CN1882357A - Therapeutic use of factor XI - Google Patents

Abstract

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 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.

Description

The treatment of factor XI, plasma thromboplastin antecedent is used

Invention field

The present invention relates to the application of people's factor XI, plasma thromboplastin antecedent in preventing and/or treating bleeding episodes, the method for purification of factor XI and factor XI, plasma thromboplastin antecedent polypeptide class from biofluid, and pharmaceutical preparation.

Background of invention

The serine protease that people's factor XI, plasma thromboplastin antecedent is formed for the identical subunit that has about 80kDa molecular weight separately by two.FXI as have～homodimer that the disulphide of 160KDa molecular weight connects circulates in blood plasma.By at Arg ₃₆₉With Ile ₃₇₀Between each monomer of cracking and activate FXI, form amino-terminal heavy chain of the 50kDa that connects by disulphide and carboxyl-terminal light chain of 35kDa.This protein (is made up of 15 exons and 14 introns the 23kb gene that is positioned on the 4th chromosome (4q35), coding is by 2, the mRNA that 097 nucleotide is formed, its encode successively 18 amino acid whose amino-terminal signal (leader region) peptide and be present in 607 aminoacid in each monomer of maturation protein.4 tandem repetitive sequences (Apple domain) of exon III-X coding and the analog structure territory homology of in human plasma PK, finding (58% homogeneity).The exon XI-XV typical trypsin-like catalyst structure domain of encoding, this domain obtains activation by at inner Arg 369-Ile 370 key places proenzyme being carried out the Proteolytic enzyme cutting, produces the heavy chain and light chain or the catalyst structure domain (238 aminoacid) that contain 4 Apple domains (369 aminoacid).

Starting a kind of mechanism solidify is to carry out the following step subsequently successively by in the damage location place contact tissue factor (TF) circulation: (i) blood plasma factor VII (FVII) combines with TF and Proteolytic enzyme changes into activated factor VII (FVIIa); (ii) factor X combines with the TF-FVIIa complex and Proteolytic enzyme changes into activated factor X (FXa); (iii) thrombinogen changes into thrombin by FXa generation Proteolytic enzyme; (iv) produce the complex of tissue factor approach restrainer (TFPI) and FXa, the TFPI:FXa complex combines with TF-FVIIa subsequently, and this makes the FXa activation of thrombin weaken and the thrombin flow of restricted passage TF approach generation.Thrombin at the relatively small amount that produces during this period causes FXI to activate into FXIa (it activates into FIXa with factors IX), and the further activation of activation of the factor V on the platelet surface and factor X.These results further promote to form the thrombin (so-called " thrombin suddenly increases ") of capacity and fibrinogen are changed into fibrin, make that thus the platelet thromboembolism at initial stage is stable and produce suitable hemostasis.

Dimerization FXI as with the non-covalent complex of cofactor high molecular weight kininogen (HK) in proenzyme in blood plasma, circulate, described cofactor high molecular weight kininogen (HK) promotes FXI and electronegative surface combination and correlates the activation that Protease F XIIa, FXIa and thrombin carry out by it.The binding site of HK and FXI comprises a plurality of Apple domains (A1, A2, A4), and wherein the A2 domain is most important.Verified, Zn is being arranged ²⁺Forming complex with HK under ion exists can promote FXI to combine with activatory platelet.Verified, the interaction on FXI and activated blood platelet surface is by the residue Ser in the A3 domain of FXI ₂₄₈-Val ₂₇₁Mediation; Residue Ser ₂₄₈, Arg ₂₅₀, Lys ₂₅₅, Phe ₂₆₀And Gln ₂₆₃Also relate to this interaction.The A3 domain of FXI also contains residue Thr ₂₄₉-Phe ₂₆₀Interior heparin binding site, and residue Lys ₂₅₂And Lys ₂₅₃Relate to hematoblastic and combining.Although FXI and the HK form with non-covalent complex in blood plasma circulates, and has confirmed HK and activated blood platelet surface combination, FXI does not obviously need combining of HK-FXI complex with the interaction of platelet surface.And seem that FXI dimer high-affinity, specificity site direct and on the activated blood platelet combine (about 1500 site/platelet; K _dBe about 10nM).The isolating reorganization A3 domain of FXI is with the site of the affinity identical with the FXI dimer in conjunction with equal number on the activated blood platelet.

Confirm that also activatory enzyme FXIa combines with high-affinity saturable site on the activated blood platelet that (Kd is about 800pM; 500 site/platelet), and can with to the activation of observed similar speed in solution FIX.Substrate FIX binding site among the FXI comprises the subdomain (Ala in the A2 domain ₁₃₄-Leu ₁₇₂) and the A3 domain in two subdomain (Ile ₁₈₄-Val ₁₉₂And Ser ₂₅₉-Ser ₂₆₅).With combining of platelet surface is compound-mediated by the glycoprotein 1b-V-IX that uses a polypeptide chain in the FXI dimer, thus another monomer is presented as the substrate binding site of FIX.Generation that might FIXa can make the catalytic FX activation of FIXa-be confined to also promote carry out the activatory platelet surface of thrombinogen by FXa.

Except that formation causes the membrane-binding complex of the local explosive generation of thrombin on the platelet surface, FXIa has also experienced the adjusting of various blood plasma and blood platelet albumen enzyme inhibitor, and the functional activity of described various blood plasma and blood platelet albumen enzyme inhibitor seems to depend on whether FXIa combines with platelet surface or whether it is free in solution.Therefore, verified a large amount of serpins comprise that α-1-protease inhibitor, Antithrombin III, C1 inhibitor, α-2-antiplasmin, plasminogen activator inhibitor 1 (PAI-1) and protein C inhibitor all can make the FXIa inactivation in the blood plasma compartment.Yet, in the environment of activated blood platelet, appear to have may FXIa physiology on maximally related inhibitor be that protease connects protein I I (PNII), found that its concentration in blood plasma is extremely low, but secretion from platelet α-granule (per 108 platelet discharge 1-1.5nM PNII), thereby the plasma concentration of prompting under normal physiological conditions is 3-5nM.PNII is effective inhibitor of FXIa, and the Ki value is 300-500pM, and it is significantly promoted having in the presence of the heparin.FXI and platelet surface are having HK and Zn ²⁺Ion exists or thrombinogen and Ca is arranged ²⁺Exist combination down to avoid inactivation, show the FXIa activity that produces on the platelet surface thrombosis place that is confined to stop blooding, and the site of PNII and other protease inhibitor adjusting FXIa appears in the solution because of PNII and α-1-protease inhibitor.Possible in addition situation is that the endotheliocyte that contains heparan sulfate glycosaminoglycans can promote the assembling of FXIa/PNII complex, strengthens the inhibition of FXIa on the endothelium thus.

Think also on the activated blood platelet surface that FXI participates in thrombin generation in that activable fibrinolysis inhibitor (TAFI) suppresses working in the fibrinolysis by thrombin, described inhibitor is removed in the plasminogen combination from fibrin and the carboxyl-terminal lysine residue that works in activating by the Proteolytic enzyme mode.Think that complete FXI feedback loop is to produce the capacity thrombin to be used for remarkable TAFI activation institute requisite.

It should be noted that, platelet and megalokaryocyte be second kind of form of synthetic FXI obviously, the FXI that its called after is platelet-derived (pd-FXI), it is exon V and be different from circulation form for want of, this exon be the coding second Apple domain two exons in first exon, and in vitro study has confirmed that the preferred substrate of platelet factor XIa may be plasma F XI, but not FIX.Have been found that platelet FXI (Mr 220KDa) combines with the platelet plasma membrane.Platelet contains 300 the pd-FXI molecule/cells of having an appointment.

The FXI defective is the autosomal recessive inheritance, AR syndrome that is characterised in that variable bleeding tendency.Although serious, this defective may be asymptomatic clinically, is subjected to up to the patient till the attack of surgery operating wound; Yet, in some cases, take place under the hemorrhage situation that may have nothing to do in seriousness with this defective.Optimal Control to the patient that has the FXI defective should be noted that the many features that comprise the FXI level.At first, importantly assessment exists the bleeding tendency and the other factors of the individuality of part defective whether to form remarkable influence.This class evaluation should comprise measures FVIIIC and Feng's von willebrand's factor level, bleeding time and platelet aggregation.FFP has been used for the treatment of first kind of known FXI defective case and before research and development FXI concentrate, has been main treatment means.The probability that the major defect of blood plasma is that required volume is big, anaphylaxis and infectious agent are propagated.In addition, it is quite variable to have reported in this product FXI content.That can buy at present has two kinds of FXI concentrate.Antithrombase (referring to 102iu/ml) and heparin (10u/ml) with high concentration are prepared from BioProducts Laboratory (BPL) FXI concentrate (England), and the latter is considered to resist any FXIa that retains.Second kind of FXI concentrate produced by Hemoleven (France), and this product is prepared with 3-5u/ml heparin, 2-3iu/ml antithrombase and C1 inhibitor.In addition, reported that the FFP of collecting is carried out pasteurization is possible, kept the active 75-95% of FXI.Generally treat patient with slight FXI defective with FFP.But, can treat patient with the FXI concentrate with serious FXI defective.

How because of relevant with surgical operation or big wound excessively hemorrhage and need patient's (comprising the patient that there is not congenital FXI defective in those) of blood transfusion can take place than without the successive hemorrhage more complication of those patients.Even need administration of human blood or blood products (such as: for example be used for the treatment of the platelet, leukocyte of blood coagulation defective, platelet-derived concentrate etc.) hemorrhage also can causing and disseminator's virus (hepatitis, HIV, parvovirus and other unknown at present virus) complication that risk is relevant of moderate.What need a large amount of blood transfusions extensive hemorrhagely can cause taking place the multiple organ failure, MOF, comprises lung and renal dysfunction.In case these severe complications have taken place in the experimenter, will start a series of cascade of events reactions, comprise many cytokines and inflammatory reaction, thereby make treatment extremely difficult or unsuccessful usually.Therefore, the main purpose in surgical operation and the treatment of main tissue injury is to avoid hemorrhage or it is minimized.Hemorrhage or it is minimized for fear of this class, importantly guarantee to form and be difficult for by the dissolved stable solid-state hemostasis thromboembolism of fibrinoclase.In addition, importantly guarantee to form fast and effectively this class thromboembolism or grumeleuse.

Disclosed the application for the treatment of in the bleeding episodes that is combined in of factor VIIa and FXI among the WO2003007983.

Therefore, there is demand in this area to the improved hemostatic treatment method that causes stablizing the quick controlled formation of fibrin grumeleuse.

Summary of the invention

The invention provides the method and composition that is used for the treatment of bleeding episodes.These methods are by carrying out the preparation that comprises factor XI, plasma thromboplastin antecedent (FXI) polypeptide that has this patient who needs to be effective to the consumption of this class treatment.Method of the present invention produces one or more among the following result in described patient: clotting time shortens; Promote hemostasis; EGCT increases; Clot strength increases; And/or total grumeleuse quality (overall clot quality, OCQ) increase.In certain embodiments, after giving the FXI polypeptide, described patient shows the effective FXI plasma concentration at least about 5nM, 10nM, 30nM, 60nM or 120nM.

In certain embodiments, the FXI polypeptide comprises that the sequence of SEQ ID NO:1 or its keep the active fragment of at least a FXI-associated biomolecule.In certain embodiments, the FXI polypeptide comprises the sequence of SEQ ID NO:2 or it keeps the active fragment of at least a FXI-associated biomolecule.In certain embodiments, the FXI polypeptide comprise SEQ ID NO:1 or SEQ ID NO:2 chemical modification derivant or contain SEQ ID NO:1 or the SEQ ID NO:2 variant that one or more aminoacid sequences change.In certain embodiments, the FXI polypeptide has the sequence of SEQ ID NO:1.In certain embodiments, the FXI polypeptide has the sequence of SEQ ID NO:2.

In certain embodiments, there is not congenital FXI defective in described patient.In certain embodiments, described hemorrhage be surgical operation, dental procedure, wound or hemodilution institute secondary.In certain embodiments, described patient suffers from acquired FXI defective.

The present invention also provides the method and composition that is used to prevent bleeding episodes.These methods are by carrying out with the preparation that the hemorrhage consumption of effective prevention comprises the FXI polypeptide patient that these needs are arranged.

In certain embodiments, before giving the FXI polypeptide, method of the present invention further comprises: (a) obtain blood sample from described patient; (b) measure at least a in the following parameters: the ratio of FXI concentration, FXIa: FXI or recover the amount of the necessary exogenous FXI of blood coagulation; (c) measure the amount of the described FXI that is effective to treat based on the result of step (b).

In one embodiment, the inventive method does not comprise and gives factor VII/ factor VIIa coagulant.

Factor VII/ factor VIIa coagulant used herein is factor VII polypeptides or the factor VII-related polypeptide described in WO2003007983.

The present invention also provides the method and composition that is used for the treatment of bleeding episodes, wherein the patient is given: (i) preparation that comprises the FXI polypeptide of first kind of consumption; The (ii) preparation that comprises non-factor VII/ factor VIIa coagulant of second kind of consumption, the condition that gives are that the combination of consumption in first kind and second can be effective to this class treatment.The limiting examples of non--factor VII/ factor VIIa coagulant comprises: factor XI, plasma thromboplastin antecedent I; Phospholipid; Factor XI, plasma thromboplastin antecedent II; Tissue factor approach restrainer (TFPI) inhibitor; Factors IX; The activable fibrinolysis inhibitor of thrombin (TAFI); Plasminogen activator inhibitor-1 (PAI-1); Factor V; Protein C inhibitor; The Protein S inhibitor; Tissue plasminogen activator (tPA) inhibitor; Thrombinogen; Factor IX; Plasminogen; With factor X.

The present invention also provides the pharmaceutical preparation that comprises (i) isolating reorganization FXI polypeptide and (ii) pharmaceutically acceptable carrier or excipient.

The present invention further provides the method that is used for from biomaterial purification of factor XI polypeptide, this method comprises makes described material carry out the sequence color spectrum on cation-exchange chromatography material, hydrophobic interaction chromatograph material and hydroxyapatite chromatography material.

The accompanying drawing summary

Accompanying drawing 1 is to increase the FXI of consumption to the graphic representation available from the influence of total grumeleuse quality in the blood of patient before and after the cardiac operation.

Accompanying drawing 2 is to increase the FXI of consumption to the graphic representation available from the influence of total grumeleuse quality in normal subjects's the blood.

Accompanying drawing 3 is 5 ℃ of bioactive graphic representations of storage different FXI preparations after 96 days down.

Accompanying drawing 4 is from using the preparative scale chromatography figure of the fraction that contains the factor XI, plasma thromboplastin antecedent polypeptide of the cation-exchange chromatography first of Obelix ST CIEX (catalog number (Cat.No.) 11-0010) as described in example 7 above.

Accompanying drawing 5 is from using the preparative scale chromatography figure of the fraction that contains the factor XI, plasma thromboplastin antecedent polypeptide of the hydrophobic interaction chromatograph of Butyl Sepharose HighPerformance High Substitution (catalog number (Cat.No.) 17-3100) as described in example 8 above.

Accompanying drawing 6 is from using the preparative scale chromatography figure of the fraction that contains the factor XI, plasma thromboplastin antecedent polypeptide of the hydroxyapatite chromatography of CHT hydroxyapatite I type BioRad (catalog number (Cat.No.) 157-0020) as described in example 9 above.

Detailed Description Of The Invention

The present invention is based on following unexpected discovery, namely the exogenous factor XI, plasma thromboplastin antecedent that gives (FXI) can be effective as the general styptic in the human blood and not need to give factor VII/ factor VIIa coagulating agent. The treatment application of FXI of the present invention can provide one or more among the following result: the clotting time shortens; Grumeleuse is more firm; And formed grumeleuse reduces to the resistance increase of fibrinolysis with hemorrhage relevant complication.

The invention provides the treatment of FXI in human patient use in useful method and composition, be used for the treatment of or prevent bleeding episodes; Be used for promoting hemostasis; For increasing EGCT; And/or for increasing clot strength. These methods are undertaken by the factor XI, plasma thromboplastin antecedent that described patient is given effective dose, in order to realize in these required therapeutic purposes one or more. Described composition comprises the pharmaceutical preparation that comprises FXI of using for the FXI therapeutic. In one embodiment, described composition comprises the pharmaceutical preparation of the FXI that comprises separation that uses for the FXI therapeutic. In one embodiment, described composition comprises the pharmaceutical preparation that comprises the FXI that recombinates of using for the FXI therapeutic. In one embodiment, described composition comprises the pharmaceutical preparation of the restructuring FXI that comprises separation that uses for the FXI therapeutic.

In a series of embodiments, the present invention relates to FXI to normal human patient's administration. " normally " used herein people is the people who does not have congenital factor XI, plasma thromboplastin antecedent defective (be congenital XI factor deficiency, referring to Seligsohn (1993), " Thrombosis and hemostasis " be 70:68-71 (Thromb.Haemost.)); Include, but are not limited to normal person described as follows: show thrombocytopenic patient (platelet count or activity decreased), prepare or carry out the patient of operation or dental operation operation and experienced wound or organ damage and therefore may show the low platelet counting and/or hang down the patient of fibrinogen, FVIII and/or other coagulated protein level. For example, normal person patient comprise because of hemorrhage, wound, chemotherapy, hepatopathy, hemodilution (such as: for example may be because keep due to blood volume or prevention shock infusion plasma extender or the salting liquid) or with birth defects in the FXI gene the temporarily-depressed patient of blood plasma level of FXI (or arbitrarily other with solidify relevant albumen or the factor) occurs without any other situation of direct correlation.

In another serial embodiment, the present invention relates to the people patient who suffers from congenital FXI defective is separated and/or the restructuring FXI.

In another serial embodiment, the present invention relates to the people patient who suffers from acquired FXI defective is separated and/or the restructuring FXI.

In implementing process of the present invention, can use and effectively to prevent or to treat hemorrhage any FXI polypeptide. It comprises and derives from blood or blood plasma or derive from blood platelet or FXI polypeptide class that those produce in the host organisms of any appropriate or cell by recombination form. Comprise that also the FXI polypeptide class of its not cracking form (proenzyme) and those have experienced the FXI polypeptide class (called after FXIa) that the proteolysis mode is processed into its corresponding biologically active form.

FXI polypeptide class used herein includes, but are not limited to FXI and FXI-related polypeptide class. Term " FXI " is in order to comprise, but be not limited to have as such as Fujikawa etc. at " biochemistry " (Biochem.) the polypeptide class of the amino acid sequence of wild type human plasma FXI described in the 25:2417 (1986) and the wild type FXI that derives from other kind, such as for example ox, pig, dog, mouse, rabbit and salmon FXI. In general, the FXI albumen of preferred use and experimenter's homology is in order to reduce the risk of induction of immunity reaction. For example, Gailani (1997) (Blood) has described the Preparation and characterization of non--people FXI among the 90:1055 at " blood ". The present invention also comprises the application of this class factor XI, plasma thromboplastin antecedent albumen in animal doctor's operation.

In certain embodiments, FXI polypeptide class is wild type human plasma FXI (SEQ ID NO:1). In other embodiments, FXI is platelet-derived FXI (pd-FXI) (SEQ ID NO:2), for example, such as (1998) such as Hsu in " biochemistry " (J.Biol.Chem.) described in the 273:13787-93.

FXI polypeptide class further comprises the natural allelic variation of the FXI that may exist, differ from one another between the Different Individual. In addition, the degree of glycosylation or other posttranslational modification and position can change in some cases, and this depends on the source of the nucleic acid of the FXI that encodes, the condition that produces the host cell of FXI and keep the cell that produces FXI.

FXI-related polypeptide class is including, but not limited to respect to chemical modification for the people FXI (being the FXI derivative) and/or contain the FXI polypeptide class that one or more amino acid sequences change (being the FXI variant) for people FXI. This class FXI-related polypeptide class for people FXI can bioactive one or more aspect in show change, the immunogenicity that the enzyme-specific of the stability that includes, but are not limited to change, the phospholipids incorporate of change, change is active, change, the bioavilability of change, change and combinations one or more FXI binding partners, change and the combination FXI inhibitor etc. FXI-related polypeptide class comprises that this class polypeptide class of its not cracking (proenzyme) form and those have been processed into the polypeptide class of its corresponding biologically active form by proteolysis, can be with their called afters " FXIa-related polypeptide class " or " the FXI-related polypeptide class of activation ".

The limiting examples of FXI derivant comprises: wild type FXI or by no matter being in vivo or external phosphorylation of carrying out, sulphation, PEGization or the FXI variant modified by the effect of one or more glycosyl transferases and/or glycosidase (for example, referring to (1999) such as Ekdahl " thrombosis and hemostasis " (Thromb.Haemost.) 82:1283-8).

The limiting examples of FXI variant comprises: the total adorned FXI in site of glycosylation that wherein one or more N-connect or that O-connects; Strand FXI (being that the monomer polypeptide does not carry out the FXI as Proteolytic enzyme cutting in the chain in the wild type); With following cysteine variant, wherein one or more cysteine residues are eliminated or reorientate, and include, but are not limited to change the change of monomer or dimeric disulfide bond syntype.In one embodiment, Cys ₁₁(it is considered to not participate in interchain or intramolecular disulfide bonding) is eliminated or replaces.

In a series of embodiments, the FXI variant decreased with the half-life that wild type people FXI compares in blood plasma.In one embodiment, the FXI variant has and is lower than 50 hours half-life.In one embodiment, the FXI variant has and is lower than 24 hours half-life.In one embodiment, the FXI variant has and is lower than 12 hours half-life.In one embodiment, the FXI variant has and is lower than 6 hours half-life.In one embodiment, the FXI variant has and is lower than 3 hours half-life.

In a series of embodiments, the FXI variant is such polypeptide, wherein the connection of the N-on one or more sites glycosylation is destroyed by the modification in the total site of the N-of associated connection glycosylation, such as, for example replace independently by arbitrary amino acid or any above-mentioned combination with N72, N108, N335, N432, N473.The limiting examples of this class variant comprises: 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.For example, can also destroy one or more locational N-by following manner and connect glycosylations: (i) the independently disappearance of residue (be one or more residues on each site can be lacked and not by other aminoacid replacement arbitrarily) arbitrarily among residue 72-74,108-110,335-337,432-434 and the 473-475; The (ii) independently replacement of N+2 residue (such as: the residue that for example T74 is replaced to any non-S; S110 is replaced to the residue of any non-T; S337 is replaced to the residue of any non-S; S434 is replaced to the residue of any non-T; T475 is replaced to the residue of any non-T; (iii) ((P) is the typical case with proline, but is not limited to it with destroying glycosylated aminoacid replacement N+1 residue.Be appreciated that combination in any in the aforesaid way can be used for destroying independently the glycosylation on the FXI polypeptide different loci.

The present invention also comprises the chimeric or fused polypeptide between all or part of FXI sequence and other heterologous peptides sequence.For example, one or more can (for example the replacement in 4 Apple domains by similar apple domain from other polypeptide class, referring to (Blood) 94:621a of (1999) " blood " such as Gailani), perhaps one or more can the disappearance in the Apple domain with its complete form.In another embodiment, with ldl receptor associated protein (LRP) (such as: for example, the verified interactional peptide that comprises residue Phe342-Asn346 among the factors IX a that provides with LRP, Rohlena etc. (2003) " journal of biological chemistry " are 278:9394 (J.Biol.Chem.)) binding site be connected to change its pharmacokinetic properties with the sequence of FXI polypeptide.

The dimerization characteristic (with two kind monomers asymmetric function in for example platelet combination and FIX activate) of FXI in its activity form can also be used in preparation of the present invention and comprise the FXI heterodimer, i.e. the combination of two kinds of FXI inequality (or FXI-is relevant) monomer polypeptide.Unique requirement is that described heterodimer shows the bioactive one or more useful aspects of FXI.

Being used for FXI polypeptide class of the present invention includes, but are not limited to show identical with wild type people FXI basically or the bioactive polypeptide class improved and FXI biological activity polypeptide class of change or reduction basically with respect to wild type people FXI is active for wherein.

In implementing process of the present invention, when wild type FXI equivalent be a kind of available from identical source or the FXI that in the same cell type, produces and when undertaken by parallel testing in identical FXI activity test specific activity than the time, the useful composition that comprises FXIa or FXIa-related polypeptide (comprising variant) comprises this based composition, they show the compositions that only comprises wild type FXI specific activity 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%.Term used herein " activity " and " specific activity " refer to bioactive any aspect of FXI or many aspects separately or with compound mode.

In certain embodiments, when testing in FXI amide hydrolysis (amidolytic) test, the ratio of the specific proteins hydrolysing activity of FXI-related polypeptide and the proteolytic activity of wild type people FXI is at least about 1.25; In other embodiments, this ratio is at least about 2.0; In other embodiments, this ratio is at least about being at least about 4.0.

The FXI biological activity

In implementing process of the present invention, for example, can be in following situation quantitatively and use FXI bioactive one or more different aspect: (i) be used for the treatment of administration suitable FXI compositions, preparation, be used for the selection of the method etc. of FXI production or purification; And/or; The (ii) effect evaluation of different therapeutic modalities.Be appreciated that the FXIa " activity specific (specific activity) " with any item in bioactive these aspects is expressed as the FXIa polypeptide class of active unit/unit mass.These aspects comprise following content:

I. proteolytic activity:

(a) can be as (1999) such as Ekdahl at " thrombosis and hemostasis " (Thromb.Haemost.) the suitable chromogenic substrate of use as described in the 82:1283-8, such as, for example S2355 (Chromogenix) carries out quantitatively amidohydrolase activity external.Activity of measuring and the standard FXIa preparation (Enzyme Research Laboratories) with definite specific activity are compared, and be the active AU value of FXIa numeric representation.

(b) for example, can, transform to the Proteolytic enzyme of IXa (Blood) described in the 97:3117-3122 at " blood ", the FXI activating activities directly be carried out quantitatively external as (2001) such as Gailani by measuring factors IX.

II. in conjunction with activity and inhibitor

Wild type people FXI has many binding partners, comprises the relevant FXI receptor of platelet of prekallikrein (PK), high molecular weight kininogen (HK), thrombin/thrombinogen, factors IX (FIX) and called after GP1b-V-IX.In implementing process of the present invention, can being used for FXI polypeptide class in conjunction with test the affinity of these (or other) binding partners is arbitrarily carried out quantitatively of any routine.This class includes, but are not limited to the CBA of the arbitrary binding partners of labelling in conjunction with test.

FXI polypeptide binding partners also comprises FXI avtive spot inhibitor, includes, but are not limited to Antithrombin III, C1 inhibitor, α 2 antitrypsins, PAI-1, protein C inhibitor and is connected protein I I (PNII) with protease.Can be by using the conventional affinity of these chemical compounds at FXI polypeptide class being carried out quantitatively in conjunction with test; Perhaps, can use the inhibition activity of this compounds of amide hydrolysis or FIX-activation test determination to the proteolytic activity of specific FXI polypeptide formulations.

III. solidify parameter:

Clotting time, EGCT and clot strength are to be used to detect the stop blooding clinical parameter of system mode of patient.After giving the FXI polypeptide, from the patient, take a blood sample and detect one or more in these parameters with appropriate intervals.Perhaps, FXI polypeptide or preparation can be used for the external/ex vivo treatment of the blood that extracts from the human body experimenter.

Can detect clotting time by standard P T or aPTT test.

For example, can pass through at " blood coagulation and fibrinolysis " (Blood coagulation ﹠amp as (2001) such as Vig; Fibrinolysis), Vol.12 (7) pp.555-561. and Sorensen (2003) measure EGCT and clot strength at the thromboelastography described in " thrombosis and hemostasis " (Throm Haemost) 1:551-558.Perhaps, can (Am.J.Med.Sci.) detect clot strength described in the 302:13-8 at (1991) " united states drug science magazine " as Carr etc.

A parameter of the coagulation activity of reflection as the FXI that measures by thromboelastography for " always solidifying quality " (OCQ).In case grumeleuse begins to form (t=0), then the clot strength measured value as time function will disclose the maximal rate (max vel) of grumeleuse formation and reach the required time (f of maximal rate _{Max vel}).Add tissue plasminogen activator (tPA) subsequently and reach the required time (t of fibrinolysis maximal rate so that measure fibrinolysis and derive _{Min vel}).Following calculating OCQ:

(Max vel/t _{max vel})×(t _{min vel}-t _{max vel}).

IV. pharmacokinetic parameter

Wild type people FXI is considered to have in blood plasma about 50 hours half-life, this to small part be because the interaction of it and HK mediates.In implementing process of the present invention, can use the FXI polypeptide class that shows the pharmacokinetic properties that is different from natural FXI.Limiting examples comprises: handle with sialidase and the FXI polypeptide class of removing one or more terminal sialic acid residueses from the bonded oligosaccharide apoplexy due to endogenous wind of FXI-; The FXI polypeptide class by the PEGization modification; With show change and interactional FXI polypeptide class HK.In implementing process of the present invention, can use, for example (CA USA) calculates pharmacokinetic properties to WinNonlin Professional Version 3.1 for Pharsight Inc., Mountain View.If there is more than one value, use the mean intensity value at each time point place to calculate so.

The pharmacokinetic parameter that can use following formula to be calculated as follows: AUC, AUC _%Extrap, C _Max, t _Max, λ _z, t _1/2, CL and V _z:

AUC is under from the time 0 to the plasma concentration-time graph the infinity

Area.Use linearity/log trapezoidal rule and be extrapolated to infinity

Row calculates.

At 0-t _MaxUse linear trapezoidal rule in time:

$\mathrm{AUC}(0-t_{\max })=(\underset{i=1}{\overset{x-1}{\mathrm{\Σ}}}\frac{C\left(i\right)+C(i+1)}{2}\·(t(i+1)-t\left(i\right)))$

t _MaxUse the log trapezoidal rule in the-final time point t:

$\mathrm{AUC}(t_{\max }-t)=(\underset{i-1}{\overset{N-1}{\mathrm{\Σ}}}\frac{C\left(i\right)-C(i+1)}{\ln \left(\frac{C\left(i\right)}{C(i+1)}\right)}\·(t(i+1)-t\left(i\right)))$

Use following formula extrapolation to infinitely great:

$\mathrm{AUC}(t-\∞)=\frac{C\left(t\right)}{{\mathrm{\λ}}_z}$

AUC _%ExtrapBecause of be extrapolated to the infinitely great AUC percentage ratio that produces from ultimate density:

${\mathrm{AUC}}_{\%\mathrm{Extrap}=}\frac{\mathrm{AUC}(t-\∞)}{\mathrm{AUC}}\·100\%$

C _MaxThe anti-maximal plasma concentration that is pushed into 0 o'clock time

The CL TBC

$\mathrm{CL}=\frac{\mathrm{Dose}}{\mathrm{AUC}}$

t _MaxObserve the time at maximal plasma concentration place.

t _1/2

Half-life: $t_{1/2}=\frac{\ln 2}{{\mathrm{\λ}}_z}$

λ _zTerminal speed constant.By to (on average) concentration and time relation

The log-linear regression calculate.

V _zDistribution volume based on latter stage:

$V_z=\frac{\mathrm{Dose}}{\mathrm{AUC}\·{\mathrm{\λ}}_z}$

The production of FXI and purification:

Can use the method for any appropriate as known in the art to be used for FXI polypeptide class of the present invention by blood plasma or by the recombinant sources preparation.Term used herein " isolating " refers to from the cell that is synthesized therein or isolating FXI polypeptide class from their culture medium of natural existence (for example blood plasma or blood).

Can include, but are not limited to from the attached cell culture, take out the cell culture medium that contains required product by any means well known in the art isolated polypeptide from its cell source; Centrifugal or filter to remove non-adherent cell etc.Alternatively, can be further purified FXI polypeptide class.Can use the various known any means in this area to carry out purification, comprise, but be not limited to: affinity chromatography, such as, for example on anti--FXI antibody column or peptide affinity chromatographic column (its limiting examples comprises heparin, Blue, Red, L-arginine, benzamidine peptide, other dyestuff or RP-chromatography); Hydrophobic interaction chromatography; Ion exchange chromatography; The size exclusion chromatography; Electrophoresis operation (for example preparation type isoelectrofocusing (IEF), difference dissolving (for example, using for example any precipitation or the crystallization of salt, pH, ammonium sulfate or other additive); Or extraction etc., more specifically describe as mentioned.Behind the purification, described preparation preferably contains and is lower than about 10% weight, more preferably less than about 5%, and most preferably is lower than the about 1% non--FXI polypeptide class that derives from host cell.

Can also be by known method purification FXI from blood plasma: comprise, but (Biochem.) 16:2279 and Bouma etc. (1977) are incorporated herein by reference these documents in the method that " journal of biological chemistry " (J.Biol.Chem.) discloses among the 252:6432 at " biochemistry " by (1977) such as Koide to be not limited to those.Being used to prepare the recombinate method of FXI is known in the art.For example, referring to (Gene) 139:275 of (1994) " genes " such as Kemball-Cook; Fujikawa etc. (1986) " biochemistry " are 25:2417 (Biochem.); With (Blood) 79:1435 of (1992) " blood " such as Meijers, the full content of these documents is incorporated herein by reference.FXIa is also available from Enzyme Research Laboratories, South Bend, IN.

The invention further relates to purification FXI polypeptide from other biomaterial, such as the method for reorganization FXI, this method comprises makes described material chromatograph on the cation-exchange chromatography material.

The invention further relates to purification FXI polypeptide from other biomaterial, such as the method for reorganization FXI, this method comprises makes described material in the enterprising circumstances in which people get things ready for a trip spectrum of hydrophobic interaction chromatograph material.

The invention further relates to purification FXI polypeptide from other biomaterial, such as the method for reorganization FXI, this method comprises makes described material in the enterprising circumstances in which people get things ready for a trip spectrum of hydroxyapatite chromatography material.

The invention further relates to purification FXI polypeptide from other biomaterial, such as the method for reorganization FXI, this method comprises makes described material carry out the sequence color spectrum on cation-exchange chromatography material, hydrophobic interaction chromatograph material and hydroxyapatite chromatography material.Should be appreciated that chromatograph is carried out according to described order in proper order.

Term used herein " hydroxyapatite chromatography material " refer to well known in the art can be in conjunction with any hydroxyapatite chromatography material of FXI polypeptide class, such as hydroxyapatite substrate.

Term used herein " cation-exchange chromatography material " refer to known in this field can be in conjunction with any cation-exchange chromatography material of FXI polypeptide class, such as cation exchange substrate.

Term used herein " hydrophobic interaction chromatograph material " refer to known in this field can be in conjunction with any hydrophobic interaction chromatograph material of FXI polypeptide class, such as hydrophobic interaction substrate.

The present invention relates to the method for purification FXI polypeptide from biomaterial in one embodiment, and this method comprises the following steps:

Make the biomaterial that comprises the FXI polypeptide first kind of enterprising circumstances in which people get things ready for a trip spectrum of cation-exchange chromatography material, described chromatography comprises:

(i) with described biomaterial application of sample to described first kind of cation-exchange chromatography material;

(ii) with buffer A unconjugated material of eluting from this first kind of cation-exchange chromatography material, this buffer A be suitable for eluting not with first kind of bonded material of cation-exchange chromatography material; With

(iii) by using buffer A ' the unconjugated material of eluting from first kind of cation-exchange chromatography material, this buffer A ' be suitable for eluting not with this first kind of bonded material of cation-exchange chromatography material; With

(iv) use buffer A " the described FXI polypeptide of eluting from first kind of cation-exchange chromatography material, this buffer A " be suitable for the described FXI polypeptide of eluting from described first kind of cation-exchange chromatography material.

The present invention relates to the method for purification FXI polypeptide from biomaterial in one embodiment, and this method comprises the following steps:

Make from step (iv) eluent or by using the chromatograph of using hydrophobic interaction chromatograph material from the fluid of step eluent preparation (iv), described chromatography comprises:

(v) will from step (iv) eluent or by using fluid application of sample from step eluent preparation (iv) to described hydrophobic interaction chromatograph material;

(vi) with buffer B unconjugated material of eluting from described hydrophobic interaction chromatograph material, this buffer B be suitable for eluting not with the bonded material of described hydrophobic interaction chromatograph material; With

(vii) use buffer B ', this buffer B ' be suitable for eluting FXI polypeptide from described hydrophobic interaction chromatograph material by gradient elution described FXI polypeptide of eluting from described hydrophobic interaction chromatograph material.

The present invention relates to the method for purification FXI polypeptide from biomaterial in one embodiment, and this method comprises the following steps:

Make from step (eluent vii) or by use from step (fluid of eluent preparation vii) uses the chromatograph of hydroxyapatite chromatography material, and described chromatography comprises:

(viii) will make from step (eluent vii) or by using (the fluid application of sample of eluent preparation vii) is to described hydroxyapatite chromatography material from step;

(ix) with buffer C unconjugated material of eluting from the hydroxyapatite chromatography material, this buffer C be suitable for eluting not with the bonded material of hydroxyapatite chromatography material; With

(x) use buffer C ' by gradient elution described FXI polypeptide of eluting from described hydroxyapatite chromatography material, this buffer C ' is suitable for eluting FXI polypeptide from described hydroxyapatite chromatography material.

The present invention relates to the method for purification FXI polypeptide from biomaterial in one embodiment, and this method comprises the following steps:

(a) biomaterial that comprises the FXI polypeptide is composed first kind of enterprising circumstances in which people get things ready for a trip of cation exchange material, described chromatography comprises:

(i) with described biomaterial application of sample to described first kind of cation-exchange chromatography material;

(ii) with buffer A unconjugated material of eluting from first kind of cation-exchange chromatography material, this buffer A be suitable for eluting not with first kind of bonded material of cation-exchange chromatography material; With

(iii) use buffer A ' the unconjugated material of eluting from first kind of cation-exchange chromatography material, this buffer A ' be suitable for eluting not with first kind of bonded material of cation-exchange chromatography material; With

(iv) use buffer A " the described FXI polypeptide of eluting from first kind of cation-exchange chromatography material, this buffer A " be suitable for the described FXI polypeptide of eluting from described first kind of cation-exchange chromatography material;

(b) make from step (iv) eluent or by using the chromatograph of using hydrophobic interaction chromatograph material from the fluid of step eluent preparation (iv), described chromatography comprises:

(v) will from step (iv) eluent or by using fluid application of sample from step eluent preparation (iv) to described hydrophobic interaction chromatograph material;

(vi) with buffer B unconjugated material of eluting from described hydrophobic interaction chromatograph material, this buffer B be suitable for eluting not with the bonded material of described hydrophobic interaction chromatograph material; With

(vii) use buffer B ', this buffer B ' be suitable for eluting FXI polypeptide from described hydrophobic interaction chromatograph material by gradient elution described FXI polypeptide of eluting from described hydrophobic interaction chromatograph material;

(c) make from step (eluent vii) or by use from step (fluid of eluent preparation vii) uses the chromatograph of hydroxyapatite chromatography material, and described chromatography comprises:

(viii) will from step (eluent vii) or by using (the fluid application of sample of eluent preparation vii) is to described hydroxyapatite chromatography material from step;

(ix) with buffer C unconjugated material of eluting from the hydroxyapatite chromatography material, this buffer C be suitable for eluting not with the bonded material of hydroxyapatite chromatography material; With

(x) use buffer C ' by gradient elution described FXI polypeptide of eluting from described hydroxyapatite chromatography material, this buffer C ' is suitable for eluting FXI polypeptide from described hydroxyapatite chromatography material.

Thereby the purification of FXI polypeptide is the process that the concentration of FXI polypeptide increases FXI polypeptide purity in this sample of raising for other composition in the sample.Should understand FXI polypeptide concentration with respect to other composition in the described sample in sample and not be equal to the concentration of FXI polypeptide in sample.Subsequently can be by using method well known in the art, for example by using SDS-PAGE (SDS-PAGE), HPLC (high performance liquid chromatography) or Berichrome algoscopy (Dade Behring Diagnostics) or clotting activity algoscopy to measure the increase of FXI polypeptide purity.

Biomaterial can be for deriving from or contain any materials of cell, cell component or cellular products.Biomaterial can be biofluid.

Biofluid can be for deriving from or contain any fluid of cell, cell component or cellular products.Biofluid comprises, but be not limited to cell culture, cell culture supernatant liquid, cell lysate, the cell lysate of purification, cell extract, tissue extract, blood, blood plasma, serum, they can also be homogenate and filtrate and for example by the biofluid of fractionated is not carried out the fraction that chromatograph is gathered.

FXI polypeptide class can purification from various biomaterials, the cell culture supernatant liquid that comprises natural generation FXI polypeptide, also comprise by the cell of genetic modification, such as the mammalian cell (for example Chinese hamster ovary celI) of the DNA conversion of using coding FXI polypeptide with generation FXI polypeptide.

Can handle biomaterial in many ways by before application of sample to the first kind of cation-exchange chromatography material, making.Centrifugal, filtration that these class methods include, but are not limited to.In one embodiment, described biomaterial is a biofluid.In one embodiment of the invention, described biofluid is the supernatant of cell lysate.In one embodiment of the invention, described biofluid is the supernatant of yeast cells lysate.

In one embodiment of the invention, as mentioned above, from cell culture, such as purification FXI polypeptide the mammalian cell cultures.Before the chromatograph in step (a), can from cell culture supernatant liquid, separate mammalian cell by centrifugal and/or filtration.Chromatographic step (a) is preceding carrying out, and can comprise inhibitor, such as EDTA (ethylenediaminetetraacetic acid) and benzamidine HCl.

Buffer is the solution that comprises following material, and the pH that this material can prevent to add the solution of small amount of acid or alkali takes place significantly to change, and keeps primary acidity of this solution or alkalescence thus basically.Buffer generally includes weak acid or weak base and its salt.

Before the chromatograph in carrying out step (a), for example by use 1M HCl or 1M NaOH or by the various known alternate manners in this area with the pH regulator of described biofluid pH to buffer A.

First kind of cation-exchange chromatography material can for known in this field can be at any cation-exchange chromatography material that discharges it under the set condition in conjunction with the FXI polypeptide and under on the same group condition not, such as the cation-exchange chromatography material that comprises sulfopropyl.Other limiting examples of cation-exchange chromatography material comprises deutero-glucosan, agarose, cellulose, polyacrylamide and private-use class silicate, such as carboxymethyl (carboxymetyl).Can identify suitable cation-exchange chromatography material through the following steps: make the biofluid that comprises the FXI polypeptide in the enterprising circumstances in which people get things ready for a trip spectrum of the cation-exchange chromatography material of selecting, collect fraction, and for example by using SDS-PAGE (SDS-PAGE), HPLC (high performance liquid chromatography), clotting activity or Berichrome algoscopy (Dade Behring Diagnostics) to measure the purity and the content of fraction, at the 280nm place, by using the absorbance of other method monitoring eluent well known in the art.The example of suitable cation-exchange chromatography material includes, but are not limited to Streamline SP XL (Amersham Biosciences cat no17-5073), Obelix ST CIEX (Amersham Biosciences catalog number (Cat.No.) 11-0010), Streamline Direct CST (Amersham Biosciences 17-5266), S-Support Unosphere, BioRad catalog number (Cat.No.) 156-0113 or Toyopearl SP-550CToso Haas catalog number (Cat.No.) 14028.In one embodiment, use Obelix ST CIEX.

Can be with first kind of cation-exchange chromatography material of buffer A pre-equilibration before described biomaterial application of sample.

Buffer A can comprise protease inhibitor, such as EDTA (ethylenediaminetetraacetic acid) and benzamidine HCl, but also can use other commercially available protease inhibitor.

In one embodiment of the invention, the pH of buffer A is at 6.5-9.In another embodiment, the pH of buffer A is at 7-9.In another embodiment, the pH of buffer A is about 8.

In one embodiment of the invention, the conductivity of buffer A is lower than about 40mS/cm.

Buffer A " be used to eluting FXI.In general, the concentration that is used for one or more compositions in the buffer (being buffer A and A ' in this case) that step (ii) washs increases in elution process or reduces, or joins new component in this buffer and the concentration of this composition increases or reduces.This increase or reduction can be recurred or take place in dispersive step, as known in the art.For eluting and the bonded material of cation-exchange chromatography material, usually with salt, for example NaCl joins in the buffer A.This concrete cationite can also be used as the hydrophobic interaction chromatography resin, with regard to this resinoid, usually propylene glycol/glycerol is joined and produces buffer A in the buffer A '.If NaCl and propylene glycol/glycerol are joined in the buffer A, so can eluting FXI.Determine which kind of fraction contains the FXI polypeptide so that merging is used for further processing, for example get rid of the unwanted impurity of eluting when FXI polypeptide eluting begins or finishes, belong to those skilled in the art's the ken.Equally, carrying out cation-exchange chromatography is well-known at for example general technology of aspects such as pre-equilibration, elution time, washing, the reconstruct of cation-exchange chromatography material.

Step (iv) in behind the eluting FXI polypeptide, add protease inhibitor to the eluate that contains the FXI polypeptide, such as EDTA (ethylenediaminetetraacetic acid) and benzamidine, carry out step (v) then.Eluent for example can also be remained on 4 ℃ of following 24 hours or longer times, or for example remain under-80 ℃.

Be used for step (b) hydrophobic interaction chromatograph material can for known in the art can be at any hydrophobic interaction chromatograph material that discharges it under the set condition in conjunction with the FXI polypeptide and under on the same group condition not, such as with phenyl, butyl or octyl group deutero-hydrophobic interaction chromatograph material or polyacrylic resin.The limiting examples of suitable hydrophobic interaction chromatograph material is Amberchrom ^TMCG 71 (Tosoh Bioscience), PhenylSepharose ^TMHigh Performance (Amersham, catalog number (Cat.No.) 17-1082), PhenylSepharose ^TM6 Fast Flow High Substitution (Amersham, catalog number (Cat.No.) 17-0973), Toyopearl  Butyl 650 (Tosoh Bioscience), Toyopearl  Phenyl (Tosoh Bioscience), Source ^TM15Phe (Amersham, catalog number (Cat.No.) 17-0147), Butyl Sepharose ^TMHigh Performance High Substitution (Amersham, catalog number (Cat.No.) 17-3100), Octyl-Sepharose ^TM(Amersham, catalog number (Cat.No.) 17-0946) and Phenyl Sepharose ^TMHigh Performance HighSubstitution (Amersham) etc.In one embodiment of the invention, described hydrophobic interaction chromatograph material uses butyl as part.

Can with buffer B and NaCl with about 1-2 volume or 2 above consumptions of volume join from step (iv) eluent or by using in the fluid from step eluent preparation (iv), after this carry out the chromatograph in the step (b); Maybe will comprise the buffer B same composition but concentration for for example 2 times conc forms with the consumption that is equivalent to concentrate buffer strength join from step (iv) eluent or by using in the fluid for preparing from step eluent (iv) (the buffer addition of 2 times of concentration is 1,5 volume).

Buffer B can have the pH of about 5-about 9, for example is about 8.In one embodiment of the invention, buffer B has the conductivity greater than 25mS/cm, for example greater than 70mS/cm.For example, can be by using phosphate buffer or passing through alternate manner well known in the art, for example NaCl reaches this purpose.In one embodiment of the invention, will from step (iv) eluent or be at least about 60mS/cm by using fluid from step eluent preparation (iv) to be adjusted to conductivity.

Buffer B ' be used for the FXI polypeptide being carried out eluting by gradient elution.In gradient elution, in elution process, change buffer B ' composition.In general, (vi) the concentration of one or more compositions in Xi Di the buffer (being buffer B in this case) increases in elution process or reduces to be used for step, or new component joined in this buffer, and the concentration of this composition increases in elution process subsequently.This increase or reduction can be recurred or take place in dispersive step, as known in the art.For eluting and the bonded material of hydrophobic interaction chromatograph material, the dilute with water lavation buffer solution goes out most of at least bonded FXI polypeptide up to eluting usually.Determine which kind of fraction contains the FXI polypeptide so that merge and be used for further processing, for example in case get rid of when FXI polypeptide eluting begins or finishes eluting do not need impurity, belong to those skilled in the art's the ken.Equally, carrying out the hydrophobic interaction chromatograph is well-known at for example general technology of aspects such as pre-equilibration, elution time, washing, the reconstruct of hydrophobic interaction chromatograph material.

In one embodiment of the invention, the method that comprises the following steps by use handle from step (eluent vii) or by use from step (fluid of eluent preparation vii):

(1) adds the stabilizing agent that one or more can increase FXI polypeptide stability can significantly improve its stable consumption effectively; And/or

This step and other processing back step as known in the art alternatively can be carried out separately or carry out with compound mode, and it is not crucial to carry out the order of described step.Those skilled in the art can determine how and when to carry out these steps.

In one embodiment of the invention, can increase the physics of FXI polypeptide and/or the stabilizing agent of chemical stability joins in the fraction that contains FXI.

" physical stability " of term FXI polypeptide used herein refer to because of this protein contact thermal-mechanical stress and/or with interface and surface (such as hydrophobic surface and interface) interaction of stabilization removal, that this protein forms non-activity biologically and/or protein insoluble aggregate or polymeric undertone.The physical stability of FXI polypeptide in being present in buffer A the time can be exposed to machinery/physical stress (for example stirring) visual inspection and/or the turbidimetric analysis turbidimetry value of different time after the time limit and estimate according to be filled in preparation in the suitable containers (for example cartridge case or bottle) under different temperatures.

The visual inspection of FXI polypeptide in being present in buffer the time can carry out in having the vernier focusing light of dark-background.The feature of the turbidity of compositions can be the vision scoring to turbidity deciding grade and level, for example the grade of 0-3 (it is 0 vision scoring that the compositions that does not show turbidity is equivalent to, and the vision turbidity that compositions shows in daylight is equivalent to 3 vision scoring).When compositions shows the vision turbidity in daylight, with regard to protein aggregation, said composition is categorized as physically unsettled.Perhaps, can pass through the well-known simple turbidimetric analysis turbidimetry of those skilled in the art, for example by measure the optical density (OD of solution at 405nm wavelength place ₄₀₅) estimate the turbidity of said composition.Can also estimate the physical stability of aqueous protein compositions by using reagents for spectrometry or protein conformation state probes.Described probe is preferably the micromolecule of this proteinic non-natural conformer of preferential combination.An example of the micromolecule spectral probe of protein structure is thioflavin T.Thioflavin T is a kind of fluorescent dye, has been widely used in to detect the amyloid fibril.Having fibril to exist and may also have in the presence of other protein configuration, thioflavin T has produced new excitation maximum at about 450nm place when combining with fibrillin matter form, and has produced enhanced emission at about 482nm place.Unconjugated thioflavin T is at these essentially no fluorescence in wavelength place.

Other micromolecule can be as protein structure from the natural probe that changes over the non-natural state.For example, " hydrophobicity sticking patch " probe of the hydrophobicity sticking patch (patch) of preferential conjugated protein exposure.These hydrophobicity sticking patch generally are embedded in the tertiary protein structure of native state, but expose when protein begins to separate folding or degeneration.The example of these micromolecule spectral probes is the aromatics hydrophobic dye, such as anthracene (antrhacene), acridine, phenanthroline etc.Other spectral probe is the metal-aminoacid complex, such as the cobalt metal composite of hydrophobic amino acid, described hydrophobic amino acid such as phenylalanine, leucine, isoleucine, methionine and valine etc.

" chemical stability " of term FXI polypeptide refers to when using in this article and causes forming the protein structure chemistry covalency of comparing the immunogenic chemical degradation product that has potential lower biological effect and/or may increase with the native protein structure and change.Can form various chemical degradation products, this type that depends on native protein and character and the environment that this protein contacted.Eliminate chemical degradation and under most of situation, can not avoid fully, and can in the storage of protein compositions and use, observe the increase of chemical degradation product amount, just as well known to the skilled person.Most of protein is easy to desamidization, and promptly the amide side chain base on glutamy amido or the asparaginyl group residue is hydrolyzed into free carboxy acid's process.Other degradation pathway comprises formation high molecular converted product, wherein two or more protein molecules are each other by transmidation and/or disulphide interaction covalent bond, cause forming covalently bound dimer, oligomer and polymer degradation products (" stability of pharmaceutical grade protein " (Stability of Protein Pharmaceuticals), Ahern.T.J.﹠amp; ManningM.C., Plenum Press, New York 1992).Oxidation (for example oxidation of methionine residues) is the another kind of version of chemical degradation.The FXI polypeptide is being present in buffer B ' in the time the chemical stability amount that can measure the chemical degradation product by the different time points place after contact varying environment condition estimate; For example, can form by elevated temperature accelerated degradation product usually.Usually by using various chromatographic techniques (for example SEC-HPLC and/or RP-HPLC) separation catabolite to measure each catabolite amount separately according to molecular size and/or electric charge.

Be present in buffer B ' in the time can significantly improve the physics of FXI polypeptide and/or any agent of chemical stability (for example by in time limit a period of time at OD ₄₀₅It is determined that the place measures turbidity) can use used as stabilizers.

For example, be suitable for to be salt (for example sodium chloride), sugar, pure (such as C with the reagent of used as stabilizers ₄-C ₈Alcohol), one or more mixture of alditol, aminoacid (for example glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), Polyethylene Glycol (for example PEG400) or its.Can use arbitrarily sugar, such as single-, two-or polysaccharide or water-soluble glucan.Alditol is structure HO CH ₂-[CH (OH)] _n-CH ₂The polyhydric alcohol of OH, wherein n is 1,2,3 .... etc.Limiting examples as the material of saccharide, alcohols or aldose alcohols is fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, glucosan, Pullulan, dextrin, cyclodextrin, soluble starch, hetastarch, carboxymethyl cellulose-Na, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, 1,2,3,4,5-pentanepentol, glycerol, the third-1,2-glycol (propylene glycol), the third-1,3-two pure and mild fourths-1, the 3-glycol.Can use above-mentioned saccharide, alcohols and aldose alcohols separately or with compound mode.Use amount there is not fixed restriction, as long as described material dissolves in liquid preparation and improves the physical stability of FXI polypeptide in solution.In this respect, can be with reference to Remington: " pharmacy science with put into practice " (The Science and Practice of Pharmacy), the 19th edition, 1995.

In one embodiment of the invention, one or more stabilizing agents that add the polyhydric alcohol type.

In one embodiment of the invention, adding is selected from glycerol (the third-1,2, the 3-triol), propylene glycol (the third-1, the 2-glycol), the third-1, one or more stabilizing agents in the group of 3-glycol, propanol (1-propanol) and isopropyl alcohol (2-propanol) composition.In one embodiment of the invention, adding is selected from glycerol, propylene glycol and the third-1, one or more stabilizing agents in the group of 3-glycol composition.

In another embodiment of the invention, when stabilizing agent was liquid alcohol or liquid polyol [such as glycerol, propylene glycol, the third-1,3-glycol, propanol or isopropyl alcohol], the concentration that exists of this stabilizing agent was about 5% volume (v/v)-Yue 50% (v/v).In another embodiment, the concentration that exists of the stabilizing agent of liquid alcohol or liquid polyhydric alcohols is about 10% (v/v)-Yue 50% (v/v).In another embodiment, the concentration that exists of the stabilizing agent of liquid alcohol or liquid polyhydric alcohols is about 10% (v/v)-Yue 20% (v/v).In another embodiment, the concentration that exists of the stabilizing agent of liquid alcohol or liquid polyhydric alcohols is about 10% (v/v).In another embodiment, the concentration that exists of the stabilizing agent of liquid alcohol or liquid polyhydric alcohols is about 20% (v/v).

As mentioned above, described stabilizing agent should be able to increase the physics and/or the chemical stability of FXI polypeptide.Can significantly improve the physics of FXI polypeptide and/or any agent of chemical stability (for example passes through in time limit a period of time at OD ₄₀₅It is determined that the place measures turbidity) can use used as stabilizers.

(eluent vii) can be used for pharmaceutical compositions from step.This process can comprise change buffer and/or adjustment conductivity and/or pH to biological value, and/or makes acceptable other effect of the use of eluate in mammal (such as the people); Making this class eluent is that this area is well-known to being used for the acceptable method of this mode.For example eluent can also be remained on 4 ℃ following more than 24 hours or 24 hours or for example remain under-80 ℃.

In one embodiment of the invention, this method further comprise make from step (eluent vii) or by use from step (fluid of eluent preparation vii) carries out stratographic step on the hydroxyapatite chromatography material, described chromatography comprises:

(viii) will from step (eluent vii) or by using (the fluid application of sample of eluent preparation vii) is to described hydroxyapatite chromatography material from step;

(ix) with buffer C unconjugated material of eluting from the hydroxyapatite chromatography material, this buffer C be suitable for eluting not with the bonded material of hydroxyapatite chromatography material; With

(x) with buffer C ' described FXI polypeptide of eluting from described hydroxyapatite chromatography material, this buffer C ' is suitable for being eluted in the step (ix) and the bonded described FXI polypeptide of described hydroxyapatite chromatography material.

For example, can before last sample, prepare by using from the step (fluid of eluent preparation vii).

In one embodiment of the invention, by add water will from step (eluent vii) or by using (the fluidic conductivity that eluent vii) prepares is adjusted to and is lower than about 20mS/cm from step.PH is adjusted to 5.8-9.In one embodiment, pH is adjusted to 6.0.

Can select the composition of buffer C and buffer C ' according to required final FXI polypeptide drug composition.This class considers to belong to those skilled in the art's the ken.

In one embodiment of the invention, as mentioned above, buffer C comprises one or more stabilizing agents, and these stabilizing agents can increase the physics and/or the chemical stability of FXI polypeptide.Can significantly improve the physics of FXI polypeptide and/or any agent of chemical stability in the time of in being present in buffer C (for example passes through in time limit a period of time at OD ₄₀₅It is determined that the place measures turbidity) can be as the stabilizing agent among buffer C or the buffer C '.

For example, be suitable for to be salt (for example sodium chloride), sugar, pure (such as C as the reagent of the stabilizing agent among the buffer C ₄-C ₈Alcohol), one or more mixture of alditol, aminoacid (for example glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), Polyethylene Glycol (for example PEG400) or its.Can use arbitrarily sugar, such as single-, two-or polysaccharide or water-soluble glucan.Limiting examples as the material of saccharide, alcohols or aldose alcohols is fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, glucosan, Pullulan, dextrin, cyclodextrin, soluble starch, hetastarch, carboxymethyl cellulose-Na, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, 1,2,3,4,5-pentanepentol, glycerol, the third-1,2-glycol (propylene glycol), the third-1,3-two pure and mild fourths-1, the 3-glycol.Can use above-mentioned saccharide, alcohols and aldose alcohols separately or with compound mode.Use amount there is not fixed restriction, as long as described material dissolves in liquid preparation and improves the physical stability of FXI polypeptide in solution.In this respect, can be with reference to Remington: " pharmacy science with put into practice " (The Science and Practice ofPharmacy), the 19th edition, 1995.

In one embodiment of the invention, buffer C comprises one or more stabilizing agents of polyhydric alcohol type.

In one embodiment of the invention, buffer C comprises NaCl.

To be selected from by glycerol (the third-1,2, the 3-triol), propylene glycol (the third-1, the 2-glycol), the third-1, one or more stabilizing agents in the group of 3-glycol, propanol (1-propanol) and isopropyl alcohol (2-propanol) composition join in the fluid from (x).

In one embodiment of the invention, adding is selected from glycerol, propylene glycol and the third-1, one or more stabilizing agents in the group of 3-glycol composition.In one embodiment of the invention, add propylene glycol.

In another embodiment of the invention, when stabilizing agent existed, the concentration that exists of this stabilizing agent was about 5% (v/v)-Yue 50% (v/v).In another embodiment, the concentration that exists of the stabilizing agent of liquid alcohol or liquid polyol type is about 10% (v/v)-Yue 50% (v/v).In another embodiment, the concentration that exists of the stabilizing agent of used liquid alcohol or liquid polyol type is about 10% (v/v)-Yue 20% (v/v).In another embodiment, the concentration that exists of the stabilizing agent of liquid alcohol or liquid polyol type is about 10% (v/v).

Buffer C ' is used for by gradient elution FXI polypeptide eluting, wherein changes the composition of buffer C ' in elution process.In general, the concentration that is used for one or more compositions in the buffer (being buffer C in this case) of step (ix) washing increases in elution process or reduces; Or new component joined in this buffer, and the concentration of this composition increases in elution process.This increase or reduction can be recurred or take place in dispersive step, as well-known in the art.For eluting and the bonded material of hydroxyapatite chromatography material, in buffer C, add salt usually, for example K-PO ₄Or NaCl, increase the concentration of this salt then, till eluting goes out most of at least bonded FXI polypeptide.Which kind of fraction that definite merging contains the FXI polypeptide is used for further processing, and for example the impurity that do not need of eluting belongs to those skilled in the art's the ken when FXI polypeptide eluting begins or finishes so that get rid of.Equally, carrying out the hydroxyapatite chromatography method is well-known in for example pre-equilibration, elution time, washing, cation in conjunction with the general technology of aspects such as chromatograph material reconstruct.

In a series of embodiments, in purification FXI used arbitrarily or use one or more stabilizing agents in all solution and can make physics and/or the chemical stability increase at least 10% of the physics of FXI and/or chemical stability than reference substance (promptly carry out same treatment but do not use the FXI of stabilizing agent), 25%, 50%, or 100%.In another serial embodiment, in purification FXI used arbitrarily or use one or more stabilizing agents in all solution and can make the physics of FXI and/or chemical stability 2-is doubly at least than the physics of reference substance (promptly carry out same treatment but do not use the FXI of stabilizing agent) and/or chemical stability increase, 5-doubly, 10-times, or 20-is doubly.

The activation of FXI

General passing through at Arg ₃₆₀With Ile ₃₇₀Between carry out Proteolytic enzyme cutting and activate wild type people FXI, this process can be by FXIa, FXIIa or catalyzed by thrombin.If desired, can use FXIa or FXIIa (all from Enzyme Research Laboratories, South Bend, IN) or thrombin (Sigma) activate being used for FXI of the present invention.For example, referring to (1999) " journal of biological chemistry " (J.Biol Chem) 51:36373-36373 and Baglia (2003) " journal of biological chemistry " (J.Biol Chem) 24:21744-21750 such as Sun.Use other protease activated FXI polypeptide, particularly FXI-related polypeptide also within the scope of the present invention.

The present invention includes the method and composition of the therapeutic administration that is used for FXI, they use the preparation with different FXI activation levels.In certain embodiments, described method and composition uses the FXI polypeptide class of not carrying out any activation act.In certain embodiments, the activation that the preparation of FXI or FXI-related polypeptide shows: the ratio (by mass) of proenzyme FXI or FXI-related polypeptide is about 1: about 99: 1 of 99-, such as, for example about 5: about 95: 5 of 95-; About 10: about 90: 10 of 90-; About 20: about 80: 20 of 80-; About 30: about 70: 30 of 70-; About 40: about 60: 40 of 60-; With about 50: 50.In certain embodiments, be benchmark with the mole, to compare with total FXI, described preparation contains and is not more than about 5% FXIa; More preferably no more than about 2.5%, even, most preferably be not more than about 0.5% or 0.1% more preferably no more than about 1%.In certain embodiments, be benchmark with the mole, described preparation contains the FXIa that is not more than about 0.01-0.05%.In certain embodiments, be benchmark with the mole, described preparation contains the FXIa that is not more than about 0.01-0.04%.In certain embodiments, be benchmark with the mole, described preparation contains the FXIa that is not more than about 0.01-0.03%.

The invention still further relates to FXI-related polypeptide class, they compare the different ability that is activated that shows with wild type FXI, such as, for example be easier to by FXIIa rather than by the FXI-related polypeptide class of activated by thrombin, vice versa; Even in the polypeptide class that does not have also to be activated in the presence of the Proteolytic enzyme cutting in the composing type mode; The heterodimer that wherein a kind of monomer (by sudden change or chemical modification) can not be activated by the Proteolytic enzyme mode etc.

In addition, the invention still further relates to the FXI-related polypeptide class of anti-autoactivation, promptly thrombin (and/or FXIIa) active rate and the ratio of FXIa active rate are higher than the variant of wild type FXI.

Method and composition of the present invention can also and/or promote activatory extra activating agent processing, pretreatment FXI polypeptide with inhibition, the FXI polypeptide is therewith stored or co-administered.The limiting examples that suppresses activatory activating agent comprises that C1 esterase inhibitor (C1Inb), α-2 antiplasmin (α 2AP), alpha1-antitrypsin (α 1AT), protease connect protein I I, benzamidine, heparin and Antithrombin III; Promote the limiting examples of activatory activating agent to comprise FXIa, FXIIa and thrombin.

Comprise the pharmaceutical preparation of FXI:

The present invention includes the pharmaceutical composition that is used to prevent and/or treat Sex therapy, they comprise the goods of FXI or FXI-related polypeptide.

Pharmaceutical composition of the present invention or preparation comprise the FXI polypeptide, and for example concentration preferably is dissolved in it pharmaceutically acceptable carrier at 0.001-100mg/ml, in preferred aqueous carrier or the diluent.Briefly, the goods by will comprising FXI and/or FXI-related polypeptide (preferred purified form) and suitable adjuvant and suitable carriers or mixing diluents prepare and are applicable to pharmaceutical composition of the present invention.Can use various aqueous carriers, such as water, buffered water, 0.4% saline, 0.3% glycine, saccharide, detergent, salt, buffer agent, glycerol, antiseptic, protease inhibitor, glycols etc.Can also use non-aqueous carrier to prepare preparation of the present invention, such as, for example be used to send or the gel form or the Liposomal formulation of targeting damage location.The Liposomal formulation general description is at for example U.S. Pat 4,837,028, US4,501,728 and US 4,975,282 in.Can be by conventional well-known sterilization technology to these compositions sterilizations.Can be with obtained aqueous solution packing so that use or it filtered and lyophilizing under aseptic condition, before administration with described lyophilized formulations and aseptic aqueous solution merging.

Described compositions can contain pharmaceutically acceptable auxiliary substance or adjuvant, includes, but are not limited to pH regulator agent and buffer agent, tension regulator, antiseptic, stabilizing agent, surfactant, chelating agen etc.Those skilled in the art can prepare compositions of the present invention in a suitable manner and according to acceptable practice, be described in " RemingtonShi pharmaceutical science " (Remington ' s Pharmaceutical Sciences) Gennaro such as those, ed., MackPublishing Co., Easton, PA, the method in 1990.

In one embodiment of the invention, the pharmaceutical composition that comprises FXI or FXI-related polypeptide goods further comprises pH regulator agent and buffer agent.In one embodiment of the invention, the pharmaceutical composition that comprises FXI or FXI-related polypeptide goods further comprises tension regulator.In one embodiment of the invention, the pharmaceutical composition that comprises FXI or FXI-related polypeptide goods further comprises antiseptic.In one embodiment of the invention, the pharmaceutical composition that comprises FXI or FXI-related polypeptide goods further comprises stabilizing agent.In one embodiment of the invention, the pharmaceutical composition that comprises FXI or FXI-related polypeptide goods further comprises surfactant.In one embodiment of the invention, the pharmaceutical composition that comprises FXI or FXI-related polypeptide goods further comprises chelating agen.

The limiting examples of suitable reducing comprises acetate buffer, carbonate buffer solution, citrate buffer, the glycylglycine buffer, histidine buffering liquid, glycine buffer, the lysine buffer, the arginine buffer, phosphate buffer (contains for example sodium dihydrogen phosphate, sodium hydrogen phosphate or tertiary sodium phosphate), TRIS[three (methylol) aminomethane] buffer, N, N-two (ethoxy) glycine (bicine) buffer, N-(methylol) methylglycine (tricine) buffer, the malate buffer, the succinate buffer, the maleate buffer, the fumarate buffer, tartrate buffer, aspartic acid salt buffer and composition thereof.

The limiting examples of pharmaceutically acceptable antiseptic comprise phenol, neighbour-cresol ,-cresol, p-Cresol, chlorocresol, methyl parahydroxybenzoate, aethyl parabenum, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, 2-phenyl phenol, 2-phenylethanol, benzylalcohol, methaform, thimerosal, bronopol, benzoic acid, miaow urea, chlorhexidine (chlorohexidine), sodium dehydroacetate, benzethonium chloride, chlorphenesin (3-is right-chlorophenoxy the third-1,2-glycol), benzamidine and composition thereof.In another embodiment of the invention, the concentration that exists of described antiseptic is 0.1mg/ml-20mg/ml.In another embodiment of the invention, the concentration that exists of described antiseptic is 0.1mg/ml-5mg/ml.In another embodiment of the invention, the concentration that exists of described antiseptic is 5mg/ml-10mg/ml.In another embodiment of the invention, the concentration that exists of described antiseptic is 10mg/ml-20mg/ml.The application of antiseptic in pharmaceutical composition is those skilled in the art's well-known (for example, referring to: " pharmacy science with put into practice " (The Science andPractice of Pharmacy), 19th edition, 1995).

The limiting examples of tension regulator (be introduced into usually so that make preparation is first-class substantially ooze) comprises that salt (for example sodium chloride), saccharide, alcohols are (such as C ₄-C ₈Alcohols), aldose alcohols, aminoacid (for example glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), polyethylene glycols (for example PEG400) and composition thereof.Can use arbitrarily sugar, such as single-, two-or polysaccharide or water-soluble glucan.Limiting examples as saccharide, alcohols or aldose alcohols material is fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, glucosan, Pullulan, dextrin, cyclodextrin, soluble starch, hetastarch, carboxymethyl cellulose-Na, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, 1,2,3,4,5-pentanepentol, glycerol, the third-1,2-glycol (propylene glycol), the third-1,3-two pure and mild fourths-1, the 3-glycol.Can use above-mentioned saccharide, alcohols and aldose alcohols separately or with compound mode.Use amount there is not fixed restriction, as long as described material dissolves in liquid preparation.

In one embodiment, the concentration that exists of described tension regulator is about the about 150mg/ml of 1mg/ml-.In another embodiment of the invention, the concentration that exists of described tension regulator is about the about 50mg/ml of 1mg/ml-.In one embodiment, described tension regulator is NaCl.In one embodiment, described tension regulator is the NaCl that has the about 150mg/ml of the about 1mg/ml-of concentration.In another embodiment of the invention, described tension regulator is the NaCl that has the about 50mg/ml of the about 1mg/ml-of concentration.

The limiting examples of chelating agen comprises salt of EDTA, citric acid and aspartic acid and composition thereof.In certain embodiments, the concentration that exists of chelating agen is 0.1mg/ml-5mg/ml; 0.1mg/ml-2mg/ml; Or 2mg/ml-5mg/ml.

Pharmaceutical composition of the present invention can be included in and may show polypeptide that aggregation forms in the composition of liquid medicine in the storage process as the therapeutic activity composition.Physics between the peptide molecule that term " aggregation formation " causes keeping the oligomer of solubility in order to expression or sedimentary big visible aggregation forms from solution interacts.Term " in the storage process " refers to composition of liquid medicine or preparation in case after the preparation, not with it immediately to experimenter's administration.But after preparation, with it with liquid form, freezing state or be dried forms (being dissolved into liquid form subsequently again) or be suitable for other packaged of experimenter's administration is stored.It [is lyophilizing that term " dried forms " refers to by lyophilization; For example, referring to Williams and Polli (1984) " non-intestinal science and technology magazine " (J.Parenteral Sci.Technol.) 38:48-59], by spray drying [referring to Masters (1991) " spray drying handbook( Spray-Drying Handbook) (5th ed; Longman Scientific and Technical, Essex, U.K.), pp.491-676; Broadhead etc. (1992) " medicament research and development of India's medicine " (Drug Devel.Ind.Pharm.) 18:1169-1206; With (Pharm.Res.) 11:12-20 of (1994) " drug researches " such as Mumenthaler] or by air-dry [Carpenter and Crowe (1988) " cryobiology " be 25:459-470 (Cryobiology); And (Biopharm.) 4:47-53 of Roser (1991) " bio-pharmaceutical "] exsiccant composition of liquid medicine of method or preparation.The aggregation of polypeptide forms and may produce harmful effect to the biological activity of this polypeptide in the storage process of composition of liquid medicine, thereby causes the therapeutic efficiency forfeiture of pharmaceutical composition.In addition, aggregation forms and can produce other problem, blocks such as the pipeline, film or the pump that take place when the use infusion system contains the pharmaceutical composition of polypeptide.

In one embodiment of the invention, pharmaceutical composition comprises the amino soda acid of the consumption that the aggregation that is enough to reduce polypeptide described in the said composition storage process forms.Term " amino soda acid " expression aminoacid or aminoacid combination, wherein any specified aminoacid exists with its free alkali form or its salt form.When using the aminoacid combination, all aminoacid all can exist with its free alkali form, also can exist with its salt form, or some exists with its free alkali form, and other exists with its salt form.In one embodiment, the aminoacid that is used to prepare the present composition carries the aminoacid of electrically charged side chain for those, such as arginine, lysine, aspartic acid or glutamic acid.Any stereoisomer of specific amino acids (for example glycine, methionine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine or its one or more mixture) or the combination of these stereoisomers may reside in the pharmaceutical composition of the present invention, as long as described specific amino acids exists with its free alkali form or its salt form.In one embodiment, use the L-stereoisomer.Can also use these amino acid whose analog to prepare compositions of the present invention.So-called " amino acid analogue " refers to naturally occurring amino acid whose derivant, and it can produce the required effect that the minimizing aggregation that polypeptide causes in composition of liquid medicine storage process of the present invention forms.Suitable arginine analog comprises: for example aminoguanidine, ornithine and N-one ethyl L-arginine, suitable methionine analog comprises ethionine and fourth methyllanthionine (buthionine), suitable cysteine analogs comprises S-methyl-L cysteine.As other aminoacid, amino acid analogue is mixed compositions with its free alkali form or its salt form.In the context of the invention, the chemical compound imidazoles can also be considered as amino acid analogue.In general, the working concentration of aminoacid or amino acid analogue is enough to prevent or delay protein aggregation.

In one embodiment, pharmaceutical preparation comprises methionine (or the aminoacid of another kind of sulfur-bearing or amino acid analogue), so that suppress the form that methionine residues is oxidized to its sulfoxide when the factor XI, plasma thromboplastin antecedent polypeptide is the polypeptide of the methionine residues that comprises that at least one is easy to take place this class oxidation.Term " inhibited oxidation " is minimized oxidized in time accumulating of kind (methionine) in order to expression.Suppressing methionine oxidation meeting causes polypeptide to keep manyly with its suitable molecular forms.Can use any stereoisomer (L, D or DL isomer) or its combination of methionine.Addition should be the amount that is enough to suppress the methionine residues oxidation, makes the amount of sulfoxide form of methionine can accept administrative organization.In general, this means that compositions contains the methionine sulfoxide form that is not more than about 10%-about 30%.This purpose generally can be by adding a certain amount of methionine, and make the ratio of the methionine that added and methionine residues about 1: about 1000: 1 of 1-, such as 10: 1-realized in about 100: 1.

The limiting examples of stabilizing agent comprises heavy polymer or low molecular weight compound, such as, for example: polyethylene glycols (for example PEG 3350); Polyvinyl alcohol (PVA); Polyvinylpyrrolidone; Carboxyl-/hydroxylated cellulose and derivant (comprising HPC, HPC-SL, HPC-L and HPMC) thereof; Cyclodextrin; The material of sulfur-bearing is as monothioglycerol, TGA and 2-methyl mercapto ethanol; Various salt (for example sodium chloride); Glycerol; Propylene glycol; The third-1, the 3-glycol; Propanol (1-propanol) and isopropyl alcohol (2-propanol).

The limiting examples of surfactant comprises: detergent; Ethoxylated castor oil; Polyglycolyzed glycerides; Acetylated monoglyceride; The sorbitan fatty ester class; (poloxamer class for example is such as Pluronic for polyoxypropylene-polyoxyethylene blocks polymer ^F68, poloxamer 188 and 407, Triton X-100); The polyoxyethylene sorbitan fatty acid esters class; Polyoxyethylene and polyoxyethylene deriv are such as alkylation and alkoxy derivative (" Tweens ", for example Tween-20, Tween-40, Tween-80 and Brij-35); Monoglyceride class and ethoxylated derivative thereof; Di-glycerides and polyoxyethylene deriv thereof; Alcohols; Glycerol; Lectin and phospholipid (Phosphatidylserine for example, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, phosphatidylinositols, cardiolipin and sphingomyelins), phospholipid derivative (for example two palmityls-phosphatidic acid) and lysophosphatide (palmityl hemolytic phosphatidyl-L-serine and ethanolamine for example, choline, the 1-acyl group of serine or threonine-sn-glycerol-3-phosphate esters), and the alkyl of LYSO-PHOSPHATIDYLCHOLINE LYSOPC and phosphatidylcholine-, alkoxyl-(Arrcostab) and alkoxyl-(alkyl ether) derivant, LYSO-PHOSPHATIDYLCHOLINE LYSOPC for example, the lauroyl of dipalmitoyl phosphatidyl choline and myristoyl derivant, and the trim of this polarity headgroup group, i.e. choline, ethanolamines, phosphatidic acid, serine, threonine, glycerol, inositol and positively charged DODAC, DOTMA, DCP, BISHOP, hemolytic phosphatidylserine and hemolytic phosphatidyl threonine and phosphoglyceride class (for example cephalin class); Glycerose lipid (for example galactopyranoside); Sphingoglycolipid class (for example ceramide type, gangliosides); Dodecylphosphoric acid gallbladder alkali; The egg LYSOLECITHIN SUNLECITHIN A; Fusidic acid derivatives (for example sodium taurodihydrofusidate etc.); Long-chain fat acids [C for example ₆-C ₁₂Fatty acid (such as oleic acid or sad)] and salt; Acylcarnitines and derivant thereof; The N of lysine, arginine and histidine ^α-acylated derivatives; Lysine and arginic side chain acylated derivatives; The N of dipeptides that comprises the combination in any of lysine, arginine and histidine and neutrality or acidic amino acid ^α-acylated derivatives; The N of tripeptides that comprises the combination in any of neutral amino acid and two charge residues ^α-acylated derivatives; DSS (docusate sodium, CAS registration number [577-11-7]); Calcium dioctyl sulfosuccinate, CAS registration number [128-49-4]); Docusate potassium, CAS registration number [7491-09-0]); SDS (sodium lauryl sulphate or sodium lauryl sulfate); Sodium caprylate; Cholic acid and derivant thereof; Bile acid and salt thereof; With glycine or taurine conjugate; Ursodesoxycholic acid; Sodium cholate; Sodium deoxycholate; Sodium taurocholate; NaGC; N-cetyl-N, N-dimethyl-3-ammonium-1-propane sulfonic acid salt; Anionic (alkyl-aryl-sulfonic acid salt) monovalence surfactant; Zwitterionic surfactant (N-alkyl-N for example, N-dimethylammonio-1-propane sulfonic acid salt; 3-gallbladder acylamino-(cholamido)-1-propyl-dimethyl ammonium-1-propane sulfonic acid salt); Cationic surface active agent (quaternary ammonium base; For example cetab, cetylpyridinium chloride); Nonionic surfactant (for example dodecyl β-D-pyranglucoside); And poloxamines (for example Tetronic ' s), promptly derive from expoxy propane and oxirane and ethylenediamine four functional blocks copolymers of addition successively; Or described surfactant can be selected from the group of imidazolidine derivatives or its mixture.In one embodiment, described pharmaceutical preparation comprises that concentration is about the surfactant of the about 50mg/ml of 0.01mg/ml-.In one embodiment, described pharmaceutical preparation comprises Tween-80.In one embodiment, described pharmaceutical preparation comprises poloxamer (poloxamer) 188.

In one embodiment, described pharmaceutical preparation comprises electrolyte.In one embodiment, described pharmaceutical preparation comprises electrolyte, such as NaCl.In one embodiment, described pharmaceutical preparation comprises electrolyte, such as KCl.

In one embodiment, when Tween 80 is used used as stabilizers, use such as the electrolyte of concentration, such as NaCl as 150mM.In one embodiment, avoided the gathering after the storage.

In a series of embodiments, the application that comprises one or more stabilizing agents in the pharmaceutical preparation of FXI or FXI-related polypeptide goods makes physics and/or the chemical stability increase at least 10% than reference substance (promptly carry out same treatment but do not have the FXI of stabilizing agent) of the physics of FXI and/or chemical stability, 25%, 50%, or 100%.In another serial embodiment, the application that comprises one or more stabilizing agents in the pharmaceutical preparation of FXI or FXI-related polypeptide goods makes the physics of FXI and/or chemical stability, and 2-is doubly at least than the physics of reference substance (promptly carry out same treatment but do not have the FXI of stabilizing agent) and/or chemical stability increase, 5-doubly, 10-times, or 20-is doubly.

The limiting examples of appropriate formulation is provided in the following table.When this class preparation contains 1.7mg/ml FXI, do not observe gathering after at least one month at ambient temperature.

ID	pH	Buffer agent	Isotonic agent	Stabilizing agent	Anti-microbial preservative
						A1	8.5	50mM three (methylol) aminomethane (TRIS), pH 8.5	na	na	na
A2	8.5	50mM three (methylol) aminomethane (TRIS), pH 8.5	150mM NaCl	na	na
						A3	8.5	50mM three (methylol) aminomethane (TRIS), pH 8.5	na	0.001％w/v Tween 80	na
A4	8.5	50mM three (methylol) aminomethane (TRIS), pH 8.5	na	0.1％w/v Tween 80	na
						A5	8.5	50mM three (methylol) aminomethane (TRIS), pH 8.5	150mM NaCl	0.1％Tween 80	na
A6	8.5	50mM three (methylol) aminomethane (TRIS), pH 8.5	16.0mg/ml glycerol	na	0.5w/v % phenol

A7	8.5	50mM three (methylol) aminomethane (TRIS), pH 8.5	na	The 5w/v% HP-	na
						A8	8.5	50mM three (methylol) aminomethane (TRIS), pH 8.5	na	The 0.1w/v% human serum albumin	na
A9	8.5	50mM three (methylol) aminomethane (TRIS), pH 8.5	na	0.5M sucrose	na
						A10	8.5	50mM three (methylol) aminomethane (TRIS), pH 8.5	na	0.3w/v% poloxamer 188	na
A11	8.5	50mM three (methylol) aminomethane (TRIS), pH 8.5	na	18.6mg/ml EDTA
						B1	8.0	50mM three (methylol) aminomethane (TRIS), pH 8.0	na	na	na
B2	8.0	50mM three (methylol) aminomethane (TRIS), pH 8.0	150mM NaCl	na	na
						B3	8.0	50mM three (methylol) aminomethane (TRIS), pH 8.0	na	0.001％w/v Tween 80	na
B4	8.0	50mM three (methylol) aminomethane (TRIS), pH 8.0	na	0.1％w/v Tween 80	na
						B5	8.0	50mM three (methylol) aminomethane (TRIS), pH 8.0	150mM NaCl	0.1％Tween 80	na
B6	8.0	50mM three (methylol) aminomethane (TRIS), pH 8.0	16.0mg/ml glycerol	na	0.5w/v % phenol
						B7	8.0	50mM three (methylol) aminomethane (TRIS), pH 8.0	na	5w/v% hydroxypropyl group-beta-cyclodextrin	na
B8	8.0	50mM three (methylol) aminomethane (TRIS), pH 8.0	na	The 0.1w/v% human serum albumin	na
						B9	8.0	50mM three (methylol) aminomethane (TRIS), pH 8.0	na	0.5M sucrose	na
B10	8.0	50mM three (methylol) aminomethane (TRIS), pH 8.0	na	0.3w/v% poloxamer 188	na
						B11	8.0	50mM three (methylol) aminomethane (TRIS), pH 8.0	na	18.6mg/ml EDTA
B12	6.1	Histidine 1.36mg/ml, pH 6.1	The 40mg/ml mannitol	0.3w/v% poloxamer 188	6mg/ml phenol
						C1	7.4	50mM phosphate, pH 7.4	na	na	na
C2	7.4	50mM phosphate, pH 7.4	150mM NaCl	na	na
						C3	7.4	50mM phosphate, pH 7.4	na	0.001％w/v Tween 80	na

C4	7.4	50mM phosphate, pH 7.4	na	0.1％w/v Tween 80	na
						C5	7.4	50mM phosphate, pH 7.4	150mM NaCl	0.1％Tween 80	na
C6	7.4	50mM phosphate, pH 7.4	16.0mg/ml glycerol	na	0.5w/v % phenol
						C7	7.4	50mM phosphate, pH 7.4	na	5w/v% hydroxypropyl group-beta-cyclodextrin	na
C8	7.4	50mM phosphate, pH 7.4	na	The 0.1w/v% human serum albumin	na
						C9	7.4	50mM phosphate, pH 7.4	na	0.5M sucrose	na
C10	7.4	50mM phosphate, pH 7.4	na	0.3w/v% poloxamer 188	na
						C11	7.4	50mM phosphate, pH 7.4	na	18.6mg/ml EDTA
C12	7.7	50mM phosphate, pH 7.7	The 14mg/ml propylene glycol	na	5.5 mg/ml phenol
						D1	6.0	The 50mM citrate, pH 6.0	na	na	na
D2	6.0	The 50mM citrate, pH 6.0	150mM NaCl	na	na
						D3	6.0	The 50mM citrate, pH 6.0	na	0.001％w/v Tween 80	na
D5	6.0	The 50mM citrate, pH 6.0	150mM NaCl	0.1％Tween 80	na
						D6	6.0	The 50mM citrate, pH 6.0	16.0mg/ml glycerol	na	0.5w/v % phenol
D7	6.0	The 50mM citrate, pH 6.0	na	The 5w/v% HP-	na
						D8	6.0	The 50mM citrate, pH 6.0	na	The 0.1w/v% human serum albumin	na
D9	6.0	The 50mM citrate, pH 6.0	na	0.5M sucrose	na
						D10	6.0	The 50mM citrate, pH 6.0	na	0.3w/v% poloxamer 188	na
D11	6.0	The 50mM citrate, pH 6.0	na	18.6mg/ml EDTA
						E2	10.0	The 50mM glycine, pH 10.0	150mM NaCl	na	na
E3	10.0	The 50mM glycine, pH 10.0	na	0.001％w/v Tween 80	na
						E4	10.0	The 50mM glycine, pH 10.0	na	0.1％w/v Tween 80	na

E5	10.0	The 50mM glycine, pH 10.0	150mM NaCl	0.1％Tween 80	na
						E6	10.0	The 50mM glycine, pH 10.0	16.0mg/ml glycerol	na	0.5w/v % phenol
E7	10.0	The 50mM glycine, pH 10.0	na	5w/v% hydroxypropyl group-beta-cyclodextrin	na
						E8	10.0	The 50mM glycine, pH 10.0	na	The 0.1w/v% human serum albumin	na
E9	10.0	The 50mM glycine, pH 10.0	na	0.5M sucrose	na
						E10	10.0	The 50mM glycine, pH 10.0	na	0.3w/v% poloxamer 188	na
F1	7.0	50mM phosphate, pH 7.0	na	na	na
						F2	7.0	50mM phosphate, pH 7.0	150mM NaCl	na	na
F3	7.0	50mM phosphate, pH 7.0	na	0.001％w/v Tween 80	na
						F5	7.0	50mM phosphate, pH 7.0	150mM NaCl	0.1％Tween 80	na
F6	7.0	50mM phosphate, pH 7.0	16.0mg/ml glycerol	na	0.5w/v % phenol
						F7	7.0	50mM phosphate, pH 7.0	na	5w/v% hydroxypropyl group-beta-cyclodextrin	na
F8	7.0	50mM phosphate, pH 7.0	na	The 0.1w/v% human serum albumin	na
						F9	7.0	50mM phosphate, pH 7.0	na	0.5M sucrose	na
F10	7.0	50mM phosphate, pH 7.0	na	0.3w/v% poloxamer 188	na
						G2	5.0	The 50mM citrate, pH 5.0	150mM NaCl	na	na
G5	5.0	The 50mM citrate, pH 5.0	150mM NaCl	0.1％Tween 80	na
						G6	5.0	The 50mM citrate, pH 5.0	16.0mg/ml glycerol	na	0.5w/v % phenol
G7	5.0	The 50mM citrate, pH 5.0	na	5w/v% hydroxypropyl group-beta-cyclodextrin	na
						G8	5.0	The 50mM citrate, pH 5.0	na	The 0.1w/v% human serum albumin	na
G9	5.0	The 50mM citrate, pH 5.0	na	0.5M sucrose	na
						G10	5.0	The 50mM citrate, pH 5.0	na	0.3w/v% poloxamer 188	na
G12	7.6	The 50mM glycylglycine, pH 7.6	na	0.05mg/ml Tween 20	0.21 mg/ml phenol

H2	3.0	The 50mM citrate, pH 3.0	150mM NaCl	na	na
						H4	3.0	The 50mM citrate, pH 3.0	na	0.1％w/v Tween 80	na
H5	3.0	The 50mM citrate, pH 3.0	150mM NaCl	0.1％Tween 80	na
						H6	3.0	The 50mM citrate, pH 3.0	16.0mg/ml glycerol	na	0.5w/v % phenol
H7	3.0	The 50mM citrate, pH 3.0	na	5w/v% hydroxypropyl group-beta-cyclodextrin	na
						H8	3.0	The 50mM citrate, pH 3.0	na	The 0.1w/v% human serum albumin	na
H9	3.0	The 50mM citrate, pH 3.0	na	0.5M sucrose	na
						H10	3.0	The 50mM citrate, pH 3.0	na	0.3w/v% poloxamer 188	na
H12	7.3	5.7mM phosphate, pH 7.3	137mM NaCl	5.4mM KCl

In non-limiting embodiments, the appropriate formulation that can reclaim active FXI after lyophilization contains:

FXI concentration: 0.2mg/ml

Buffer: 20mM buffer (histidine or TRIS) (pH 5.5,6.5 or 7.4), 25mg/ml mannitol (filler), 2.5mg/ml NaCl (filler) have 0.01%Tween 80

Preferably give described pharmaceutical composition, promptly by intravenous, subcutaneous or intramuscular by non-intestinal; Most preferably pass through intravenous administration.Can also by continuously or the infusion mode of beating with they administrations.Be understandable that and use any effective ways that give the FXI polypeptide, comprise, for example use mucosa or inhalation method.

For example, can be by spraying, perfusion, two balloon catheters, support, be incorporated in artificial blood vessel or the support, be used to apply balloon catheters hydrogel, mix the abundant method for building up local delivery of gauze or other bandage material or other preparation of the present invention, such as, for example local application.

Can give pharmaceutical composition of the present invention with various dosage forms, for example as solution, suspension, Emulsion, microemulsion, many Emulsions, foam, ointment, paste, plaster, ointment, tablet, coated tablet, irrigation, capsule (for example hard gelatin capsule or Perle), suppository, rectal capsule, drop, gel, spray, powder, aerosol, inhalant, eye drop, ophthalmic ointment, the eye irrigation, vaginal suppository, pessary, vagina ointment, injection solution, converted in-situ solution (in-situ gelling for example, original position is set (in situ setting), in-situ precipitate or in-situ crystallization), infusion solution or as implant.

Pharmaceutical composition of the present invention further can be sneaked into pharmaceutical carrier, drug delivery system or senior drug delivery system, or combine with them or put together (for example by covalency, hydrophobic or electrostatic interaction), so that further promote the stability of factor XI, plasma thromboplastin antecedent polypeptide, improve bioavailability, increase dissolubility, reduce untoward reaction, realize the well-known chronotherapy of those skilled in the art, and/or increase patient's compliance.Carrier, the example of drug delivery system and senior drug delivery system comprises, but be not limited to polymer, for example cellulose and derivant thereof, other polysaccharide (for example glucosan and derivant thereof, starch and derivant thereof), poly-(vinyl alcohol), acrylate and methacrylate polymers, polylactic acid and polyglycolic acid and block copolymer thereof, polyethylene glycols, carrier protein (for example albumin), gel (for example hot glue coagulates system, such as the well-known block copolymer of those skilled in the art system), micelle, liposome, microsphere, nanoparticle, liquid crystal and dispersion thereof, well-known L2 phase of technical staff and dispersion thereof in liquid-water system phase behaviour field, polymer micelle, multiple emulsion (from-emulsifying with from-microemulsified), cyclodextrin and derivant thereof and tree-shaped polymer (dendrimers).

The pharmaceutical composition that comprises the factor XI, plasma thromboplastin antecedent polypeptide of the application of the invention method preparation is applicable to and uses for example metered dose inhaler, powder inhaler or aerosol apparatus to carry out the preparation of solid, semisolid, powder and solution that pulmonary administration uses that all these are the well-known device of those skilled in the art.

The pharmaceutical composition that comprises the factor XI, plasma thromboplastin antecedent polypeptide of the application of the invention method preparation is applicable to controlled release, continue to discharge, prolong discharge, be obstructed discharge or the preparation of the delivery system of slow release in.For example, the pharmaceutical composition that comprises the factor XI, plasma thromboplastin antecedent polypeptide of the application of the invention method preparation can be used for the non-intestinal controlled release and the slow-released system (two kinds of systems all cause administration number of times to reduce at double) of the well-known type of those skilled in the art, such as controlled release that is used for subcutaneous administration and slow-released system.The useful controlled release system and the example of compositions are hydrogel, oleogel, liquid crystal, polymer micelle, microsphere and nanoparticle, but these not delimit the scope of the invention.

Production is used to comprise that the method for controlled release system of pharmaceutical composition of the factor XI, plasma thromboplastin antecedent polypeptide of the application of the invention method preparation includes, but are not limited to crystallization, condensation, cocrystallization, precipitation, co-precipitation, emulsifying, dispersion, high-pressure homogenization, encapsulation, spray drying, microencapsulation, condenses, is separated, solvent evaporation and produce microsphere, extrude and the supercritical fluid method.Generally referring to " Medicine Thing controlled release handbook" ( Handbook of Pharmaceutical Controlled Release) (Wise, D.L., ed., Marcel Dekker, New York, 2000) and " medicine and pharmacy science " ( Drugs and the Pharmaceutical Sciences) vol.99:ProteinFormulation and Delivery (MacNally, E.J., ed.Marcel Dekker, NewYork, 2000).

Can be by means of syringe, for example the syringe of pencil type apparatus form carries out parenterai administration by subcutaneous, intramuscular, intraperitoneal or intravenous injection.Perhaps, can carry out parenterai administration by infusion pump.The further administering selected of the solution that contains the factor XI, plasma thromboplastin antecedent polypeptide of the application of the invention method preparation or the compositions of suspension form is as nose or pulmonary's spray administration.Select as another kind, the pharmaceutical composition that contains the factor XI, plasma thromboplastin antecedent polypeptide of the application of the invention method preparation can be suitable for transdermal administration, for example by the needleless injection, by using patch (such as the iontophoresis patch) or by striding mucosa (for example sucking) administration.

In one embodiment of the invention, can stablize above useful life and the shelf life more than 3 years in 6 weeks by the pharmaceutical composition that comprises the factor XI, plasma thromboplastin antecedent polypeptide of the inventive method preparation.

In another embodiment of the invention, can stablize above useful life and the shelf life more than 3 years in 6 weeks by the pharmaceutical composition that comprises the factor XI, plasma thromboplastin antecedent polypeptide of the inventive method preparation.

In another embodiment of the invention, can stablize above useful life and the shelf life more than 2 years in 4 weeks by the pharmaceutical composition that comprises the factor XI, plasma thromboplastin antecedent polypeptide of the inventive method preparation.

In another embodiment of the invention, can stablize above useful life and the shelf life more than 2 years in 2 weeks by the pharmaceutical composition that comprises the factor XI, plasma thromboplastin antecedent polypeptide of the inventive method preparation.

In certain embodiments, the FXI polypeptide formulations has the pH of about 4.0-about 10.0.In certain embodiments, the FXI polypeptide formulations has the pH of about 4.0-about 8.0.In certain embodiments, the FXI polypeptide formulations has the pH of about 4.0-about 7.0.In certain embodiments, the FXI polypeptide formulations has the pH of about 4.0-about 6.5.In certain embodiments, the FXI polypeptide formulations has the pH of about 4.0-about 6.0.In certain embodiments, the FXI polypeptide formulations has about 6.5 or lower pH, such as, for example about pH5.0-6.5; All pH5.5-6.5 according to appointment.

The treatment administration of factor XI, plasma thromboplastin antecedent:

The invention provides and use the FXI prevention and treat hemorrhage method.

The hemorrhage blood that refers to overflows from any ingredient of blood circulation.Bleeding episodes comprises relevant unwanted, uncontrolled of the tissue injury with surgical operation, wound or other form and usually is over-drastic hemorrhage and have among the experimenter of bleeding disorder unwanted hemorrhage.Hemorrhagely can be used as spontaneous situation and take place, such as ICH (ICH).Bleeding episodes can have basically normal coagulation system, but the experimenter of experience (temporary) coagulopathy and suffer from congenital or acquired solidify or the experimenter of bleeding disorder in take place.In the experimenter who suffers from the platelet function defective, hemorrhage can compare for hemophilia cause hemorrhage, this is unusual must solidify " chemical compound " (for example platelet or Feng's von willebrand's factor albumen) because lack or exist as hemostasis system in hemophilia.Popularity is taking place, among the experimenter of for example relevant tissue injury with surgical operation or big wound, normal hemostatic mechanism may be suppressed by hemostatic requirement at once, although and hemostatic mechanism basically (wound before or surgical operation before) normal, massive hemorrhage still can take place in these experimenters.Usually need this class experimenter who further repeatedly transfuses blood (temporary) coagulopathy (promptly causing blood coagulating protein dilution, fibrinolysis to increase and the platelet counts minimizing because of hemorrhage and/or blood transfusion) to take place because of hemorrhage and/or blood transfusion.Hemorrhagely also may in organ, take place, such as brain, interior ear field and ophthalmic; These be exist surgical operation stop blooding limited probability, therefore obtaining in-problem zone aspect the gratifying hemostasis.

Similar problem also appears at from different organs (liver, lung, tumor tissues, gastrointestinal tract) and gets in the process of biopsy and laparoscopic surgery and radical retropubic prostatectomy.Commonly being difficult in all these situations provides hemostasis by surgical technic (stitching thread, suture clip etc.), also is like this when hemorrhage disperse (for example hemorrhagic gastritis and metrorrhagia).Hemorrhage can also occurring among the experimenter who implements anticoagulant therapy, in them, the therapy that gives has been brought out the hemostasis defective; These are hemorrhage normally acute and a large amount of.Usually give anticoagulant therapy in case the tampon embolism class diseases.This class therapy can comprise vitamin K-antagonist, blood coagulating protein inhibitor and aspirin and other anticoagulant of Dan Baijutang, warfarin or other form of heparin, other form, such as, for example active antibody of GP IIb/IIIa or other inhibitor.Hemorrhage can also be because of due to the so-called thrombolytic therapy, and this therapy comprises and antiplatelet drug (for example aspirin), anticoagulant (for example heparin) and fibrinolytic agent (tissue plasminogen activator for example, therapeutic alliance tPA).The implication of bleeding episodes is also including, but not limited to relevant with experimenter's Chinese and foreign department operation or wound uncontrolled and excessive hemorrhage, and described experimenter suffers from or has hemorrhage in acute hemarthrosis (intraarticular is hemorrhage), chronic hemophilic arthosis, hematoma (for example behind muscle, the peritoneum, after Sublingual and the pharynx), other tissue, hematuria (hemorrhage from the kidney road), cerebral hemorrhage, surgical operation (for example hepatectomy), exodontia and a gastrointestinal hemorrhage (for example UGI is hemorrhage).Bleeding episodes can be relevant with following situation: the inhibitor of anti-Factor IX; Haemophilia A; Use the haemophilia A of inhibitor; Haemophilia B; Factor VII defective; The factor XI, plasma thromboplastin antecedent defective; Thrombocytopenia; Feng's von willebrand's factor defective (Feng's von Willebrand's disease); Serious tissue injury; Serious wound; Surgical operation; Laparoscopic surgery; Acidosis, hemodilution, consumption coagulopathy, hyperfibrinolysis, hypothermia (hyopthermia), hemorrhagic gastritis; Get biopsy; Anticoagulant therapy; Upper gastrointestinal hemorrhage (UGI); Or stem cell transplantation.Bleeding episodes can be metrorrhagia; In using the limited organ of machinery hemostasis probability, take place; In brain, take place; In interior ear field, take place; Or take place within the eye.

Low counting of platelet or low activity refer to platelet (blood platelet) quantity that is present in experimenter's blood plasma and this class hematoblastic with solidify relevant biological activity.For example, low counting may be because of platelet destruction increases, platelet produces and reduces and due to platelet is concentrated more than normal part in spleen.For example, thrombocytopenia is defined as platelet count and is lower than 150,000 platelet/microlitres; The upper limit that it is generally acknowledged orthoplastocyte counting is 150,000-450,000 platelet/microlitre.Can measure platelet count by automatic platelet counter; This is the well-known methods of those skilled in the art.The syndrome that causes because of the low platelet counting includes, but are not limited to thrombocytopenia, coagulopathy.The aspect of biologically active pdgf includes, but are not limited to hematoblastic gathering, adhesion and CA.For example, active reduction may be that unusual because of glycoprotein, unusual film-cytoskeleton interacts, the granule of platelet is unusual, the platelet CA is unusual, due to signal transduction and the diacrisis.Can measure biologically active pdgf by well known to a person skilled in the art standard method, comprise gathering, adhesion and CA.For example, referring to " platelet-practical means " (Platelets.A Practical Approach), Ed.S.P.Watson ﹠amp; K.S.Authi: " clinicing aspects of blood platelet disorders) " (ClinicalAspects of Platelet Disorders) be 15:299-318 (K.J.Clemetson), and 1996, Oxford University Press; Williams Hematology, SixthEdition, Eds.Beutler, Lichtman, Coller, Kipps ﹠amp; Seligsohn, 2001, McGraw-Hill.The syndrome that causes because of the low platelet activity includes, but are not limited to the graceful Thromboasthenia of Glan thatch (Glanzmann thrombathenis), Bernard Soulier syndrome, storage poll disease, anticoagulant therapy and thrombolytic therapy.

In the context of the present invention, treatment comprises that prevention is hemorrhage, comprise, but what be not limited to prevent to estimate is hemorrhage, such as, for example estimate in operative process or take place subsequently hemorrhage, and regulate taken place hemorrhage, such as for example in wound, its purpose is to suppress hemorrhage or minimizes hemorrhage.Hemorrhage can be on the position of determining or can be on uncertain position.Therefore, the preparation that comprises the preventative FXI of comprising polypeptide in the treatment.

In certain embodiments, can be to normal person patient, promptly do not exist the people of FXI birth defect to give FXI and/or FXI-related polypeptide, dosage is equivalent to every day or the each about 500mg wild type of the about 0.05mg-of bleeding episodes FXI, for example for the experimenter of 70-kg, every day or each about 200mg of the about 1mg-of bleeding episodes, or the about 175mg of for example about 1mg-, as load and maintenance dose, this depends on the order of severity of experimenter's body weight, the state of an illness and the state of an illness.

In certain embodiments, from need be: (i) blood plasma level of FXI with extracting blood the patient of FXI polypeptide treatment and testing (before the administration of FXI polypeptide) to estimate one or more in the following parameters; (ii) activated form: the ratio of zymogen forms FXI; And/or (iii) for recovering effectively to solidify the concentration of the FXI that needs exogenous interpolation; Based on the result of this test, use predetermined scheme to give an amount of FXI polypeptide.The test of any appropriate can be used for these mensuration, comprising: ELISA or based on the method for gel for example.Use the appropriate calibration reference material so that will measure level and the common level (about 30nM) of FXI in human plasma compares.In general, it is desirable to the FXI level is supplemented at least about 5nM, all 10nM according to appointment, all 15nM according to appointment, all 20nM according to appointment are such as at least about 30nM FXI, such as 60nM, such as 120nM.

When the FXI-related polypeptide being used for replenish patient FXI activity, described FXI-related polypeptide shows at least a FXI biological activity of specified level, and the purpose of treatment is to provide the biological activity that is equivalent to scheduled volume wild type FXI (i.e. " effectively FXI plasma concentration ").

In certain embodiments, the present invention includes and give FXI polypeptide to patient treatment, this patient's FXI blood plasma level is lower than about 3nM; 5nM; Or 10nM.

Conjoint therapy

The present invention also comprises the method and composition that conjoint therapy is provided, wherein with FXI polypeptide and non-factor VII/ factor VIIa coagulant co-administered.Suitable non--factor VII/ factor VIIA coagulant includes, but are not limited to factor XI, plasma thromboplastin antecedent II (for example, referring to WO 01/85198); Tissue factor approach restrainer (TFPI inhibitor) (for example, referring to WO 01/85199); Factors IX (for example, referring to WO 02/062376); The activable fibrinolysis inhibitor of thrombin (TAFI) is (for example, referring to PCT/DK02/00734; PAI-1 is (for example, referring to PCT/DK02/00735; Factor V (for example, referring to PCT/DK02/00736); Protein C inhibitor (for example, referring to PCT/DK02/00737); Thrombomodulin (for example, referring to PCT/DK02/00738); Protein S inhibitor (for example, referring to PCT/DK02/00739); Tissue plasminogen activator's inhibitor (for example, referring to PCT/DK02/00740); α 2-antiplasmin (for example, referring to PCT/DK02/00741); Aprotinin (for example, referring to PCT/DK02/00742); Tranexamic acid (for example, referring to PCT/DK02/00751); Episilon amino caproic acid (for example, referring to PCT/DK02/00752); Thrombinogen, thrombin, factor VII, factor X and Fibrinogen.

Following is a series of embodiment of the present invention:

Embodiment 1: the method for treatment bleeding episodes, described method comprise the preparation that the patient that these needs are arranged is comprised factor XI, plasma thromboplastin antecedent (FXI) or FXI-related polypeptide with the consumption that is effective to this class treatment.

Embodiment 2: the method described in enforcement scheme 1, wherein said administration cause the clotting time among the described patient to shorten.

Embodiment 3: the method described in enforcement scheme 1 or embodiment 2, wherein said administration cause promoting the hemostasis among the described patient.

Embodiment 4: as implementing any described method among the scheme 1-3, wherein said administration causes the EGCT among the described patient to increase.

Embodiment 5: as implementing any described method among the scheme 1-4, wherein said administration causes the clot strength among the described patient to increase.

Embodiment 6: as implementing any described method among the scheme 1-5, wherein said administration causes the total grumeleuse quality (OCQ) among the described patient to increase.

Embodiment 7: as implementing any described method among the scheme 1-6, wherein after described administration, effective FXI plasma concentration that described patient shows is at least about 5nM.

Embodiment 8: the method described in enforcement scheme 7, wherein said effective FXI plasma concentration is at least about 10nM.

Embodiment 9: the method described in enforcement scheme 8, wherein said effective FXI plasma concentration is at least about 30nM, such as at least about 60nM, such as at least about 120nM.

Embodiment 10: as implementing among the scheme 1-9 a described method arbitrarily, wherein said FXI or FXI-related polypeptide comprise that the sequence of SEQ ID NO:1 or its keep the active fragment of at least a FXI-associated biomolecule.

Embodiment 11: as implementing among the scheme 1-9 a described method arbitrarily, wherein said FXI or FXI-related polypeptide comprise that the sequence of SEQ ID NO:2 or its keep the active fragment of at least a FXI-associated biomolecule.

Embodiment 12: as implementing any described method among the scheme 1-11, there is not congenital FXI defective in wherein said patient.

Embodiment 13: as implementing among the scheme 1-12 a described method arbitrarily, wherein said hemorrhage is situation institute secondary in being selected from the group of surgical operation, dental procedure, wound or hemodilution composition.

Embodiment 14: as implementing any described method among the scheme 1-13, taking a step forward in described administration comprises the following steps:

(a) from described patient, obtain blood sample; (b) measure at least a in the following parameters: the ratio of FXI concentration, FXIa: FXI or recover the amount of the necessary exogenous FXI of blood coagulation; (c) measure the amount of the described FXI that is effective to treat based on the result of step (b).

Embodiment 15: the method for treatment bleeding episodes, described method comprises described patient is given: (i) preparation that comprises the FXI polypeptide of first consumption; The (ii) preparation that comprises non-factor VII/ factor VIIa coagulant of second consumption, the combination of wherein said first and second consumptions can be effective to this class treatment.

Embodiment 16: the method described in enforcement scheme 15, wherein said non--factor VII/ factor VIIa coagulant is selected from the group of following composition: factor XI, plasma thromboplastin antecedent II; Tissue factor approach restrainer (TFPI) inhibitor; Factors IX; The activable fibrinolysis inhibitor of thrombin (TAFI); Plasminogen activator inhibitor-1 (PAI-1); Factor V; Protein C inhibitor; The Protein S inhibitor; And tissue plasminogen activator (tPA) inhibitor.

Embodiment 17: the method described in enforcement scheme 15 or embodiment 16, wherein said administration cause the clotting time among the described patient to shorten.

Embodiment 18: as implementing among the scheme 15-17 a described method arbitrarily, wherein said administration causes promoting the hemostasis among the described patient.

Embodiment 19: as implementing any described method among the scheme 15-18, wherein said administration causes the EGCT among the described patient to increase.

Embodiment 20: as implementing any described method among the scheme 15-19, wherein said administration causes the clot strength among the described patient to increase.

Embodiment 21: as implementing any described method among the scheme 15-20, wherein said administration causes the total grumeleuse quality (OCQ) among the described patient to increase.

Embodiment 22: as implementing any described method among the scheme 15-21, wherein after described administration, effective FXI plasma concentration that described patient shows is at least about 5nM.

Embodiment 23: the method described in enforcement scheme 22, wherein said effective FXI plasma concentration is at least about 10nM.

Embodiment 24: the method described in enforcement scheme 23, wherein said effective FXI plasma concentration is at least about 30nM, such as at least about 60nM, such as at least about 120nM.

Embodiment 25: as implementing among the scheme 15-14 a described method arbitrarily, wherein said FXI or FXI-related polypeptide comprise that the sequence of SEQ ID NO:1 or its keep the active fragment of at least a FXI-associated biomolecule.

Embodiment 26: as implementing among the scheme 15-24 a described method arbitrarily, wherein said FXI or FXI-related polypeptide comprise that the sequence of SEQ ID NO:2 or its keep the active fragment of at least a FXI-associated biomolecule.

Embodiment 27: as implementing any described method among the scheme 15-26, there is not congenital FXI defective in wherein said patient.

Embodiment 28: as implementing among the scheme 15-27 a described method arbitrarily, wherein said hemorrhage is situation institute secondary in being selected from the group of surgical operation, dental procedure, wound or hemodilution composition.

Embodiment 29: as implementing any described method among the scheme 15-28, taking a step forward in described administration comprises:

(a) from described patient, obtain blood sample; (b) measure at least a in the following parameters: the ratio of FXI concentration, FXIa: FXI or recover the amount of the necessary exogenous FXI of blood coagulation; (c) measure the amount of the described FXI that is effective to treat based on the result of step (b).

Embodiment 30: the method described in enforcement scheme 1, wherein said method do not comprise and give factor VII/ factor VIIa coagulant.

Embodiment 31: pharmaceutical preparation wherein comprises (i) isolating reorganization FXI polypeptide and (ii) pharmaceutically acceptable carrier or excipient.

The application of embodiment 32:FXI polypeptide in the treatment bleeding episodes.

Embodiment 33: the application of embodiment 32, wherein said bleeding episodes are to be selected from situation institute secondary in the group of surgical operation, dental procedure, wound or hemodilution composition.

Embodiment 34: the application of embodiment 32 or embodiment 33, wherein treat described bleeding episodes without factor VII/ factor VIIa coagulant.

Embodiment 35:FXI polypeptide promotes hemostatic to use in the patient of needs.

Embodiment 36:FXI polypeptide increases the application of EGCT in the patient of needs.

Embodiment 37:FXI polypeptide increases the application of clot strength in the patient of needs.

Embodiment 38:FXI polypeptide increases the application of total grumeleuse quality (OCQ) in the patient of needs.

Embodiment 39:FXI polypeptide shortens the application of clotting time in the patient of needs.

Embodiment 40: the application of item arbitrarily among the embodiment 32-39 wherein increases to the effective FXI plasma concentration among the patient and is at least about 5nM.

Embodiment 41: the application of embodiment 40 wherein increases to effective FXI plasma concentration and is at least about 10nM.

Embodiment 42: the application of embodiment 41 wherein increases to effective FXI plasma concentration and is at least about 30nM, such as at least about 60nM, such as at least about 120nM.

Embodiment 43: the application of item arbitrarily among the embodiment 32-42, wherein without factor VII/ factor VIIa coagulant treatment patient.

Embodiment 44:FXI polypeptide is used for the treatment of application in the pharmaceutical preparation of bleeding episodes in preparation.

Embodiment 45: the application of embodiment 44, wherein said bleeding episodes are to be selected from situation institute secondary in the group of surgical operation, dental procedure, wound or hemodilution composition.

Embodiment 46: the application of embodiment 44 or embodiment 45, wherein treat described bleeding episodes without factor VII/ factor VIIa coagulant.

Embodiment 47:FXI polypeptide is used for promoting having application in patient's hemostatic pharmaceutical preparation of these needs in preparation.

Embodiment 48:FXI polypeptide is used for increasing application in the pharmaceutical preparation that the patient of these needs EGCT is arranged in preparation.

Embodiment 49:FXI polypeptide is used for increasing the application that has in the patient of these needs pharmaceutical preparation of clot strength time in preparation.

Embodiment 50:FXI polypeptide is used for increasing application in the pharmaceutical preparation of the total grumeleuse quality (OCQ) of the patient that these needs are arranged in preparation.

Embodiment 51:FXI polypeptide is used for shortening application in the pharmaceutical preparation that the patient of these needs clotting time is arranged in preparation.

Embodiment 52: the application of item arbitrarily among the embodiment 47-51 wherein increases to the effective FXI plasma concentration among the patient and is at least about 5nM.

Embodiment 53: the application of embodiment 52 wherein increases to effective FXI plasma concentration and is at least about 10nM.

Embodiment 54: the application of embodiment 53 wherein increases to effective FXI plasma concentration and is at least about 30nM, such as at least about 60nM, such as at least about 120nM.

Embodiment 55: the application of item arbitrarily among the embodiment 44-54, wherein without factor VII/ factor VIIa coagulant treatment patient.

Embodiment 56: the application of item arbitrarily among the embodiment 32-55, wherein there is not congenital FXI defective in the patient who is treated.

Embodiment 57: any application among the embodiment 32-56, wherein said FXI polypeptide comprise that the sequence of SEQ ID NO:1 or its keep the active fragment of at least a FXI-associated biomolecule.

Embodiment 57: any application among the embodiment 32-56, wherein said FXI polypeptide comprise that the sequence of SEQ ID NO:2 or its keep the active fragment of at least a FXI-associated biomolecule.

Embodiment 58: the application of item arbitrarily among the embodiment 32-57, wherein with described FXI polypeptide and non-factor VII/ factor VIIa coagulant administering drug combinations.

Embodiment 59: the application of embodiment 58, wherein said non--factor VII/ factor VIIa coagulant is selected from the group of following composition: factor XI, plasma thromboplastin antecedent II; Tissue factor approach restrainer (TFPI) inhibitor; Factors IX; The activable fibrinolysis inhibitor of thrombin (TAFI); Plasminogen activator inhibitor-1 (PAI-1); Factor V; Protein C inhibitor; The Protein S inhibitor; And tissue plasminogen activator (tPA) inhibitor.

The method of embodiment 60. purification FXI polypeptide from biomaterial, this method comprise makes described material carry out the sequence color spectrum on cation-exchange chromatography material, hydrophobic interaction chromatograph material and hydroxyapatite chromatography material.

The method of embodiment 61. embodiments 60, wherein the FXI polypeptide is reorganization FXI.

The method of embodiment 62. embodiments 60 or embodiment 61, wherein FXI polypeptide behaviour FXI.

The method of embodiment 63. embodiments 60 or embodiment 61, wherein the FXI polypeptide is a dimer.

The method of embodiment 64. embodiments 63, the wherein dimer of FXI polypeptide behaviour subunit.

Any method among the embodiment 65. embodiment 60-64, wherein said biomaterial is a biofluid.

The method of embodiment 66. embodiments 65, wherein said biofluid are the mammalian cell supernatant.

The method of embodiment 67. embodiments 66, wherein said biofluid are the CHO culture supernatants.

Any method among the embodiment 68. embodiment 60-67, wherein this method comprises the following steps:

(a) biomaterial that comprises the FXI polypeptide is composed first kind of enterprising circumstances in which people get things ready for a trip of cation-exchange chromatography material, described chromatography comprises:

(i) with described biomaterial application of sample to described first kind of cation-exchange chromatography material;

(ii) use buffer A ' the unconjugated material of eluting from first kind of cation-exchange chromatography material, this buffer A be suitable for eluting not with first kind of bonded material of cation-exchange chromatography material; With

(iii) use buffer A ' the unconjugated material of eluting from first kind of cation-exchange chromatography material, this buffer A ' be suitable for eluting not with first kind of bonded material of cation-exchange chromatography material; With

(iv) by using buffer A " eluting described FXI polypeptide of eluting from first kind of cation-exchange chromatography material, this buffer A " be suitable for the described FXI polypeptide of eluting from described first kind of cation-exchange chromatography material;

(b) make from step (iv) eluent or by using the chromatograph of using hydrophobic interaction chromatograph material from the fluid of step eluent preparation (iv), described chromatography comprises:

(v) will from step (iv) eluent or by using fluid application of sample from step eluent preparation (iv) to described hydrophobic interaction chromatograph material;

(vi) with buffer B unconjugated material of eluting from described chromatograph material, this buffer B be suitable for eluting not with the bonded material of described hydrophobic interaction chromatograph material; With

(vii) by with buffer B ' gradient elution described FXI polypeptide of eluting from described chromatograph material, this buffer B ' be suitable for eluting FXI polypeptide from described hydrophobic interaction chromatograph material.

The method of embodiment 69. embodiments 68, wherein buffer A comprises one or more stabilizing agents that can increase FXI polypeptide stability.

The method of embodiment 70. embodiments 69, wherein buffer A comprises stabilizing agent, and this stabilizing agent is sugar, alcohol or alditol.

The method of embodiment 71. embodiments 70, wherein buffer A comprises stabilizing agent, and this stabilizing agent is sugar, C ₄-C ₈-alcohol or alditol.

The method of embodiment 72. embodiments 71, wherein buffer A comprises stabilizing agent, and this stabilizing agent is a polyhydric alcohol.

The method of embodiment 73. embodiments 72, wherein buffer A comprises stabilizing agent, and this stabilizing agent is selected from glycerol, propylene glycol, the third-1, the group that 3-glycol, propanol and isopropyl alcohol are formed.

The method of embodiment 74. embodiments 73, wherein buffer A comprises and is selected from glycerol, propylene glycol and the third-1, stabilizing agent in the group of 3-glycol composition.

Any method among the embodiment 75. embodiment 72-74, the concentration that exists of wherein said stabilizing agent is about 5% (v/v)-Yue 50% (v/v).

The method of embodiment 76. embodiments 75, the concentration that exists of wherein said stabilizing agent is about 10% (v/v)-Yue 50% (v/v).

The method of embodiment 77. embodiments 76, the concentration that exists of wherein said stabilizing agent is about 10% (v/v)-Yue 20% (v/v).

The method of embodiment 78. embodiments 77, the concentration that exists of wherein said stabilizing agent is about 10% (v/v).

The method of embodiment 79. embodiments 78, the concentration that exists of wherein said stabilizing agent is about 20% (v/v).

Any method among the embodiment 80. embodiment 68-79, wherein the pH of buffer A is about 6.5-about 9.

The method of embodiment 81. embodiments 80, wherein the pH of buffer A is about 7-about 9.

The method of embodiment 82. embodiments 81, wherein the pH of buffer A is about 8.

Any method among the embodiment 83. embodiment 68-82, wherein buffer A has the conductivity that is lower than about 50mS/cm.

Any method among the embodiment 84. embodiment 60-83, wherein hydrophobic interaction chromatograph material uses butyl or phenyl as part.

The method of embodiment 85. embodiments 84, wherein hydrophobic interaction chromatograph material are Phenyl Sepharose High Performance High Substitution.

The method of embodiment 86. embodiments 84, wherein hydrophobic interaction chromatograph material are Butyl Sepharose High Performance High Substitution.

Any method among the embodiment 87. embodiment 68-86, wherein the pH of buffer B is about 6-about 9.

The method of embodiment 88. embodiments 87, wherein the pH of buffer B is about 8.

The method of any Xu among the embodiment 89. embodiment 68-88, wherein buffer B has the conductivity greater than 50mS/cm.

Method in embodiment 90. embodiments 89, wherein buffer B has the conductivity greater than 70mS/cm.

Any method among the embodiment 91. embodiment 68-90, wherein the method that comprises the following steps by use handle from step (eluent vii) or by use from step (fluid of eluent preparation vii):

(1) adds the stabilizing agent that one or more can increase FXI polypeptide stability can significantly improve its stable consumption effectively; And/or

(2) will from step (eluent vii) or by using (the fluidic pH regulator of eluent preparation vii) is about 7-9 to pH from step.

The method of embodiment 92. embodiments 91, wherein the stabilizing agent that uses in the step (1) is sugar, alcohol or alditol.

The method of embodiment 93. embodiments 92, wherein the stabilizing agent that uses in the step (1) is sugar, C ₄-C ₈-alcohol or alditol.

The method of embodiment 94. embodiments 93, wherein the stabilizing agent that uses in the step (1) is polyhydric alcohol.

The method of embodiment 95. embodiments 94, wherein the stabilizing agent that uses in the step (1) is selected from glycerol, propylene glycol, the third-1, the group that 3-glycol, propanol and isopropyl alcohol are formed.

The method of embodiment 96. embodiments 95, wherein the stabilizing agent that uses in the step (1) is selected from glycerol, propylene glycol and the third-1, the group that the 3-glycol is formed.

Any method among the embodiment 97. embodiment 94-96, wherein the interpolation concentration of the stabilizing agent that uses in the step (1) is about 5% (v/v)-Yue 50% (v/v).

The method of embodiment 98. embodiments 97, wherein the interpolation concentration of the stabilizing agent that uses in the step (1) is about 10% (v/v)-Yue 50% (v/v).

The method of embodiment 99. embodiments 98, wherein the interpolation concentration of the stabilizing agent that uses in the step (1) is about 10% (v/v)-Yue 20% (v/v).

Any method among the embodiment 100. embodiment 60-99, wherein this method further comprises the following steps: to make from the eluent of hydrophobic interaction chromatography or by use and composes in the enterprising circumstances in which people get things ready for a trip of hydroxyapatite chromatography material from the material of the eluent preparation of hydrophobic interaction chromatography.

Any method among the embodiment 101. embodiment 68-100, wherein this method further comprises the following steps:

Make from step (eluent vii) or by use from step (fluid of eluent preparation vii) is in the enterprising circumstances in which people get things ready for a trip spectrum of hydroxyapatite chromatography material, and described chromatography comprises:

(viii) will from step (eluent vii) (dilute and regulated pH) or by using (the fluid application of sample of eluent preparation vii) is to described hydroxyapatite chromatography material from step;

(ix) with buffer C unconjugated material of eluting from the hydroxyapatite chromatography material, this buffer C be suitable for eluting not with the bonded material of hydroxyapatite chromatography material; With

(x) with buffer C ' eluting FXI polypeptide from described hydroxyapatite chromatography material, wherein buffer C ' is suitable for eluting and step (the bonded FXI polypeptide of the hydroxyapatite chromatography material class viii).

The method of embodiment 102. embodiments 101, wherein buffer C and/or buffer C ' comprise one or more stabilizing agents that can increase FXI polypeptide stability.

The method of embodiment 103. embodiments 101 wherein joins stabilizing agent in the fraction that contains FXI, and this stabilizing agent is sugar, alcohol or alditol.

The method of embodiment 104. embodiments 103 wherein adds stabilizing agent, and this stabilizing agent is sugar, C ₄-C ₈-alcohol or alditol.

The method of embodiment 105. embodiments 104 wherein adds stabilizing agent, and this stabilizing agent is a polyhydric alcohol.

The method of embodiment 106. embodiments 105 wherein adds stabilizing agent, and this stabilizing agent is selected from glycerol, propylene glycol, the third-1, the group that 3-glycol, propanol and isopropyl alcohol are formed.

The method of embodiment 107. embodiments 106 wherein adds stabilizing agent, and this stabilizing agent is selected from glycerol, propylene glycol and the third-1, the group that the 3-glycol is formed.

Any method among the embodiment 108. embodiment 105-107, the interpolation concentration of wherein said stabilizing agent is about 5% (v/v)-Yue 50% (v/v).

The method of embodiment 109. embodiments 108, the interpolation concentration of wherein said stabilizing agent are about 10% (v/v)-Yue 50% (v/v).

The method of embodiment 110. embodiments 109, the interpolation concentration of wherein said stabilizing agent are about 10% (v/v)-Yue 20% (v/v).

The method of embodiment 111. embodiments 110, the interpolation concentration of wherein said stabilizing agent is about 10% (v/v).

Any method among the embodiment 112. embodiment 101-111, wherein buffer C and/or buffer C ' have about 5, the pH of 8-about 7,8.

Any method among the embodiment 113. embodiment 101-112, wherein buffer C and/or buffer C ' have about 6,0 pH.

Embodiment 114. pharmaceutical compositions wherein comprise the FXI polypeptide by the method preparation of using any item among the embodiment 60-113.

Be non-limiting example of the present invention below.

Embodiment

Embodiment 1:FXI influences hemostatic among the cardiac:

Implement to get blood at perioperatively 5 patients of cardiac operation from using cardiopulmonary bypass.Use roTEG (rotation thromboelastography), (2001) such as application Vig are at " blood coagulation and fibrinolysis " (Blood Coagulation ﹠amp; Fibrinolysis) method among the 12:555 is estimated the influence of FXI to grumeleuse formation and stability.Briefly, by (HTI/Enzyme Research Laboratories Essen) exists and adds Innovin (final dilution factor: 1: 50,0000) (Dade Behring) and CaCl down being with or without FXI (2.5,10 or 25nM) ₂(final concentration: 15nM) start blood coagulation and start fibrinolysis by adding 4nM tPA (AmeericanDiagnostica).Use ROTEG-04 whole blood hemostasis system's rotation thrombus elasticity recording instrument (Whole Blood Haemostasis System RotationThrombelastography apparatus) (Pentapharm GmBH) to measure.Total grumeleuse quality (OCQ) is calculated as:

Max vel/t _{max vel})×(t _{min vel}-t _{max vel})

Then OCQ is calibrated control sample (being incubated in the presence of without any hemorrhage).

The result is as shown in accompanying drawing 1.FXI has significantly improved total grumeleuse and has formed, and is having the grumeleuse that forms in the presence of the FXI fibrinolysis to be had the resistance of increase.

Embodiment 2:FXI is to hemostatic effect in the normal blood

Blood is estimated the influence that FXI forms grumeleuse by ROTEG as described in example 1 above available from 4 normal subjectses.

Accompanying drawing 2 illustrates FXI and causes the dose dependent of OCQ in normal blood to increase.

Embodiment 3: the activity of the destructive FXI polypeptide of glycosylation

Use standard method to make up the FXI variant that contains following replacement, and dye the back at the HEK293 transit cell and express.From collecting the granular cell culture supernatants 96 hours the cell of growth down at 37 ℃.Measure the FXI activity by ROTEG as described in example 1 above.

The result is as shown in following table.

Protein	FXI activity in the % of predicted value
		NHP (human normal plasma) (31nM FXI)	106
FXI N72Q-1,2nM	42
		FXI N108Q-1,3nM	62
FXI N335Q-0,4nM	75
		FXI N432Q-1,2nM	33
FXI N473Q-0,6nM	83

The bin stability of embodiment 4:FXI preparation

Prepare the solution of following FXI and be stored in 5 ℃ of following 5 weeks, after this measure the FXI activity as described in example 1 above.

1.384nM FXI is at the 4mM acetate, 150mM NaCl is among the pH 5.4

2.190nM FXI is at the 50mM acetate buffer, 150mM NaCl is among the pH 5.4

3.190nM FXI is at the 50mM acetate buffer, 150mM NaCl, and pH 5.4, among the 1mM CaCl2

4.190nM FXI is at the 50mM acetate buffer, 75mM NaCl, and pH 5.4, in the 300mg/ml sucrose

5.190nM FXI is at 50mM MES buffer, pH 6.5, among the 150mM NaCl

6.190nM FXI is at 50mM MES buffer, pH 6.5, and 150mM NaCl is among the 1mMCaCl2

7.190nM FXI is at 50mM MES buffer, pH 6.5, and 75mM NaCl is in the 300mg/ml sucrose

The result is as shown in accompanying drawing 3.

The binding peptide class of embodiment 5:FXI

Carry out following experiment so that identify peptide class in conjunction with FXI.

I. peptide library is synthetic:

On Tentagel resin bead, use the Fmoc solid phase method of peptide synthesis to synthesize following library from Rapp Polymere (Germany).In screening, use three kinds of different peptide pearl libraries.With them called after BL121, BL122 and BL123.

The form in BL121 library is:

O ₁-O ₂-O ₃-O ₄-O ₅-O ₆-O ₇-O ₈-O ₉-O ₁₀-O ₁₁-O ₁₂-O ₁₃-O ₁₄-Tentagel resin, wherein On is a L-aminoacid, and n=1,2 and 11,12 can be the arbitrary protein matter source property L-aminoacid except that methionine and cysteine, and n=4,5 and 7,8 and 10,11 and 13,14 can be the arbitrary protein matter source property L-aminoacid except that methionine and cysteine and disappearance

And n=3,6,9,12 can be Phe, Trp, Tyr, Leu.

The form of library BL122 is:

O ₁-O ₂-O ₃-O ₄-O ₅-O ₆-O ₇-O ₈-O ₉-O ₁₀-O ₁₁-O ₁₂-Tentagel resin, wherein On is a L-aminoacid, and n=1-12 can be the arbitrary protein matter source property L-aminoacid except that methionine and cysteine.

The form of library BL124 is:

O ₁-O ₂-O ₃-O ₄-O ₅-O ₆-O ₇-Asp-Phe-Pro-O ₈-O ₉-O ₁₀-O ₁₁-Tentagel resin, wherein On is a L-aminoacid, and n=1-11 can be the arbitrary protein matter source property L-aminoacid except that methionine and cysteine.

2. screen peptide pearl library:

Be purchased recombinant factor XI and make its biotinylation from Heamatologic Technologies according to the standard laboratory scheme.Then with the 5ul factor XI, plasma thromboplastin antecedent (1,2uM) and 1ul streptavidin-alkali phosphatase (1mg/ml Sigma) joins respectively among three kinds of synthetic peptide pearl library BL121, BL122 and the BL124 and with its insulation about 2-3 hour.The insulation buffer is 15mMTRIS-HCl, pH=7,4,0,15M NaCl, 0,5% bovine serum albumin (BSA) and 0,05%Tween20.Using lavation buffer solution (M TRIS-HCl, pH=7,4,0,15M NaCl and 0 is 05%Tween20) after the washing, at colour developing buffer (50mM TRIS-HV1 pH=8,8,0,15M NaCl, 0,05%Tween20 and 15mM MgCl2) middle BCIP and the NBT of adding, and colour developing was carried out 30 minutes-1,5 hour.

3. sequencing

From the library, take out active blue beads and use Edman sequenator (Procise, AppliedBiosystems) order-checking.

The result:

Atopen XI that finds in library BL121 of the present invention, BL122 and BL123 and factor XI, plasma thromboplastin antecedent sample binding peptide comprise the peptide that contains following generalized aminoacid sequence:

The sequence of finding among the BL121

SEQ ID NO：03：SRWPWSVFPDFPD

SEQ ID NO：04：DVWDYVVFDDFPS

SEQ ID NO：05：QRWVPYDDFPSLRS

SEQ ID NO：06：RHFHVFPDFPFVH

SEQ ID NO：07：HHFPPFSHFPDLPQ

SEQ ID NO：08：RRLPLSRLPDFP

SEQ ID NO：09：HPFFRGYPDFPD

SEQ ID NO：10：HPWHLVYPDFPS

SEQ ID NO：11：HDWLVRWPDFPS

SEQ ID NO：12：SHFWRQWPDFSD

SEQ ID NO：13：PQLRWHDFPDFGS

SEQ ID NO：14：VVWRHWQDFDQFVV

SEQ ID NO：15：VDWQWSRFDDFPS

SEQ ID NO：16：HPWFDDFPHLFQ

From the sequence among the BL122 of library

SEQ ID NO：17：YKWIHHDDFPLV

SEQ ID NO：18：FDRKRVHPDFPH

SEQ ID NO：19：DVWDYVVFDDFPS

SEQ ID NO：20：QQPIQRFPDFP

SEQ ID NO：21：QAIFTRFPDFPN

SEQ ID NO：22：EWFPDFPEGSDG

SEQ ID NO：23：HTHAFPDFPPH

SEQ ID NO：24：LVKGFPDFPNHN

SEQ ID NO：25：GPFPYAYEDFPE

SEQ ID NO：26：FYLKTRYYDFPE

SEQ ID NO：27：FQARHTIGDFPA

SEQ ID NO：28：RIKDFPSDSNTV

SEQ ID NO：29：IWESHKVIEDFP

SEQ ID NO：30：QWFSVSRYQDFD

SEQ ID NO：31：QKDFHWRILPDF

SEQ ID NO：32：KIVKFPHTFPDL

SEQ ID NO：33：HLYDFDLDNEY

SEQ ID NO：34：KTILGDVDFDI

SEQ ID NO：35：RQLHPFHHFHG

SEQ ID NO：36：RSWLRYGYGH

SEQ ID NO：37：FNWNNVDEYYDW

SEQ ID NO：38：DQWDWEDYDEAW

SEQ ID NO：39：YDIYDDYEIWA

The sequence of finding among the BL124

SEQ ID NO：40：YPKHIYADFPSTRL

SEQ ID NO：41：YPRHIYPDFPTDTT

SEQ ID NO：42：YLKHAWPDFPKLQQ

SEQ ID NO：43：YVRHRFEDFPTALP

SEQ ID NO：44：FPWHKYEDFPSPRT

SEQ ID NO：45：QPAHRYPDFPRNNH

SEQ ID NO：46：LPKTRFLDFPHVSF

SEQ ID NO：47：LPPARYPDFPAAKK

SEQ ID NO：48：IPKNRFSDFPDAQG

SEQ ID NO：49：LPSFRFPDFPATKT

SEQ ID NO：50：RVLNRYPDFPTTNQ

SEQ ID NO：51：FFKKTYADFPTSQT

SEQ ID NO：52：IFKKTYEDFPRFVY

SEQ ID NO：53：VLHNKYDDFPRVKK

SEQ ID NO：54：KVKHRFNDFPVWGN

Infer that useful FXI-binding peptide comprises that those have the peptide of the core aminoacid primitive of Asp-Phe-Pro.

Embodiment 6

By centrifugal or filter the cell that from supernatant, separates from mammalian cell cultures.Add benzamidine and EDTA to final concentration be 1mM.

Embodiment 7

Use first kind of cation-exchange chromatography of Obelix ST CIEX (catalog number (Cat.No.) 11-0010)

With the buffer A balance Obelix substrate of 5 column volumes (cv), and to be equivalent to the applied sample amount upper prop of the every ml packed column of 150ml supernatant.With the buffer A (30mM Tris pH8,0) of 4cv, use the buffer A of 5cv then ' (50mM Tris 50% glycerol 87%pH 9,0) wash this post.Use the 5cv buffer A then " (50mM Tris 50% glycerol 87%1M NaClpH 9,0) carry out eluting.Flow velocity is 16cv/h, and temperature is 0-10 ℃.Make column regeneration with 1M NaOH.Peak height from about 50% is collected fraction, discard peak eluent first, and ensuing main peak contains FXI.Use C4 Jupiter Phenomonex catalog number (Cat.No.) OOG-4167-EO by HPLC (vide infra), 4.6 * 250mm and the SDS-PAGE (using the MOPS electrophoretic buffer, under reducing condition) that passes through on NUpage 4-12%Bis/TrisGel (Invitrogen) analyze the fraction that contains the FXI polypeptide.Benzamidine and EDTA joined (to 1mM) in the fraction that contains the FXI polypeptide and be kept in the cold closet under about 4 ℃ or be chilled under-80 ℃, till further use.

The another kind of alternative Obelix ST CIEX is chosen as Streamline DirectCST Amersham catalog number (Cat.No.) 17-5266-03.(accompanying drawing 4, preparative scale chromatography figure)

Embodiment 8

Use the hydrophobic interaction chromatograph of Butyl Sepharose High Performance High Substitution (catalog number (Cat.No.) 17-3100)

The buffer that contains 2M NaCl 40mM Tris pH 8 of 1,5 volume is joined in the merging fraction that contains the FXI polypeptide from embodiment 7, and, if pH as yet not 8,0-8, between 4, so with pH regulator to 8,2.With buffer B (1M NaCl20mM Tris pH 8,0) the balance Butyl Sepharose High Performance HighSubstitution substrate of 3cv, and will be equivalent to the applied sample amount upper prop of about 1mg/ml.Wash this post with the buffer B of 2cv then, carry out gradient elution then, go through 20cv and begin to 100% elution buffer B ' (20mM Tris pH 8,0), follow 100% elution buffer B ' by 2cv from buffer B.Flow velocity is 12cv/h, and temperature is 0-10 ℃.Behind the about 10cv of eluting, collect fraction, till 15cv.Use C4 JupiterPhenomonex catalog number (Cat.No.) OOG-4167-EO (4.6 * 250mm) and the fraction that contains the FXI polypeptide is analyzed by HPLC (vide infra) by go up SDS-PAGE (using the MOPS electrophoretic buffer, under reducing condition) at NUpage4-12%Bis/Tris Gel (Invitrogen).Immediately the propylene glycol of 1/4 volume is joined in the gleanings of the fraction that contains the FXI polypeptide to final concentration be 20% (v/v) propylene glycol, then gained is merged thing about 4 ℃ down or be refrigerated to further use.

The another kind of Butyl Sepharose High Performance High Substitution is chosen as Phenyl Sepharose High Performanc High Substitution.This alternative substrate causes back eluting.

(accompanying drawing 5, preparative scale chromatography figure)

Embodiment 9

Use the hydroxyapatite chromatography method of CHT I type hydroxyapatite BioRad catalog number (Cat.No.) 157-0020)

Will be from the pH regulator to 6 of the merging thing of the fraction that contains the FXI polypeptide of embodiment 8,0, and water to the conductivity that adds 1 volume is lower than 20mS/cm.With the buffer C balance I type hydroxyapatite 20 μ m substrate of 6cv, then to be equivalent to the applied sample amount upper prop of 5mg/ml gel.Use the buffer C (20mM K-PO4 pH 6,0) of 15cv to wash this post subsequently, and operate the buffer that contains 95% buffer C and 5% elution buffer C ' (20mM K-PO42M NaCl pH 6,0) as washing step.Carry out the gradient elution of 5%C '-100%C ', be used for little fraction eluting FXI polypeptide.The conductivity that contains the gleanings of FXI polypeptide fraction is about 60mS/cm, and pH is about 6,0.Use C4 JupiterPhenomonex catalog number (Cat.No.) OOG-4167-EO (4.6 * 250mm) and the fraction that contains the FXI polypeptide is analyzed by HPLC (vide infra) by the SDS-PAGE on NUpage4-12%Bis/Tris Gel (Invitrogen) (using the MOPS electrophoretic buffer, under reducing condition).HPLC purity＞97%, the concentration of FXI is about 1,2mg/ml.

Collection contains the high-purity fraction of FXI, and to add propylene glycol to final concentration be 10%v/v, is stored in below-20 ℃.(accompanying drawing 6, preparative scale chromatography figure)

Embodiment 10

HPLC analysis operation step

Use C4 Jupiter Phenomonex catalog number (Cat.No.) OOG-4167-EO (4.6 * 250mm) and use following buffer to carry out high performance liquid chromatography (HPLC; Above mention among the embodiment 7-9):

Buffer I: at H ₂Among the O 0,1%TFA

Buffer II: at CH ₃Among the CN 0,07%TFA

Use the mixture coupled columns of 75% (v/v) buffer I and 25% (v/v) buffer II to carry out balance 5 minutes (flow velocity 1ml/ minute).

Use 75% buffer I/25% buffer II-39% buffer I/61% buffer II coupled columns in 18 minute time limit to carry out eluting (flow velocity 1ml/ minute).

By washing 2 minutes with 100% buffer II, coupled columns is regenerated.(flow velocity 0.5ml/ minute).Used detection wavelength is 214nm.Temperature is 50 ℃.With 2-50 μ g sample upper prop.

The full content of all patents, patent application and the list of references that this paper is mentioned is incorporated herein by reference.

Many versions of the present invention are clearly to those skilled in the art under the instruction of foregoing detailed description.The conspicuous version of this class belongs in the scope of expecting fully of claims.

Sequence table

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＜120〉treatment of factor XI, plasma thromboplastin antecedent is used

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Claims (47)

1. treat the method for bleeding episodes, described method comprises the preparation that the patient that these needs are arranged is comprised factor XI, plasma thromboplastin antecedent (FXI) or FXI-related polypeptide with the consumption that is effective to this class treatment.

2. the method described in claim 1, wherein said administration cause clotting time to shorten in described patient.

3. the method described in claim 1, wherein said administration cause hemostatic to promote in described patient.

4. the method described in claim 1, wherein said administration cause EGCT to increase in described patient.

5. the method described in claim 1, wherein said administration cause clot strength to increase in described patient.

6. the method described in claim 1, wherein said administration cause total grumeleuse quality (OCQ) to increase in described patient.

7. the method described in claim 1, wherein after described administration, described patient shows the effective FXI plasma concentration at least about 5nM.

8. the method described in claim 7, wherein said effective FXI plasma concentration is at least about 10nM.

9. the method described in claim 8, wherein said effective FXI plasma concentration is at least about 30nM.

10. the method described in claim 1, wherein said FXI or FXI-related polypeptide comprise the sequence of SEQ ID NO:1 or it keeps the active fragment of at least a FXI-associated biomolecule.

11. the method described in claim 1, wherein said FXI or FXI-related polypeptide comprise the sequence of SEQ ID NO:2 or it keeps the active fragment of at least a FXI-associated biomolecule.

12. the method described in claim 1, there is not congenital FXI defective in wherein said patient.

13. the method described in claim 1, wherein said bleeding episodes are to be selected from situation institute secondary in the group of surgical operation, dental procedure, wound or hemodilution composition.

14. the method described in claim 1, taking a step forward in described administration comprises:

(a) from described patient, obtain blood sample; (b) measure at least a in the following parameters: the ratio of FXI concentration, FXIa: FXI or recover the amount of the necessary exogenous FXI of blood coagulation; (c) measure the amount of the described FXI that is effective to treat based on the result of step (b).

15. the method for treatment bleeding episodes, described method comprise described patient is given: (i) preparation that comprises the FXI polypeptide of first consumption; The (ii) preparation that comprises non-factor VII/ factor VIIa coagulant of second consumption, the combination of wherein said first and second consumptions can be effective to this class treatment.

16. the method described in claim 15, wherein said non--factor VII/ factor VIIa coagulant is selected from the group of following composition: factor XI, plasma thromboplastin antecedent II; Tissue factor approach restrainer (TFPI) inhibitor; Factors IX; The activable fibrinolysis inhibitor of thrombin (TAFI); Plasminogen activator inhibitor-1 (PAI-1); Factor V; Protein C inhibitor; The Protein S inhibitor; And tissue plasminogen activator (tPA) inhibitor.

17. not comprising, the method described in claim 1, wherein said method do not give factor VII/ factor VIIa coagulant.

18. pharmaceutical preparation wherein comprises: (i) isolating reorganization FXI polypeptide; (ii) pharmaceutically acceptable carrier or excipient.

19.FXI the application of polypeptide in the treatment bleeding episodes.

20. the application of claim 19, wherein said bleeding episodes are to be selected from situation institute secondary in the group of surgical operation, dental procedure, wound or hemodilution composition.

21. the application of claim 19 or claim 20 is wherein treated described bleeding episodes without factor VII/ factor VIIa coagulant.

22.FXI polypeptide promotes hemostatic to use in the patient who needs is arranged.

23.FXI polypeptide increases the application of EGCT in the patient who needs is arranged.

24.FXI polypeptide increases the application of clot strength in the patient who needs is arranged.

25.FXI polypeptide increases the application of total grumeleuse quality (OCQ) in the patient who needs is arranged.

26.FXI polypeptide shortens the application of clotting time in the patient who needs is arranged.

27. the application of item arbitrarily wherein increases to the effective FXI plasma concentration among the patient and is at least about 5nM among the claim 19-25.

28. the application of claim 26 wherein increases to effective FXI plasma concentration and is at least about 10nM.

29. the application of claim 27 wherein increases to effective FXI plasma concentration and is at least about 30nM.

30. the application of item is arbitrarily wherein treated described patient to be treated without factor VII/ factor VIIa coagulant among the claim 19-28.

31.FXI polypeptide is used for the treatment of application in the pharmaceutical preparation of bleeding episodes in preparation.

32. the application of claim 31, wherein said bleeding episodes are to be selected from situation institute secondary in the group of surgical operation, dental procedure, wound or hemodilution composition.

33. the application of claim 31 or claim 32 is wherein treated described bleeding episodes without factor VII/ factor VIIa coagulant.

34.FXI polypeptide is used for having the patient of needs to promote application in the hemostatic pharmaceutical preparation in preparation.

35.FXI polypeptide is used for having the patient of needs to increase application in the pharmaceutical preparation of EGCT in preparation.

36.FXI polypeptide is used for having the patient of needs to increase application in the pharmaceutical preparation of clot strength in preparation.

37.FXI polypeptide is used for having the patient of needs to improve application in the pharmaceutical preparation of total grumeleuse quality (OCQ) in preparation.

38.FXI polypeptide is used for having the patient of needs to shorten application in the pharmaceutical preparation of clotting time in preparation.

39. the application of item arbitrarily wherein increases to about 5nM with the effective FXI plasma concentration among the patient among the claim 31-38.

40. the application of claim 39 wherein increases to about 10nM with effective FXI plasma concentration.

41. the application of claim 40 wherein increases to about 30nM with effective FXI plasma concentration.

42. the application of item arbitrarily among the claim 31-41 is wherein without factor VII/ factor VIIa coagulant treatment patient.

43. the application of item arbitrarily among the claim 31-42, there is not congenital FXI defective in wherein said patient to be treated.

44. any application among the claim 19-43, wherein said FXI polypeptide comprise the sequence of SEQ ID NO:1 or it keeps the active fragment of at least a FXI-associated biomolecule.

45. any application among the claim 19-43, wherein said FXI polypeptide comprise the sequence of SEQ ID NO:2 or it keeps the active fragment of at least a FXI-associated biomolecule.

46. the application of item arbitrarily among the claim 19-45 is wherein with described FXI polypeptide and non-factor VII/ factor VIIa coagulant administering drug combinations.

47. the application of claim 46, wherein said non--factor VII/ factor VIIa coagulant is selected from the group of following composition: factor XI, plasma thromboplastin antecedent II; Tissue factor approach restrainer (TFPI) inhibitor; Factors IX; The activable fibrinolysis inhibitor of thrombin (TAFI); Plasminogen activator inhibitor-1 (PAI-1); Factor V; Protein C inhibitor; The Protein S inhibitor; And tissue plasminogen activator (tPA) inhibitor.

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