Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/36—Blood coagulation or fibrinolysis factors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
  • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • 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
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21027—Coagulation factor XIa (3.4.21.27)

Definitions

• the present invention relates to the therapeutic use of human Factor XI for the prevention and/or treatment of bleeding episodes, methods for the purification of factor XI and factor XI polypeptides from biological fluids, as well as pharmaceutical formulations.
• Human Factor XI is a serine protease consisting of two identical subunits, each having a molecular mass of about 80 kDa.
• FXI circulates in plasma as a disulfide-linked homodimer having a molecular mass of ⁇ 160KDa.
• FXI is activated by cleavage of each monomer between Arg 36 g and lle 3 7o to form an amino-terminal heavy chain of 50 kDa and a carboxy-terminal light chain of 35 kDa, which are disulfide-linked.
• the protein is encoded by a 23 kb gene located on chromosome 4 (4q35) 15 exons and 14 introns coding for a mRNA consisting of 2,097 nucleotides, which in turn encodes an amino-terminal signal (leader) peptide of 18 amino acids and the 607 amino acids present in each monomer of the mature protein.
• Exons lll-X encode four tandem repeats sequences (Apple domains) homologous to similar domains found in human plasma PK (58% identity).
• Exons XI-XV encode the typical trypsin-like catalytic domain, which is activated by proteolytic cleavage of the zymogen at an internal Arg 369-lle 370 bond to yield a heavy chain containing four Apple domains (369 amino acids) and the light chain or catalytic domain (238 amino acids).
• TF tissue factor
• FVII plasma Factor VII
• FVIIa plasma Factor VII
• FVIIa plasma Factor VII
• FXa proteolytic conversion to activated Factor X
• FXa proteolytic conversion by FXa of prothrombin to thrombin
• TFPI tissue factor pathway inhibitor
• thrombin burst The relatively small amount of thrombin produced during this phase results in the activation of FXI to FXIa (which activates Factor IX to FIXa) and the activation of Factor V on the surface of platelets and the further activation of Factor X. These events further promote the formation of sufficient amounts of thrombin (the so-called "thrombin burst") to convert fibrinogen into fibrin, thereby stabilizing an initial platelet plug and resulting in appropriate hemostasis.
• Dimeric FXI circulates in plasma as a zymogen in a non-covalent complex with the cofactor high molecular weight kininogen (HK) that promotes the binding of FXI to negatively charged surfaces and its activation by its cognate proteases, FXIIa, FXIa, and thrombin.
• HK cofactor high molecular weight kininogen
• the HK binding site to FXI involves multiple Apple domains (A1 , A2, A4), with the A2 domain being the most important.
• Complex formation with HK in the presence of Zn 2+ ions has been shown to promote the binding of FXI to activated platelets.
• FXI The interaction of FXI with the surface of activated platelets has been shown to be mediated via residues Ser 248 -Val 271 within the A3 domain of FXI; residues Ser 248 , Arg 250 , Lys 255 , Phe 26 o and Gln 26 3 have also been implicated in this interaction.
• the A3 domain of FXI also contains a heparin binding site within residues Thr 249 -Phe 26 o and residues Lys 252 and Lys 253 have been implicated in the binding to platelets.
• FXI and HK circulate in plasma in a non-covalent complex, and HK has been shown to bind to the surface of activated platelets, the interaction of FXI with the platelet surface apparently does not require binding of HK-FXI complex. Instead, it appears that the FXI dimer binds directly to a high-affinity, specific site on activated platelets (approx. 1500 sites/platelet; K d at approx 10 nM). The isolated recombinant A3 domain of FXI binds to the same number of sites on activated platelets and with the same affinity as the FXI dimer.
• the activated enzyme, FXIa has also been shown to bind to high-affinity, saturable sites on activated platelets (Kd at approx 800 pM; 500 sites/platelet) and can activate FIX at rates similar to those observed in solution.
• the substrate FIX binding site in FXI involves both a subdomain (Ala ⁇ 3 -Leu ⁇ 72 ) in the A2 domain and two subdomains (lle ⁇ 84 -Val 92 and Ser 259 -Ser 2 65) within the A3 domain. Binding to the platelet surface is mediated by the glycoprotein 1 b-V-IX complex utilizing one polypeptide chain of the FXI dimer, thereby presenting the other monomer as a substrate binding site for FIX.
• FXIa generation serves to localize FIXa-catalyzed FX activation to the platelet surface which also promotes prothrombin activation by FXa.
• FXIa is also subject to regulation by a variety of plasma and platelet protease inhibitors whose functional activity appears to depend on whether FXIa is bound to the platelet surface or whether it is free in solution.
• PNII is a potent inhibitor of FXIa with a Ki of 300-500 pM that is significantly enhanced in the presence of heparin. Binding of FXI to the platelet surface in the presence of HK and Zn 2+ ions or in the presence of prothrombin and Ca 2+ is protected from inactivation by both PNII and ⁇ -1 -protease inhibitor showing that FXIa activity generated on the surface of platelet is localized to the haemostatic thrombus whereas the site of regulation of FXIa by PNII and other protease inhibitors occurs in solution.
• endothelial cells which contain heparan sulphate glycosaminoglycans, might promote the assembly of FXIa/PNII complexes thereby potentiating the inhibition of FXIa on the endothelium.
• the participation of FXI in thrombin generation on the surface of of the activated platelet is also thought to play a role in inhibiting fibrinolysis via thrombin-activatable fibrinolysis inhibitor (TAFI), which proteolytically removes the carboxy-terminal lysine residues from fibrin that play a role in plasminogen binding and activation.
• TAFI thrombin-activatable fibrinolysis inhibitor
• platelets and megakaryocytes apparently synthesize a second form of FXI, designated platelet-derived FXI (pd-FXI), which differs from the circulating form in lacking Exon V, which is the first exon of the two exons encoding the second Apple domain, and in vitro studies have shown that the preferred substrate for platelet factor Xla may be plasma FXI and not FIX.
• Platelet FXI (Mr 220 KDa) has been found to be associated with the platelet plasma membrane. Platelets contain about 300 molecules of pd-FXI/cell. FXI deficiency is an autosomal recessive syndrome characterized by a variable tendency to bleed.
• the main disadvantages of plasma are the large volumes required, allergic reactions and the potential for transmission of infectious agents.
• FXI concentrates are currently available.
• the FXI concentrate from Bio Products Laboratory (BPL) (England) is formulated with a high concentration of antithrombin (mean 102 iu/ml) and heparin (10u/ml) which is thought to protect against any residual FXIa.
• a second FXI concentrate is produced by Hemoleven (France) and the product is formulated with 3-5 u/ml heparin, 2-3 iu/ml of antithrombin and 01 inhibitor.
• a major goal in surgery as well as in the treatment of major tissue damage is to avoid or minimise the bleeding.
• WO2003007983 discloses the use of a combination of factor Vila and FXI for treatment of bleeding episodes.
• the present invention provides methods and compositions for treating bleeding episodes.
• the methods are carried out by administering to a patient in need thereof a preparation comprising a factor XI (FXI) polypeptide, in an amount effective for such treatment.
• the methods of the invention result in one or more of: reduced clotting time; enhancement of hemostasis; increase in clot lysis time; increase in clot strength; and/or increase in overall clot quality (OCQ) in said patient.
• the patient following administration of a FXI polypeptide, the patient exhibits an effective FXI plasma concentration of at least about 5 nM, 10 nM, 30 nM, 60 nM, or 120 nM.
• the FXI polypeptide comprises the sequence of SEQ ID NO: 1 , or a fragment thereof that retains at least one FXI-associated biological activity. In some embodiments, the FXI polypeptide comprises the sequence of SEQ ID NO:2, or a fragment thereof that retains at least one FXI-associated biological activity. In some embodiments, the FXI polypeptide comprises a chemically modified derivative of SEQ ID NO:1 or SEQ ID NO:2, or a variant of either SEQ ID NO:1 or SEQ ID NO:2 containing one or more amino acid sequence alterations. In some embodiments, the FXI polypeptide has the sequence of SEQ ID NO:1. In some embodiments, the FXI polypeptide has the sequence of SEQ ID NO:2.
• the patient does not suffer from a congenital FXI deficiency.
• the bleeding episodes are secondary to surgery, a dental procedure, trauma, or hemodilution.
• the patient suffers from aquired FXI deficiency.
• the invention also provides methods and compositions for preventing bleeding episodes. The methods are carried out by administering to a patient in need thereof a preparation comprising a FXI polypeptide, in an amount effective to prevent bleeding.
• the methods of the invention further comprise, prior to administration of a FXI polypeptide: (a) obtaining a sample of blood from said patient; (b) determining at least one of: FXI concentration, ratio of FXIa:FXI, or amount of exogenous FXI necessary to restore coagulation; and (c) based on the results of step (b), determining said amount of FXI effective for treatment.
• the methods of the invention does not comprise administration of a Factor Vll/Factor Vila coagulation agent.
• a Factor Vll/Factor Vila coagulation agent is a Factor VII polypeptide or a Factor VI l-related polypeptide as described in WO2003007983.
• the invention also provides methods and compositions for treating bleeding episodes in which a patient is administered (i) a first amount of a preparation comprising a FXI polypeptide and (ii) a second amount of a preparation comprising a non-Factor Vll/Factor Vila coagulation agent, under conditions in which the first and second amounts in combination are effective for such treatment.
• Non-limiting examples of non-Factor Vll/Factor Vila coagulation agents include: Factor XII, phospholipids, Factor XIII; tissue factor pathway inhibitor (TFPI) inhibitor; Factor IX; thrombin activatable fibrinolysis inhibitor (TAFI); plasminogen activator inhibitor-1 (PAI-1 ); Factor V; protein C inhibitor; protein S inhibitor; tissue plasminogen activator (tPA) inhibitor; prothrombin, Factor VIII, fibrinogen, and Factor X.
• the invention also provides pharmaceutical formulations comprising (i) isolated recombinant FXI polypeptide and (ii) a pharmaceutically acceptable carrier or excipient.
• the invention further provides methods for purifying a factor XI polypeptide from a biological material, the method comprising subjecting the material to sequential chromatography on an cation-exchange chromatographic material, a hydrophobic interaction chromatographic material, and a Hydroxyapatite chromatographic material.
• Figure 1 is a graphic representation of the effect of increasing amounts of FXI on overall clot quality in blood obtained from patients before and after cardiac surgery.
• Figure 2 is a graphic representation of the effect of increasing amounts of FXI on overall clot quality in blood obtained from normal subjects.
• Figure 3 is a graphic representation of the biological activity of different FXI formulations after storage at 5°C for 96 days.
• Figure 4 is the preparative chromatogram of factor XI polypeptide-containing fractions from first cation-exchange chromatography using Obelix ST CIEX (cat no 11-0010) as described in example 7.
• Figure 5 is the preparative chromatogram of factor XI polypeptide-containing fractions from Hydrophobic interaction chromatography using Butyl Sepharose High Performance High Substitution (cat no 17-3100) as described in example 8.
• Figure 6 is the preparative chromatogram of factor XI polypeptide-containing fractions from Hydroxyapatite chromatography using CHT Hydroxyapatite Type I BioRad cat no 157-0020) as described in example 9.
• the present invention is based on the surprising finding that exogenously administered Factor XI (FXI) can be effective as a general hemostatic agent in human blood without the administration of Factor Vll/Factor Vila coagulation agents.
• the therapeutic use of FXI according to the invention may provide one or more of: a shortened clotting time, a firmer clot, and an increased resistance of the formed clots to fibrinolysis and a reduction of bledding-associated complications.
• the present invention provides methods and compositions useful in the therapeutic use of FXI in human patients for treating or preventing bleeding episodes, for enhancing hemostasis, for increasing clot lysis time, and/or for increasing clot strength.
• compositions include pharmaceutical formulations for the therapeutic use of FXI that comprise FXI.
• compositions include pharmaceutical formulations for the therapeutic use of FXI that comprise isolated FXI.
• compositions include pharmaceutical formulations for the therapeutic use of FXI that comprise recombinant FXI.
• compositions include pharmaceutical formulations for the therapeutic use of FXI that comprise isolated recombinant FXI.
• present invention relates to administration of FXI to normal human patients.
• a "normal" human is one who does not suffer from a congenital deficiency in Factor XI (i.e., Hemophilia C, see, Seligsohn (1993), Thromb. Haemost. 70:68-71 ); normal humans include, without limitation, patients exhibiting thrombocytopenia (lowered count or activity of platelets), patients contemplating or undergoing a surgical or dental procedure, and patients who have been subjected to trauma or organ damage and who, as a consequence, may exhibit lowered platelet counts and/or lowered levels of fibrinogen, FVIII, and/or other coagulation proteins.
• Factor XI i.e., Hemophilia C, see, Seligsohn (1993), Thromb. Haemost. 70:68-71 )
• normal humans include, without limitation, patients exhibiting thrombocytopenia (lowered count or activity of platelets), patients contemplating or undergoing a surgical or dental procedure, and patients who have been subjected to trauma or organ damage and who
• Normal human patients encompass patients experiencing a transient decrease in the plasma levels of FXI (or any other coagulation-related protein or factor) due to bleeding, trauma, chemotherapy, liver disease, hemodilution (such as, e.g., may result from the infusion of plasma expanders or salt solutions to maintain blood volume or prevent shock), or any other circumstances not directly related to a congenital defect in a FXI gene.
• the present invention relates to administration of isolated and/or recombinant FXI to human patients suffering from a congenital FXI deficiency.
• the present invention relates to administration of isolated and/or recombinant FXI to human patients suffering from aquired FXI deficiency.
• any FXI polypeptide may be used that is effective in preventing or treating bleeding. This includes FXI polypeptides derived from blood or plasma or from platelets or those produced by recombinant means in any suitable host organism or cell. Also encompassed are FXI polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms (designated FXIa).
• FXI polypeptides encompass, without limitation, FXI as well as FXI- related polypeptides.
• FXI is intended to encompass, without limitation, polypeptides having the amino acid sequence of wild-type human plasma FXI, as described, e.g., in Fujikawa et al., Biochem. 25:2417 (1986), as well as wild-type FXI derived from other species, such as, e.g., bovine, porcine, canine, murine, rabbit, and salmon FXI.
• FXI proteins syngeneic with the subject, in order to reduce the risk of inducing an immune response. Preparation and characterization of non-human FXI has been described, e.g., by Gailani (1997), Blood 90:1055.
• the FXI polypeptide is wild-type human plasma FXI (SEQ ID NO:1 ).
• the FXI is platelet-derived FXI (pd-FXI) (SEQ ID NO:2), as described, e.g., in Hsu et al. (1998), J. Biol. Chem. 273:13787-93.
• FXI polypeptides further encompass natural allelic variations of FXI that may exist and differ from one individual to another.
• FXI-related polypeptides include, without limitation, FXI polypeptides that have either been chemically modified relative to human FXI (i.e., FXI derivatives) and/or contain one or more amino acid sequence alterations relative to human FXI (i.e., FXI variants).
• FXI-related polypeptides may exhibit an alteration in one or more aspects of biological activity relative to human FXI, including, without limitation, altered stability, altered phospholipid binding, altered specific enzymatic activity, altered immunogenicity, altered bioavailability, altered binding to one or more FXI binding partners, altered binding to FXI inhibitors, and the like.
• FXI-related polypeptides encompass such polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated "FXIa-related polypeptides" or "activated FXI-related polypeptides".
• FXI derivatives include: wild-type FXI or FXI variants that have been modified by phosphorylation, sulfation, PEGylation, or by the action of one or more glycosyltransferases and/or glycosidases, whether in vivo or in vitro (see, e.g., Ekdahl et al. (1999), Thromb. Haemost. 82:1283-8).
• FXI variants include: FXI in which one or more N-linked or O-linked glycosylation consensus sites have been modified, single-chain FXI (i.e., FXI in which the monomer polypeptides are not subject to intrachain proteolytic cleavage as in the wild-type), and cysteine variants in which one or more cysteine residues are eliminated or relocated, including, but not limited to, alterations that change the disulfide bonding pattern of the monomer or dimer.
• Cys-n which is not believed to participate in inter- or intramolecular disulfide bonding is eliminated or substituted.
• the FXI variant has decreased half-life in plasma relative to wild-type human FXI. In one embodiment the FXI variant has a half-life lower than 50 hours. In one embodiment the FXI variant has a half-life lower than 24 hours. In one embodiment the FXI variant has a half-life lower than 12 hours. In one embodiment the FXI variant has a half-life lower than 6 hours. In one embodiment the FXI variant has a half-life lower than 3 hours.
• FXI variants are polypeptides in which N-linked glycosylation at one or more sites has been disrupted by modified of the cognate N-linked glycosylation consensus sites, such as, e.g., by independent substitution with any amino acid of N72, N108, N335, N432, N473, or combinations of any of the foregoing.
• Non-limiting examples of such variants include FXI-N72Q; FXI-N108Q; FXI-N335Q, FXI-N432Q, FXI- N473Q; FXI-N72Q/N108Q; FXI-N72Q/N108Q/N335Q; FXI-N72Q/N108Q/N335Q/N432Q; FXI-N72Q/N108Q/N335Q/N432Q/N473Q; FXI-N72Q/N432Q; FXI-N72Q/N473Q; FXI- N108Q/N432Q; FXI-N108Q/N473Q; and FXI-N432Q/N473Q.
• Disruption of N-linked glycosylation at one or more of the sites may also be achieved, e.g., by: (i) independent deletion of any of residues 72-74, 108-1 10, 335-337, 432-434, and 473-475 (i.e., one or more residues at each site may be deleted and not substituted with any another amino acid) (ii) independent substitution of the N+2 residue (such as, e.g., substituting T74 to any residue other than S, substitution of S110 to any residue other than T, substitution of S337 to any residue other than S, substitution of S434 to any residue other than T, substitution of T475 to any residue other than T; (Hi) substitution of the N+1 residues with a glycosylation-disrupting amino acid (exemplified by, but not limited to, proline (P).
• P proline
• any combination of the above means may be used to independently disrupt glycosylation at different sites within the FXI polypeptide.
• Also encompassed by the invention are chimeric or fusion polypeptides between all or part of the FXI sequence and other heterologous peptide sequences.
• one or more of the four Apple domains may be substituted by similar apple domains from other polypeptides (see, e.g., Gailani et al.(1999) Blood 94:621a) or one or more of the Apple domains may be deleted in its entirety.
• a binding site for LDL Receptor-associated protein (such as, e.g., a peptide comprising residues Phe 342 - Asn 346 of Factor IXa, which has been shown to contribute to the interaction with LRP, Rohlena et al. (2003), J. Biol. Chem. 278:9394) is attached to the sequence of a FXI polypeptide to modify its pharmacokinetic properties.
• LRP LDL Receptor-associated protein
• FXI polypeptides for use in the present invention include, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wild- type human FXI, as well as polypeptides in which the FXI biological activity has been substantially modified or reduced relative to the activity of wild-type human FXI.
• compositions comprising FXIa or FXIa- related polypeptides encompass those that exhibit at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, or at least about 130%, of the specific activity of compositions comprising solely wild-type FXI, when the wild-type FXI equivalent is one that has been obtained from the same source or produced in the same cell type, and when the activity comparison is made by parallel testing in an identical FXI activity assay.
• the terms “activity” and “specific activity” apply, individually or in aggregate, to any aspect or aspects of FXI bioactivity.
• the ratio between the specific proteolytic activity of a FXI- related polypeptide and the proteolytic activity of wild-type human FXI is at least about 1.25 when tested a FXI amidolytic assay; in other embodiments, the ratio is at least about 2.0; in further embodiments, the ratio is at least about 4.0.
• FXI Biological Activity In practicing the present invention, one or more different aspects of FXI bioactivity may be quantified and used, e.g., in (i) selection of: appropriate FXI compositions for therapeutic administration, formulations, methods for FXI production or purification, and the like; and/or (ii) assessment of the efficacy of different therapeutic modalities. It will be understood that "specific activity" of FXIa for any of these aspects of bioactivity is expressed as units of activity per unit mass of FXIa polypeptides. These aspects include the following:
• Amidolytic activity may be quantified in vitro using a suitable chromogenic substrate, such as, e.g., S2355 (Chromogenix), as described in Ekdahl et al. (1999), Thromh.
• a suitable chromogenic substrate such as, e.g., S2355 (Chromogenix), as described in Ekdahl et al. (1999), Thromh.
• FXI activation activity may be quantified directly in vitro by measuring the proteolytic conversion of factor IX to IXa as described for example, in Gailani et al. (2001 ),
• Wild-type human FXI has a number of binding partners, including prekallikrein (PK), high-molecular weight kininogen (HK), thrombin/prothrombin, Factor IX (FIX), and the platelet-associated FXI receptor designated GP1 b-V-IX.
• PK prekallikrein
• HK high-molecular weight kininogen
• FIX Factor IX
• GP1 b-V-IX the platelet-associated FXI receptor designated GP1 b-V-IX.
• any conventional binding assay may be used to quantify the affinity of FXI polypeptides for any of these (or other) binding partners.
• binding assays include, but are not limited to, competition binding assays in which either binding partner is labelled.
• FXI polypeptide binding partners are FXI active site inhibitors, including, without limitation, antithrombin III, C1 inhibitor, ⁇ 2 antitrypsin, PAI-1 , protein C inhibitor, and protease nexin II (PNII).
• FXI active site inhibitors including, without limitation, antithrombin III, C1 inhibitor, ⁇ 2 antitrypsin, PAI-1 , protein C inhibitor, and protease nexin II (PNII).
• the affinity of these compounds for FXI polypeptides may be quantified by use of conventional binding assays; alternatively, the inhibitory activity of such compounds for the proteolytic activity of particular FXI polypeptide preparations may be measuring using an amidolytic or FIX-activation assay.
• Clotting parameters are clinical parameters used for assaying the status of patient's haemostatic system. Blood samples are drawn from the patient at suitable intervals after administration of a FXI polypeptide and one or more of these parameters are assayed. Alternatively, a FXI polypeptide or preparation may be used for in vitro/ex vivo treatment of blood that has been drawn from a human subject. Clotting time may be assayed by means of standard PT or aPTT assays. Clot lysis time and clot strength may be measured by thromboelastograpy as described by, e.g., Vig et al.
• clot strength may be assayed as described by Carr et al, (1991 ), Am. J. Med. Sci. 302: 13-8.
• One parameter that reflects the clotting activity of FXI as measured by thromboelastography is the "overall clot quality" (OCQ).
• tissue plasminogen activator tPA
• tPA tissue plasminogen activator
• FXI polypeptides which exhibit pharmacokinetic properties that differ from native FXI, may be used.
• Non-limiting examples include FXI polypeptides that have been treated with sialidase to remove one or more terminal sialic acid residues from FXI-associated oligosaccharides, FXI polypeptides that have been modified by PEGylation, and FXI polypeptides exhibiting an altered interaction with HK.
• pharmacokinetic properties may be calculated using, e.g., WinNonlin Professional Version 3.1 (Pharsight Inc., Mountain View, CA, USA). Calculations are performed using mean concentration values at each time point, if more than one value was present.
• the following pharmacokinetic parameters may be calculated: AUC, AUC% E xtr aP , Cm a x, tm a x, ⁇ z , t 1 2 , CL, and V z using the following formulas: AUC Area under the plasma concentration-time curve from time 0 to infinity. Calculated using the linear/log trapezoidal rule with extrapolation to infinity.
• the linear trapezoidal rule is used from time 0 to t max :
• the log trapezoidal rule is used from time t max to the last time point t:
• AUC ⁇ i Extrapolation to infinity is performed using: AUC oEx trap Percentage of AUC that is due to extrapolation from the last concentration to infinity: A Ti .... A ⁇ »• *U'- C v U v -txi) I ⁇ IW AUC Maximum plasma concentration back extrapolated to time zero CL Total body clearance
• FXI polypeptides for use in the present invention may be prepared from plasma or from recombinant sources using any suitable method known in the art.
• isolated refers to FXI polypeptides that have been separated from the cell in which they were synthesized or the medium in which they are found in nature (e.g., plasma or blood). Separation of polypeptides from their cell of origin may be achieved by any method known in the art, including, without limitation, removal of cell culture medium containing the desired product from an adherent cell culture; centrifugation or filtration to remove non- adherent cells; and the like.
• FXI polypeptides may be further purified.
• Purification may be achieved using any method known in the art, including, without limitation, affinity chromatography, such as, e.g., on an anti-FXI antibody column or a peptide affinity column (non-limiting examples of which include Heparin, Blue, Red, L-arginine, Benzamidine peptide, other dyes, or RP-chromatography); hydrophobic interaction chromatography; ion- exchange chromatography; size exclusion chromatography; electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., any precipitation or crystallization using, e.g., salt, pH, ammonium sulphate, or other additives), or extraction and the like, as described in more detail above.
• affinity chromatography such as, e.g., on an anti-FXI antibody column or a peptide affinity column (non-limiting examples of which include Heparin, Blue, Red, L-arginine, Benzamidine peptide,
• the preparation preferably contains less than about 10% by weight, more preferably less than about 5% and most preferably less than about 1 %, of non-FXI polypeptides derived from the host cell.
• Purification of FXI from plasma may also be achieved by known methods, including, without limitation, those disclosed by Koide et al. (1977), Biochem. 16: 2279 and Bouma et al. (1977), J. Biol. Chem. 252:6432, incorporated herein by reference. Methods for preparing recombinant FXI are known in the art. See, for example, Kemball-Cook et al. (1994), Gene 139:275, Fujikawa et al. (1986), Biochem.
• FXIa is also commercially available from Enzyme Research Laboratories, South Bend, IN.
• the present invention further concerns a method for purifying a FXI polypeptide, such as recombinant FXI, from other biological material, the method comprising subjecting the material to chromatography on a cation-exchange chromatographic material.
• the present invention further concerns a method for purifying a FXI polypeptide, such as recombinant FXI, from other biological material, the method comprising subjecting the material to chromatography on a a hydrophobic interaction chromatographic material.
• the present invention further concerns a method for purifying a FXI polypeptide, such as recombinant FXI, from other biological material, the method comprising subjecting the material to chromatography on a hydroxyapatite chromatographic material.
• the present invention further concerns a method for purifying a FXI polypeptide, such as recombinant FXI, from other biological material, the method comprising subjecting the material to sequential chromatography on a cation-exchange chromatographic material, a hydrophobic interaction chromatographic material and Hydroxyapatite chromatographic material. It is to be understood that a sequential chromatography is performed in the order as described.
• Hydroxyapatite chromatographic material as used herein means any Hydroxyapatite chromatographic material known in the art which is capable of binding a FXI polypeptides, such as a Hydroxyapatite matrix.
• cation-exchange chromatographic material as used herein means any cation-exchange chromatographic material known in the art which is capable of binding a FXI polypeptides, such as a cation-exchange matrix.
• hydrophobic interaction chromatographic material as used herein means any hydrophobic interaction chromatographic material known in the art which is capable of binding a FXI polypeptides, such as a hydrophobic interaction matrix.
• the present invention concerns a method for purifying a FXI polypeptide from a biological material, the method comprising the step of: subjecting a biological material comprising a FXI polypeptide to chromatography on a first cation-exchange chromatographic material, said chromatography comprising: (i) applying said biological material to said first cation-exchange chromatographic material; (ii) eluting unbound material from the first cation-exchange chromatographic material with a buffer A, which buffer A is suitable for eluting material not bound to the first cation-exchange chromatographic material; and (iii) eluting unbound material from the first cation-exchange chromatographic material with a buffer A', which buffer A' is suitable for eluting material not bound to the first cation-exchange chromatographic material; and (iv) eluting said FXI polypeptide from the first cation-exchange chromatographic material by elution with buffer A",
• the present invention concerns a method for purifying a FXI polypeptide from a biological material, the method comprising the steps of: subjecting the eluate from step (iv) or a fluid prepared by use of the eluate from step (iv) to chromatography using a hydrophobic interaction chromatographic material, said chromatography comprising: (v) applying the eluate from step (iv) or a fluid prepared by use of the eluate from step (iv) to said hydrophobic interaction chromatographic material; (vi) eluting unbound material from said hydrophobic interaction chromatographic material with buffer B, which buffer B is suitable for eluting material not bound to the hydrophobic interaction chromatographic material; and (vii) eluting said FXI polypeptide from said hydrophobic interaction chromatographic material by gradient-elution with buffer B', which buffer B' is suitable for eluting FXI polypeptide from said hydrophobic interaction chromatographic material.
• the present invention concerns a method for purifying a FXI polypeptide from a biological material, the method comprising the steps of: subjecting the eluate from step (vii) or a fluid prepared by use of the eluate from step (vii) to chromatography using a Hydroxyapatite chromatographic material, said chromatography comprising: (viii) applying the eluate from step (vii) or a fluid prepared by use of the eluate from step (vii) to said hydroxyapatite chromatographic material; (ix) eluting unbound material from the hydroxyapatite chromatographic material with buffer C, which buffer C is suitable for eluting material not bound to the hydroxyapatite chromatographic material; and (x) eluting said FXI polypeptide from said hydroxyapatite chromatographic material by gradient-elution with buffer C, which buffer C is suitable for eluting FXI polypeptide from said hydroxyapatite chromatographic material.
• step (b) subjecting the eluate from step (iv) or a fluid prepared by use of the eluate from step (iv) to chromatography using a hydrophobic interaction chromatographic material, said chromatography comprising: (v) applying the eluate from step (iv) or a fluid prepared by use of the eluate from step (iv) to said hydrophobic interaction chromatographic material; (vi) eluting unbound material from said hydrophobic interaction chromatographic material with buffer B, which buffer B is suitable for eluting material not bound to the hydrophobic interaction chromatographic material; and (vii) eluting said FXI polypeptide from said hydrophobic interaction chromatographic material by gradient-elution with buffer B', which buffer B' is suitable for eluting FXI polypeptide from said hydrophobic interaction chromatographic material;
• FXI polypeptide in a sample in relation to other components of said sample resulting in an increase of the purity of the FXI polypeptide.
• concentration of a FXI polypeptide in a sample in relation to other components of said sample is not equivalent to the concentration of FXI polypeptide in the sample.
• the increase in the purity of the FXI polypeptide may be followed measured by use of methods known in the art, such as for instance by use of SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), HPLC (High Performance Liquid Chromatography) or Berichrome assays (Dade Behring Diagnostics), or Clot activity assay.
• Biological material may be any material derived from or containing cells, cell components or cell products.
• a biological material may be a biological fluid.
• a biological fluid may be any fluid derived from or containing cells, cell components or cell products.
• Biological fluids include, but are not limited to cell cultures, cell culture supernatants, cell lysates, cleared cell lysates, cell extracts, tissue extracts, blood, plasma, serum, all of which may also be homogenizates and filtrates, and fractions thereof, for instance collected by chromatography of unfractionated biological fluids.
• the FXI polypeptides may be purified from a wide variety of biological materials, including cell culture supernatants, which naturally produce a FXI polypeptide, but also of cells which have been genetically modified to produce a FXI polypeptide, such as mammalian cells (for instance CHO cells) transformed with DNA coding for a FXI polypeptide.
• the biological material may be treated by use of a number of methods prior to application on the first cation-exchange chromatographic material. Such methods include, but a not limited to, centrifugation, filtration.
• the biological material is a biological fluid.
• the biological fluid is the supernatant of a cell lysate.
• the biological fluid is the supernatant of a yeast cell lysate.
• the FXI polypeptide is purified from a cell culture, such as a mammalian cell culture, as described above. Prior to the chromatography in step (a), the mammalian cells may be separated from cell culture supernatant by centrifugation and / or filtration. Inhibitors such as EDTA (ethylenediamine tetraacetic acid) and benzamidineHCI may be included before being subjected to chromatographic step (a).
• EDTA ethylenediamine tetraacetic acid
• benzamidineHCI may be included before being subjected to chromatographic step (a).
• a buffer is a solution comprising a substance, which substance is capable of preventing significant changes in the pH of solutions to which small amounts of acids or bases are added and thereby of maintaining largely the original acidity or basicity of the solution.
• a buffer usually comprises a weak acid or weak base together with a salt thereof.
• the pH of the biological fluid may be adjusted to the pH of buffer A prior the chromatography in step (a), for instance by using 1 M HCI or 1 M NaOH or by other means known in the art.
• the first cation-exchange chromatographic material may be any cation-exchange chromatographic material known in the art which is capable of binding a FXI polypeptide under one set of conditions and releasing it under a different set of conditions, such as an cation-exchange chromatographic material comprising a sulphopropyl group.
• cation-exchange chromatographic materials include derivatised dextrans, agarose, cellulose, polyacrylamide, and specialty silicas, such as carboxymetyl.
• Suitable cation-exchange chromatographic material may be identified by subjecting a biological fluid comprising FXI polypeptide to chromatography on the cation-exchange chromatographic material of choice, collecting fractions and determining the purity and content of the fractions, for instance by use of SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), HPLC (High Performance Liquid Chromatography), clotactivity or Berichrome assays (Dade Behring Diagnostics), monitoring the absorbance of the eluate at 280 nm and by use of other methods known in the art.
• SDS-PAGE sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
• HPLC High Performance Liquid Chromatography
• Berichrome assays Dade Behring Diagnostics
• Suitable cation-exchange chromatographic materials include, but are not limited to Streamline SP XL (Amersham Biosciences cat no 17-5073), Obelix ST CIEX (Amersham Biosciences cat no 11-0010), Streamline Direct CST (Amersham Biosciences 17-5266), S-Support Unosphere, BioRad cat no 156-0113 or Toyopearl SP-550C Toso Haas cat no 14028. In one embodiement Obelix ST CIEX is used.
• the first cation-exchange chromatographic material may be pre-equilibrated with buffer A prior to application of the biological material.
• Buffer A may comprise protease inhibitors such as EDTA (ethylenediamine tetraacetic acid) and benzamidineHCI, but other commercially available protease inhibitors may also be used.
• the pH of buffer A is between 6.5 and 9.
• the pH of buffer A is between 7 and 9.
• the pH of buffer A is about 8.
• the conductivity of buffer A is less than about 40 mS/cm. Buffer A" is used for the elution of the FXI .
• the concentration of one or more of the components of the buffer used for washing in step (ii), in this case buffer A and A' is increased or decreased during the course of elution or a new component is added to the buffer, and the concentration of this component .
• This increase or decrease may take place continuously or in discrete steps as it is known in the art.
• a salt for instance NaCI
• This specific cation exchanger can also be used as a hydrophobic interaction chromatographic resin, for this kind of resin it is customary to add a propandiol / glycerol to buffer A creating buffer A'.
• FXI can be eluted.
• the determination of which fractions containing FXI polypeptide to pool for further processing, for instance to exclude undesired impurities eluting at the beginning or the end of the FXI polypeptide elution, is within the knowledge of a person skilled in the art.
• the general art of performing an cation-exchange chromatography with regard to for instance pre-equilibration, elution time, washing, reconstitution of the cation-exchange chromatographic material etc is well-known.
• the eluate containing the FXI polypeptides protease inhibitors such as EDTA (ethylenediamine tetraacetic acid) and Benzamidine is added and then taken to step (v) .
• the eluate may also be kept at, for instance, 4°C for 24 hours or longer, or at, for instance, -80°C.
• the hydrophobic interaction chromatographic material for use in step (b) may be any hydrophobic interaction chromatographic material known in the art, which is capable of binding a FXI polypeptide under one set of conditions and releasing it under a different set of conditions, such as a hydrophobic interaction chromatographic material derivatised with phenyl, butyl or octyl groups, or polyacrylic resins.
• Non-limiting examples of suitable hydrophobic interaction chromatographic material are AmberchromTM CG 71 (Tosoh Bioscience), Phenyl SepharoseTM High Performance (Amersham, cat no 17-1082), Phenyl SepharoseTM 6 Fast Flow High Substitution (Amersham, cat no 17-0973), Toyopearl® Butyl 650 (Tosoh Bioscience), Toyopearl® Phenyl (Tosoh Bioscience), SourceTM 15Phe (Amersham, cat no 17-0147), Butyl SepharoseTM High Performance High Substitution (Amersham, cat no 17-3100), Octyl-SepharoseTM (Amersham, cat no 17-0946) and Phenyl SepharoseTM High Performance High Substitution (Amersham), and the like.
• AmberchromTM CG 71 Tosoh Bioscience
• Phenyl SepharoseTM High Performance Amersham, cat no 17-1082
• the hydrophobic interaction chromatographic material uses butyl as a ligand.
• Buffer B and NaCI may be added to the eluate from stage (iv) or a fluid prepared by use of the eluate from stage (iv) prior to the chromatography in step (b) in an amount of about one to two volumes or more, or a concentrated version of buffer B, comprising the same ingredients as buffer B, but in, e.g., twice the concentration, is added to the eluate from stage (iv) or a fluid prepared by use of the eluate from stage (iv) in an amount corresponding to the strength of the concentrated buffer (a twice-concentrated buffer is added in the amount of 1 ,5 volumes).
• Buffer B may have a pH from about 5 to about 9, for instance about 8.
• buffer B has a conductivity of more than 25 mS/cm, for instance more than 70 mS/cm. This may be achieved, for example, by use of a phosphate buffer or by other means known in the art, e.g. NaCI.
• the conductivity of the eluate from step (iv) or a fluid prepared by use of the eluate from step (iv) is adjusted to a conductivity of at least about 60 mS/cm. Buffer B' is used for the elution of the FXI polypeptide by gradient elution.
• the composition of buffer B' is changed during the course of elution.
• concentration of one or more of the components of the buffer used for washing in step (vi), in this case buffer B is increased or decreased during the course of elution, or a new component is added to the buffer and the concentration of this component is then increased during the course of elution.
• This increase or decrease may take place continuously or in discrete steps, as is well known in the art.
• the eluate from stage (vii) or a fluid prepared by use of the eluate from stage (vii) is treated by use of a method comprising a step of (1 ) addition of one or more stabilizing agents which are capable of increasing the stability of the FXI polypeptide in an amount effective to significantly improve the stability thereof, and/or
• This step, and optionally other steps of post-processing known in the art may be carried out alone or in combination, and the order in which the steps are performed is not critical. The person skilled in the art will be able to determine how and when to perform these steps.
• a stabilizing agents which are capable of increasing the physical and/or chemical stability of the FXI polypeptide is added to the fractions containing FXI.
• the term "physical stability" of the FXI polypeptide as used herein refers to the potential tendency of the protein to form biologically inactive and/or insoluble aggregates or multimers of the protein as a result of exposure of the protein to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces.
• Physical stability of the FXI polypeptide when present in buffer A may be evaluated by means of visual inspection and/or turbidity measurements after exposing the formulation filled in suitable containers (e.g. cartridges or vials) to mechanical/physical stress (e.g.
• the turbidity of the composition may be characterized by a visual score ranking the degree of turbidity, for instance on a scale from 0 to 3 (a composition showing no turbidity then corresponding to a visual score 0, and a composition showing visual turbidity in daylight corresponding to visual score 3).
• a composition is classified as physically unstable with respect to protein aggregation when it shows visual turbidity in daylight.
• the turbidity of the composition may be evaluated by simple turbidity measurements well-known to the skilled person, for instance by measuring the optical density of the solution at a wavelength of 405 nm (OD 405 ).
• Physical stability of the aqueous protein compositions may also be evaluated by using a spectroscopic agent or probe of the conformational status of the protein.
• the probe is preferably a small molecule that preferentially binds to a non-native conformer of the protein.
• Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils.
• Thioflavin T gives rise to a new excitation maximum at about 450 nm and enhanced emission at about 482 nm when bound to a fibril protein form. Unbound Thioflavin T is essentially non-fluorescent at the wavelengths.
• Other small molecules can be used as probes of the changes in protein structure from native to non-native states. For instance the "hydrophobic patch" probes that bind preferentially to exposed hydrophobic patches of a protein. The hydrophobic patches are generally buried within the tertiary structure of a protein in its native state, but become exposed as a protein begins to unfold or denature.
• spectroscopic probes examples include aromatic, hydrophobic dyes, such as antrhacene, acridine, phenanthroline or the like.
• Other spectroscopic probes are metal-amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids, such as phenylalanine, leucine, isoleucine, methionine, and valine, or the like.
• chemical stability of the FXI polypeptide when used herein refers to chemical covalent changes in the protein structure leading to formation of chemical degradation products with potentially lower biological potency and/or potentially increased immunogenic properties compared to the native protein structure.
• the amount of each individual degradation product is often determined by separation of the degradation products depending on molecule size and/or charge using various chromatographic techniques (e.g. SEC-HPLC and/or RP-HPLC).
• Any agent which is capable of significantly improving the physical and/or chemical stability of FXI polypeptide when present in buffer B' may be used as a stabilizing agent.
• An agent suitable for use as stabilizing agent for instance, be a salt (e.g. sodium chloride), a sugar, an alcohol (such as an C 4 -C 8 alcohol), an alditol, an amino acid (e.g.
• glycine histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), a polyethyleneglycol (e.g. PEG400), or a mixture of one or more thereof.
• Any sugar such as a mono-, di-, or polysaccharide, or a water-soluble glucan, may be used.
• An alditol is a polyalcohol of structure HOCH 2 -[CH(OH)] n -CH 2 OH, where n is 1, 2, 3.... etc.
• Non-limiting examples of substances which are sugars, alcohols or alditols are fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, carboxymethylcellulose-Na, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol, glycerol (glycerine), propan-1 ,2-diol (propylene glycol), propan-1 ,3-diol, and butan-1 ,3-diol.
• the sugars, alcohols and alditols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the substance is soluble in the liquid preparation and improves the physical stability of a FXI polypeptide in solution. In this respect, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995. In one embodiment of the present invention, one or more stabilizing agents of the polyalcohol type is added.
• one or more stabilizing agents selected from the group consisting of glycerol (propan-1 ,2,3-triol), propylene glycol (propan-1 ,2-diol), propan-1 ,3-diol, propyl alcohol (1-propanol) and isopropyl alcohol (2-propanol) is added.
• one or more stabilizing agents selected from the group consisting of glycerol, propylene glycol and propan-1 ,3-diol is added.
• the stabilizing when the stabilizing is a liquid alcohol or liquid polyalcohol [such as, e.g., glycerol, propylene glycol, propan-1 ,3-diol, propyl alcohol or isopropyl alcohol], the stabilizing agent is present in a concentration of from about 5% by volume (v/v) to about 50% (v/v). In a further embodiment, a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of from about 10% (v/v) to about 50% (v/v). In a further embodiment, a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of from about 10% (v/v) to about 20% (v/v).
• a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of from about 10% (v/v) to about 20% (v/v).
• a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of about 10% (v/v). In a still further embodiment, a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of about 20% (v/v).
• the stabilizing agent mentioned should be capable of increasing the physical and/or chemical stability, as described above, of the FXI polypeptide. Any agent which is capable of significantly improving the physical and/or chemical stability of FXI polypeptide (e.g. as determined by measuring turbidity at OD 40 s over a period of time) may be used as a stabilizing agent.
• the eluate from step (vii) may be used for the preparation of a pharmaceutical composition.
• the eluate may also be kept at, e.g., 4°C for 24 hours or longer, or at, e.g., -80°C.
• the method further comprises a step of subjecting the eluate from stage (vii), or a fluid prepared by use of the eluate from stage (vii), to chromatography on a hydroxyapatite chromatographic material, said chromatography comprising: (viii) applying the eluate from stage (vii), or a fluid prepared by use of the eluate from stage (vii), to said hydroxyapatite chromatographic material; (ix) eluting unbound material from the hydroxyapatite chromatographic material with buffer C, which buffer C is suitable for eluting material not bound to the hydroxyapatie chromatographic material; and (x) eluting said FXI polypeptide from the hydroxyapatite chromatographic material with buffer C, which buffer C is suitable for eluting FXI polypeptides which bind to the hydroxyapatite chromatographic material in step (ix).
• a fluid prepared by use of the eluate from stage (vii) may, for instance, be prepared before application.
• the conductivity of the eluate from stage (vii), or a fluid prepared by use of the eluate from stage (vi) is adjusted to less than about 20 mS/cm by adding water. pH is adjusted to 5,8 to 9. In one embodiment pH is adjusted to 6,0
• the components of buffer C and buffer C may be chosen with a view to the desired final pharmaceutical composition of the FXI polypeptide. Such considerations are within the knowledge of a person skilled in the art.
• buffer C comprises one or more stabilizing agents, which stabilizing agents are capable of increasing the physical and/or chemical stability, as described above, of the FXI polypeptide.
• Any agent which is capable of significantly improving the physical and/or chemical stability of FXI polypeptide when present in buffer C may be used as a stabilizing agent in buffer C or buffer C
• An agent suitable for use as stabilizing agent in buffer C may, for instance, be a salt
• a sugar e.g. sodium chloride
• a sugar e.g. a sugar, an alcohol (such as an C 4 -C 8 alcohol), an alditol, an amino acid (e.g. glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), a polyethyleneglycol (e.g. PEG400), or a mixture of one or more thereof.
• Any sugar such as a mono-, di-, or polysaccharide, or a water-soluble glucan, may be used.
• Non-limiting examples of substances which are sugars, alcohols or alditols are fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, carboxymethylcellulose-Na, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol, glycerol (glycerine), propan-1, 2- diol (propylene glycol), propan-1 ,3-diol, and butan-1 ,3-diol.
• buffer C comprises one or more stabilizing agents of the polyalcohol type.
• buffer C comprises NaCI.
• One or more stabilizing agents selected from the group consisting of glycerol (propan-1, 2,3-triol), propylene glycol (propan-1 ,2-diol), propan-1 ,3-diol, propyl alcohol (1-propanoI) and isopropyl alcohol (2-propanol) is added to the fluid from (x).
• one or more stabilizing agents selected from the group consisting of glycerol, propylene glycol and propan-1 ,3-diol is added.
• propylene glycol is added.
• a stabilizing agent of the liquid alcohol or liquid polyalcohol type when it is present in a concentration of from about 5% (v/v) to about 50% (v/v). In a further embodiment, a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of from about 10% (v/v) to about 50% (v/v). In a further embodiment, a stabilizing agent of the liquid alcohol or liquid polyalcohol type used is present in a concentration of from about 10% (v/v) to about 20% (v/v). In a further embodiment, a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of about 10% (v/v).
• Buffer C is used for the elution of the FXI polypeptide by gradient elution, wherein the composition of buffer C is changed during the course of elution.
• concentration of one or more of the components of the buffer used for washing in step (ix), in this case buffer C is increased or decreased during the course of elution, or a new component is added to the buffer and the concentration of this component is then increased during the course of elution. This increase or decrease may take place continuously or in discrete steps, as is well known in the art.
• a salt e.g.
• the use of one or more stabilizing agents in any or all of the solutions used in purification of FXI results in an increase in the physical and/or chemical stability of FXI by at least 10%, 25%, 50%, or 100% over the physical and/or chemical stability of a control (i.e., FXI subjected to the same treatment but in the absence of the stabilizing agent).
• the use of one or more stabilizing agents in any or all of the solutions used in purification of FXI results in an increase in the physical and/or chemical stability of FXI by at least 2-fold, 5-fold, 10-fold, or 20-fold over the physical and/or chemical stability of a control (i.e., FXI subjected to the same treatment but in the absence of the stabilizing agent).
• FXI Wild-type human FXI is normally activated by proteolytic cleavage between Arg 360 and lle 3 0 , which may be catalyzed by FXIa, FXIIa, or thrombin. If desired, activation of FXI for use in the present invention may be achieved using FXIa or FXIIa (both from Enzyme Research Laboratories, South Bend, IN) or thrombin (Sigma). See, e.g., Sun et al. (1999) J. Biol Chem 51 :36373-36373 and Baglia (2003) J. Biol Chem 24:21744-21750.
• the present invention encompasses methods and compositions for the therapeutic administration of FXI that utilize preparations having different FXI activation levels. In some embodiments, the methods and compositions employ FXI polypeptides that have not been subjected to any activation procedure.
• the preparation of FXI or FXI- related polypeptide exhibits a ratio (by mass) of activated:zymogen FXI or FXI-related polypeptide of between about 1 :99 to about 99:1 , such as, e.g., between about 5:95 to about 95:5; about 10:90 to about 90:10; about 20:80 to about 80:20; about 30:70 to about 70:30; about 40:60 to about 60:40; and about 50:50.
• the preparation contains not more than about 5% FXIa relative to the total FXI on a molar basis; more preferably, not more than about 2.5%, even more preferably, not more than about 1 %, most preferably not more than about 0.5% or 0.1 %. In some embodiments, the preparation contains not more than about 0.01-0.05% FXIa on a molar basis. In some embodiments, the preparation contains not more than about 0.01-0.04% FXIa on a molar basis. In some embodiments, the preparation contains not more than about 0.01-0.03% FXIa on a molar basis.
• the invention also relates to FXI-related polypeptides that exhibit a differential capacity to be activated relative to wild-type FXI, such as, e.g., FXI-related polypeptides that are more easily activated by FXIIa than by thrombin, and vice versa; polypeptides that are constitutively activated, even in the absence of proteolytic cleavage; heterodimers in which one monomer (by virtue of mutation or chemical modification) cannot be proteolytically activated; and the like. Furthermore, the invention also relates to FXI-related polypeptides that are resistant to autoactivation, i.e.
• the methods and compositions of the invention may also employ treatment, pre- treatment, storage, or co-administration of a FXI polypeptide with additional agents that inhibit and/or promote activation.
• agents that inhibit activation include C1 esterase inhibitor (C1 lnb), ⁇ -2 antiplasmin, ( ⁇ 2AP), ⁇ 1-antitrypsin ( ⁇ 1AT), protease Nexin II, benzamidine, heparin, and antithrombin III; non-limiting examples of agents that promote activation include FXIa, FXIIa, and thrombin.
• compositions comprising FXI encompasses pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide for prophylactic and/or therapeutic treatment.
• Pharmaceutical compositions or formulations according to the invention comprise a a FXI polypeptide, such as, e.g., at concentrations between 0.001-100 mg/ml, that is preferably dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent.
• pharmaceutical compositions suitable for use according to the present invention are made by mixing a preparation comprising FXI and/or a FXI-related polypeptide, preferably in purified form, with suitable adjuvants and a suitable carrier or diluent.
• aqueous carriers such as water, buffered water, 0.4% saline, 0.3% glycine, sugars, detergents, salts, buffers, glycerols, preservatives, protease inhibitors, glycols, and the like.
• the preparations of the invention can also be formulated using non-aqueous carriers, such as, e.g., in the form of a gel or as liposome preparations for delivery or targeting to the sites of injury. Liposome preparations are generally described in, e.g., U.S. Patents Nos. 4,837,028, 4,501,728, and 4,975,282.
• the compositions may be sterilised by conventional, well-known sterilisation techniques.
• compositions may contain pharmaceutically acceptable auxiliary substances or adjuvants, including, without limitation, pH adjusting and buffering agents, tonicity adjusting agents, preservatives, stabilizers, surfactants, chelating agents, and the like.
• auxiliary substances or adjuvants including, without limitation, pH adjusting and buffering agents, tonicity adjusting agents, preservatives, stabilizers, surfactants, chelating agents, and the like.
• One skilled in this art may formulate the compositions of the invention an appropriate manner, and in accordance with accepted practices, such as those disclosed in Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton, PA, 1990.
• the pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide further comprises a pH adjusting and buffering agent. In one embodiment of the invention, the pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide further comprises a tonicity adjusting agent. In one embodiment of the invention, the pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide further comprises a preservative. In one embodiment of the invention, the pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide further comprises a stabilizer. In one embodiment of the invention, the pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide further comprises a surfactant. In one embodiment of the invention, the pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide further comprises a chelating agent.
• Non-limiting examples of suitable buffers include acetate buffers, carbonate buffers, citrate buffers, glycylglycine buffers, histidine buffers, glycine buffers, lysine buffers, arginine buffers, phosphate buffers (containing, e.g., sodium dihydrogen phosphate, disodium hydrogen phosphate or trisodium phosphate), TRIS [tris(hydroxymethyl)aminomethane] buffers, bicine buffers, tricine buffers, malate buffers, succinate buffers, maleate buffers, fumarate buffers, tartrate buffers, aspartate buffers, and mixtures thereof.
• phosphate buffers containing, e.g., sodium dihydrogen phosphate, disodium hydrogen phosphate or trisodium phosphate
• TRIS [tris(hydroxymethyl)aminomethane] buffers bicine buffers, tricine buffers, malate buffers, succinate buffers, maleate buffers, fuma
• Non-limiting examples of pharmaceutically acceptible preservatives include phenol, o-cresol, m-cresol, p-cresol, chlorocresol, methyl p-hydroxybenzoate, ethyl p- hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, 2-phenoxyethanol, 2- phenylethanol, benzyl alcohol, chlorobutanol, thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, benzethonium chloride, chlorphenesine (3-p- chlorphenoxypropane-1 ,2-diol), benzamidine and mixtures thereof.
• the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In one further embodiment of the present invention the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In another further embodiment of the present invention the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In another further embodiment of the present invention the preservative is present in a concentration from 10 mg/ml to 20 mg/ml.
• the use of a preservative in pharmaceutical compositions is well-known to the skilled person (see, e.g., Remington: The Science and Practice of Pharmacy, 19th edition, 1995).
• Non-limiting examples of tonicity-adjusting agents (which are normally incorporated for the purpose of rendering the formulation substantially isotonic include salts (e.g. sodium chloride), sugars, alcohols (such as C 4 -C 8 alcohols), alditols, amino acids (e.g. glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), polyethyleneglycols (e.g. PEG400), and mixtures thereof. Any sugar, such as a mono-, di-, or polysaccharide, or a water-soluble glucan, may be used.
• salts e.g. sodium chloride
• sugars e.g. sodium chloride
• alcohols such as C 4 -C 8 alcohols
• alditols e.glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threon
• Non-limiting examples of substances which are sugars, alcohols or alditols are fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, carboxymethylcellulose-Na, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol, glycerol (glycerine), propan-1 , 2-diol (propylene glycol), propan-1 ,3- diol, and butan-1 ,3-diol.
• the sugars, alcohols and alditols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the substance is soluble in the liquid preparation.
• the tonicity-adjusting agent is present in a concentration of from about 1 mg/ml to about 150 mg/ml. In a further embodiment of the present invention, the tonicity-adjusting agent is present in a concentration of from about 1 mg/ml to about 50 mg/ml.
• the tonicity-adjusting agent is NaCI. In one embodiment, the tonicity-adjusting agent is NaCI present in a concentration of from about 1 mg/ml to about 150 mg/ml.
• the tonicity-adjusting agent is NaCI present in a concentration of from about 1 mg/ml to about 50 mg/ml.
• chelating agents include salts of EDTA, citric acid and aspartic acid, and mixtures thereof.
• a chelating agent is present in a concentration from 0.1 mg/ml to 5 mg/ml; from 0.1 mg/ml to 2 mg/ml; or from 2 mg/ml to 5 mg/ml.
• the pharmaceutical compositions of the present invention may include as a therapeutically active component a polypeptide that possibly may exhibit aggregate formation during storage in liquid pharmaceutical compositions.
• the term “aggregate formation” is intended to indicate a physical interaction between the polypeptide molecules that results in formation of oligomers which may remain soluble, or of large visible aggregates that precipitate from the solution.
• the term “during storage” refers to a liquid pharmaceutical composition or formulation which, once prepared, is not immediately administered to a subject. Rather, following preparation, it is packaged for storage in a liquid form, in a frozen state, or in a dried form for later reconstitution into a liquid form or other form suitable for administration to a subject.
• the term “dried form” refers to a liquid pharmaceutical composition or formulation dried by freeze-drying [i.e. lyophilization; see, for example, Williams and Polli (1984), J. Parenteral Sci. Technol.
• the pharmaceutical composition comprises an amount of an amino acid base sufficient to decrease aggregate formation by the polypeptide during storage of the composition.
• amino acid base indicates an amino acid or a combination of amino acids where any given amino acid is present either in its free base form or in its salt form. When a combination of amino acids is used, all of the amino acids may be present in their free base forms, all may be present in their salt forms, or some may be present in their free base forms while others are present in their salt forms.
• amino acids for use in preparing compositions of the present invention are those carrying a charged side chain, such as arginine, lysine, aspartic acid or glutamic acid.
• Any stereoisomer of a particular amino acid e.g. glycine, methionine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine or a mixture of one or more thereof), or combinations of these stereoisomers, may be present in pharmaceutical compositions of the present invention so long as the particular amino acid is present either in its free base form or its salt form.
• the L-stereoisomer is used.
• compositions of the present invention may also be formulated with analogues of these amino acids.
• amino acid analogue is intended a derivative of the naturally occurring amino acid that brings about the desired effect of decreasing aggregate formation by the polypeptide during storage of the liquid pharmaceutical compositions of the present invention.
• Suitable arginine analogues include, for example, aminoguanidine, ornithine and N-monoethyl L-arginine, suitable methionine analogues include ethionine and buthionine and suitable cysteine analogues include S-methyl-L cysteine.
• the amino acid analogues are incorporated into the compositions in either their free base form or their salt form.
• the compound imidazole is also to be regarded as an amino acid analogue in the context of the present invention.
• the amino acids or amino acid analogues are used in a concentration which is sufficient to prevent or delay aggregation of the protein.
• the pharmaceutical formulation comprises methionine (or another sulfur-containing amino acid or amino acid analogue) to inhibit oxidation of methionine residues to their sulfoxide form when the factor XI polypeptide is a polypeptide comprising at least one methionine residue susceptible to such oxidation.
• the term "inhibit oxidation” is intended to indicate minimization of accumulation of oxidized species (of methionine) with time.
• the amount to be added should be an amount sufficient to inhibit oxidation of the methionine residues such that the amount of sulfoxide form of methionine is acceptable to regulatory agencies. Typically, this means that the composition contains no more than from about 10% to about 30% methionine sulfoxide form. This can in general be achieved by adding methionine in an amount such that the ratio of added methionine to methionine residues ranges from about 1 :1 to about 1000:1 , such as 10:1 to about 100:1.
• Non-limiting examples of stabilizers include high-molecular-weight polymers or low- molecular-weight compounds, such as, e.g., polyethylene-glycols (e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose and derivatives thereof (including HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulfur-containing substances as monothioglycerol, thioglycolic acid and 2-methylthioethanol, various salts (e.g., polyethylene-glycols (e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose and derivatives thereof (including HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulfur-containing substances as monothioglycerol, thioglycolic acid and 2-methylthioethanol, various salts (e.
• Non-limiting examples of surfactants include detergents, ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers (e.g.
• poloxamers such as Pluronic ® F68, poloxamer 188 and 407, Triton X-100 ), polyoxyethylene sorbitan fatty acid esters, polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives ("Tweens", e.g. Tween-20, Tween-40, Tween-80 and Brij-35), monoglycerides and ethoxylated derivatives thereof, diglycerides and polyoxyethylene derivatives thereof, alcohols, glycerol, lectins and phospholipids (eg.
• phosphatidyl-serine phosphatidyl-choline, phosphatidyl-ethanolamine, phosphatidyl-inositol, diphosphatidyl-glycerol and sphingomyelin
• derivatives of phospholipids e.g. dipalmitoyl-phosphatidic acid
• lysophospholipids e.g.
• cholines ethanolamines, phosphatidic acid, serines, threonines, glycerol, inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, and glycerophospholipids (e.g. cephalins), glyceroglycolipids (e.g. galactopyranoside), sphingoglycolipids (e.g. ceramides, gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic acid derivatives (e.g.
• long-chain fatty acids e.g. C 6 -C ⁇ 2 fatty acids (such as oleic acid or caprylic acid)] and salts thereof, acylcarnitines and derivatives thereof, N ⁇ -acylated derivatives of lysine, arginine and histidine, side-chain acylated derivatives of lysine and arginine, N ⁇ -acylated derivatives of dipeptides comprising any combination of lysine, arginine and histidine and a neutral or acidic amino acid, N ⁇ -acylated derivatives of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, DSS (docusate sodium, CAS registry no [577-11-7]), docusate calcium, CAS registry no [128-49-4]), docusate potassium, CAS registry no [7491-09-0]), SDS (sodium dodecyl
• the pharmaceutical formulation comprises a surfactant in a concentration of about 0.01 mg/ml to about 50 mg/ml.
• the pharmaceutical formulation comprises Tween-80.
• the pharmaceutical formulation comprises poloxamer 188.
• the pharmaceutical formulation comprises an electrolyte.
• the pharmaceutical formulation comprises an electrolyte, such as NaCI.
• the pharmaceutical formulation comprises an electrolyte, such as KCI.
• an electrolyte such as NaCI, such as in a concentration of 150 mM, is employed when Tween 80 is employed as stabiliser.
• aggregation following storage is avoided.
• the use of one or more stabilizing agents used in a pharmaceutical formulation comprising a preparation of FXI or FXI-related polypeptide results in an increase in the physical and/or chemical stability of FXI by at least 10%, 25%, 50%), or 100% over the physical and/or chemical stability of a control (i.e., FXI subjected to the same treatment but in the absence of the stabilizing agent).
• the use of one or more stabilizing agents in a pharmaceutical formulation comprising a preparation of FXI or FXI-related polypeptide results in an increase in the physical and/or chemical stability of FXI by at least 2-fold, 5-fold, 10-fold, or 20-fold over the physical and/or chemical stability of a control (i.e., FXI subjected to the same treatment but in the absence of the stabilizing agent).
• a suitable formulation that allows recovery of active FXI after freeze-drying contains: FXI cone: 0.2 mg/ml Buffer: 20 mM buffer (Histidine or TRIS) (pH 5.5, 6.5 of 7.4), 25 mg/ml Mannitol (bulking agent), 2.5 mg/ml NaCI (bulking agent), with 0.01 % Tween 80
• the pharmaceutical compositions are administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly; intravenously being most preferred. They may also be administered by continuous or pulsatile infusion. It will be understood that any effective method for administering a FXI polypeptide may be used, including, e.g., using mucosal or inhalation methods of administration.
• compositions of the present invention may be administered in various dosage forms, e.g. as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules (e.g.
• compositions of the present invention may further be compounded in, or bound or conjugated to (e.g.
• a drug carrier e.g. cellulose and derivatives thereof, other polysaccharides (e.g. dextran and derivatives thereof, starch and derivatives thereof), poly(vinyl alcohol), acrylate and methacrylate polymers, polylactic acid and polyglycolic acid and block co-polymers thereof, polyethyleneglycols, carrier proteins (e.g.
• albumin e.g. thermogelling systems, such as block co-polymeric systems well known to those skilled in the art
• micelles e.g. liposomes, microspheres, nanoparticulates, liquid crystals and dispersions thereof, L2 phase and dispersions thereof well known to those skilled in the art of phase behaviour in lipid-water systems, polymeric micelles, multiple emulsions (self-emulsifying and self-microemulsifying), cyclodextrins and derivatives thereof, and dendrimers.
• compositions comprising a factor XI polypeptide prepared by use of a method according to the present invention are suitable for use in the formulation of solids, semisolids, powders and solutions for pulmonary administration using, for example, a metered dose inhaler, dry powder inhaler or a nebulizer, all of which are devices well known to those skilled in the art.
• Pharmaceutical compositions comprising a factor XI polypeptide prepared by use of a method according to the present invention are suitable for use in the formulation of controlled-release, sustained-release, protracted-release, retarded-release or slow-release drug delivery systems.
• compositions comprising a factor XI polypeptide prepared by use of a method according to the present invention are, for instance, useful in formulation of parenteral controlled-release and sustained-release systems (both systems leading to a many-fold reduction in number of administrations) of types well known to those skilled in the art, such as controlled-release and sustained-release systems for subcutaneous administration.
• examples of useful controlled-release systems and compositions are hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres and nanoparticles
• Methods for producing controlled release systems useful for pharmaceutical compositions comprising a factor XI polypeptide prepared by use of a method according to the present invention include, but are not limited to, crystallization, condensation, co- crystallization, precipitation, co-precipitation, emulsification, dispersion, high-pressure homogenisation, encapsulation, spray-drying, microencapsulation, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes.
• Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, for example a syringe in a device of the pen type. Alternatively, parenteral administration can be performed by means of an infusion pump.
• a further option for administration of a composition in the form of a solution or suspension containing a factor XI polypeptide prepared by use of a method according to the present invention is administration as a nasal or pulmonary spray.
• pharmaceutical compositions containing a factor XI polypeptide prepared by use of a method according to the present invention may be adapted to transdermal administration, e.g. by needleless injection, by application of a patch (such as an iontophoretic patch) or by transmucosal (e.g. buccal) administration.
• a pharmaceutical composition comprising a factor XI polypeptide prepared by use of a method according to the present invention is stable for more than 6 weeks of usage and for more than 3 years of storage.
• a pharmaceutical composition comprising a factor XI polypeptide prepared by use of a method according to the present invention is stable for more than 4 weeks of usage and for more than 3 years of storage.
• a pharmaceutical composition comprising a factor XI polypeptide prepared by use of a method according to the present invention is stable for more than 4 weeks of usage and for more than 2 years of storage.
• a pharmaceutical composition comprising a factor XI polypeptide prepared by use of a method according to the present invention is stable for more than 2 weeks of usage and for more than 2 years of storage.
• FXI polypeptide formulations have a pH from about 4.0 to about 10.0.
• FXI polypeptide formulations have a pH from about 4.0 to about 8.0. In some embodiments, FXI polypeptide formulations have a pH from about 4.0 to about 7.0. In some embodiments, FXI polypeptide formulations have a pH from about 4.0 to about 6.5. In some embodiments, FXI polypeptide formulations have a pH from about 4.0 to about 6.0. In some embodiments, FXI polypeptide formulations have a pH of about 6.5 or below, such as, e.g., between about pH 5.0 and about 6.5; such as between about 5.5 and 6.5.
• Bleeding refers to extravasation of blood from any component of the circulatory system.
• a bleeding episode encompasses unwanted, uncontrolled and often excessive bleeding in connection with surgery, trauma, or other forms of tissue damage, as well as unwanted bleedings in subjects having bleeding disorders.
• Bleedings may occur as a spontaneous events, such as intra-cerebral hemorrhage (ICH).
• ICH intra-cerebral hemorrhage
• Bleeding episodes may occur in subjects having a basically normal coagulation system but experiencing a (temporary) coagulophathy, as well as in subjects having congenital or acquired coagulation or bleeding disorders.
• the bleedings may be likened to bleedings caused by haemophilia because the haemostatic system, as in haemophilia, lacks or has abnormal essential clotting "compounds" (e.g., platelets or von Willebrand factor protein).
• compounds e.g., platelets or von Willebrand factor protein.
• the normal haemostatic mechanism may be overwhelmed by the demand of immediate haemostasis and they may develop excessive bleeding in spite of a basically (pre-trauma or pre-surgery) normal haemostatic mechanism.
• Such subjects who further often are multi transfused, develop a (temporary) coagulopathy as a result of the bleeding and/or transfusions (i.e., a dilution of coagulation proteins, increased fibrinolysis and lowered number of platelets due to the bleeding and/or transfusions).
• Bleedings may also occur in organs such as the brain, inner ear region and eyes; these are areas with limited possibilities for surgical haemostasis and thus problems with achieving satisfactory haemostasis. Similar problems may arise in the process of taking biopsies from various organs (liver, lung, tumour tissue, gastrointestinal tract) as well as in laparoscopic surgery and radical retropubic prostatectomy.
• Such therapy may include heparin, other forms of proteoglycans, warfarin or other forms of vitamin K-antagonists, inhibitors of coagulation proteins, as well as aspirin and other platelet aggregation inhibitors, such as, e.g., antibodies or other inhibitors of GP llb/IIIa activity.
• the bleeding may also be due to so-called thrombolytic therapy which comprises combined treatment with an antiplatelet agent (e.g., acetylsalicylic acid), an anticoagulant (e.g., heparin), and a fibrinolytic agent (e.g., tissue plasminogen activator, tPA).
• an antiplatelet agent e.g., acetylsalicylic acid
• an anticoagulant e.g., heparin
• a fibrinolytic agent e.g., tissue plasminogen activator, tPA
• Bleeding episodes are also meant to include, without limitation, uncontrolled and excessive bleeding in connection with surgery or trauma in subjects having acute haemarthroses (bleedings in joints), chronic haemophilic arthropathy, haematomas, (e.g., muscular, retroperitoneal, sublingual and retropharyngeal), bleedings in other tissue, haematuria (bleeding from the renal tract), cerebral haemorrhage, surgery (e.g., hepatectomy), dental extraction, and gastrointestinal bleedings (e.g., UGI bleeds).
• acute haemarthroses bleedings in joints
• chronic haemophilic arthropathy haematomas, (e.g., muscular, retroperitoneal, sublingual and retropharyngeal)
• bleedings in other tissue e.g., haematuria (bleeding from the renal tract), cerebral haemorrhage, surgery (e.g., hepatectomy), dental extraction, and gastrointestinal bleedings (e.
• the bleeding episodes may be associated with inhibitors against factor VIII; haemophilia A; haemophilia A with inhibitors; haemophilia B; deficiency of factor VII; deficiency of factor XI; thrombocytopenia; deficiency of von Willebrand factor (von Willebrand's disease); severe tissue damage; severe trauma; surgery; laparoscopic surgery; acidosis, hemodilution, consumption coagulopathies, hyperfibrinolysis, hyopthermia, haemorrhagic gastritis; taking biopsies; anticoagulant therapy; upper gastroentestinal bleedings (UGI); or stem cell transplantation.
• the bleeding episodes may be profuse uterine bleeding; occurring in organs with a limited possibility for mechanical haemostasis; occurring in the brain; occurring in the inner ear region; or occurring in the eyes.
• a lowered count or activity of platelets refers to the number of platelets (thrombocytes) present in the subject's plasma and to the biological, coagulation-related activity of such platelets. Lowered counts may be due, e.g., to increased platelet destruction, decreased platelet production, and pooling of a larger than normal fraction of platelets in the spleen.
• Thrombocytopenia for example, is defined as a platelet count less than 150,000 platelets per microliter; the upper limit of the normal platelet count is generally considered to be between 150,000 and 450,000 platelets per microliter. Platelet count may be measured by automated platelet counters; this is a well known method to the skilled worker. Syndromes due to lowered platelet count include, without limitation, thrombocytopenia, coagulophathy. Aspects of platelet activity include, without limitation, aggregation, adhesion, and coagulant activity of the platelets.
• Decreased activity may be due, e.g., to glycoprotein abnormalities, abnormal membrane-cytoskeleton interaction, abnormalities of platelet granules, abnormalities of platelet coagulant activity, abnormalities of signal transduction and secretion.
• Platelet activity including aggregation, adhesion, and coagulant activity, are measured by standard methods known to the skilled worker, see e.g..Platelets. A Practical Approach, Ed. S.P. Watson & K.S. Authi: Clinical Aspects of Platelet Disorders (K.J. Clemetson) 15:299-318, 1996, Oxford University Press; Williams Hematology, Sixth Edition, Eds.
• treatment encompasses both prevention of bleeding, including, without limitation, prevention of an expected bleeding, such as, for example, might be expected to occur during or consequent to a surgical procedure, as well as regulation of an already occurring bleeding, such as, for example, in trauma, with the purpose of inhibiting or minimizing the bleeding.
• the bleeding may be at an identified site or may be at an undetermined site.
• Prophylactic administration of a preparation comprising a FXI polypeptide is thus included in treatment.
• a normal human patient i.e., one not suffering from a congenital deficiency of FXI
• blood is drawn from a patient in need of treatment with a FXI polypeptide and an assay is performed (prior to FXI polypeptide administration) to assess one or more of: (i) the plasma level of FXI; (ii) the ratio of activated :zymogen FXI; and/or (iii) the concentration of FXI needed to be added exogenously in order to restore effective coagulation; based on the results of the assay, an appropriate amount of FXI polypeptide is administered using a predetermined regimen. Any suitable assay may be used for these determinations, including, e.g., an ELISA or a gel-based method.
• Appropriate calibration standards are used in order to allow the comparison of the measured level with the usual level of FXI in human plasma (about 30 nM). Typically, it will be desired to replenish FXI levels to at least about 5 nM, such as about 10 nM, such as about 15 nM, such as about 20 nM, and such as at least about 30 nM FXI, such as 60 nM, such as 120 nM.
• the FXI-related polypeptide When a FXI-related polypeptide is being used to replenish FXI activity in a patient, the FXI-related polypeptide will exhibit a particular level of at least one FXI bioactivity and the goal of the treatment is to provide an amount of that bioactivity that corresponds to a predetermined amount of wild-type FXI (i.e., an "effective FXI plasma concentration").
• the present invention encompasses therapeutic administration of FXI polypeptide to patients whose plasma level of FXI is below about 3 nM; 5 nM; or 10 nM.
• non-Factor VI I/Factor VI I A coagulation agents include, without limitation, Factor XIII (see, e.g., WO 01/85198); inhibitors of tissue factor pathway inhibitor (TFPI inhibitors) (see, e.g., WO 01/85199); Factor IX (see, e.g., WO 02/062376); thrombin activatable fibrinolysis inhibitor (TAFI) (see, e.g., PCT/DK02/00734; PAI-1 (see, e.g., PCT/DK02/00735; Factor V (see, e.g., PCT/DK02/00736); protein C inhibitors (see, e.g., PCT/DK02/00737); thrombomodul
• PCT/DK02/00740 ); ⁇ 2-antiplasmin (see, e.g., PCT/DK02/00741 ); aprotinin (see, e.g., PCT/DK02/00742); tranexamic acid (see, e.g., PCT/DK02/00751); ⁇ -aminocaproic acid (see, e.g., PCT/DK02/00752); prothrombin, thrombin, Factor VII, Factor X, and fibrinogen.
• Embodiment 1 A method for treating bleeding episodes, said method comprising administering to a patient in need thereof a preparation comprising Factor XI (FXI) or FXI- related polypeptide, in an amount effective for such treatment.
• Embodiment 2 A method as defined in embodiment 1 , wherein said administering results in a reduced clotting time in said patient.
• Embodiment 3 A method as defined in embodiment 1 or embodiment 2, wherein said administering results in an enhancement of hemostasis in said patient.
• Embodiment 4 A method as defined in any of embodiments 1 to 3, wherein said administering results in an increase in clot lysis time in said patient.
• Embodiment 5 A method as defined in any of embodiments 1 to 4, wherein said administering results in an increase in clot strength in said patient.
• Embodiment 6 A method as defined in any of embodiments 1 to 5, wherein said administering results in an increase in overall clot quality (OCQ) in said patient.
• Embodiment 7 A method as defined in any of embodiments 1 to 6, wherein, following said administration, said patient exhibits an effective FXI plasma concentration of at least about 5 nM.
• Embodiment 8 A method as defined in embodiment 7, wherein said effective FXI plasma concentration is at least about 10 nM.
• Embodiment 9 A method as defined in embodiment 8, wherein said effective FXI plasma concentration is at least about 30 nM, such as at least about 60 nM, such as at least about 120 nM.
• Embodiment 10 A method as defined in any of embodiments 1 to 9, wherein said FXI or FXI-related polypeptide comprises the sequence of SEQ ID NO:1 , or a fragment thereof that retains at least one FXI-associated biological activity.
• Embodiment 11 A method as defined in any of embodiments 1 to 9, wherein said FXI or FXI-related polypeptide comprises the sequence of SEQ ID NO:2, or a fragment thereof that retains at least one FXI-associated biological activity.
• Embodiment 12 A method as defined in any of embodiments 1 to 11 , wherein said patient does not suffer from a congenital FXI deficiency.
• Embodiment 13 A method as defined in any of embodiments 1 to 12, wherein said bleeding episodes are secondary to a condition selected from the group consisting of: surgery, a dental procedure, trauma, or hemodilution.
• Embodiment 14 A method as defined in any of embodiments 1 to 13, further comprising, prior to said administering:
• Embodiment 15 A method for treating bleeding episodes, said method comprising administering to said patient (i) a first amount of a preparation comprising a FXI polypeptide and (ii) a second amount of a preparation comprising a non-Factor Vll/Factor Vila coagulation agent, wherein said first and second amounts in combination are effective for such treatment.
• Embodiment 16 A method as defined in embodiment 15, wherein said non-Factor Vll/Factor Vila coagulation agent is selected from the group consisting of: Factor XIII; tissue factor pathway inhibitor (TFPI) inhibitor; Factor IX; thrombin activatable fibrinolysis inhibitor (TAFI); plasminogen activator inhibitor-1 (PAI-1 ); Factor V; protein C inhibitor; protein S inhibitor; and tissue plasminogen activator (tPA) inhibitor.
• Embodiment 17 A method as defined in embodiment 15 or embodiment 16, wherein said administering results in a reduced clotting time in said patient.
• Embodiment 18 A method as defined in any of embodiments 15 to 17, wherein said administering results in an enhancement of hemostasis in said patient.
• Embodiment 19 A method as defined in any of embodiments 15 to 18, wherein said administering results in an increase in clot lysis time in said patient.
• Embodiment 20 A method as defined in any of embodiments 15 to 19, wherein said administering results in an increase in clot strength in said patient.
• Embodiment 21 A method as defined in any of embodiments 15 to 20, wherein said administering results in an increase in overall clot quality (OCQ) in said patient.
• Embodiment 22 A method as defined in any of embodiments 15 to 21 , wherein, following said administration, said patient exhibits an effective FXI plasma concentration of at least about 5 nM.
• Embodiment 23 A method as defined in embodiment 22, wherein said effective FXI plasma concentration is at least about 10 nM.
• Embodiment 24 A method as defined in embodiment 23, wherein said effective FXI plasma concentration is at least about 30 nM, such as at least about 60 nM, such as at least about 120 nM.
• Embodiment 25 A method as defined in any of embodiments 15 to 14, wherein said FXI or FXI-related polypeptide comprises the sequence of SEQ ID NO:1 , or a fragment thereof that retains at least one FXI-associated biological activity.
• Embodiment 26 A method as defined in any of embodiments 15 to 24, wherein said FXI or FXI-related polypeptide comprises the sequence of SEQ ID NO:2, or a fragment thereof that retains at least one FXI-associated biological activity.
• Embodiment 27 A method as defined in any of embodiments 15 to 26, wherein said patient does not suffer from a congenital FXI deficiency.
• Embodiment 28 A method as defined in any of embodiments 15 to 27, wherein said bleeding episodes are secondary to a condition selected from the group consisting of: surgery, a dental procedure, trauma, or hemodilution.
• Embodiment 29 A method as defined in any of embodiments 15 to 28, further comprising, prior to said administering: (a) obtaining a sample of blood from said patient; (b) determining at least one of: FXI concentration, ratio of FXIa:FXI, or amount of exogenous FXI necessary to restore coagulation; and (c) based on the results of step (b), determining said amount of FXI effective for treatment.
• Embodiment 30 A method as defined in embodiment 1 , wherein said method does not comprise administration of a Factor Vll/Factor Vila coagulation agent.
• Embodiment 31 A pharmaceutical formulation comprising (i) isolated recombinant a
• Embodiment 32 Use of a FXI polypeptide for treating bleeding episodes.
• Embodiment 33 Use according to embodiment 32, wherein said bleeding episodes are secondary to a condition selected from the group consisting of: surgery, a dental procedure, trauma, or hemodilution.
• Embodiment 34 Use according to embodiment 32 or embodiment 33, wherein said bleeding episodes are not treated with a Factor Vll/Factor Vila coagulation agent.
• Embodiment 35 Use of a FXI polypeptide for enhancement of hemostasis in a patient in need thereof .
• Embodiment 36 Use of a FXI polypeptide for increasing clot lysis time in a patient in need thereof.
• Embodiment 37 Use of a FXI polypeptide for increasing clot strength in a patient in need thereof.
• Embodiment 38 Use of a FXI polypeptide for increasing overall clot quality (OCQ) in a patient in need thereof.
• Embodiment 39 Use of a FXI polypeptide for reducing clotting time in a patient in need thereof.
• Embodiment 40 Use according to any of embodiments 32 to 39, wherein the effective FXI plasma concentration in the patient is increased to at least about 5 nM.
• Embodiment 41 Use according to embodiment 40, wherein the effective FXI plasma concentration is increased to at least about 10 nM.
• Embodiment 42 Use according to embodiment 41, wherein the effective FXI plasma concentration is increased to at least about 30 nM, such as at least about 60 nM, such as at least about 120 nM.
• Embodiment 43 Use according to any of embodiments 32 to 42, wherein the patient to be treated is not treated with a Factor Vll/Factor Vila coagulation agent.
• Embodiment 44 Use of a FXI polypeptide for preparation of a pharmaceutical formulation for treating bleeding episodes.
• Embodiment 45 Use according to embodiment 44, wherein said bleeding episodes are secondary to a condition selected from the group consisting of: surgery, a dental procedure, trauma, or hemodilution.
• Embodiment 46 Use according to embodiment 44 or embodiment 45, wherein said bleeding episodes are not being treated with a Factor Vll/Factor Vila coagulation agent.
• Embodiment 47 Use of a FXI polypeptide for preparation of a pharmaceutical formulation for enhancement of hemostasis in a patient in need thereof .
• Embodiment 48 Use of a FXI polypeptide for preparation of a pharmaceutical formulation for increasing clot lysis time in a patient in need thereof.
• Embodiment 49 Use of a FXI polypeptide for preparation of a pharmaceutical formulation for increasing clot strength in a patient in need thereof.
• Embodiment 50 Use of a FXI polypeptide for preparation of a pharmaceutical formulation for increasing overall clot quality (OCQ) in a patient in need thereof.
• Embodiment 51 Use of a FXI polypeptide for preparation of a pharmaceutical formulation for reducing clotting time in a patient in need thereof..
• Embodiment 52 Use according to any of embodiments 47 to 51 , wherein the effective FXI plasma concentration in the patient is increased to at least about 5 nM.
• Embodiment 53 Use according to embodiment 52, wherein the effective FXI plasma concentration is increased to at least about 10 nM.
• Embodiment 54 Use according to embodiment 53, wherein the effective FXI plasma concentration is increased to at least about 30 nM, such as at least about 60 nM, such as at least about 120 nM.
• Embodiment 55 Use according to any of embodiments 44 to 54, wherein the patient to be treated is not treated with a Factor Vll/Factor Vila coagulation agent.
• Embodiment 56 Use according to any of embodiments 32 to 55, wherein the patient to be treated does not suffer from a congenital FXI deficiency.
• Embodiment 57 Use according to any of embodiments 32 to 56, wherein said FXI polypeptide comprises the sequence of SEQ ID NO:1 , or a fragment thereof that retains at least one FXI-associated biological activity.
• Embodiment 57 Use according to any of embodiments 32 to 56, wherein said FXI polypeptide comprises the sequence of SEQ ID NO:2, or a fragment thereof that retains at least one FXI-associated biological activity.
• Embodiment 58 Use according to any of embodiments 32 to 57, wherein said FXI polypeptide is to be administered in combination with a non-Factor Vll/Factor Vila coagulation agent.
• Embodiment 59 Use according to embodiment 58, wherein said non-Factor Vll/Factor Vila coagulation agent is selected from the group consisting of: Factor XIII; tissue factor pathway inhibitor (TFPI) inhibitor; Factor IX; thrombin activatable fibrinolysis inhibitor (TAFI); plasminogen activator inhibitor-1 (PAI-1 ); Factor V; protein C inhibitor; protein S inhibitor; and tissue plasminogen activator (tPA) inhibitor.
• TFPI tissue factor pathway inhibitor
• TAFI thrombin activatable fibrinolysis inhibitor
• PAI-1 plasminogen activator inhibitor-1
• tPA tissue plasminogen activator
• Embodiment 60 Use according to embodiment 58, wherein said non-Factor Vll/Factor Vila coagulation agent is selected from the group consisting of: Factor XIII; tissue factor pathway inhibitor (TFPI) inhibitor; Factor IX; thrombin activatable fibrinolysis inhibitor (TAFI); plasminogen
• a method for purifying a FXI polypeptide from a biological material comprising subjecting the material to sequential chromatography on an cation- exchange chromatographic material, a hydrophobic interaction chromatographic material and a hydroxyapatite chromatographic material.
• Embodiment 61. A method according to embodiment 60, wherein the FXI polypeptide is a recombinant FXI.
• Embodiment 62. A method according to embodiment 60 or embodiment 61 , wherein the FXI polypeptide is human FXI.
• Embodiment 63. A method according to embodiment 60 or embodiment 61 , wherein the FXI polypeptide is a dimer.
• Embodiment 64 is
• Embodiment 65 A method according to any of embodiments 60 to 64, wherein the biological material is a biological fluid.
• Embodiment 66. A method according to embodiment 65, wherein the biological fluid is the supernatant of a mammalian cell.
• Embodiment 67. A method according to embodiment 66, wherein the biological fluid is the supernatant of a CHO culture.
• Embodiment 68. A method according to any of embodiments 60 to 67, wherein the method comprises the steps of:
• step (b) subjecting the eluate from step (iv), or a fluid prepared by use of the eluate from step (iv), to chromatography using a hydrophobic interaction chromatographic material, said chromatography comprising: (v) applying the eluate from step (iv), or a fluid prepared by use of the eluate from step (iv), to said hydrophobic interaction chromatographic material; (vi) eluting unbound material from the chromatographic material with buffer B, which buffer B is suitable for eluting material not bound to the hydrophobic interaction chromatographic material; and (vii) eluting said FXI polypeptide from said chromatographic material by gradient- elution with buffer B', which buffer B' is suitable for eluting FXI from said hydrophobic interaction chromatographic material.
• Embodiment 69 A method according to embodiment 68, wherein buffer A comprises one or more stabilizing agents which are capable of increasing the stability of the FXI polypeptide.
• Embodiment 70 A method according to embodiment 69, wherein buffer A comprises a stabilizing agent, which stabilizing agent is a sugar, an alcohol or an alditol.
• Embodiment 71 A method according to embodiment 70, wherein buffer A comprises a stabilizing agent, which stabilizing agent is a sugar, a C -C 8 -alcohol or an alditol.
• Embodiment 72 A method according to embodiment 71 , wherein buffer A comprises a stabilizing agent, which stabilizing agent is a polyalcohol.
• Embodiment 73 A method according to embodiment 68, wherein buffer A comprises one or more stabilizing agents which are capable of increasing the stability of the FXI polypeptide.
• Embodiment 70 A method according to embodiment 69, wherein buffer A comprises a stabilizing agent, which stabilizing agent is
• buffer A comprises a stabilizing agent selected from the group consisting of glycerol, propylene glycol, propan-1 ,3-diol, propyl alcohol and isopropyl alcohol.
• buffer A comprises a stabilizing agent selected from the group consisting of glycerol, propylene glycol and propan-1 ,3-diol.
• Embodiment 75 A method according to any of embodiments 72 to 74, wherein said stabilizing agent is present in a concentration of from about 5% (v/v) to about 50% (v/v).
• Embodiment 76 Embodiment 76.
• Embodiment 77. A method according to embodiment 76, wherein said stabilizing agent is present in a concentration of from about 10% (v/v) to about 20% (v/v).
• Embodiment 78. A method according to embodiment 77, wherein said stabilizing agent is present in a concentration of about 10% (v/v).
• Embodiment 79. A method according to embodiment 78, wherein said stabilizing agent is present in a concentration of about 20% (v/v).
• Embodiment 80. A method according to any of embodiments 68 to 79, wherein the pH of buffer A is between about 6.5 and about 9.
• Embodiment 80 wherein the pH of buffer A is between about 7 and about 9.
• Embodiment 82 A method according to embodiment 81 , wherein the pH of buffer A is about 8.
• Embodiment 83 A method according to any of embodiments 68 to 82, wherein buffer A has a conductivity of less than about 50 mS/cm.
• Embodiment 84 A method according to any of embodiments 60 to 83, wherein the hydrophobic interaction chromatographic material uses butyl or phenyl as the ligand.
• Embodiment 85 A method according to embodiment 84, wherein the hydrophobic interaction chromatographic material is Phenyl Sepharose High Performance High Substitution.
• Embodiment 86 A method according to embodiment 80, wherein the pH of buffer A is between about 7 and about 9.
• Embodiment 82 A method according to embodiment 81 , wherein the pH of buffer A is about 8.
• Embodiment 83 A method according to any of embodiments 68 to 82,
• Embodiment 87. A method according to any of embodiments 68 to 86, wherein the pH of buffer B is from about 6 to about 9.
• Embodiment 88. A method according to embodiment 87, wherein the pH of buffer B is about 8.
• Embodiment 89. A method according to any of embodiments 68 to 88, wherein buffer B has a conductivity of more than 50 mS/cm.
• Embodiment 90. A method according to embodiment 89, wherein buffer B has a conductivity of more than 70 mS/cm.
• Embodiment 91. A method according to any of embodiments 68 to 90, wherein the eluate from stage (vii), or a fluid prepared by use of the eluate from stage (vii), is treated by use of a method comprising a step of
• Embodiment 92 A method according to embodiment 91 , wherein the stabilizing agent used in step (1 ) is a sugar, an alcohol or an alditol.
• Embodiment 93 A method according to embodiment 92, wherein the stabilizing agent used in step (1 ) is a sugar, a C -C 8 -alcohol or an alditol.
• Embodiment 94 A method according to embodiment 92, wherein the stabilizing agent used in step (1 ) is a sugar, a C -C 8 -alcohol or an alditol.
• a method according to embodiment 93, wherein the stabilizing agent used in step (1 ) is a polyalcohol.
• Embodiment 95. A method according to embodiment 94, wherein the stabilizing agent used in step (1 ) is selected from the group consisting of glycerol, propylene glycol, propan-1 ,3-diol, propyl alcohol and isopropyl alcohol.
• Embodiment 96. A method according to embodiment 95, wherein the stabilizing agent used in step (1 ) is selected from the group consisting of glycerol, propylene glycol and propan-1 ,3-diol.
• Embodiment 98. A method according to embodiment 97, wherein the stabilizing agent used in step (1 ) is added to a concentration of from about 10% (v/v) to about 50% (v/v).
• Embodiment 99. A method according to embodiment 98, wherein the stabilizing agent used in step (1 ) is added to a concentration of from about 10% (v/v) to about 20% (v/v).
• Embodiment 100 Embodiment 100.
• Embodiment 101 A method according to any of embodiments 60 to 99, wherein the method further comprises a step of subjecting the eluate from the hydrophobic interaction chromatography, or a material prepared by use of the eluate from the hydrophobic interaction chromatography, to chromatography on a Hydroxyapatite chromatographic material.
• a method according to any of embodiments 68 to 100, wherein the method further comprises a step of: subjecting the eluate from stage (vii), or a fluid prepared by use of the eluate from stage (vii), to chromatography on a hydroxyapatite chromatographic material, said chromatography comprising: (viii) applying the eluate (diluted and pH adjusted) from stage (vii), or a fluid prepared by use of the eluate from stage (vii), to said hydroxyapatite chromatographic material; (ix) eluting unbound material from the hydroxyapatite chromatographic material with buffer C, which buffer C is suitable for eluting material not bound to the hydroxyapatite chromatographic material; and (x) eluting said FXI polypeptide from the hydroxyapatite chromatographic material with buffer C, wherein buffer C is suitable for eluting FXI polypeptides which bind to the hydroxyapatite chromatographic material in step (viii).
• Embodiment 102 A method according to embodiment 101 , wherein buffer C and/or buffer C comprises one or more stabilizing agents which are capable of increasing the stability of the FXI polypeptide.
• Embodiment 103 A method according to embodiment 101 , wherein a stabilizing agent is added to the fXI containing fractions, which stabilizing agent is a sugar, an alcohol or an alditol.
• Embodiment 104 A method according to embodiment 103, wherein a stabilizing agent is added, which stabilizing agent is a sugar, a C 4 -C 8 -aIcohol or an alditol.
• Embodiment 105 A method according to embodiment 101 , wherein buffer C and/or buffer C comprises one or more stabilizing agents which are capable of increasing the stability of the FXI polypeptide.
• Embodiment 103 A method according to embodiment 101 , wherein a stabilizing agent is added to the fXI containing fractions, which stabilizing agent is a sugar, an alcohol or
• Embodiment 106. A method according to embodiment 105 wherein a stabilizing agent selected from the group consisting of glycerol, propylene glycol, propan-1 ,3-diol, propyl alcohol and isopropyl alcohol is added.
• Embodiment 107. A method according to embodiment 106, wherein a stabilizing agent selected from the group consisting of glycerol, propylene glycol and propan-1 ,3-diol is added.
• Embodiment 110. A method according to embodiment 109, wherein said stabilizing agent is added to a concentration of from about 10% (v/v) to about 20% (v/v).
• a method according to any of embodiments 101 to 11 1 wherein buffer C and/or buffer C has a pH from about 5,8 to about 7,8.
• Embodiment 1 13 A method according to any of embodiments 101 to 112, wherein buffer C and/or buffer C has a pH of about 6,0.
• Embodiment 114 A pharmaceutical composition comprising a FXI polypeptide prepared by use of a method according to any of embodiments 60 to 113. The following are intended as non-limiting examples of the present invention.
• Example 1 Effect of FXI on hemostasis in cardiac patients: Blood was obtained before and after surgery from 5 patients undergoing cardiac surgery with cardiopulmonary bypass. The effect of FXI on clot formation and stability was evaluated using roTEG (rotational thromboelastography), using the method of Vig et al. (2001 ), Blood Coagulation & Fibrinolysis 12:555. Briefly, coagulation was initiated by adding Innovin (final dilution: 1 :50,0000) (Dade Behring) and CaCI 2 (final concentration: 15 nM), in the presence or absence of FXI (2.5, 10, or 25 nM) (HTI/Enzyme Research Laboratories, Essen). Fibrinolysis was initiated by addition of 4 nM tPA (American Diagnostica).
• Example 2 Effect of FXI on hemostasis in normal blood Blood was obtained from 4 normal subjects, and the effect of FXI on clot formation was evaluated by ROTEG as described in Example 1.
• Figure 2 illustrates that FXI caused a dose-dependent increase is OCQ in normal blood.
• Example 3 Activity of glycosylation-disrupted FXI polypeptides FXI variant containing the following substitutions were constructed using standard methodologies and were expressed after transfection in HEK293 cells. Crude cell culture supernatants were collected from cells grown for 96 h at 37°C. FXI activity was measured by ROTEG as described in Example 1. The results are shown in the following Table.
• Example 4 Storage stability of FXI formulations The following solutions of FXI were prepared and stored for 5 weeks at 5°C, after which FXI activity was measured as described in Example 1. 1. 384 nM FXI in 4 mM acetate, 150 mM NaCI, pH 5.4 2. 190 nM FXI in 50 mM acetate buffer, 150 mM NaCI, pH 5.4 3. 190 nM FXI in 50 mM acetate buffer, 150 mM NaCI, pH 5.4, 1 mM CaCI2 4. 190 nM FXI in 50 mM acetate buffer, 75 mM NaCI, pH 5.4, 300 mg/ml sucrose 5.
• Example 5 Binding peptides for FXI The following experiments were performed to identify peptides that bind FXI. /. Synthesis of peptide libraries: The following libraries were synthesized using Fmoc solid phase peptide synthesis on Tentagel resin bead from Rapp Polymere (Germany). Three different peptide bead libraries were used in the screening. They are named BL121 , BL122 and BL123.
• the format of the library BL121 is:
• the format of the library BL122 is
• Or0 2 -0 3 -0 - ⁇ 5-0 6 -0 7 -Os- ⁇ 9-O ⁇ o-O ⁇ -O ⁇ 2 -Tentagel resin, where On is a L-amino acid and n 1 -12 can be any proteinogenic L-amino acid except methionine and cysteine.
• the format of the library BL124 is:
• Example 6 The cells from mammalian cell culture was separated from the supernatant by centrifugation or filtration. Benzamidine and EDTA was added to final concentration 1 mM.
• Example 7 The cells from mammalian cell culture was separated from the supernatant by centrifugation or filtration. Benzamidine and EDTA was added to final concentration 1 mM.
• Example 9 Hydroxyapatite chromatography using CHT Hydroxyapatite Type I BioRad cat no 157-0020
• the pH of the pool of FXI polypeptide-containing fractions from Example 8 was adjusted to 6,0 and 1 volume water was added to a conductivity of below 20 mS/cm.
• a Hydroxyapatite Type I 20 ⁇ m matrix was equilibrated with 6 cv of buffer C, and then a load corresponding to 5 mg/ml gel was applied to the column.
• the column was then washed with 15 cv of buffer C (20mM K-P04 pH 6,0), and a buffer containing 95% buffer C and 5% elution buffer C (20mM K-P04 2M NaCI pH 6,0) was performed as a washing step.
• a gradient elution from 5%C to 100%C was performed and used to elute the FXI polypeptide in small fractions.
• the conductivity of the pool containing the FXI polypeptide fractions was about 60 mS/cm and the pH about 6,0.
• Analysis of FXI polypeptide-containing fractions was performed by HPLC (vide infra) using C4 Jupiter Phenomonex cat no OOG-4167-EO,
• HPLC Analysis Procedure High-Performance Liquid Chromatography (HPLC; referred to in Examples 7-9, above) was performed using C4 Jupiter Phenomonex cat no OOG-4167-EO, 4.6x250 mm and employing buffers as follows: Buffer l: 0,1 %TFA in H 2 O
• Buffer II 0,07%TFA in CH 3 CN Equilibration of the column was carried out using a mixture of 75% (v/v) Buffer I with 25% (v/v) Buffer II for 5 minutes (flow rate 1 ml/min.). Elution of the column took place using a gradient going from 75% Buffer I / 25% Buffer II to 39% Buffer I / 61 % Buffer II over a period of 18 minutes (flow rate 1 ml/min.). Regeneration of the column was performed by washing with 100% Buffer II for 2 minutes, (flow rate 0.5 ml/min.). The detection wavelength employed was 214 nm. Temperature was 50°C. Samples of from 2 to 50 ⁇ g were loaded onto the column.

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