the invention, the FVIII molecule is full-length Factor VIII. The FVIII molecule is a protein which is encoded for by DNA sequences capable of hybridizing to DNA encoding Factor VIILC. Such a protein may contain amino acid deletions at various sites between or within the domains A1-A2-B-A3- C1-C2 (U.S. Pat. No. 4,868,112). The FVIII molecule may also be an analog of native FVIII wherein one or more amino acid residues have been replaced by site-directed mutagenesis.
[0031] The FVIII molecules useful for the present invention include the full-length protein, precursors of the protein, biologically active or functional subunits or fragments of the protein, and functional derivatives thereof, as well as variants thereof as described herein below. Reference to FVIII is meant to include all potential forms of such proteins and wherein each of the forms of FVIII has at least a portion or all of the native B domain sequence intact.
[0032] According to the present invention, the term "recombinant Factor VIII " (rFVIII) may include any rFVIII, heterologous or naturally occurring, obtained via recombinant DNA technology, or a biologically active derivative thereof. In certain embodiments, the term encompasses proteins as described above and nucleic acids, encoding a rFVIII of the invention. Such nucleic acids include, for example and without limitation, genes, pre-mRNAs, mRNAs, polymorphic variants, alleles, synthetic and naturally-occurring mutants. Proteins embraced by the term rFVIII include, for example and without limitation, those proteins and polypeptides described hereinabove, proteins encoded by a nucleic acid described above, interspecies homologs and other polypeptides that: (1) have an amino acid sequence that has greater than about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or greater amino acid sequence identity, over a region of at least about 25, about 50, about 100, about 200, about 300, about 400, or more amino acids (up to the full length sequence of 406 amino acids for the mature native protein), to a polypeptide encoded by a referenced nucleic acid or an amino acid sequence described herein; and/or (2) specifically bind to antibodies, e.g., polyclonal or monoclonal antibodies, generated against an immunogen comprising a referenced amino acid sequence as described herein, an immunogenic fragment thereof, and/or a conservatively modified variant thereof.
[0033] Polynucleotides encoding a rFVIII of the invention include, without limitation, those that (1) specifically hybridize under stringent hybridization conditions to a nucleic acid encoding a referenced amino acid sequence as described herein, and conservatively modified variants thereof; (2) have a nucleic acid sequence that has greater than about 95%, about 96%, about 97%, about 98%, about 99%, or higher nucleotide sequence identity, over a region of at least about 25, about 50, about 100, about 150, about 200, about 250, about 500, about 1000, or more nucleotides (up to the full length sequence of 1218 nucleotides of the mature protein), to a reference nucleic acid sequence as described herein.
[0034] As used herein, "endogenous FVIII " includes FVIII which originates from the mammal intended to receive treatment. The term also includes FVIII transcribed from a transgene or any other foreign DNA present in said mammal. As used herein, "exogenous FVIII " includes FVIII which does not originate from said mammal. [0035] Variant (or analog) polypeptides include insertion variants, wherein one or more amino acid residues are added to an FVIII amino acid sequence of the invention. Insertions may be located at either or both termini of the protein, and/or may be positioned within internal regions of the FVIII amino acid sequence. Insertion variants, with additional residues at either or both termini, include for example, fusion proteins and proteins including amino acid tags or other animo acid labels. In one aspect, the FVIII molecule may optionally contain an N-terminal Met, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli.
[0036] In deletion variants, one or more amino acid residues in a FVIII polypeptide as described herein are removed. Deletions can be effected at one or both termini of the FVIII polypeptide, and/or with removal of one or more residues within the FVIII amino acid sequence. Deletion variants, therefore, include all fragments of a FVIII polypeptide sequence.
[0037] In substitution variants, one or more amino acid residues of a FVIII polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature and conservative substitutions of this type are well known in the art. Alternatively, the invention embraces substitutions that are also non- conservative. Exemplary conservative substitutions are described in Lehninger, [Biochemistry, 2nd Edition; Worth Publishers, Inc., New York (1975), pp.71-77] and set out immediately below.
CONSERVATIVE SUBSTITUTIONS
SIDE CHAIN AMINO ACID
CHARACTERISTIC
Non-polar (hydrophobic):
A. Aliphatic A L I V P
B. Aromatic F W
C. Sulfur-containing M
D. Borderline G Uncharged-polar:
A. Hydroxyl S T Y
B. Amides N Q C. Sulfhydryl C
D. Borderline G Positively charged (basic) KRH Negatively charged (acidic) D E
Alternatively, exemplary conservative substitutions are set out immediately below.
CONSERVATIVE SUBSTITUTIONS II
VAL RESIDUE EXEMPLARY
SUBSTITUTION
Ala (A) VaI, Leu, He
Arg (R) Lys, GIn, Asn
Asn (N) GIn, His, Lys, Arg
Asp (D) GIu
Cys (C) Ser
GIn (Q) Asn
GIu (E) Asp
His (H) Asn, GIn, Lys, Arg
He (I) Leu, VaI, Met, Ala, Phe,
Leu (L) He, VaI, Met, Ala, Phe
Lys (K) Arg, GIn, Asn
Met (M) Leu, Phe, He
Phe (F) Leu, VaI, He, Ala
Pro (P) GIy
Ser (S) Thr
Thr (T) Ser
Trp (W) Tyr
Tyr (Y) Trp, Phe, Thr, Ser
VaI (V) He, Leu, Met, Phe, Ala
[0038] A "naturally-occurring" polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or any mammal. The nucleic acids and proteins of the invention can be recombinant molecules (e.g., heterologous and encoding the wild type sequence or a variant thereof, or non-naturally occurring). Reference polynucleotide and polypeptide sequences include, e.g., UniProtKB/Swiss-Prot P00451 (FA8_HUMAN); Gitschier J et al., Characterization of the human Factor VIII gene, Nature, 312(5992): 326-30 (1984); Vehar GH et al., Structure of human Factor VIII, Nature, 312(5992):337-42 (1984); and Thompson AR. Structure and Function of the Factor VIII gene and protein, Semin Thromb Hemost, 2003:29;ll-29 (2002), (references incorporated herein in their entireties). [0039] As used herein "biologically active derivative" or "biologically active variant" includes any derivative or variant of a molecule having substantially the same functional and/or biological properties of said molecule, such as binding properties, and/or the same structural basis, such as a peptidic backbone or a basic polymeric unit.
[0040] As used herein, "plasma-derived FVIII " or "plasmatic" includes all forms of the protein found in blood obtained from a mammal having the property of activating the coagulation pathway.
[0041] In various aspects, production of rFVIII includes any method known in the art for (i) the production of recombinant DNA by genetic engineering, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by, for example and without limitation, transfection, electroporation or microinjection, (iii) cultivating said transformed cells, (iv) expressing rFVIII, e.g. constitutively or upon induction, and (v) isolating said rFVIII, e.g. from the culture medium or by harvesting the transformed cells, in order to (vi) obtain purified rFVIII.
[0042] In other aspects, the rFVIII is produced by expression in a suitable prokaryotic or eukaryotic host system characterized by producing a pharmacologically acceptable rFVIII molecule. Examples of eukaryotic cells are mammalian cells, such as CHO, COS, HEK 293, BHK, SK-Hep, and HepG2.
[0043] In still other aspects, a wide variety of vectors are used for the preparation of the rFVIII and are selected from eukaryotic and prokaryotic expression vectors. Examples of vectors for prokaryotic expression include plasmids such as, and without limitation, pRSET, pET, and pBAD, wherein the promoters used in prokaryotic expression vectors include one or more of, and without limitation, lac, trc, trp, recA, or araBAD. Examples of vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as, and without limitation, pAO, pPIC, pYES, or pMET, using promoters such as, and without limitation, AOXl, GAP, GALl, or AUGl; (ii) for expression in insect cells, vectors such as and without limitation, pMT, pAc5, pIB, pMIB, or pBAC, using promoters such as and without limitation PH, plO, MT, Ac5, OpIE2, gp64, or polh, and (iii) for expression in mammalian cells, vectors such as and without limitation pSVL, pCMV, pRc/RSV, pcDNA3, or pBPV, and vectors derived from, in one aspect, viral systems such as and without limitation vaccinia virus, adeno-associated viruses, herpes viruses, or retroviruses, using promoters such as and without limitation CMV, SV40, EF-I, UbC, RSV, ADV, BPV, and β- actin.
[0044] . In certain aspects, FVIII molecules are conjugated to a water soluble polymer by any of a variety of chemical methods (Roberts JM et al., Advan Drug Delivery Rev 2002;54:459-76). For example, in one embodiment FVIII is PEGylated by the conjugation of PEG to free amino groups of the protein using N-hydroxysuccinimide (NHS) esters. In another embodiment the water soluble polymer, for example PEG, is coupled to free SH groups using maleimide chemistry or the coupling of PEG hydrazides or PEG amines to carbohydrate moieties of the FVIII after prior oxidation.
[0045] In other embodiments, FVIII is conjugated to other water soluble polymers, where the water soluble polymers are, for example, polyalkylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, polyoxazoline, a poly acryloylmorpholine, carbohydrate or a polysaccharide such as polysialic acid (PSA) or dextran. The coupling of the water soluble polymer can be carried out by direct coupling to the protein or via linker molecules. One example of a chemical linker is MBPH (4-[4-N-Maleimidophenyl]butyric acid hydrazide) containing a carbohydrate-selective hydrazide and a sulfhydryl-reactive maleimide group (Chamow et al., J Biol Chem 1992;267: 15916-22).
[0046] The conjugation can be performed by direct coupling (or coupling via linker systems) of the water soluble polymer to Factor VIII under formation of stable bonds. In addition degradable, releasable or hydrolysable linker systems can be used in the present invention (Tsubery et al. J Biol Chem 2004;279:38118-24 / Greenwald et al., J Med Chem 1999;42:3657-67 / Zhao et al., Bioconj Chem 2006;17:341-51 / WO2006/138572A2 / US7259224B2 / US7060259B2).
[0047] As discussed herein, an embodiment of the invention is the coupling of the activated soluble polymer to the oxidized carbohydrate moiety of FVIII. The term "activated water soluble polymer" is used herein to refer to water soluble polymers used for coupling to FVIII having an active functional group, which allows chemical conjugation of the water soluble polymer to a linker or directly to FVIII (which contains an active aldehyde group). The term "oxidized carbohydrate moiety" as used herein refers to FVIII containing free aldehyde groups, which are generated by an oxidative agent such as NaIO4. In one aspect of the invention, aldehyde- activated dextran (containing an active aldehyde groups) is coupled to the aldehyde groups of FVIII via a dihydrazide linker. [0048] According to the glycosylation pattern of FVIII (Lenting et al; Blood, 92:3983-96(1998)), conjugation of FVII via carbohydrate moieties should likely take place in the B domain of FVIII. Targeting the B domain for such conjugation reactions is desired since the B domain does not play a role in the activity of FVIII. Enzymatic glycoconjugation is described in US 2008/00700275.
[0049] In one embodiment of the invention, FVIII was modified via lysine residues by use of polyethylene glycol derivatives containing an active N-hydroxysuccinimide ester (NHS) such as succinimidyl succinate, succinimidyl glutarate or succinimidyl propionate. These derivatives react with the lysine residues of FVIII under mild conditions by forming a stable amide bond. In one embodiment of the invention, the chain length of the PEG derivative is 5,000 Da. Other PEG derivatives with chain lengths of 500 to 2,000 Da, 2,000 to 5,000 Da, greater than 5,000 up to 10,000 Da or greater than 10,000 up to 20,000 Da, or greater than 20,000 up to 150,000 Da are used in various embodiments, including linear and branched structures.
[0050] Alternative methods for the PEGylation of amino groups are the chemical conjugation with PEG carbonates by forming urethane bonds, or the reaction with aldehydes or ketones by reductive amination forming secondary amide bonds.
[0051] In the present invention an FVIII molecule is chemically modified using PEG derivatives that are commercially available. These PEG derivatives can have a linear or branched structures. Examples of PEG-derivatives containing NHS groups are listed below.
[0052] The following PEG derivatives are examples of those commercially available from Nektar Therapeutics (Huntsville, Ala.; see www.nektar.com/PEG reagent catalog; Nektar Advanced PEGylation, price list 2005-2006): mPEG- Succinimidyl propionate (mPEG-SPA)
Figure imgf000013_0001
mPEG-Succinimidyl α-methylbutanoate (mPEG-SMB)
Figure imgf000014_0001
mPEG-CM-HB A-NHS (CM=carboxymethyl; HBA=Hydroxy butyric acid)
Figure imgf000014_0002
Structure of a Branched PEG-derivative (Nektar Therapeutics): Branched PEG N-Hydroxysuccinimide (mPEG2-NHS)
Figure imgf000014_0003
[0053] This reagent with branched structure is described in more detail by Kozlowski et al. (BioDrugs 2001;5:419-29).
[0054] Other examples of PEG derivatives are commercially available from NOF Corporation (Tokyo, Japan; see www.nof.co.jp/english: Catalogue 2005)
General Structure of Linear PEG-derivatives (NOF Corp.):
Figure imgf000014_0004
X=carboxymethyl —
Figure imgf000015_0001
X=carboxypentyl
—
Figure imgf000015_0002
x=succinate
Figure imgf000015_0003
mPFG Succinimidyl succinate
x=glutarate
Figure imgf000015_0004
mPEG Succinimidyl glutarate
Structures of Branched PEG-derivatives (NOF Corp.): 2,3- Bis(methylpolyoxyethylene-oxy)-l-(l,5-dioxo-5-succinimidyloxy, pentyloxy)propane
Figure imgf000015_0005
2,3-Bis(methylpolyoxyethylene-oxy)-l -(succinimidyl carboxypentyloxy)propane
Figure imgf000016_0001
[0055] These propane derivatives show a glycerol backbone with a 1,2 substitution pattern. In the present invention branched PEG derivatives based on glycerol structures with 1,3 substitution or other branched structures described in US2003/0143596A1 can also be used.
[0056] PEG derivatives with degradable (for example, hydrolysable linkers) as described by Tsubery et al. (J Biol Chem 2004;279:38118-24) and Shechter et al. (WO04089280A3) can also be used in the present invention.
[0057] Surprisingly, the PEGylated FVIII of this invention exhibits full functional activity, combined with an extended FVIII half- life in vivo. In addition the PEGylated rFVIII seems to be more resistant against thrombin inactivation. This was shown by a variety of in vitro and in vivo methods, and is illustrated by the following examples.
[0058] As used herein, "sialic acid moieties" includes sialic acid monomers or polymers ("polysaccharides") which are soluble in an aqueous solution or suspension and have little or no negative impact, such as side effects, to mammals upon administration of the PSA- FVIII -conjugate in a pharmaceutically effective amount. There is no particular limitation to the sialic acid unit used according to the present invention. The polymers are characterized, in one aspect, as having from 1 to 4 units. In certain aspects, different sialic acid units are combined in a chain.
[0059] In various aspects of the invention, sialic acid moieties are bound to FVIII for example by the method described in US Patent No. 4,356,170, which is herein incorporated by reference. In various embodiments of the invention, the polysaccharide compound is a naturally occurring polysaccharide, a derivative of a naturally occurring polysaccharide, or a naturally occurring polysaccharide derivative. Generally, all of the saccharide residues in the compound are sialic acid residues.
[0060] Other techniques for coupling PSA to polypeptides are also known. For example, US Publication No. 2007/0282096 describes conjugating an amine or hydrazide derivative of, e.g., PSA, to proteins. In addition, US Publication No. 2007/0191597 describes PSA derivatives containing an aldehyde group for reaction with substrates (e.g., proteins) at the reducing terminal end.
[0061] In one embodiment of the invention, the polysialic acid portion of the polysaccharide compound is highly hydrophilic, and in another embodiment the entire compound is highly hydrophilic. Hydrophilicity is conferred primarily by the pendant carboxyl groups of the sialic acid units, as well as the hydroxyl groups. The saccharide unit may contain other functional groups, such as, amine, hydroxyl or sulphate groups, or combinations thereof. These groups may be present on naturally occurring saccharide compounds, or introduced into derivative polysaccharide compounds.
[0062] Polysaccharide compounds of particular use for the invention are, in one aspect, those produced by bacteria. Some of these naturally occurring polysaccharides are known as glycolipids. In one embodiment, the polysaccharide compounds are substantially free of terminal galactose units.
[0063] In one embodiment of the present invention, the in vivo half- life of the proteinaceous construct is prolonged. In a related embodiment, the in vivo half- life of the proteinaceous construct is prolonged by at least a factor of two, while in another embodiment the in vivo half-life is prolonged by at least a factor of three, as compared to FVIII which is not bound to a water soluble polymer.
[0064] In one embodiment the proteinaceous construct of the present invention may be administered by injection, such as intravenous, intramuscular, or intraperitoneal injection.
[0065] To administer compositions comprising a proteinaceous construct of the present invention to human or test animals, in one aspect, the compositions comprise one or more pharmaceutically acceptable carriers. The terms "pharmaceutically" or "pharmacologically acceptable" refer to molecular entities and compositions that are stable, inhibit protein degradation such as aggregation and cleavage products, and in addition do not produce allergic, or other adverse reactions when administered using routes well-known in the art, as described below. "Pharmaceutically acceptable carriers" include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like, including those agents disclosed above.
[0066] As used herein, "effective amount" includes a dose suitable for treating a mammal having a bleeding disorder as outlined above. [0067] The compositions may be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and or surgical implantation at a particular site is contemplated as well. Generally, compositions are essentially free of pyrogens, as well as other impurities that could be harmful to the recipient.
[0068] Single or multiple administrations of the compositions can be carried out with the dose levels and pattern being selected by the treating physician. For the prevention or treatment of disease, the appropriate dosage will depend on the type of disease to be treated, as described above, the severity and course of the disease, whether drug is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the drug, and the discretion of the attending physician.
[0069] The present invention also relates to a pharmaceutical composition comprising an effective amount of a proteinaceous construct as defined above. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, salt, buffer, or excipient. The pharmaceutical composition can be used for treating the above-defined bleeding disorders. The pharmaceutical composition of the invention may be a solution or a lyophilized product. Solutions of the pharmaceutical composition may be subjected to any suitable lyophylization process.
[0070] As an additional aspect, the invention includes kits which comprise a composition of the invention packaged in a manner which facilitates its use for administration to subjects. In one embodiment, such a kit includes a compound or composition described herein (e.g., a composition comprising a proteinaceous construct), packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition in practicing the method. In one embodiment, the kit contains a first container having a composition comprising a proteinaceous construct and a second container having a physiologically acceptable reconstitution solution for the composition in the first container. In one aspect, the compound or composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the composition according to a specific route of administration. Preferably, the kit contains a label that describes use of the therapeutic protein or peptide composition.
EXAMPLES
Example 1 PEGylation of Lysine Residues in rFVIII with mPEG Succinimidyl Succinate
[0071] A solution of a rFVIII bulk derived from the Advate manufacturing process (3,400 U/ml) was gel filtrated by use of Econo-Pac 10DG columns (Bio-Rad) using 20 mM Hepes buffer, 150 mM NaCl, pH 7.4, containing 0.5% sucrose and 0.1% Polysorbate 80. Then mPEG Succinimidyl succinate (Abuchowski et al. Cancer Biochim Biophys 1984;7: 175-86) with a chain length of 5,000 Da (PEG-SS 5000) was added to this solution under gentle stirring (5 mg PEG-SS/mg protein) and the pH value was adjusted to 7.4 by drop wise addition of 0.5 M NaOH. Then the PEGylation was carried out under gentle stirring for 1 hour at room temperature.
[0072] Subsequently the reaction mixture was applied onto an equilibrated ion- exchange chromatography resin (Fractogel EMD TMAE 650M/Pharmacia XK-10 column, bed height: 15.0 cm) in 20 mM Hepes buffer, 150 mM NaCl, pH 7.4, containing 0.5% sucrose and 0.1% Polysorbate 80. Then the column was washed with 20 CV equilibration buffer to remove excess reagent and the PEGylated rFVIII was eluted with elution buffer (20 mM Hepes, 1.0 M NaCl, 0.5% sucrose, 0.1% Polysorbate 80, pH 7.4). The eluate was concentrated by ultrafiltration/diafiltration with a membrane consisting of regenerated cellulose and with a molecular weight cut-off of 30 kD using a buffer system consisting of 20 mM Hepes, 150 mM NaCl, 0.5% sucrose, pH 7.4.
Example 2 Biochemical Characterization of PEGylated rFVIII in vitro
[0073] RFVIII derived from the Advate manufacturing process was PEGylated according to Example 1 and the PEGylated FVIII product was biochemically characterized. The functional activity of the PEG-rFVIII was determined by use of the FVIII chromogenic assay (Rosen S, Scand J Haematol 1984;33 (Suppl 40): 139-45). The method is based on Ph. Eur. 5th edition (5.05) 2.7.4 Assay of Blood Coagulation Factor VIII.
[0074] A sample, containing factor VIII (FVIILC) is mixed with thrombin, activated factor IX (FIXa), phospholipids and factor X (FX) in a buffer containing calcium. FVIII is activated by thrombin and subsequently forms a complex with phospholipids, FIXa and calcium ions. This complex activates factor X to factor Xa, which in turn cleaves the chromogenic substrate FXa-I (AcOH*CH3OCO-D-CHA-Gly-Arg-pNA). The time course of para-nitroaniline (pNA) released is measured with a micro plate reader at 405 nm. The slope of the reaction is proportional to the factor VIII concentration in the sample. The FVIII antigen value was measured by use of an ELISA system commercially available (Cedarlane, Hornby, Ontario, Canada) with minor modifications. From these values the ratios FVIII chromogen/FVIII antigen were calculated. The protein content in the preparations was determined by measuring the optical density at 280 nm. From these data the protein content was calculated (Hoyer LW in: Human Protein Data. Installments 1-6; Heberli Ed.; Wiley V C H, Weinheim, Germany, 1998) and expressed in mg/ml.
TABLE 1
PEG-rFVIII
Native rFVIII PEG-SS 5K (5 mg per mg protein)
FVIII:Chr activity 3,430 64 [U/ml]
FVIILAg 4,067 81
[U/ml]
Ratio 0.84 0.79 FVIII:Chr/FVIII:Ag
Recovery of biological 100 94 activity (%)
[0075] The data in Table 1 shows that in the PEGylated rFVIII preparation, the biological activity (expressed by the ratio FVIII chromogenic activity to FVIII antigen) is recovered to more than 90% in comparison to the biological activity of the native rFVIII (100%).
Example 3 Characterization of PEGylated rFVIII by SDS-PAGE and Immunoblotting Techniques
[0076] Native rFVIII was characterized by SDS PAGE under reducing conditions by using a 4-12% polyacrylamide gradient gel obtained from Invitrogen (Carlsbad, Calif., USA) according to the instructions of the manufacturer. As molecular weight markers (MW) Precision Plus markers (10 kD-250 kD) obtained from Bio-Rad (Hercules, Calif., USA) were used. Then the proteins were transferred on a PVDF membrane obtained from Bio-Rad (Hercules, Calif., USA) by electroblotting and subsequently incubated with a polyclonal sheep anti human FVIILC antibody obtained from Cedarlane (Hornby, Ontario, Canada). The last steps of the immuno staining procedure were the incubation with an alkaline phosphatase (ALP) conjugated anti-sheep antibody obtained from Accurate (Westbury, N. Y., USA) followed by the final visualization by use of an ALP substrate kit (Bio-Rad, Hercules, Calif., USA). The results are summarized in FIG. 1. The blot demonstrates the domain structure of native and PEGylated rFVIII. It is shown that the PEGylated rFVIII has broader bands and high molecular masses than the native recombinant protein.
Example 4 Pharmacokinetics of PEGylated rFVIII in a FVIII Deficient Knock Out Mouse Model
[0077] FVIII deficient mice described in detail by Bi et al. (Nat Genet 1995;10:119-21) were used as a model of severe human hemophilia A. Groups of 5 mice received a bolus injection (10 ml/kg) via the tail vein with either PEG-rFVIII (PEG-SS, 5K) prepared according to Example 1 or native rFVIII in a dose of 200 IU FVIII/kg bodyweight. Citrate plasma by heart puncture after anesthesia was prepared from the respective groups, 5 minutes, 3, 6, 9 and 24 hours after injection. FVIII activity levels were measured in plasma samples. The results of this experiment are summarized in FIG. 2. Mean half life increased from 1.9 hours (for native rFVIII) to 4.9 hours (for PEGylated rFVIII), area under curve (AUC) increased from 13.0 to 25.2 hours*IU/ml. Half-life calculation was performed with MicroMath Scientist, model 1 from pharmacokinetic library (MicroMath, Saint Louis, Mo., USA).
Example 5 Detailed Analysis of PEGylation of rFVIII by SDS-PAGE and Immunoblotting Techniques
[0078] Native and PEGylated rFVIII was digested with 1 nM thrombin for 60 minutes at 6OT!, which resulted in specific cleavage of the FVIII molecule with well defined degradation products. These heavy- and light chain fragments were separated by SDS-PAGE followed by electroblotting, as described in Example 3. To visualize the cleaved fragments, a polyclonal antibody and monoclonal antibodies against the heavy chain Al and A2 domains, the B domain and the light chain N-terminal A3 domain were applied. [0079] As seen in FIG. 3 all domains were PEGylated, albeit to a different extent. The B domain was strongly PEGylated. Both the Al and A2 domains of the heavy chain were partially PEGylated. Various PEGylation-degrees (mono-, di-, tri- . . . ) could be observed in the light chain A3 -domain. In agreement with Example 6, the PEGylated FVIII seemed to be more resistant to thrombin.
Example 6 Thrombin-resistancy of PEGylated rFVIII
[0080] In vitro thrombin treatment of FVIII results in a rapid increase and subsequent decrease in its procoagulant activity. The rate of activation and inactivation, which depends on the thrombin concentration and on the integrity of FVIII, was monitored by a FIXa cof actor assay, as follows:
[0081] FVIII was incubated at 37 T! with 0.5 or 1 nM thrombin. Subsamples were withdrawn at time intervals between 0.5 to 40 minutes and added to a mixture of FIXa, FX, PL-vesicles and CaCl2 also containing a specific thrombin inhibitor to stop the further thrombin-mediated reactions and incubated for 3 minutes. A subsample was added to a chromogenic substrate, which is selectively cleaved by FXa and contained EDTA to stop further Xa activation. After a 15 min incubation, the reaction was terminated by acetic acid. The absorbance (A405) values, which are proportional to the FXa concentrations, were measured in an ELISA reader and converted to FXa concentrations using a purified FXa reference curve. The generated FXa concentrations were plotted against the incubation time with thrombin.
[0082] Pseudo-first order inactivation rate of FVIII was determined by fitting the declining part of the curves with a single exponential fit.
TABLE 2
First order inactivation Rate k' (1/min)
Relative k'
Thrombin Native FVIII PEG-FVIII PEG/native
0.5 nM 0.14 0.08 0.57
1 nM 0.24 0.14 0.58 [0083] As shown in FIG. 4 and Table 2, PEGylated rFVIII showed a slower inactivation rate at both applied thrombin concentrations.
Example 7
PEGylation of Lysine Residues in rFVIII with Branched 2,3-Bis(methylpolyoxyethylene- oxy)-l-(l, 5-dioxo-5-succinimidyloxy, pentyloxy)propane
[0084] A solution of rFVIII in 20 mM Hepes buffer pH 7.4 containing 150 mM NaCl, 0.5% sucrose and 0.1% Polysorbate 80 was prepared from bulk material derived from the Advate manufacturing process containing 489 IU FVIII/ml. A branched PEG succinimidyl glutarate (PEG-SG) reagent (2,3-Bis(methylpolyoxyethylene-oxy)-l-(l,5- dioxo-5-succinimidyloxy, pentyloxy) propane) obtained from NOF Corporation (Tokyo, Japan) with a molecular weight of 20 kD was added to 153 ml of this solution under gentle stirring (5 mg reagent/mg protein) and the pH value was adjusted to 7.4 by drop wise addition of 0.5 M NaOH after 10 minutes. Then the PEGylation of rFVIII was performed under gentle stirring for 1 hour at room temperature.
[0085] Subsequently the reaction mixture was applied onto an equilibrated ion- exchange chromatography resin (Fractogel EMD TMAE 650M/Pharmacia XK-50 column, bed height: 14.5 cm) in 20 mM Hepes buffer, 150 mM NaCl, pH 7.4, containing 0.5% sucrose and 0.1% Polysorbate 80 using a linear flow rate of 1 cm/min. The column was washed with 25 CV equilibration buffer to remove excess reagent (linear flow rate: 2 cm/min) and the PEGylated rFVIII was eluted with elution buffer (20 mM Hepes, 1.0 M NaCl, 0.5% sucrose, 0.1% Polysorbate 80, pH 7.4) at a linear flow rate of 0.5 cm/min. Then the eluate was concentrated by ultrafiltration/diafiltration with a membrane consisting of regenerated cellulose and with a molecular weight cut-off of 30 kD using a buffer system consisting of 20 mM Hepes, 150 mM NaCl, 0.5% sucrose, pH 7.4.
Example 8 In- vitro Characterization of rFVIII PEGylated with Branched PEG-SG 20 kD
[0086] RFVIII derived from the Advate manufacturing process was PEGylated via lysine residues using a branched PEG-SG reagent according to Example 7 and the PEGylated rFVIII product was biochemically characterized as described in Example 2. TABLE 3
PEG-rFVIII
Native rFVIII PEG-SG 2OK (5 mg per mg protein)
FVIII:Chr activity 9,950 1,040 [U/ml]
FVIILAg 20,807 1,763
[U/ml]
Ratio 0.48 0.59 FVIII:Chr/FVIII:Ag
Recovery of biological 100 120 activity (%)
[0087] The data in Table 3 show that in the PEGylated rFVIII preparation the biological activity (expressed by the ratio FVIII chromogenic activity to FVIII antigen) completely recovered in comparison to the biological activity of the native rFVIII (100%).
[0088] The PEGylated rFVIII was characterized by SDS-PAGE and immunoblotting techniques under reducing conditions using a 4-12% polyacrylamide gradient gel as described in Example 3. The results are summarized in FIG. 5. The blot demonstrates the domain structure of native and PEGylated rFVIII. It is shown that the PEGylated rFVIII has broader bands and high molecular masses than the native recombinant protein.
[0089] For more detailed analysis of PEGylation of the rFVIII preparation by SDS- PAGE and immunoblotting techniques, the native and PEGylated rFVIII was digested with 1 nM thrombin for 60 minutes at 60 °, which resulted in specific cleavage of the FVIII molecule with well defined degradation products, as described in Example 5. The fragments were separated by SDS-PAGE followed by electroblotting and visualized by different anti-FVIII antibodies. As seen in FIG. 6, all domains were PEGylated, albeit to a different extent. The B domain was strongly PEGylated. Various PEGylation-degrees (mono-, di-, tri-PEGylation) could be observed in the light chain A3-domain. The results indicate that the PEGylated rFVIII seemed to be more resistant to thrombin. [0090] The rate of activation and inactivation by thrombin was monitored by a FIXa cofactor assay as described in Example 6. Pseudo-first order inactivation rate of FVIII was determined by fitting the declining part of the curves with a single exponential fit.
TABLE 4
First order inactivation Rate k' (1/min)
Relative k'
Thrombin Native FVIII PEG-FVIII PEG/native
0.5 nM 0.13 0.09 0.67
1 nM 0.21 0.15 0.71
[0091] As shown in FIG. 7 and Table 4, the PEGylated rFVIII showed a slower inactivation rate at both applied thrombin concentrations.
Example 9 PEGylation of rFVIII via carbohydrate moiety
[0092] For preparation of a PEG-rFVIII conjugate via carbohydrate residues, a solution of rFVIII (final concentration: 1.2 mg/ml) is prepared in 25 mM phosphate buffer, pH 6.7. NaIO4 is added (final concentration 0.3 mM) for the oxidation of carbohydrate residues (Roberts et al.; Advanced Drug Del Rev.; 54:459-76 (2002); Meir and Wilchek; Meth Enzymol;l38: 429-42(1987)). The reaction was quenched by addition of glycerol in a final concentration of 10%, and the excess reagents were separated by repeated centrifugation using Amicon Micron-10 devices (Amicon, Billerica, MA). PEG-hydrazide (MW 3300 Da / Nektar, Huntsville, Alabama) was added to give a final concentration of 1.5 mM reagent. The PEGylation was then performed for 2h at room temperature. Subsequently, the conjugate obtained and the excess reagent was separated by repeated centrifugation on Amicon Micron-10 devices using 25 mM phosphate buffer, pH 6.7.
Example 10 Polysialylation of rFVIII with PSA-hydrazine
[0093] For preparation of a PSA-rFVIII conjugate via carbohydrate residues, a solution of rFVIII (final concentration: 1 mg/ml) is prepared in 20 mM sodium acetate buffer, pH 6.0. NaIO4 is added (final concentration 0.25 mM) for the oxidation of carbohydrate residues. The oxidation is carried out for 60 min at 40C in the dark. Sodium bisulfite (final concentration 25 rnM) is added to stop the reaction. The excess sodium periodate is separated by gelfiltration on DG-10 columns (Bio-Rad). Subsequently, PSA-hydrazine with a chain length of 20 kD (prepared according to WO2006/016168) is added (final concentration 10 mM). The polysialylation procedure is carried out for 2 h at room temperature. The polysialylated rFVIII is purified by HIC on Butyl-Sepharose (GE- Healthcare). A 5 M NaCl solution is added to the mixture to give a final concentration of 3M NaCl. This mixture is applied to the column filled with Butyl-Sepharose (GE-Healthcare) and the elution of the rFVIII-PSA conjugate is carried out with 50 mM Hepes-buffer, pH 7.4, containing 6.7 mM CaCl2. After elution of the conjugate, the pH is adjusted to pH 6.9.
Example 11 Purification and derivatization of Polysialic Acid
[0094] Polysialic Acid was purified by anion-exchange chromatography on Q- Sepharose FF as described in WO06016161A1. Five grams of PSA were dissolved in 50 mL 10 mM Triethanolamine buffer, pH 7.4 containing 25 mM NaCl (= starting buffer). This solution was applied onto a Pharmacia XK50 column filled with Q-Sepharose FF (GE Healthcare, Munich, Germany), which was equilibrated with starting buffer. The column was next washed with 8 column volumes (CV) starting buffer and the bound PSA was eluted stepwise with 3CV 200 mM NaCl, 350 mM NaCl and 500 mM NaCl in starting buffer. The fraction eluted with 350 mM NaCl showed a molecular weight of 20 kDa as indicated by SDS gel electrophoresis. This fraction was concentrated by ultrafiltration using a 5 kD membrane made of regenerated cellulose (Millipore, Billerica, MA) and subsequently diafiltrated against 50 mM phosphate buffer, pH 7.2. The PSA was oxidized with NaIO4 and a terminal primary amino group was introduced by reductive amination as described in WO05016973A1. For reductive amination, 11 mL of a 2 M NH4Cl solution were added to 20 mL of a solution containing 58 mg oxidized PSA / ml in 50 mM phosphate buffer, pH 7.2. A solution of 5M NaCNBH3 in IM NaOH was then added to give a final concentration of 75 mM. The reaction was performed for 5d at room temperature at pH 8.0.
[0095] The mixture was then dialyzed against a (NH4)2Cθ3 solution (50 mg/L) containing 10 mM NaCl and subsequently against 50 mM phosphate buffer, pH 8.0, containing 5 mM EDTA. A sulfhydryl group was next introduced by reaction of the terminal primary amino group with 2-iminothiolane (Traut's reagent / Pierce, Rockford, IL). The reaction was carried out in 50 mM phosphate buffer, pH 8.0, containing 5 mM EDTA with 20 fold molar excess of reagent for Ih at room temperature. Finally the PSA solution containing a terminal free SH - group was subjected to ultrafiltration/diafiltration using a membrane with a cut-off of 5 kD and made of regenerated cellulose (Millipore, Billerica, MA).
Example 12 Polysialylation of rFVIII by use of a heterobifunctional cross-linker
[0096] For coupling of PSA-SH to rFVIII, the heterobifunctional cross-linker MBPH (4-[4-N-Maleimidophenyl]butyric acid hydrazide-HCl/Pierce, Rockford, IL) containing a carbohydrate-selective hydrazide and a sulfhydryl-reactive maleimide group was used (Chamow et al., J Biol Chem; 267:15916-22(1992)). PSA-SH containing an active sulfhydryl group was prepared according to Example 11.
[0097] Two ml rFVIII (638 mg, 3.856 mg/ml protein concentration) were transferred to oxidation buffer (50 mM sodium acetate, pH 6) using desalting columns (Bio- Rad Econopac 10 DG) according to the instructions of the manufacturer. The protein was then oxidized with 0.25mM NaIO4 (Merck) (Ih at 40C in the dark). The oxidation reaction was quenched with glycerol in a final concentration of 10%. Glycerol and NaIO4 were removed and the protein was transferred into reaction buffer (50 mM sodium phosphate pH 6.5) using desalting columns (Bio-Rad Econopac 10 DG) according to the manufacturer's instructions. A mixture containing 1 mg MBPH/mg protein and PSA-SH (200 fold molar excess to protein) were next incubated for 2h at RT at pH 6.5. The excess of linker was removed using desalting columns (Bio-Rad Econopac 10 DG) according to the instructions of the manufacturer and the linker-PSA conjugate was transferred into reaction buffer.
[0098] The MPBH-PSA conjugate was added to the oxidized rFVIII (0.105 mg/ml protein) and the reaction mixture was incubated for 2h at RT under gentle shaking. The rFVIII-PSA conjugate was purified by HIC using a prepacked Butyl Sepharose column (GE Healthcare, Butyl HiTrap FF 5 ml). To allow hydrophobic interactions of the conjugate with Butyl Sepharose the sample was cooled to 2-80C and the ionic strength of the reaction mixture was increased to a conductivity of approx. 185 mS/cm by adding a buffer solution containing 5 M NaCl (50 mM Hepes, 5 M NaCl, 6.7 mM CaCl2, 0.01% Tween, pH 6.9). The reaction mixture was loaded onto the column that was equilibrated with equilibration buffer pH 6.9 (containing 50 mM Hepes, 3 M NaCl, 6.7 mM CaCl2, 0.01% Tween 80) with a flow rate of 1.2 cm/min. Unbound sample was washed out with 10 column volumes (CV) of equilibration buffer. The conjugate was eluted with a buffer of low ionic strength, pH 7.4 (50 niM Hepes, 6.7 niM CaCl2) with a flow rate of 1.2 cm /min. During the chromatography process, samples and buffers were cooled using an ice bath. Finally, the pH of the eluate was adjusted to 6.9.
Example 13 Conjugation of rFVIII with Dextran
[0099] For conjugation of rFVIII with dextran, 2 ml rFVIII (638 mg, 3.4 mg/ml protein) were transferred to oxidation buffer (50 mM sodium acetate, pH 6) using desalting columns (Bio-Rad Econopac 10 DG) according to the manufacturer's instruction. The protein was then oxidated with 0.25mM NaIO4 (Ih at 40C in the dark). The oxidated protein was first concentrated using vivaspin ultrafiltration spin columns (Sartorius Stedim Biotech GmbH) with a MWCO of 30 kDa according to the manufacturer's instructions. The sample was next dialyzed against reaction buffer (50 mM sodium phosphate pH 7) over night at 40C.
[00100] After dialysis, 26.58 mg adipic acid dihydrazide (ADH) (Sigma) was added (500 fold molar excess) and the reaction mixture was incubated 2 h at RT at pH 7 under gentle shaking. ADH was removed using desalting columns (Bio-Rad Econopac 10 DG) according to the instructions of the manufacturer. Ten mg aldehyde- activated dextran (Pierce) was added (17 fold molar excess to protein) and the mixture was incubated for 2 h at RT, pH 7.
[00101] The conjugate was purified by IEX chromatography on Q-Sepharose HP (GE-Healthcare). The sample was loaded onto a column (6.4 mm x 3 cm, V = ImI) that was equilibrated with buffer A (50 mM sodium phosphate pH 6.8) with a flow rate of 0.5 ml/min. Unbound sample was washed out with 5 CV buffer A. Finally, the conjugate was eluted with a linear salt gradient (0-100% buffer B [50 mM sodium phosphate pH 6.8 + IM NaCl] in 10 CV) with a flow rate of 0.5 ml/min.

Claims

CLAIMS:
1. A method of conjugating a water soluble polymer to an oxidized carbohydrate moiety of Factor VIII comprising contacting the oxidized carbohydrate moiety with an activated water soluble polymer under conditions that allow conjugation.
2. The method according to claim 1 wherein the water soluble polymer is selected from the group consisting of PEG, PSA and dextran.
3. The method according to claim 1 wherein the activated water soluble polymer is selected from the group consisting of PEG-hydrazide, PSA-hydrazine and aldehyde- activated dextran.
4. The method according to claim 1 wherein the carbohydrate moiety is oxidized by incubation in a buffer comprising NaIO4.
5. The method according to claim 1 wherein the oxidized carbohydrate moiety of FVIII is located in the B domain of Factor VIII.
6. A modified Factor VIII produced by the method according to any one of claims 1-5.
7. A proteinaceous construct comprising:
(a) a Factor VIII molecule; and
(b) at least one water soluble polymer bound to said Factor VIII molecule, wherein said water soluble polymer is attached to the Factor VIII via one or more carbohydrate moieties located in the B domain Factor VIII.
8. The proteinaceous construct of claim 7 wherein the water soluble polymer is selected from the group consisting of PEG, PSA and dextran..
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013519636A (en) * 2010-02-16 2013-05-30 ノヴォ ノルディスク アー／エス Conjugate FVIII variant
JP2013536180A (en) * 2010-07-30 2013-09-19 バクスター・インターナショナル・インコーポレイテッド Nucleophilic catalysts for oxime ligation
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Families Citing this family (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004099231A2 (en) 2003-04-09 2004-11-18 Neose Technologies, Inc. Glycopegylation methods and proteins/peptides produced by the methods
US9005625B2 (en) * 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
US20080305992A1 (en) 2003-11-24 2008-12-11 Neose Technologies, Inc. Glycopegylated erythropoietin
EP1771066A2 (en) 2004-07-13 2007-04-11 Neose Technologies, Inc. Branched peg remodeling and glycosylation of glucagon-like peptide-1 glp-1
US20080176790A1 (en) 2004-10-29 2008-07-24 Defrees Shawn Remodeling and Glycopegylation of Fibroblast Growth Factor (Fgf)
PT2371856T (en) * 2004-11-12 2022-08-12 Bayer Healthcare Llc Site-directed modification of fviii
US9029331B2 (en) * 2005-01-10 2015-05-12 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
EP1871795A4 (en) * 2005-04-08 2010-03-31 Biogenerix Ag Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
US20070105755A1 (en) * 2005-10-26 2007-05-10 Neose Technologies, Inc. One pot desialylation and glycopegylation of therapeutic peptides
WO2007056191A2 (en) 2005-11-03 2007-05-18 Neose Technologies, Inc. Nucleotide sugar purification using membranes
US7982010B2 (en) * 2006-03-31 2011-07-19 Baxter International Inc. Factor VIII polymer conjugates
US7645860B2 (en) * 2006-03-31 2010-01-12 Baxter Healthcare S.A. Factor VIII polymer conjugates
US7683158B2 (en) 2006-03-31 2010-03-23 Baxter International Inc. Pegylated factor VIII
US7985839B2 (en) * 2006-03-31 2011-07-26 Baxter International Inc. Factor VIII polymer conjugates
US20080242607A1 (en) * 2006-07-21 2008-10-02 Neose Technologies, Inc. Glycosylation of peptides via o-linked glycosylation sequences
US20100075375A1 (en) * 2006-10-03 2010-03-25 Novo Nordisk A/S Methods for the purification of polypeptide conjugates
JP5457185B2 (en) * 2006-10-04 2014-04-02 ノヴォ ノルディスク アー／エス Glycerol-linked PEGylated sugars and glycopeptides
WO2008074032A1 (en) 2006-12-15 2008-06-19 Baxter International Inc. Factor viia- (poly) sialic acid conjugate having prolonged in vivo half-life
DK2144923T3 (en) * 2007-04-03 2013-05-13 Biogenerix Ag METHODS OF TREATMENT WITH USING GLYCOPEGYLATED G-CSF
WO2008154639A2 (en) 2007-06-12 2008-12-18 Neose Technologies, Inc. Improved process for the production of nucleotide sugars
JP5647899B2 (en) * 2008-01-08 2015-01-07 ラツィオファルム ゲーエムベーハーｒａｔｉｏｐｈａｒｍ ＧｍｂＨ Glycoconjugation of polypeptides using oligosaccharyltransferase
CA2715465C (en) 2008-02-27 2017-03-21 Novo Nordisk A/S Conjugated factor viii molecules
CN102348715B (en) 2009-02-03 2017-12-08 阿穆尼克斯运营公司 Extension recombinant polypeptide and the composition for including the extension recombinant polypeptide
NZ597600A (en) 2009-07-27 2014-05-30 Lipoxen Technologies Ltd Glycopolysialylation of non-blood coagulation proteins
US8642737B2 (en) 2010-07-26 2014-02-04 Baxter International Inc. Nucleophilic catalysts for oxime linkage
DK2459224T3 (en) * 2009-07-27 2016-07-25 Baxalta GmbH Blodstørkningsproteinkonjugater
US8809501B2 (en) * 2009-07-27 2014-08-19 Baxter International Inc. Nucleophilic catalysts for oxime linkage
PL2459224T3 (en) * 2009-07-27 2017-08-31 Baxalta GmbH Blood coagulation protein conjugates
AU2010290077C1 (en) 2009-08-24 2015-12-03 Bioverativ Therapeutics Inc. Coagulation factor IX compositions and methods of making and using same
EP2616486B1 (en) 2010-09-15 2019-01-02 Stichting Sanquin Bloedvoorziening Factor viii variants having a decreased cellular uptake
HUE049352T2 (en) 2010-12-22 2020-09-28 Baxalta GmbH Materials and methods for conjugating a water soluble fatty acid derivative to a protein
EP3412314A1 (en) 2011-05-27 2018-12-12 Baxalta GmbH Therapeutic proteins conjugated to polysialic acid and methods of preparing same
CN102584933A (en) * 2012-02-13 2012-07-18 汪志友 Method for improving separation efficiency, purity and biological specific activity of blood coagulation factor VIII and analog thereof by using affine aqueous two-phase system
NZ628014A (en) 2012-02-15 2016-09-30 Biogen Ma Inc Recombinant factor viii proteins
RS63870B1 (en) 2012-02-15 2023-01-31 Bioverativ Therapeutics Inc Factor viii compositions and methods of making and using same
AU2013204754C1 (en) 2012-05-16 2018-10-11 Takeda Pharmaceutical Company Limited Nucleophilic Catalysts for Oxime Linkage
GB201210770D0 (en) * 2012-06-18 2012-08-01 Polytherics Ltd Novel conjugation reagents
EP3033097B1 (en) 2013-08-14 2021-03-10 Bioverativ Therapeutics Inc. Factor viii-xten fusions and uses thereof
JP2015155469A (en) * 2015-06-01 2015-08-27 バクスター・インターナショナル・インコーポレイテッドＢａｘｔｅｒ Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｉｎｃｏｒｐ０Ｒａｔｅｄ Fviii-polymer conjugates
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AU2016362606A1 (en) 2015-12-03 2018-06-28 Takeda Pharmaceutical Company Limited Factor VIII with extended half-life and reduced ligand-binding properties
MA45473A (en) 2016-04-04 2019-02-13 Shire Human Genetic Therapies CONJUGATE C1 ESTERASE INHIBITOR AND ITS USES
AU2017296195A1 (en) 2016-07-11 2019-01-24 Translate Bio Ma, Inc. Nucleic acid conjugates and uses thereof
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JP2018115170A (en) * 2018-03-02 2018-07-26 バクスアルタ ゲーエムベーハー Factor fviii-polymer conjugates
WO2019219049A1 (en) * 2018-05-18 2019-11-21 北京辅仁瑞辉生物医药研究院有限公司 Improved fviii fusion protein and use thereof
JP2021523878A (en) 2018-05-18 2021-09-09 バイオベラティブ セラピューティクス インコーポレイテッド How to treat hemophilia A
WO2020099513A1 (en) 2018-11-13 2020-05-22 Lipoxen Technologies Limited Glycopolysialylation of blinatumomab
AU2023232562A1 (en) 2022-03-08 2024-09-05 Equashield Medical Ltd Fluid transfer station in a robotic pharmaceutical preparation system

Family Cites Families (87)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4356170A (en) 1981-05-27 1982-10-26 Canadian Patents & Development Ltd. Immunogenic polysaccharide-protein conjugates
US4757006A (en) * 1983-10-28 1988-07-12 Genetics Institute, Inc. Human factor VIII:C gene and recombinant methods for production
US4965199A (en) 1984-04-20 1990-10-23 Genentech, Inc. Preparation of functional human factor VIII in mammalian cells using methotrexate based selection
US4970300A (en) * 1985-02-01 1990-11-13 New York University Modified factor VIII
AU591671B2 (en) 1985-04-11 1989-12-14 Children's Medical Center Corporation Von willebrand factor
CA1341174C (en) 1985-04-12 2001-01-30 John J. Toole Jr. Procoagulant proteins derived from factor viii: c
US5250421A (en) * 1986-01-03 1993-10-05 Genetics Institute, Inc. Method for producing factor VIII:C-type proteins
US5198349A (en) * 1986-01-03 1993-03-30 Genetics Institute, Inc. Method for producing factor VIII:C and analogs
CA1339946C (en) 1987-03-31 1998-07-07 Michael J. Griffith Ultrapurification process for polypeptides
JPH0387173A (en) 1987-09-10 1991-04-11 Teijin Ltd Preparation of human active natural type factor viii c and transformant using the same
US5605884A (en) 1987-10-29 1997-02-25 Rhone-Poulenc Rorer Pharmaceuticals Inc. Factor VIII formulations in high ionic strength media
US4877608A (en) 1987-11-09 1989-10-31 Rorer Pharmaceutical Corporation Pharmaceutical plasma protein formulations in low ionic strength media
US5153265A (en) * 1988-01-20 1992-10-06 Cetus Corporation Conjugation of polymer to colony stimulating factor-1
US5252421A (en) * 1988-07-18 1993-10-12 Fuji Xerox Co., Ltd. Electrophotographic toner
US5122614A (en) * 1989-04-19 1992-06-16 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
SE465222C5 (en) 1989-12-15 1998-02-10 Pharmacia & Upjohn Ab A recombinant human factor VIII derivative and process for its preparation
DE4001451A1 (en) 1990-01-19 1991-08-01 Octapharma Ag STABLE INJECTABLE SOLUTIONS OF FACTOR VIII AND FACTOR IX
SE466754B (en) * 1990-09-13 1992-03-30 Berol Nobel Ab COVALENT BINDING POLYMERS TO HYDROPHILIC SURFACES
CA2073511A1 (en) * 1990-11-14 1992-05-29 Matthew R. Callstrom Conjugates of poly(vinylsaccharide) with proteins for the stabilization of proteins
EP0576589A4 (en) 1991-03-18 1994-07-27 Enzon Inc Hydrazine containing conjugates of polypeptides and glycopolypeptides with polymers
US5595732A (en) 1991-03-25 1997-01-21 Hoffmann-La Roche Inc. Polyethylene-protein conjugates
DE4111393A1 (en) 1991-04-09 1992-10-15 Behringwerke Ag STABILIZED FACTOR VIII PREPARATIONS
US5846951A (en) 1991-06-06 1998-12-08 The School Of Pharmacy, University Of London Pharmaceutical compositions
MX9203763A (en) 1991-06-28 1993-08-01 Rhone Poulenc Rorer Int THERAPEUTIC POLYPEPTIDES BASED ON THE VON WILLEBRAND FACTOR
US6037452A (en) * 1992-04-10 2000-03-14 Alpha Therapeutic Corporation Poly(alkylene oxide)-Factor VIII or Factor IX conjugate
AU5098193A (en) 1992-09-01 1994-03-29 Berlex Laboratories, Inc. Glycolation of glycosylated macromolecules
CA2124690C (en) * 1992-10-02 2007-09-11 Thomas Osterberg Composition comprising coagulation factor viii formulation, process for its preparation and use of a surfactant as stabilizer
NZ250375A (en) 1992-12-09 1995-07-26 Ortho Pharma Corp Peg hydrazone and peg oxime linkage forming reagents and protein derivatives
US5298643A (en) * 1992-12-22 1994-03-29 Enzon, Inc. Aryl imidate activated polyalkylene oxides
FR2700268B1 (en) 1993-01-13 1995-03-31 Lvmh Rech Cosmetic or pharmaceutical composition, especially dermatological, containing an extract of Vismia.
WO1994015625A1 (en) 1993-01-15 1994-07-21 Enzon, Inc. Factor viii - polymeric conjugates
US5621039A (en) * 1993-06-08 1997-04-15 Hallahan; Terrence W. Factor IX- polymeric conjugates
US5919455A (en) 1993-10-27 1999-07-06 Enzon, Inc. Non-antigenic branched polymer conjugates
DE4435485C1 (en) 1994-10-04 1996-03-21 Immuno Ag Process for obtaining high-purity von Willebrand factor
US5700873A (en) * 1995-03-07 1997-12-23 Adhesives Research, Inc. Method of preparation of water-soluble copolymer
US6127153A (en) 1995-06-07 2000-10-03 Neose Technologies, Inc. Method of transferring at least two saccharide units with a polyglycosyltransferase, a polyglycosyltransferase and gene encoding a polyglycosyltransferase
WO1996040731A1 (en) 1995-06-07 1996-12-19 Mount Sinai School Of Medicine Of The City University Of New York Pegylated modified proteins
SE9503380D0 (en) * 1995-09-29 1995-09-29 Pharmacia Ab Protein derivatives
US6005077A (en) 1995-11-10 1999-12-21 Immuno Aktiengesellschaft Use of von willebrand factor and pharmaceutical formulation
US6562781B1 (en) 1995-11-30 2003-05-13 Hamilton Civic Hospitals Research Development Inc. Glycosaminoglycan-antithrombin III/heparin cofactor II conjugates
US6214966B1 (en) 1996-09-26 2001-04-10 Shearwater Corporation Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution
AT405740B (en) 1996-12-13 1999-11-25 Immuno Ag FROM WILLEBRAND FACTOR DERIVATIVE AND A METHOD FOR ISOLATING PROTEINS
AT406867B (en) 1997-02-27 2000-10-25 Immuno Ag METHOD FOR OBTAINING HIGH PURITY VWF OR FACTOR VIII / VWF COMPLEX
WO1998048837A1 (en) * 1997-04-30 1998-11-05 Enzon, Inc. Polyalkylene oxide-modified single chain polypeptides
US6183738B1 (en) * 1997-05-12 2001-02-06 Phoenix Pharamacologics, Inc. Modified arginine deiminase
AT405485B (en) 1997-05-28 1999-08-25 Immuno Ag A PHARMACEUTICAL PREPARATION CONTAINING THE VWF PROPEPTIDE
WO1998055607A2 (en) * 1997-06-04 1998-12-10 Oxford Biomedica (Uk) Limited Tumor targeted vector
AU2855499A (en) 1998-03-24 1999-10-18 Nof Corporation Oxirane derivatives and process for producing the same
DK1121156T3 (en) 1998-10-16 2006-06-06 Biogen Idec Inc Polymer conjugates of interferon-beta-1a and their use
DE19852729A1 (en) * 1998-11-16 2000-05-18 Werner Reutter Recombinant glycoproteins, processes for their preparation, medicaments containing them and their use
AT407750B (en) 1999-02-19 2001-05-25 Immuno Ag METHOD FOR PRODUCING A VWF PREPARATION
DK2130554T3 (en) 1999-02-22 2012-12-03 Univ Connecticut Albumin-free factor VIII preparations
AU6357900A (en) 1999-07-20 2001-02-05 Amgen, Inc. Hyaluronic acid-protein conjugates, pharmaceutical compositions and related methods
US6531122B1 (en) * 1999-08-27 2003-03-11 Maxygen Aps Interferon-β variants and conjugates
CN1309423C (en) * 1999-11-12 2007-04-11 马克西根控股公司 Interferon gamma conjugates
SE515295C2 (en) 1999-11-23 2001-07-09 Medicarb Ab Process for the preparation of conjugates of a biologically active polysaccharide and a solid substrate, and such conjugates
US7074878B1 (en) 1999-12-10 2006-07-11 Harris J Milton Hydrolytically degradable polymers and hydrogels made therefrom
US6413507B1 (en) 1999-12-23 2002-07-02 Shearwater Corporation Hydrolytically degradable carbamate derivatives of poly (ethylene glycol)
WO2001058935A2 (en) * 2000-02-11 2001-08-16 Maxygen Aps FACTOR VII OR VIIa-LIKE MOLECULES
US6586398B1 (en) * 2000-04-07 2003-07-01 Amgen, Inc. Chemically modified novel erythropoietin stimulating protein compositions and methods
US7118737B2 (en) * 2000-09-08 2006-10-10 Amylin Pharmaceuticals, Inc. Polymer-modified synthetic proteins
EP1333036B1 (en) 2000-10-16 2009-01-21 Chugai Seiyaku Kabushiki Kaisha Peg-modified erythropoietin
AUPR610501A0 (en) 2001-07-04 2001-07-26 Smart Drug Systems Inc Treatment of parasitic disease
US6913915B2 (en) * 2001-08-02 2005-07-05 Phoenix Pharmacologics, Inc. PEG-modified uricase
US7265084B2 (en) 2001-10-10 2007-09-04 Neose Technologies, Inc. Glycopegylation methods and proteins/peptides produced by the methods
US7157277B2 (en) 2001-11-28 2007-01-02 Neose Technologies, Inc. Factor VIII remodeling and glycoconjugation of Factor VIII
CN101724075B (en) * 2001-10-10 2014-04-30 诺和诺德公司 Remodeling and glycoconjugation of peptides
US7026440B2 (en) 2001-11-07 2006-04-11 Nektar Therapeutics Al, Corporation Branched polymers and their conjugates
US6473908B1 (en) * 2002-01-09 2002-11-05 Thomas A. Bontems Garment having a buttocks cleavage revealing feature
CN102139114A (en) 2003-02-26 2011-08-03 尼克塔治疗公司 Polymer factor VIII moiety conjugates
US20050176108A1 (en) 2003-03-13 2005-08-11 Young-Min Kim Physiologically active polypeptide conjugate having prolonged in vivo half-life
WO2004089280A2 (en) 2003-04-08 2004-10-21 Yeda Research And Development Co. Ltd. Reversible pegylated drugs
ES2294535T3 (en) 2003-08-12 2008-04-01 Lipoxen Technologies Limited DERIVATIVES OF POLISIALIC ACID.
EP1654290B1 (en) 2003-08-12 2019-03-13 Lipoxen Technologies Limited Sialic acid derivatives for protein derivatisation and conjugation
EP1673453A2 (en) 2003-10-07 2006-06-28 Novo Nordisk Health Care AG Hybrid molecules having factor vii/viia activity
JP2007513965A (en) 2003-12-10 2007-05-31 ネクター セラピューティクス アラバマ，コーポレイション Composition comprising two populations of polymer-active agent complex
JP5026266B2 (en) 2004-08-12 2012-09-12 リポクセン テクノロジーズ リミテッド Separation
WO2006016168A2 (en) 2004-08-12 2006-02-16 Lipoxen Technologies Limited Sialic acid derivatives
EP1835938B1 (en) 2004-12-27 2013-08-07 Baxter International Inc. Polymer-von willebrand factor-conjugates
PT2279758E (en) 2005-06-16 2015-05-27 Nektar Therapeutics Conjugates having a degradable linkage and polymeric reagents useful in preparing such conjugates
US7645860B2 (en) * 2006-03-31 2010-01-12 Baxter Healthcare S.A. Factor VIII polymer conjugates
US7683158B2 (en) * 2006-03-31 2010-03-23 Baxter International Inc. Pegylated factor VIII
WO2008025856A2 (en) * 2006-09-01 2008-03-06 Novo Nordisk Health Care Ag Modified glycoproteins
WO2008074032A1 (en) * 2006-12-15 2008-06-19 Baxter International Inc. Factor viia- (poly) sialic acid conjugate having prolonged in vivo half-life
US8642737B2 (en) 2010-07-26 2014-02-04 Baxter International Inc. Nucleophilic catalysts for oxime linkage
AU2016362606A1 (en) 2015-12-03 2018-06-28 Takeda Pharmaceutical Company Limited Factor VIII with extended half-life and reduced ligand-binding properties

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2349342B1 (en) * 2008-10-17 2018-06-27 Baxalta GmbH Modified blood factors comprising a low degree of water soluble polymer
EP3437661A1 (en) * 2008-10-17 2019-02-06 Baxalta GmbH Modified blood factors comprising a low degree of water soluble polymer
JP2013519636A (en) * 2010-02-16 2013-05-30 ノヴォ ノルディスク アー／エス Conjugate FVIII variant
US9018166B2 (en) 2010-02-16 2015-04-28 Novo Nordisk A/S Conjugated FVIII variants
JP2021038256A (en) * 2010-07-30 2021-03-11 バクスアルタ インコーポレイテッド Nucleophilic catalyst for oxime linkage
JP7258999B2 (en) 2010-07-30 2023-04-17 武田薬品工業株式会社 Nucleophilic catalyst for oxime ligation
JP2022046676A (en) * 2010-07-30 2022-03-23 武田薬品工業株式会社 Nucleophilic catalysts for oxime linkage
JP7054407B2 (en) 2010-07-30 2022-04-13 武田薬品工業株式会社 Nucleophilic catalyst for oxime linkage
JP2019116500A (en) * 2010-07-30 2019-07-18 バクスアルタ インコーポレイテッド Nucleophilic catalyst for oxime linkage
JP2013536180A (en) * 2010-07-30 2013-09-19 バクスター・インターナショナル・インコーポレイテッド Nucleophilic catalysts for oxime ligation
US9486506B2 (en) 2011-09-23 2016-11-08 Novo Nordisk A/S Glucagon analogues
US9486505B2 (en) 2011-09-23 2016-11-08 Novo Nordisk A/S Glucagon analogues
JP2015517506A (en) * 2012-05-14 2015-06-22 ノヴォ ノルディスク アー／エス Stabilized protein solution
US11466051B2 (en) 2012-05-14 2022-10-11 Novo Nordisk A/S Stabilised protein solutions
US9751927B2 (en) 2013-04-18 2017-09-05 Novo Nordisk A/S Stable, protracted GLP-1/glucagon receptor co-agonists for medical use
US9474790B2 (en) 2013-04-18 2016-10-25 Novo Nordisk A/S Stable, protracted GLP-1/glucagon receptor co-agonists for medical use
US10570184B2 (en) 2014-06-04 2020-02-25 Novo Nordisk A/S GLP-1/glucagon receptor co-agonists for medical use
US10238718B2 (en) 2014-08-04 2019-03-26 Csl Limited Factor VIII formulation
WO2016142288A1 (en) 2015-03-06 2016-09-15 Csl Behring Recombinant Facility Ag Modified von willebrand factor having improved half-life
US11155601B2 (en) 2015-03-06 2021-10-26 CSL Behring Lengnau AG Modified von Willebrand factor having improved half-life
WO2021001522A1 (en) 2019-07-04 2021-01-07 CSL Behring Lengnau AG A truncated von willebrand factor (vwf) for increasing the in vitro stability of coagulation factor viii
WO2021094344A1 (en) 2019-11-11 2021-05-20 CSL Behring Lengnau AG Polypeptides for inducing tolerance to factor viii

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