CN101535339B

CN101535339B - Modified proteins

Info

Publication number: CN101535339B
Application number: CN200780040932.7A
Authority: CN
Inventors: C·贝伦斯
Original assignee: Novo Nordisk Health Care AG
Current assignee: Novo Nordisk Health Care AG
Priority date: 2006-09-01
Filing date: 2007-09-03
Publication date: 2014-11-12
Anticipated expiration: 2027-09-03
Also published as: CN101535339A

Abstract

Method of conjugating glycoproteins by means of chemical modification is provided as well as new modified glycoproteins.

Description

Modified proteins

Invention field

The present invention relates to improve the preparation of medicine, especially preparation has the modified proteins of pharmacodynamics and the pharmacokinetics character of improvement.

Background of invention

Because biogenic albumen has efficient and highly selective to its native ligand conventionally, so their utmost points promise to be medicine.Because organism has had very clear and definite pathways metabolism and removed mechanism and can use, biogenic essence has improved the nontoxic possibility of biogenic albumen, thereby uses safer than conventional small-molecule drug.It is combined with actual, and albumen can be prepared by recombinant DNA technology in many different expression systems at present, has realized large-scale preparation, causes albumen to become ideal candidates medicine.Yet usually circulating half-life is shorter in vivo as hormone, solvable acceptor, cytokine, enzyme etc. for interesting treatment albumen, has conventionally weakened their treatment effectiveness.

Treatment albumen is eliminated from circulation by many approach.Some are had to the albumen of pharmacologically active, have its special receptor being removed of mediation from circulation.Glycosylated protein can be removed by the agglutinin receptor in liver, and this receptor only has specificity to the glycosyl part in these molecules.Proved albumen and peptide (particularly nonglycosylated albumen and peptide) through the non-specific removing of kidney lower than about 50kDa.It has been observed that asialoglycoprotein removes faster (Bocci (1990) Advanced DrugDelivery Reviews 4:149) in kidney than the albumen of natural sugar albumen or sugar based.In the situation that treatment albumen and autologous protein are incomplete same, they also can be eliminated by immunity system from circulate, even the small differences of aminoacid sequence or three-dimensional structure can cause treating the immunogenicity of albumen.By the immune response that causes for the treatment of albumen except making it accelerates to be eliminated from circulation, also have other different side effects: the sterically hindered antibody that makes that approaches binding site on treatment albumen may disturb or hinder result for the treatment of, artificial antibody can with autologous protein cross reaction, thereby produce autoimmune response etc.Albumen is interested is also to make them to take specific cells, tissue or organ as target in modification treatment for people.Albumen is puted together or merged with the ligandin molecule existing in specific cells to high affinity is one of known way reaching this effect.

Therefore, generally need to provide the method for preparing modification (treatment) albumen, this albumen has the pharmacological properties of the serum half-life of prolongation and/or the immunogenicity of reduction and/or improvement.

Summary of the invention

The invention provides the prolongation of soluble sugar protein derivatives circulating half-life, thereby while reducing treatment or prevention, maintain dosage and the frequency of injection of the required injectable drug of circulation glycoprotein treatment level of significance.Due to frequency and the quantity of the soluble protein that may need in treatment or prevention, the glycoprotein of some therapeutic activities does not meet the requirements shorter plasma half-life.The present invention is by the structure of effective change glycoprotein and substantially maintain the circulating half-life that biological activity extends described glycoprotein.

The invention provides suitable method and prepare glycoprotein derivative, introduce the oximido of polymeric part on the glycosyl group of described glycoprotein, described modified proteins has the pharmacological properties of improvement with respect to initial glycoprotein.

Therefore, the present invention relates to a kind of method with following general formula modified proteins of preparing:

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I)

Joint

Wherein M and optional M ' are the polymeric part that increases modified proteins molecular weight independently, wherein L and L ' represent divalence joint (linker) independently, wherein P represents glycoprotein, the one or more oxidized dextran end that described glycoprotein comprises glycoprotein, O, N, C and H represent respectively oxygen, nitrogen, carbon and hydrogen atom, n is 1-10, and m is 0-50, and the method comprises the following steps:

A) with Periodic acid or periodate (periodate) ionic oxide formation there is glycoprotein P at least one ^*on glycan end, P wherein ^*represent a plurality of sugared types, to obtain the glycoprotein P-(CHO) that contains one or more aldehyde groups _n+m, and

B) by P-(CHO) _n+mwith M-L-O-NH ₂reaction is to be had (M-L-O-N=CH)) _n-P-(CHO) _mthe modified proteins of structure, and

C) optionally will there is structure (M-L-O-N=CH) _n-P-(CHO) _mglycoprotein in any responseless aldehyde group and M '-L '-O-NH ₂reaction, to obtain having structure (M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _mmodified proteins, wherein, with respect to the quantity of not reducing glycan end existing on glycoprotein, described Periodic acid or periodate ion exist to be less than the amount of 50 equivalents, and wherein said modified proteins is with respect to initial glycoprotein P ^*there is the pharmacological properties of improvement and keep its functionally active.

Be understandable that P-(CHO) _n+min aldehyde group can be of short duration with it/their paired glycol form or hemiacetal form exist.

It is also understood that for P-and-P-is the glycoprotein that contains one or more oxidized dextran ends, wherein chemical bond is connected with described one or more dextran ends.

The invention further relates to the modified proteins that can obtain by the inventive method.

Therefore, the invention further relates to the modified proteins with following general formula

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I)

Joint wherein M and optional M ' independently for increasing the polymeric part of modified proteins molecular weight, wherein L and L ' represent divalence joint independently, wherein P represents glycoprotein, the one or more oxidized dextran end that described glycoprotein comprises glycoprotein, O, N, C and H represent respectively oxygen, nitrogen, carbon and hydrogen atom, n is 1-10, and m is 0-50, and wherein said modified proteins is with respect to initial glycoprotein P ^*there is the pharmacological properties of improvement and keep its functionally active.

The invention still further relates to and comprise the multiple preparation (preparation) with the modified proteins of following general formula

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I)

Detailed Description Of The Invention

" multiple modified proteins " can be understood as the various modified proteins may in preparation without identical chemical structure, between each glycoprotein molecule, may have sizable difference.Initial glycoprotein P ^*conventionally with different sugar type, exist." sugared type " refers to by translating rear or translation modification, same protein and the protein isoform that homopolysaccharide is not connected.Also there is the molecule of some preparations not to be modified completely, some molecules more than one polymer part that is modified, then the different glycan ends at each glycoprotein are modified again.P ^*therefore be considered to a plurality of albumen P sugar types.

Term " non-reduced glycan end " or " do not reduce glycan end " refer to that the end points of oligose or end are not reduced by for example Fehling's solution or Tollens reagent, and different from the reducing end of oligose, they are by these reagent oxidation.In glycoprotein, oligose is connected with albumen by glucosides or glycosamine key by its reducing end conventionally.The non-reduced end of this oligose comprises such as but not limited to the end sialic acid residues on N-and the poly-carbohydrate complexes of O-; End sialic acid residues on heterozygosis N-glycan; End mannose residue on height-seminose N-glycan; Terminal galactose residues on N-and O-glycan mixture; The terminal galactose residues of heterozygosis N-glycan; End N-acetyl galactose residue on N-and O-glycan mixture; End N-acetyl galactose residue on heterozygosis N-glycan; End mannose residue in all or part of three seminose core residue that expose; End trehalose residue, comprises core trehalose residue; Terminal glucose and xylose residues.

If preferred specific glycan group end reacts with Periodic acid or periodate, initial glycoprotein P ^*optional by sialidase, tilactase, N-acetyl-glucosamine Glycosylase, mannosidase or mycoside enzyme modification.Selectable, glycan group can for example be used sialytransferase, galactotransferase, N-acetyl-glucosamine sugar transferring enzyme, mannose transferase and saccharide donor separately as CMP-Sia; UDP-Gal; The transformations such as GDP-Fu.

Use the glycan end of Periodic acid or periodate oxidation glycoprotein fully to be proved, and be used as preparing the general method of protein conjugate.Publication number is the oxidation that the international patent application of WO 06/071801 has been described von Willebrand factor glycan end, and for the preparation of albumen PEGization type.Publication number is that the international patent application of WO 00/23114 has been described the methodology of preparing interferon-beta-1a PEGization type, publication number is that the international patent application of WO 92/16555 has been described the several different methods that PEG group and albumen are puted together, and wherein relates to Periodic acid or periodate and forms glycan end aldehyde group functional group.

Except the glycol of cracking glycosyl, Periodic acid or periodate become known for being oxidized for example amino acid side chain of methionine(Met) (metheonine) equally, and cracking contains for example n terminal amino acid of Serine and Threonine of amino alcohol group.In some cases, the oxidation of methionine(Met) side chain can cause the change of protein biology profile, especially bioactive change.As Kornfelt, T; Persson, E.and Palm, L.; Archives of Biochemistry and Biophysics Vol.363, described in No.1pp.43-54 (1999), the condense activity form of the FVII factor (as FVIIa) of recombinant chou is proved to be the oxidation to methionine(Met) and has high susceptibility.With behind hydrogen peroxide oxidation methionine(Met) 298 and 306, the combination of FVIIa and solvable tissue factor (TF) is weakened, and being shown as dissociation constant increases by three times.The acyl ammonia degrading activity that forms the methionine(Met) oxidation FVIIa of mixture with solvable TF also only has 80% of natural FVIIa-TF mixture.

Not unexpected, consistent with these observationss, when we find the use Periodic acid of common description in using as these articles or the conjugation reaction of periodate mediation, ability by its cracking known peptide substrate judges, the peptidolytic activity that can observe FVIIa significantly and in some situation weakens completely.Therefore,, when when oxygenizement is had to the glycoprotein of high susceptibility, the conjugation of Periodic acid or the periodate mediation for example currently known methods of PEG group is used very limited.The invention describes the universal method addressing this problem, therefore disclose first the method that can prepare the oxime glycoconjugate albumen that keeps functionally active.

When using lower concentration, with the mode-Periodic acid of equivalent or periodate with respect to the non-reduced glycan end quantity existing in glycoprotein with close to stoichiometric amount, can keep biological activity.Unexpected, with stoichiometric Periodic acid or periodate ion, then within the acceptable time, put together chemical action, can access highly purified biological function protein conjugate, and have medium to first-class yield.

An embodiment of the invention relate to a kind of method of preparing modified proteins, and wherein said glycoprotein is that N-is glycosylated and/or O-is glycosylated and/or contains sialic acid residues.

The further embodiment of the present invention relates to a kind of method of preparing modified proteins, and the method comprises that this modified proteins of further confirmation has the step of the pharmacological properties of improvement with respect to initial glycoprotein.

The pharmacological properties improving is in one embodiment selected from the bioavailability of raising, plasma half-life in plasma half-life, the immunogenicity of reduction, the function Half-life in vivo of the albumin avidity of the protease resistant of raising, raising, the receptor affinity of improvement, the stability in storage of raising, shortening, the body of shortening in the body of the function Half-life in vivo of prolongation, prolongation.

In one embodiment, the transformation period of prolongation reduces as the group that increases molecular weight by M and/or M ' or eliminates kidney removing and/or obtain as the group of sheltering the binding partners of liver receptor by M and/or M '.

In one embodiment, the immunogenicity of reduction by M and/or M ' as blocking antibody with cause the group of being combined in immune site and obtain.

In one embodiment, improving albumin avidity obtains with the group that albumin has high affinity by M and/or M ' conduct.

In one embodiment, improving receptor affinity obtains as the group with target cell surface receptor specific binding by M and/or M '.

Further the present invention relates in one embodiment a kind of method of preparing the modified proteins with following general formula

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I),

Wherein M and/or M ' are selected from: the organic charged groups of lower molecular weight, and it can contain one or more carboxylic acids, amine, sulfonic acid, phosphonic acids or its combination; Lower molecular weight neutral hydrophilic molecule, for example Polyethylene Chain of cyclodextrin or optional branching; Low-molecular-weight lipophilic molecule, as lipid acid or cholic acid or derivatives thereof; Molecular-weight average is the polyoxyethylene glycol of 2-40kDa; Well-defined accurate polymkeric substance, as the definite molecular weight ranges dendritic macromole (dendrimer) that is 700Da to 20kDa; Substantially the polypeptide of non-immunogenic, as albumin, antibody or optionally contain the part of the antibody of Fc-structural domain; With high molecular organic polymer.

The further embodiment of the present invention relates to the method that preparation has following general formula modified proteins

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I),

Wherein M and/or M ' are selected from dendritic macromole, dendritic macromole, polyoxyalkylene (PAO), comprise polyalkylene glycol (PAG), as polyoxyethylene glycol (PEG) and polypropylene glycol (PPG), branching PEG, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyethylene-copolymerization-maleic anhydride, polystyrene-copolymerization-maleic anhydride, dextran, Sensor Chip CM 5.

In further embodiment, M and/or M ' are selected from hydroxyalkyl starch (HAS) and hydroxyethylamyle (HES), and for example Clin Pharmacokinet 2005; 44 (7): the compound of describing in 681-699, and disclosed compound in WO 2006094810A2; The activated form that HES is suitable is disclosed in WO 2005092369A2, is introduced into as a reference.

In further embodiment, M and/or M ' are poly-(the 1-hydroxymethyl ethene hydroxymethyl formaldehyde) dextran of (PHF) or similarly degrading, the described compound of WO 2006094810A2 for example, be introduced into as a reference, wherein also disclose the suitable activated form of polymkeric substance.

In further embodiment, M and/or M ' are selected from amphoteric ion polymer, and for example WO 03062290A1 is described, is incorporated herein by reference.In embodiment, this polymkeric substance is 2-methacryloxy-2 '-ethyl-trimethyl ammonium phosphate inner salt (MPC).

In further embodiment, the present invention relates to a kind of method with following general formula modified proteins of preparing

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I),

Wherein M and/or M ' are selected from serum protein binding partner and contain the organic molecule that changes the part of charge property under physiological condition, suppress the structure of glycan and receptors bind, and stop the middle substituent of glycan specific recognition.

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I),

Variant wherein P is selected from FVII, FVIII, FIX, FX, FII, FV, PROTEIN C, Protein S, tPA, PAI-1, tissue factor, FXI, FXII, FXIII and sequence variants thereof, immunoglobulin (Ig), cytokine is as interleukin, α-, β-and gamma-interferon, G CFS, comprise granulocytosis colony stimulative factor, fibroblast growth factor, Thr6 PDGF BB, Phospholipid hydrolase activator (PUP), Regular Insulin, vegetable-protein is as lectin and ricin, tumour necrosis factor and relevant allelotrope, the Tumor Necrosis Factor Receptors of solubilized form, the interleukin-2-receptor of interleukin-2-receptor and solubilized form, somatomedin is tissue growth factor for example, as TGFa ' s or TGFps and Urogastron, hormone, somatomedin, erythropoietin, pigment hormone, hypothalamic releasing factor, vassopressin, prolactin, chorionic-gonadotropin hormone, follicle stimulating hormone, thyrotropic hormone, tissue plasminogen activator, with immunoglobulin (Ig) as IgG, IgE, IgM, IgA and IgD and fragment thereof, or any fusion rotein that contains any aforementioned albumen or its fragment.

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I),

Wherein glycoprotein is VII factor polypeptide.

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I),

The aminoacid sequence that wherein glycoprotein contains the wild-type people VII factor.

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I),

Wherein glycoprotein is VIII factor polypeptide.

" the VIII factor " used herein and " FVIII polypeptide " comprise VIII:C, B-domain deletion form, amino acid variant and the combination thereof of FVIII.

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I),

The specific activity that wherein modified proteins shows in conjunction with test at one or more CAs described in specification sheets of the present invention, proteolysis effects test or TF be unmodified VII factor polypeptide specific activity at least about 10%, as at least about 20%, as at least about 40%, as at least about 60%, as at least about 80%, as at least about 100%.

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I),

The bioavailability that wherein modified proteins shows be unmodified glycoprotein bioavailability at least about 110%, as at least about 120%, approximately 130% or be unmodified glycoprotein bioavailability at least about 140%.

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I),

The serum half-life that wherein modified proteins shows be unmodified sugared egg serum half-life at least about 125%, according to appointment 150%, approximately 200% or be unmodified sugared egg serum half-life at least about 250%.

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I),

Wherein with respect to the quantity of not reducing glycan end existing on glycoprotein, Periodic acid or periodate ion are to be less than the amount of 20 equivalents, for example be less than 10 equivalents, for example, be less than 5 equivalents, the amount that is for example less than 1 equivalent exists, for example, with respect to the quantity of not reducing glycan end existing on glycoprotein, with 0.1-20 equivalent, 0.1-10 equivalent for example, 0.1-5 equivalent for example, for example the amount of 0.1-1 equivalent exists.

Therefore, in one embodiment, the amount of Periodic acid or periodate ion is the 10-20 equivalent of quantity that does not reduce glycan end with respect to existing on glycoprotein.In another embodiment, the amount of Periodic acid or periodate ion is the 5-10 equivalent of quantity that does not reduce glycan end with respect to existing on glycoprotein.In another embodiment, the amount of Periodic acid or periodate ion is the 2-5 equivalent of quantity that does not reduce glycan end with respect to existing on glycoprotein.In another embodiment, the amount of Periodic acid or periodate ion equates substantially with the quantity of glycan end of not reducing existing on glycoprotein.In another embodiment, the amount of Periodic acid or periodate ion is the 0.1-0.9 equivalent of quantity that does not reduce glycan end with respect to existing on glycoprotein.

In another embodiment, with respect to the quantity of not reducing glycan end existing on glycoprotein, use Periodic acid or periodate lower than stoichiometry.In present specification and claim, term " polypeptide " refers to the linear chain molecule that comprises peptide linkage amino-acid residue.Therefore, term cyclic peptide (2-10 amino-acid residue), oligopeptide (11-100 amino-acid residue) and pure polypeptide (thering are 100 amino-acid residues of surpassing).Polypeptide because of but have biological activity but also can be without any the structural unit of function." albumen " herein refers to have outside function or nonfunctional molecule or demonomerization, the mixture that contains at least one polypeptide, this term also comprises multimeric molecule, as or heteromultimeric.Albumen can comprise prothetic group, also can comprise different glycosylations and esterification structure." glycoprotein " refers to that some is by glycosylated albumen.In some embodiments of the present invention, glycoprotein is N-glycosylation and/or O-glycosylated protein and/or uses sialic acids groups modification.

In another embodiment, the method for preparing modification glycosyl chemoattractant molecule comprises that further confirmation modified proteins has the step of improving pharmacological properties with respect to glycosylation starting molecule.

Typically, the pharmacological properties of improvement be selected from the bioavailability of raising, in the body of the function Half-life in vivo of prolongation, prolongation plasma half-life, the immunogenicity of reduction, the albumin avidity of the protease resistant of raising, raising, the receptor affinity of improvement, the stability in storage of raising.

Term " function Half-life in vivo " refers to its common implication, that is, modified proteins or associated molecule in vivo or target organ kept for 50% bioactive time, or the activity of modified proteins or associated molecule is down to time of 50% of peak value.As other selections of measurement function Half-life in vivo, can measure " plasma half-life in body ", the time that 50% modified proteins or associated molecule circulate before being eliminated in blood or blood plasma.Measure plasma half-life conventionally simple than measurement function Half-life in vivo, the value of plasma half-life has well represented the value of function Half-life in vivo conventionally.The alternative terms of plasma half-life comprises serum half-life, circulation transformation period, circulating half-life, serum clearance rate, plasma clearance and removing transformation period.What keep is functionally selected from thromboplastic, proteolysis, cofactor conventionally in conjunction with, receptor-binding activity, or the biological activity of the other types relevant to specific protein.

Term " raising " is used to indicate function Half-life in vivo or plasma half-life, and the relevant transformation period of modified proteins is with respect to its associated molecule, but for example other identical glycoprotein growths significantly statistically of not using method of the present invention to process.Therefore, the transformation period is measured under collating condition.For example the relevant transformation period can extend at least about 25%, for example, at least about 50%, as at least about 100%, 150%, 200%, 250% or 500%.In some embodiments, the modified proteins transformation period of the present invention with respect to the Increased Plasma Half-life of unmodified glycoprotein at least about 0.25 hour, preferably at least about 0.5 hour, more preferably at least about 1 hour, with most preferably from about 2 hours.

Can adopt the several different methods of describing in document to measure biological half-life in body.Test modification FVIIa (coagulation factor VIIa) detects the example of rFVIIa and its variant Half-life in vivo described in FDA reference number 96-0597.In brief, in blood plasma, use the coagulation power of measuring FVIIa before or after modified proteins in 24 hours.Under steady state, measure the intermediate value of apparent volume of distribution and measure half elimination ratio.

" bioavailability " refers to use the ratio of detectable glycoconjugate application dosage in rear scheduled time blood plasma.Typically, bioavailability detects by use the preparation of about 25-250 μ g/kg dosage to test animal; After using, the scheduled time is got plasma sample; Use the glycoprotein concentration in suitable biological detection or immunodetection or suitable detection method working sample.Data are shown as [glycoprotein] v. time with diagram conventionally, and bioavailability is expressed as area under curve (AUC).The relative bioavailability of test formulation refers to the AUC ratio of test formulation and unmodified glycoprotein.

In some embodiments, the relative bioavailability of preparation of the present invention be corresponding unmodified glycoprotein biological utilisation at least about 110%, preferably at least about 120%, more preferably at least about 130% and most preferably at least about 140%.Bioavailability can be measured with any Mammals, and preferably dog, can comprise different increments by 10 minutes-8 hours for calculating the scheduled time of AUC.Bioavailability can for example detect by the following method in dog model: this test is divided into four groups by 12 beagles and carries out four leg exchange tests.All animals are applied test formulation A that dosage is approximately 90 μ g/kg and relevant preparation B, said preparation is contained in suitable buffered soln, as the glycyl peptide glycine buffer (pH5.5) that contains sodium-chlor (2.92mg/ml), CALCIUM CHLORIDE DIHYDRATE (1.47mg/ml), N.F,USP MANNITOL (30mg/ml) and tween 80.After administered formulation, after 10,30 and 60 minutes and 2,3,4,6 and 8 hours, get blood.By sample, obtain blood plasma, by the quantitative glycoprotein of ELISA.

" immunogenicity " of term preparation refers to that preparation uses the harmful immunoreactive ability of rear initiation to people, no matter is body fluid, cell or both have concurrently.In anyone subpopulation, may there is the individuality to specific application albumen sensitivity.Immunogenicity can detect by the anti-glycoprotein antibody with existing in the quantitative sensitive individual of prior art conventional means and/or the T-cell of glycoprotein sensitivity.In some embodiments, the immunogenicity of modified proteins of the present invention in sensitive individual reduces at least about 10%, preferably at least about 25%, more preferably at least about 40%, most preferably at least about 50% with respect to the individual immunogenicity of unmodified glycoprotein.

The immunogenicity of medicine also the medicine of finger protein matter character may be non-sensitive experimenter immunogenic phenomenon, mean that repetitive administration medicine causes the immune response of continuous rising to medicine.This is the most less desirable situation, because the activity of immune response meeting interference medicament, why Here it is needs the dosage that increases gradually medicine to reach result for the treatment of.The immunogenicity of modified protein of the present invention in non-sensitive individuality reduces at least about 10%, preferably at least about 25%, more preferably at least about 40%, most preferably at least about 50% with respect to the individual immunogenicity of unmodified glycoprotein in some embodiments.

Term " protease resistant " refers to cause after glycoprotein is chemically modified the glycoprotein of described compound to blood plasma peptase or proteolytic enzyme resistance.The known degraded that participates in many peptide hormones of plasma proteinase, has also participated in the degraded of large protein.

Glycoprotein can detect by following degraded test the resistance by for example dipeptidyl aminopeptidase IV (DPPIV) degrades: part glycoprotein (5nmol) is hatched 10-180 minute with the 1 μ L purifying dipeptidyl aminopeptidase IV that enzyme activity is 5mU accordingly at 37 ℃ in 100 μ L 0.1M triethylamine-HCl damping fluids.Enzyme reaction is by adding 5 μ L 10% trifluoroacetic acids to stop, and isolated peptides degraded product, detects quantitatively with HPLC.According to people such as Siegel., Regul.Pept.1999; The people such as 79:93-102 and Mentlein .Eur.J.Biochem.1993; 214:829-35, one of working method of this detection: mixture is injected to (30nm hole, the wide hole of Vydac C18,5 μ m particles) 250 * 4.6mm post, with containing the acetonitrile of 0.1% trifluoroacetic acid with flow velocity 1ml/ minute linear stepwise gradient wash-out (three minutes 0% acetonitrile, 17 minutes 0-24% acetonitriles, 1 minute 24-48% acetonitrile).Peptide and degraded product thereof can be monitored by the absorption of its 220nm (peptide bond) or 280nm (die aromatischen Aminosaeuren), by its peak area of integration and standard control, come quantitatively.The percent hydrolysis of the incubation period estimation dipeptidyl aminopeptidase IV being hydrolyzed at the peptide lower than 10% to peptide.

In Mammals blood circulation, the highest protein ingredient of content is serum albumin, and its concentration is generally approximately 3 to 4.5 grams of every 100 milliliters of whole bloods.Serum albumin is approximately 70,000 daltonian blood proteins, and it brings into play multiple important function in the recycle system.For example, it can be used as the vehicle of each organic molecular species of finding in blood, as various metabolites, if lipid acid and bilirubin are by the main vehicle of blood, and due to its rich content, can be used as the infiltrative conditioning agent of blood circulation.Sero-abluminous long half time is in one week, and one of method that extends peptide plasma half-life is to use the chemical entity derived peptide of being combined with serum albumin.Term " albumin bound agent " refers to that known and plasma proteins are as the chemical entities of albumin bound.The character of albumin bound can be according to J.Med.Chem, and the described detection of 43,2000,1986-1992, is introduced into as a reference.Albumin bound group can comprise derivative of fatty acid, organic peptide sulphur thiazole polycyclic aromatic hydrocarbons, for example thyroliberin, and contain the peptide that is less than 40 amino-acid residues, J.Biol Chem.277 for example, the group of describing in 38 (2002) 35035-35043, is introduced into as a reference.

Modified proteins prepared according to the methods of the invention has improved functional property with respect to unmodified glycoprotein.Improved functional property can include, without being limited to a) physical properties, for example improved stability in storage; B) improved pharmacokinetics character, the bioavailability and the transformation period that for example increase; C) the human immunity originality reducing.

When the stability in storage of glycoprotein can be stored in 25 ℃ with dry powdered form by the following Data Detection of mensuration (a), bioavailability reduces by 20% required time; (b) double the predetermined degraded product of the preparation time as needed in condensation product ratio.

In some embodiments, when the preparation that contains glycoprotein is stored in 25 ℃ with dry powdered form, the time lengthening that modified proteins bioavailability of the present invention 20% time needing of reducing needs with respect to unmodified glycoprotein is at least about 30%, preferably at least about 60%, more preferably at least about 100%.

The measuring method of bioavailability can operate according to the relevant biological activity type of specific protein, and for example, the biological activity of thrombin can detect in conjunction with mensuration or the ind zymoplasm generation of TF-mensuration with any CA, CA, TF-.

In some embodiments, when preparation is all stored in 25 ℃ with dry powdered form, preparation of the present invention doubles predetermined degraded product, and the time as required in condensation product extends at least about 30% with respect to Comparative formulation, preferably at least about 60%, more preferably at least about 100%.Condensation product concentration can for example be measured by gel infiltration HPLC or other known chromatographic processes.For thrombin, condensation product can be measured with Protein Pak 300 SW posts (7.5 * 300mm) (water, 80013) by the following method with gel infiltration HPLC.With eluent A (0.2M ammonium sulfate, 5% Virahol, regulates pH2.5 with phosphoric acid, then regulates pH7.0 with triethylamine) balance columns, then by 25 μ g sample upper props.Wash-out, with elutriant A wash-out 30 minutes under the condition of flow velocity 0.5ml/ minute, then completes detection by measuring absorbancy at 215nm.The concentration of condensation product is pressed thrombin condensation product peak area/total thrombin peak area (monomer and condensation product) and is calculated.

divalence joint L and L ':

L and L ' represent divalence chemistry joint, and it can be identical or different

In one embodiment L and L ' be independently key or

substituting group M and M '

Below substituting group M and/or M ' will be discussed.

In an embodiment of the invention, by M and/or M ', as the group that increases molecular weight, using and reduce or eliminate renal clearance and/or M and/or M ' as the group prolong half-life of sheltering liver receptor binding site.In selectable embodiment, by M and/or M ', as blocking antibody and immunogenic site conjugated group, reduce immunogenicity.In another embodiment, by M and/or M ', as group albumin to high-affinity, improve albuminous avidity.And in another embodiment, by M and/or M ', as the group with target cell surface receptor specific binding, improve the avidity to acceptor.Substituting group M and/or M ' can be revising of the functions groups, as " protractor group " (protractor group).At this, refer to combine closely to extend described albumen or peptide with respect to the group of the circulating half-life of glycoprotein not with albumen or peptide.The particular principles of protractor effect may be to be caused by the volume increasing, having covered can be by the peptide sequence of peptase or antibody recognition, thereby or sheltered glycan and make them can not be present in the glycan specific receptors identification of liver for example or scavenger cell, stop or reduced removing.The protractor effect of protractor group also can be for example causes as albumin by being bonded to blood constitutent, or is caused by specificity or non-specific adsorption to vascular tissue.Substantially the biological activity that has kept non-modified proteins in conjunction with glycoprotein.

Other possibility comprises that M and/or M ' are the groups that makes the cell or tissue of modified proteins target particular type, if for example can to bring into play its effect under very high partial concn relevant with glycoprotein.Further possibility comprises that M and/or M ' have its effective constituent, as radionuclide or toxicant-will be so suitable in the situation that unmodified glycoprotein has avidity to malignant tissue, thereby as the target group of modification molecule.

M and/or M ' are selected from an embodiment of the invention

The organic charged groups of-lower molecular weight (15-1000Da), contains one or more carboxylic acids, amine, sulfonic acid, phosphoric acid or its combination.

-lower molecular weight (15-1000Da) neutral hydrophilic molecule, for example Polyethylene Chain of cyclodextrin or optional branch;

-lower molecular weight (15-1000Da) lipophilic molecule, as lipid acid, cholic acid or derivatives thereof;

-molecular-weight average is the polyoxyethylene glycol of 2-40kDa;

-well-defined accurate polymkeric substance, as to have definite molecular weight ranges be 700Da to 20kDa or more preferably 700-10, the dendritic macromole of 000Da;

-the polypeptide of non-immunogenic substantially, as albumin, antibody or optionally contain the part of the antibody of Fc-structural domain; With

-high molecular organic polymer is as dextran.

Poly molecule is selected from dendritic macromole (if molecular weight ranges is 700-10 in embodiments of the present invention, 000Da or the disclosed dendritic macromole of International Patent Application WO 2005014049), polyoxyalkylene (PAO), comprise polyalkylene glycol (PAG), as polyoxyethylene glycol (PEG), polypropylene glycol (PPG), branching PEG, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyethylene-copolymerization-maleic anhydride, polystyrene-copolymerization-maleic anhydride and dextran, comprise Sensor Chip CM 5, HES, PHF or MPC.In an embodiment of the invention, multimeric molecule is PEG group.In an embodiment of the invention, multimeric molecule is dendritic macromole.

In an embodiment of the invention, M and/or M ' are protractor group (protactorgroup), be selected from serum protein binding partner, as serum protein binding partner, for example, with the molecule of albumin bound, as lipid acid, C5-C24 lipid acid, fat diacid (as C5-C24), the structure (as sialic acid derivative or its stand-in) that suppresses glycan and acceptor (as asialoglycoprotein receptor and mannose receptor) combination, contain the organic molecule that changes the part of charge property under physiological condition, for example carboxylic acid or amine or middle substituent, can stop glycan specific recognition, low-grade alkyl substituent (as C1-C5 alkyl) for example, the organic charged groups of lower molecular weight (as C1-C25), it contains one or more carboxylic acids, amine, sulfonic acid, phosphoric acid or its mixture, lower molecular weight (as C1-C25) neutral hydrophilic molecule, for example Polyethylene Chain of cyclodextrin or optional branch, molecular-weight average is the polyoxyethylene glycol of 2-40kDa, well-defined polymkeric substance, if definite molecular weight ranges is 700Da to 20kDa or more preferably 700-10, the dendritic macromole of 000Da, substantially the polypeptide of non-immunogenic, as albumin, antibody or optionally contain the part of the antibody of Fc-structural domain.

In one embodiment M and/or M ' independently selected from:

Wherein Q represents 10-20,10-30,10-40,20-30,20-40, the integer of 30-40, for example 10,20 or 30.

In one embodiment, M-L-ONH ₂and/or M ' L '-ONH ₂be:

In one embodiment, M-L-ONH ₂and/or M ' L '-ONH ₂independently selected from:

M and/or M ' are selected from the organic group of one of following group:

-straight chain, side chain and/or ring-type C _1-30alkyl, C _2-30thiazolinyl, C _2-30alkynyl, C _1-30assorted alkyl, C _2-30assorted thiazolinyl, C _2-30assorted alkynyl, wherein can insert one or more homoatomic cyclic aromatic compounds double-basis or heterogeneous ring compound double-basis, wherein said C _1-30or C _2-30group is optionally replaced by one or more following groups :-CO ₂h ,-SO ₃h ,-PO ₂oH ,-SO ₂nH ₂,-NH ₂,-OH ,-SH, halogen or aryl, wherein said aryl is optionally replaced by following group :-CO ₂h ,-SO ₃h ,-PO ₂oH ,-SO ₂nH ₂,-NH ₂,-OH ,-SH or halogen; Steroid class group; Lipid group;

-poly-carbohydrate group, as dextran, α-, β-or γ-cyclodextrin, polyamide-based group, as polyamino acid; PVP; Poly-(1-3-dioxane); Poly-(1,3,6-trioxane); Ethene/toxilic acid polymkeric substance;

-vapour bar (Cibacron) dyestuff, as the polymeric amide of Cibacron Blue 3GA and certain chain lengths, described in WO00/12587, is introduced into as a reference;

-the protein residues of non-immunogenic substantially, for example blood constitutent is as albumin derivant, or antibody or its region Fc-structural domain normal IgG1 that behaves for example, as Kan, the people such as people such as SK are at The Journal of Immunology 2001,166 (2), in 1320-1326 or at Stevenson, GT, The Journal of Immunology 1997, described in 158,2242-2250;

-polyoxyethylene glycol (PEG) or methoxy poly (ethylene glycol) (mPEG) group and its aminoderivative, wherein molecular-weight average can be 500-100,000Da, as 500-60,000Da, as 1000-40,000Da, as 500-40,000Da;

-known and plasma proteins is as the group of albumin bound, wherein albumin bound character can be according to J.Med.Chem, method described in 43,2000,1986-1992 is measured, be introduced into as a reference, or albumin bound group, for example contain the peptide that is less than 40 amino-acid residues, as J.Biol Chem.277, group described in 38 (2002) 35035-35043, is introduced into as a reference.

In other embodiments, M and/or M ' are C ₁-C ₂₀alkyl, as C ₁-C ₁₈alkyl.Specifically refer to C ₁₄-, C ₁₆-and C ₁₈-alkyl, is optionally replaced by special charged groups, polar group and/or halogen.Described comprise-CO of substituent example ₂h, tetrazyl and halogen.In embodiment, C ₁-C ₂₀all hydrogen of alkyl are replaced and form perfluoroalkyl by fluorine.

In embodiment, M ' is different from M.In embodiment, M ' has the lower molecular weight substantially than M.In an embodiment, M ' is Methoxyamine (MeONH ₂).

starting molecule P ^*

Below substituting group P will be discussed ^*.

Glycoprotein P ^*contain glycan end, the oxygenizement of Periodic acid is had to reactive behavior.

Therefore, P ^*on reactive group be a part for glycosyl, or derived from glycosyl group, as the N-of glycosylation glycoprotein or O-glycan.Selectable, glycoprotein P ^*reactive group can be sialic acid residues.

P in one embodiment ^*be selected from FVII, FVIII, FIX,, FX, FII, FV, PROTEIN C, Protein S, tPA, PAI-1, tissue factor, FXI, FXII, FXIII and sequence variants thereof; Immunoglobulin (Ig), cytokine as interleukin, α-, β-, gamma-interferon, G CFS comprises granulocytosis colony stimulative factor, fibroblast growth factor, Thr6 PDGF BB, Phospholipid hydrolase activator (PUP).P ^*also can be other albumen and peptides with general biology and treatment importance, comprise Regular Insulin, vegetable-protein is as Sugar receptors, ricin, tumour necrosis factor and relevant allelotrope, the Tumor Necrosis Factor Receptors of solubilized form, the interleukin-2-receptor of interleukin-2-receptor and solubilized form, somatomedin is tissue growth factor for example, as TGFa ' s or TGFps and Urogastron, hormone, somatomedin, erythropoietin, pigment hormone, hypothalamic releasing factor, vassopressin, prolactin, chorionic-gonadotropin hormone, follicle stimulating hormone, thyrotropic hormone, tissue plasminogen activator and immunoglobulin (Ig) are as IgG, IgE, IgM, , IgA, IgD and its fragment.

Can be described in people l.Glycobiology 12 (11) 762-770 (2002) such as Li Shao containing peptide and the albumen of glycan end, with glycosyltransferase, carry out enzyme glycosylation, or for example with standard peptide chemistry and glycosylation amino acid composition, carry out chemosynthesis glycosylation as N-galactosylation l-asparagine.

Selectable glycosylation site can design albumen or the peptide normally existing with non-glycosylated form in vivo.Such as repeating to insert consensus sequence Cys-XXX-Ser-XXX-Pro-Cys selectivity O-glycosylation Serine (people l.Glycobiology 12 (11) 762-770 (2002) such as Li Shao) with UDPG and glucanotransferase at EGF, and insert consensus sequence Asn-XXX-Ser/Thr, carry out N-glycosylation (R.A.Dwek, Chem.Rev.1996,96,683-720).The peptide sequence that contains Threonine or Serine also can be at UDP-GalNAc: under the existence of Polypeptide N-acetylgalactosaminyltransferase and UDP-GalNAc, with sequence, rely on mode glycosylation (referring to for example B.C.O ' Connell, F.K.Hagen and L.A.Tabak in J.Biol.Chem.267 (35), 25010-25018 (1992)).Selectable, directed mutagenesis is introduced cyophorin sudden change and be can be used for the semi-lactosi of being introduced semi-lactosi or being contained sugared structure by the formation of mixed disulfide, if D.P.Gamblin etc. is at Angew.Chem.Int.Ed., described in 43,828 (2004).The semi-lactosi that contains peptide and albumen or N-acetylgalactosamine also can be prepared by being bonded to the method that contains abiotic means, as P.G.Schultz in J.Am.Chem.Soc, 125, described in 1702 (2003), or nonspecific with glycosyl donor substrate as the direct glycosylated peptide such as trichoroacetic acid(TCA) galactoside.Add glycosidase inhibitor fermentation culture, thereby generate the Glycosylase with brachymemma glycan structures, as US 4925796A/US 5272066A1, also be to obtain the semi-lactosi contain peptide or albumen or the possible method of N-acetylgalactosamine, can also use TGase enzyme modification glutamine residue (to participate in such as people .Angew.Chem.Int.Ed.43 such as M.Sato, 1516-1520, (2004)).

Prepare obligatory gene N-glycosylated protein and be not limited to use mammalian host cell as CHO or bhk cell, can also use insect cell, yeast or with bacterial cell, as people such as M.Wacker. at Science, described in 298,1790-1793 (2002).Peptide is Trypsin inhibitor,Trasylol in an embodiment of the invention, tissue factor pathway inhibitor or other enzyme inhibitorss, Regular Insulin or insulin precurosor, people or Trobest, interleukin, glucagon, oxyntomodulin, GLP-1, GLP-2, IGF-I, IGF-II, tissue plasminogen activator, transforming growth factor γ or β, platelet-derived somatomedin, GRF (somatotropin releasing factor), human growth factor, immunoglobulin (Ig), EPO, TPA, PROTEIN C, thrombin is as FVII, FVIII, FIX, FX, FII, FV, PROTEIN C, S albumen, PAI-1, tissue factor, FXI, FXII and FXIII, exendin-3, exentidin-4 and enzyme or its functional analogue.In this article, term " functional analogue " refers to have with native protein the albumen of identity function.This albumen may with native protein structural similitude, also may be by adding one or more amino acid to one of the C-terminal of native protein or last N end or all, different loci at one or more natural acid sequences replaces one or more amino acid, in one of the end of native protein or one or more sites of aminoacid sequence or all lack one or more amino acid, or it is derivative from native protein in one or more sites of natural acid sequence, to insert one or more amino acid.Further, albumen can be acetylation in one or more positions, referring to for example WO 98/08871, wherein discloses acetylize GLP-1 and its analogue.The example of GLP-1 acetyl derivatives is Lys26 (N ε-myristoyl)-GLP-1 (7-37), and wherein the Yi Pu Shillong amino group of the Lys residue of the middle position 26 of GLP-1 (7-37) is by myristoylation.

Albumen or its part can be with the known technology of the art those of ordinary skill preparation or separated, and as tissue culture, animal source is extracted or passed through recombinant DNA method.The hereditary transfer sources such as albumen, peptide, amino acid also can be considered to use.These materials obtain from transgenic animal, and as mouse, pig, ox etc., wherein albumen is by milk, blood or tissue expression.Transgenic insect and baculovirus expression system also can be considered as source.In addition, the mutant of albumen, for example the mutant of TNF and/or interferon mutant are also within the scope of the present invention.Other interested albumen are hogweed, antigen E, meltittin venom, mite allergen etc.

Previously described biologically active peptides is suitable for and is combined according to protractor group of the present invention.Be understandable that clearly do not mention but the biological active materials with appropriate peptide also expect, within the scope of the invention.

In one embodiment, glycoprotein is FVII polypeptide.In one embodiment, polypeptide is the wild-type VIIa factor.

Any albumen (as the polypeptide that contains the U.S. patent No. 4,784,950 disclosed aminoacid sequences) of the aminoacid sequence 1-406 that term " VII factor polypeptide " or " FVII polypeptide " refer to contain the wild-type people FVII factor as used herein, and variant.

Term " the VII factor " refers to comprise the VII factor polypeptide existing with not cracking (proenzyme) form, and those corresponding biologically active forms that generate through proteolysis process, can refer in particular to the VIIa factor.Concrete, between VII factor cracking residue 152 and 153, generate the VIIa factor.These variants of the VII factor have different character with respect to the people VII factor, comprise activity specific of stability, phospholipids incorporate, change etc.

In this " wild-type people VIIa factor " used, be to there is U.S. patent No.4, the polypeptide of 784,950 disclosed aminoacid sequences.

The unrestriced example of VII factor variant comprises S52A-FVIIa, S60A-FVIIa (people such as Lino., Arch.Biochem.Biophys.352:182-192,1998); FVIIa variant shows the proteolysis stability improving, and as the U.S. patent No. 5,580,560 is disclosed; Between FVIIa misfolded proteins enzymolysis cracking residue 290 and 291 or between residue 315 and 316 (people such as Mollerup., Biotechnol.Bioeng.48:501-505,1995); The oxidised form of the VIIa factor (people such as Kornfelt., Arch.Biochem.Biophys.363:43-54,1999); FVII variant as disclosed in PCT/DK02/00189 (corresponding with WO 02/077218); And FVII variant shows the proteolysis stability increasing, as disclosed in WO 02/38162 (ScrippsResearch Institute); FVII variant has modification Gla-region and has the film combination of enhancing, as WO 99/20767, US patent US 6017882 and US 6747003, US patent application 20030100506 (University of Minnesota) and WO 00/66753, US patent application US 20010018414, US 2004220106 and US 200131005, US patent US6762286 and US 6693075 (University of Minnesota) are disclosed; With FVII variant as WO 01/58935, US patent US 6806063, US patent application 20030096338 (Maxygen ApS), WO 03/93465 (Maxygen ApS), WO 04/029091 (MaxygenApS), WO 04/083361 (Maxygen ApS) and WO 04/111242 (Maxygen ApS) and WO 04/108763 (Canadian Blood Services) are disclosed.

The non-limitative example with respect to wild-type FVIIa with the bioactive FVII variant of enhancing comprises as WO 01/83725, WO 02/22776, WO 02/077218, PCT/DK02/00635 (corresponding to WO 03/027147), Danish Patent Application PA 200201423 (corresponding to WO 04/029090), Danish Patent Application PA 200101627 (corresponding to WO 03/027147); The disclosed FVII variant of WO 02/38162 (Scripps Research Institute); And the FVIIa variant with enhanced activity of as disclosed in JP 2001061479 (Chemo-Sero-TherapeuticRes Inst.).

The example of the variant of the VII factor includes but not limited to

L305V-FVII，

L305V/M306D/D309S-FVII，L305I-FVII，L305T-FVII，F374P-FVII，V158T/M298Q-FVII，

V158D/E296V/M298Q-FVII，K337A-FVII，M298Q-FVII，V158D/M298Q-FVII，L305V/K337A-FVII，V158D/E296V/M298Q/L305V-FVII，V158D/E296V/M298Q/K337A-FVII，

V158D/E296V/M298Q/L305V/K337A-FVII，K157A-FVII，E296V-FVII，E296V/M298Q-FVII，

V158D/E296V-FVII，V158D/M298K-FVII，and S336G-FVII，L305V/K337A-FVII，L305V/V158D-FVII，L305V/E296V-FVII，L305V/M298Q-FVII，L305V/V158T-FVII，L305V/K337A/V158T-FVII，

L305V/K337A/M298Q-FVII，L305V/K337A/E296V-FVII，L305V/K337A/V158D-FVII，

L305V/V158D/M298Q-FVII，L305V/V158D/E296V-FVII，L305V/V158T/M298Q-FVII，

L305V/V158T/E296V-FVII，L305V/E296V/M298Q-FVII，L305V/V158D/E296V/M298Q-FVII，

L305V/V158T/E296V/M298Q-FVII，L305V/V158T/K337A/M298Q-FVII，

L305V/V158T/E296V/K337A-FVII，L305V/V158D/K337A/M298Q-FVII，

L305V/V158D/E296V/K337A-FVII，L305V/V158D/E296V/M298Q/K337A-FVII，

L305V/V158T/E296V/M298Q/K337A-FVII，S314E/K316H-FVII，S314E/K316Q-FVII，

S314E/L305V-FVII，S314E/K337A-FVII，S314E/V158D-FVII，S314E/E296V-FVII，

S314E/M298Q-FVII，S314E/V158T-FVII，K316H/L305V-FVII，K316H/K337A-FVII，

K316H/V158D-FVII，K316H/E296V-FVII，K316H/M298Q-FVII，K316H/V158T-FVII，

K316Q/L305V-FVII，K316Q/K337A-FVII，K316Q/V158D-FVII，K316Q/E296V-FVII，

K316Q/M298Q-FVII，K316Q/V158T-FVII，S314E/L305V/K337A-FVII，S314E/L305V/V158D-FVII，S314E/L305V/E296V-FVII，S314E/L305V/M298Q-FVII，S314E/L305V/V158T-FVII，

S314E/L305V/K337A/V158T-FVII，S314E/L305V/K337A/M298Q-FVII，

S314E/L305V/K337A/E296V-FVII，S314E/L305V/K337A/V158D-FVII，

S314E/L305V/V158D/M298Q-FVII，S314E/L305V/V158D/E296V-FVII，

S314E/L305V/V158T/M298Q-FVII，S314E/L305V/V158T/E296V-FVII，

S314E/L305V/E296V/M298Q-FVII，S314E/L305V/V158D/E296V/M298Q-FVII，

S314E/L305V/V158T/E296V/M298Q-FVII，S314E/L305V/V158T/K337A/M298Q-FVII，

S314E/L305V/V158T/E296V/K337A-FVII，S314E/L305V/V158D/K337A/M298Q-FVII，

S314E/L305V/V158D/E296V/K337A-FVII，S314E/L305V/V158D/E296V/M298Q/K337A-FVII，

S314E/L305V/V158T/E296V/M298Q/K337A-FVII，K316H/L305V/K337A-FVII，

K316H/L305V/V158D-FVII，K316H/L305V/E296V-FVII，K316H/L305V/M298Q-FVII，

K316H/L305V/V158T-FVII，K316H/L305V/K337A/V158T-FVII，K316H/L305V/K337A/M298Q-FVII，K316H/L305V/K337A/E296V-FVII，K316H/L305V/K337A/V158D-FVII，

K316H/L305V/V158D/M298Q-FVII，K316H/L305V/V158D/E296V-FVII，

K316H/L305V/V158T/M298Q-FVII，K316H/L305V/V158T/E296V-FVII，

K316H/L305V/E296V/M298Q-FVII，K316H/L305V/V158D/E296V/M298Q-FVII，

K316H/L305V/V158T/E296V/M298Q-FVII，K316H/L305V/V158T/K337A/M298Q-FVII，

K316H/L305V/V158T/E296V/K337A-FVII，K316H/L305V/V158D/K337A/M298Q-FVII，

K316H/L305V/V158D/E296V/K337A-FVII，K316H/L305V/V158D/E296V/M298Q/K337A-FVII，

K316H/L305V/V158T/E296V/M298Q/K337A-FVII，K316Q/L305V/K337A-FVII，

K316Q/L305V/V158D-FVII，K316Q/L305V/E296V-FVII，K316Q/L305V/M298Q-FVII，

K316Q/L305V/V158T-FVII，K316Q/L305V/K337A/V158T-FVII，K316Q/L305V/K337A/M298Q-FVII，K316Q/L305V/K337A/E296V-FVII，K316Q/L305V/K337A/V158D-FVII，

K316Q/L305V/V158D/M298Q-FVII，K316Q/L305V/V158D/E296V-FVII，

K316Q/L305V/V158T/M298Q-FVII，K316Q/L305V/V158T/E296V-FVII，

K316Q/L305V/E296V/M298Q-FVII，K316Q/L305V/V158D/E296V/M298Q-FVII，

K316Q/L305V/V158T/E296V/M298Q-FVII，K316Q/L305V/V158T/K337A/M298Q-FVII，

K316Q/L305V/V158T/E296V/K337A-FVII，K316Q/L305V/V158D/K337A/M298Q-FVII，

K316Q/L305V/V158D/E296V/K337A-FVII，K316Q/L305V/V158D/E296V/M298Q/K337A-FVII，

K316Q/L305V/V158T/E296V/M298Q/K337A-FVII，F374Y/K337A-FVII，F374Y/V158D-FVII，

F374Y/E296V-FVII，F374Y/M298Q-FVII，F374Y/V158T-FVII，F374Y/S314E-FVII，

F374Y/L305V-FVII，F374Y/L305V/K337A-FVII，F374Y/L305V/V158D-FVII，

F374Y/L305V/E296V-FVII，F374Y/L305V/M298Q-FVII，F374Y/L305V/V158T-FVII，

F374Y/L305V/S314E-FVII，F374Y/K337A/S314E-FVII，F374Y/K337A/V158T-FVII，

F374Y/K337A/M298Q-FVII，F374Y/K337A/E296V-FVII，F374Y/K337A/V158D-FVII，

F374Y/V158D/S314E-FVII，F374Y/V158D/M298Q-FVII，F374Y/V158D/E296V-FVII，

F374Y/V158T/S314E-FVII，F374Y/V158T/M298Q-FVII，F374Y/V158T/E296V-FVII，

F374Y/E296V/S314E-FVII，F374Y/S314E/M298Q-FVII，F374Y/E296V/M298Q-FVII，

F374Y/L305V/K337A/V158D-FVII，F374Y/L305V/K337A/E296V-FVII，

F374Y/L305V/K337A/M298Q-FVII，F374Y/L305V/K337A/V158T-FVII，

F374Y/L305V/K337A/S314E-FVII，F374Y/L305V/V158D/E296V-FVII，

F374Y/L305V/V158D/M298Q-FVII，F374Y/L305V/V158D/S314E-FVII，

F374Y/L305V/E296V/M298Q-FVII，F374Y/L305V/E296V/V158T-FVII，

F374Y/L305V/E296V/S314E-FVII，F374Y/L305V/M298Q/V158T-FVII，

F374Y/L305V/M298Q/S314E-FVII，F374Y/L305V/V158T/S314E-FVII，

F374Y/K337A/S314E/V158T-FVII，F374Y/K337A/S314E/M298Q-FVII，

F374Y/K337A/S314E/E296V-FVII，F374Y/K337A/S314E/V158D-FVII，

F374Y/K337A/V158T/M298Q-FVII，F374Y/K337A/V158T/E296V-FVII，

F374Y/K337A/M298Q/E296V-FVII，F374Y/K337A/M298Q/V158D-FVII，

F374Y/K337A/E296V/V158D-FVII，F374Y/V158D/S314E/M298Q-FVII，

F374Y/V158D/S314E/E296V-FVII，F374Y/V158D/M298Q/E296V-FVII，

F374Y/V158T/S314E/E296V-FVII，F374Y/V158T/S314E/M298Q-FVII，

F374Y/V158T/M298Q/E296V-FVII，F374Y/E296V/S314E/M298Q-FVII，

F374Y/L305V/M298Q/K337A/S314E-FVII，F374Y/L305V/E296V/K337A/S314E-FVII，

F374Y/E296V/M298Q/K337A/S314E-FVII，F374Y/L305V/E296V/M298Q/K337A-FVII，

F374Y/L305V/E296V/M298Q/S314E-FVII，F374Y/V158D/E296V/M298Q/K337A-FVII，

F374Y/V158D/E296V/M298Q/S314E-FVII，F374Y/L305V/V158D/K337A/S314E-FVII，

F374Y/V158D/M298Q/K337A/S314E-FVII，F374Y/V158D/E296V/K337A/S314E-FVII，

F374Y/L305V/V158D/E296V/M298Q-FVII，F374Y/L305V/V158D/M298Q/K337A-FVII，

F374Y/L305V/V158D/E296V/K337A-FVII，F374Y/L305V/V158D/M298Q/S314E-FVII，

F374Y/L305V/V158D/E296V/S314E-FVII，F374Y/V158T/E296V/M298Q/K337A-FVII，

F374Y/V158T/E296V/M298Q/S314E-FVII，F374Y/L305V/V158T/K337A/S314E-FVII，

F374Y/V158T/M298Q/K337A/S314E-FVII，F374Y/V158T/E296V/K337A/S314E-FVII，

F374Y/L305V/V158T/E296V/M298Q-FVII，F374Y/L305V/V158T/M298Q/K337A-FVII，

F374Y/L305V/V158T/E296V/K337A-FVII，F374Y/L305V/V158T/M298Q/S314E-FVII，

F374Y/L305V/V158T/E296V/S314E-FVII，F374Y/E296V/M298Q/K337A/V158T/S314E-FVII，

F374Y/V158D/E296V/M298Q/K337A/S314E-FVII，

F374Y/L305V/V158D/E296V/M298Q/S314E-FVII，F374Y/L305V/E296V/M298Q/V158T/S314E-FVII，F374Y/L305V/E296V/M298Q/K337A/V158T-FVII，

F374Y/L305V/E296V/K337A/V158T/S314E-FVII，F374Y/L305V/M298Q/K337A/V158T/S314E-FVII，F374Y/L305V/V158D/E296V/M298Q/K337A-FVII，

F374Y/L305V/V158D/E296V/K337A/S314E-FVII，F374Y/L305V/V158D/M298Q/K337A/S314E-FVII，F374Y/L305V/E296V/M298Q/K337A/V158T/S314E-FVII，

F374Y/L305V/V158D/E296V/M298Q/K337A/S314E-FVII，S52A-Factor VII，S60A-Factor VII；

R152E-Factor VII，S344A-Factor VII，T106N-FVII，K143N/N145T-FVII，V253N-FVII，

R290N/A292T-FVII, G291N-FVII, R315N/V317T-FVII, K143N/N145T/R315N/V317T-FVII; With

And the FVII that there is replacement in aminoacid sequence 233Thr to 240Asn, increase or lack;

The FVII that there is replacement in aminoacid sequence 304Arg to 329Cys, increases or lack; And the FVII that there is replacement in aminoacid sequence 153Ile to 233Arg, increase or lack.

Glycoprotein is FVIII polypeptide in one embodiment.In one embodiment, glycoprotein is FIX polypeptide.

other aspects of the present invention

The present invention is also relevant with the new modified proteins with following general formula:

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(general formula I)

Wherein M and optional M ' are the polymeric part that increases modified proteins molecular weight independently, wherein L and L ' represent divalence joint independently, wherein P represents glycoprotein, the one or more oxidized dextran end that described glycoprotein comprises glycoprotein, O, N, C and H represent respectively oxygen, nitrogen, carbon and hydrogen atom, n is 1-10, and m is 0-50, and wherein said modified proteins is with respect to initial glycoprotein P ^*there is the pharmacological properties of improvement and keep its functionally active.

Described modified proteins in certain embodiments of the invention is selected from FVII, FVIII, FIX,, FX, FII, FV, PROTEIN C, Protein S, tPA, PAI-1, tissue factor, FXI, FXII, FXIII and sequence variants thereof, immunoglobulin (Ig), cytokine as interleukin, α-, β-, gamma-interferon, G CFS comprise granulocytosis colony stimulative factor, fibroblast growth factor, Thr6 PDGF BB, Phospholipid hydrolase activator (PUP).

Other modified proteins are modified protein and the peptides with general biology and therapeutics importance, for example, comprise Regular Insulin, vegetable-protein is as Sugar receptors, ricin, tumour necrosis factor and relevant allelotrope, the Tumor Necrosis Factor Receptors of solubilized form, the interleukin-2-receptor of interleukin and solubilized form, somatomedin is tissue growth factor for example, as TGFa ' s or TGFps and Urogastron, hormone, somatomedin, erythropoietin, pigment hormone, hypothalamic releasing factor, vassopressin, prolactin, chorionic-gonadotropin hormone, follicle stimulating hormone, thyrotropic hormone, tissue plasminogen activator and immunoglobulin (Ig) are as IgG, IgE, IgM, , IgA, IgD and its fragment.

Modified proteins is that N-is glycosylated and/or O-is glycosylated and/or contains saliva acidic group in an embodiment of the invention.

The pharmacological properties that modified proteins has an improvement is in an embodiment of the invention selected from the bioavailability of raising, plasma half-life in plasma half-life, the immunogenicity of reduction, the function Half-life in vivo of the albumin avidity of the protease resistant of raising, raising, the receptor affinity of improvement, the stability in storage of raising, shortening, the body of shortening in the body of the function Half-life in vivo of prolongation, prolongation.Pass through in one embodiment M ' and using reduction or elimination renal clearance and/or M as the group prolong half-life of sheltering liver receptor binding site as the group that increases molecular weight.By M, as blocking antibody and immunogenic site conjugated group, reduce immunogenicity in one embodiment.In one embodiment, by M, as group albumin to high-affinity, improve albuminous avidity.In one embodiment, by M, as the group with target cell surface receptor specific binding, improve the avidity to acceptor.

In an embodiment of the invention, the M group of modified proteins is selected from: the organic charged groups of lower molecular weight, and it can contain one or more carboxylic acids, amine, sulfonic acid, phosphonic acids or its combination; Lower molecular weight neutral hydrophilic molecule, for example Polyethylene Chain of cyclodextrin or optional branching; Low-molecular-weight lipophilic molecule, as lipid acid or cholic acid or derivatives thereof; Molecular-weight average is the polyoxyethylene glycol of 2-40kDa; Well-defined accurate polymkeric substance, as the definite molecular weight ranges dendritic macromole that is 700Da to 20kDa; Substantially the polypeptide of non-immunogenic, as albumin, antibody or optionally contain the part of the antibody of Fc-structural domain; With high molecular organic polymer.

In modified proteins, M is selected from dendritic macromole in an embodiment of the invention, polyoxyalkylene (PAO), comprise polyalkylene glycol (PAG), as polyoxyethylene glycol (PEG) and polypropylene glycol (PPG), branching PEG, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyethylene-copolymerization-maleic anhydride, polystyrene-copolymerization-maleic anhydride, dextran, Sensor Chip CM 5, HES, MPC and PHF.

In modified proteins, M is selected from serum protein binding partner and contains the organic molecule that changes the part of charge property under physiological condition in an embodiment of the invention, the structure that suppresses glycan and receptors bind, and the middle substituent of prevention glycan specific recognition.

In modified proteins of the present invention, P is selected from FVII, FVIII, FIX, FX, FII, FV, PROTEIN C, Protein S, tPA, PAI-1, tissue factor, FXI, FXII, FXIII and sequence variants thereof in one embodiment, immunoglobulin (Ig), cytokine is as interleukin, α-, β-and gamma-interferon, G CFS, comprise granulocytosis colony stimulative factor, fibroblast growth factor, Thr6 PDGF BB, Phospholipid hydrolase activator (PUP), Regular Insulin, vegetable-protein is as lectin and ricin, tumour necrosis factor and relevant allelotrope, the Tumor Necrosis Factor Receptors of solubilized form, the interleukin-2-receptor of interleukin-2-receptor and solubilized form, somatomedin is tissue growth factor for example, as TGFa ' s or TGFps and Urogastron, hormone, somatomedin, erythropoietin, pigment hormone, hypothalamic releasing factor, vassopressin, prolactin, chorionic-gonadotropin hormone, follicle stimulating hormone, thyrotropic hormone, tissue plasminogen activator, with immunoglobulin (Ig) as IgG, IgE, IgM, IgA and IgD and fragment thereof, or any fusion rotein that contains any aforementioned albumen or its fragment.

The glycoprotein of modified proteins of the present invention is VII factor polypeptide in one embodiment.

The glycoprotein of modified proteins of the present invention has the aminoacid sequence of the wild-type people VII factor in one embodiment.

The specific activity that modified proteins of the present invention shows in conjunction with test at one or more CAs described in specification sheets of the present invention, proteolysis effects test or TF be in one embodiment unmodified VII factor polypeptide specific activity at least about 10%, as at least about 20%, as at least about 40%, as at least about 60%, as at least about 80%, as at least about 100%.

The bioavailability that modified proteins of the present invention shows be in one embodiment unmodified glycoprotein bioavailability at least about 110%, as at least about 120%, approximately 130% or be unmodified glycoprotein bioavailability at least about 140%.

The serum half-life that modified proteins of the present invention shows be in one embodiment unmodified sugared egg serum half-life at least about 125%, according to appointment 150%, approximately 200% or be at least about 250% of unmodified sugared egg serum half-life.

In one embodiment, the method for preparing modification glycosyl chemoattractant molecule comprises the step that further described glycosylation molecular preparation is become to pharmaceutical composition.

In one embodiment of the present invention, there is general formula (M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _mmodified proteins in m scope be 0-50, for example 0-20, for example 0-10, for example 0-5, for example 0-3.

There is general formula (M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _mmodified proteins in n be subject to the not reduction glycan end existing on concrete glycoprotein to count quantitative limitation.Therefore,, in one embodiment of the present invention, there is general formula (M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _mthe n of modified proteins equal not reduce in glycoprotein glycan end quantity.In another embodiment of the present invention, there is general formula (M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _mthe n scope of modified proteins be 1-10,1-5 for example, 1-3 for example, 1-2 for example, for example 1, for example 2, for example 3.

There is in one embodiment (M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _mthe modified proteins of (formula I) is selected from

Pharmaceutical composition

Another object of the present invention is to provide a kind of pharmaceutical composition that contains modified proteins, and its concentration is 10 ^-12mg/ml to 200mg/ml, for example 10 ^-10mg/ml to 5mg/ml and described composition have pH2.0 to 10.0.Said composition can further contain buffer system, sanitas, tonicity agents, sequestrant, stablizer and tensio-active agent.At one embodiment of the present invention Chinese traditional medicine composition, be aqueous composition, as the composition that contains water.Said composition is solution or suspension normally.Term " aqueous composition " refers to contain at least composition of 50%w/w water.Equally, term " aqueous solution " refers to contain at least solution of 50%w/w water, and term " aqueous suspension " refers to contain at least suspension of 50%w/w water.

Pharmaceutical composition is freeze-dried composition in another embodiment, and doctor or patient add solvent and/or thinner wherein before use.In another embodiment pharmaceutical composition be in use without any need for the drying composition dissolving in advance (as lyophilize or spraying dry).

The present invention further relates to the pharmaceutical composition that contains modified protein aqueous solution and buffer reagent again, described modified protein concentration range be 0.1-100mg/ml or more than, the pH of wherein said composition is approximately 2.0 to approximately 10.0.

The pH of composition is selected from 2.0 in yet another embodiment of the present invention, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 and 10.0.

In the further embodiment of the present invention, buffer reagent is selected from sodium acetate, sodium carbonate, Citrate trianion, glycyl peptide glycine, Histidine, glycine, Methionin, arginine, SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic, sodium phosphate and Trometamol, N, N-bis-(hydroxyethyl) glycine, three (methylol) methylglycine, oxysuccinic acid, succinate, toxilic acid, fumaric acid, tartrate, aspartic acid or its mixture.Each concrete buffer reagent has formed the selectable embodiment of the present invention.

In the further embodiment of the present invention, composition further contains pharmaceutically acceptable sanitas.In further embodiment of the present invention, sanitas is selected from phenol, ortho-cresol, meta-cresol, p-cresol, Tegosept M, propylben, 2-Phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethyl alcohol, phenylcarbinol, trichloro-butyl alcohol and Thiomersalate, bronopol, phenylformic acid, miaow urea, chlorhexidine, sodium dehydroacetate, parachlorometacresol, ethyl p-hydroxybenzoate, benzethonium chloride, chlorobenzene glycosides ether (3-to chlorobenzene Ethylene Oxide-1,2-glycol) or its mixture.In the further embodiment of the present invention, the concentration of sanitas is 0.1mg/ml to 20mg/ml.In the further embodiment of the present invention, the concentration of sanitas is 0.1mg/ml to 5mg/ml.In the further embodiment of the present invention, the concentration of sanitas is 5mg/ml to 10mg/ml.In the further embodiment of the present invention, the concentration of sanitas is 10mg/ml to 20mg/ml.Each concrete sanitas has formed the selectable embodiment of the present invention.In pharmaceutical composition, using sanitas is that those skilled in the art of the present technique are known.The reference being applicable to is Remington:The Science and Practice ofPharmacy, 20 ^thedition, 2000.

In the further embodiment of the present invention, composition further contains isotonic agent.Further embodiment isotonicity agent of the present invention is selected from salt (as sodium-chlor), sugar or sugar alcohol, amino acid (as L-glycine, L-Histidine, arginine, Methionin, Isoleucine, aspartic acid, tryptophane, Threonine), alditol (as glycerine (glycerol), 1,2-(dihydroxypropane) (propylene glycol), 1, ammediol, 1,3 butylene glycol), macrogol (as PEG400) or its mixture.Can use any sugar as single-, two-or polysaccharide or water-soluble glucan, comprise for example fructose, glucose, seminose, sorbose, wood sugar, maltose, lactose, sucrose, trehalose, dextran, amylopectin, dextrin, cyclodextrin, soluble starch, hydroxyethylamyle and Xylo-Mucine.Sugar additives is sucrose in one embodiment.Sugar alcohol is defined as has at least one-C of OH ₄-C ₈hydrocarbon, for example N.F,USP MANNITOL, sorbyl alcohol, inositol, galactitol, galactitol, Xylitol and arabitol.Sugar alcohol additive is seminose in one embodiment.Above-mentioned sugar or sugar alcohol may be used alone or in combination.For usage quantity, there is no fixed constraints, sugar or sugar alcohol are dissolved in liquid preparation, can not have a negative impact to the stabilising effect that uses method of the present invention to obtain.The about 1mg/ml of the concentration of Saccharide and saccharide alcohols is to about 150mg/ml in one embodiment.At the about 1mg/ml of concentration of the further embodiment isotonicity agent of the present invention to about 50mg/ml.At the about 1mg/ml of concentration of the further embodiment isotonicity agent of the present invention to about 7mg/ml.At the about 8mg/ml of concentration of the further embodiment isotonicity agent of the present invention to about 24mg/ml.At the about 25mg/ml of concentration of the further embodiment isotonicity agent of the present invention to about 50mg/ml.Each concrete isotonic agent has formed the selectable embodiment of the present invention.In pharmaceutical composition, using isotonic agent is that those skilled in the art of the present technique are known.The reference being applicable to is Remington:The Science and Practice of Pharmacy, 20 ^thedition, 2000.

In the further embodiment of the present invention, composition further contains sequestrant.In further embodiment of the present invention, sequestrant is selected from ethylenediamine tetraacetic acid (EDTA) (EDTA), citric acid and aspartic acid and its mixture.In the further embodiment of the present invention, sequestrant concentration is 0.1mg/ml to 5mg/ml.In the further embodiment of the present invention, sequestrant concentration is 0.1mg/ml to 2mg/ml.In the further embodiment of the present invention, sequestrant concentration is 2mg/ml to 5mg/ml.Each concrete sequestrant has formed the selectable embodiment of the present invention.In pharmaceutical composition, using sequestrant is that those skilled in the art of the present technique are known.The reference being applicable to is Remington:TheScience and Practice of Pharmacy, 20 ^thedition, 2000.

In the further embodiment of the present invention, composition further comprises stablizer.In pharmaceutical composition, using stablizer is that those skilled in the art of the present technique are known.The reference being applicable to is Remington:The Science and Practice of Pharmacy, 20 ^thedition, 2000.

More specifically, the present composition is stable composition of liquid medicine, and its therapeutic activity component is included in composition of liquid medicine may have aggregate to form albumen between the shelf lives.The physical action that " aggregate " forms between finger protein molecule forms oligopolymer, and this oligopolymer may be still solvable, or be a large amount of visible aggregations, in solution, precipitates." between the shelf lives " once refer to composition of liquid medicine or composition preparation after not by immediately for patient.More definite, after preparation, composition with liquid form, freezing state or after from new formation liquid form or other dried forms packing and storing that is suitable for using to patient." dried forms " refers to that composition of liquid medicine or composition adopt lyophilize (as lyophilization; Referring to for example Williams and Polli (1984) J.Parenteral Sci.Technol.38:48-59), spraying dry (referring to Masters (1991), Spray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez, U.K.), pp.491-676; The people such as Broadhead. (1992) Drug Devel.Ind.Pharm.18:1169-1206; With people such as Mumenthaler. (1994) Pharm.Res.11:12-20), or seasoning (Carpenter and Crowe (1988) Cryobiology 25:459-470; And Roser (1991) Biopharm.4:47-53) dry.The composition of liquid medicine aggregate form that albumen forms between the shelf lives can have a negative impact to the biological activity of albumen, causes the reduction of medicine composite for curing activity.Further, the formation of aggregate can cause other problems, while passing through infusion system administration as the pharmaceutical composition when containing albumen, causes tubing, film or pump to block.

Pharmaceutical composition of the present invention further contains many amino acid bases and is enough to reduce the aggregate that albumen causes during composition stores and forms." amino acid base " refers to amino acid or aminoacid mixture, and wherein any amino acid exists with the form of its free alkali form or its salt.While using aminoacid mixture, all amino acid exists with its free alkali form, or all with the form of its salt, exists, or a part ofly with its free alkali form, exists and the form of its salt of another part exists.In one embodiment, for the preparation of the amino acid of the present composition, there is charged side chain, as arginine, Methionin, aspartic acid and L-glutamic acid.When concrete amino acid or organic bases exist with free alkali form or its salt form, concrete any steric isomer (as L or D isomer or its mixture) of amino acid (methionine(Met), Histidine, arginine, Methionin, Isoleucine, aspartic acid, tryptophane, Threonine or its mixture) or the mixture of its steric isomer or glycine or organic bases may reside in pharmaceutical composition of the present invention such as but not limited to imidazoles.In one embodiment, use amino acid L-steric isomer.In one embodiment, use amino acid D-steric isomer.The present composition can be with these amino acid whose analog formulations." amino acid analogue " refers to the natural amino acid whose derivative that exists, and can between the shelf lives, produce at composition of liquid medicine of the present invention and reduce the effect that aggregate that protein causes produces.Suitable arginine analog comprises for example mono-ethyl L-arginine of aminoguanidine, ornithine and N-, and suitable methionine(Met) analogue comprises ethionine and fourth methyllanthionine, and suitable cysteine analogs comprises S-methyl-Cys.The same with other amino acid, can be used for the amino acid analogue of composition with the form of its free alkali form or its salt.The concentration that in the further embodiment of the present invention, amino acid or amino acid analogue are used is enough to stop or delay the gathering of albumen.

In the further embodiment of the present invention, when the protein as medicine is the protein of the methionine residue that contains at least one easy oxidated impact, can adds methionine(Met) (or other sulphur amino acid or amino acid analogues) to suppress methionine residue and be oxidized to methionine(Met)." inhibition " refers to methionine(Met) oxide compound lowest accumulated in time.Suppressing methionine(Met) oxidation causes protein to retain to a greater extent its normal molecular form.Can use any steric isomer or its any mixture of methionine(Met) (L or D isomer).Add-on should be the amount that is enough to suppress methionine residue oxidation, and the amount of methionine(Met) oxysulfide is that regulating effect institute is receptible like this.Concrete, refer to contain and be not more than approximately 10% to approximately 30% methionine(Met) oxysulfide in composition.Conventionally, can be by adding methionine(Met) to realize, the methionine(Met) proportional range that adds methionine residue is approximately 1: 1 to approximately 1000: 1, for example 10: 1 to approximately 100: 1.

In further embodiment of the present invention, composition further comprises stablizer, is selected from high-molecular weight polymer or low-molecular weight compound.In the further embodiment of the present invention, stablizer is selected from polyoxyethylene glycol (as PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxyl/hydroxylated cellulose or derivatives thereof (as HPC, HPC-SL, HPC-L and HPMC), cyclodextrin, S-contained substance as thioglycerin, Thiovanic acid, 2-methyl mercapto ethanol and different salt (as sodium-chlor).Each concrete stablizer has formed the selectable embodiment of the present invention.

Pharmaceutical composition also can contain additional stability agent, further increases the wherein stability of therapeutic activity albumen.The concrete useful stablizer of the present invention comprises but is not limited to methionine(Met) and EDTA, can avoid methionine(Met) oxygenizement for protected protein, and nonionogenic tenside, the gathering of can protected protein matter avoiding freeze thawing or mechanical stirring to cause.

In further embodiment of the present invention, composition further comprises tensio-active agent.In the further embodiment of the present invention, tensio-active agent is selected from sanitising agent, ethoxy castor oil, polyglycolyzed glyceride, acetylize monoglyceride, sorbitan aliphatic ester, polyoxypropylene-polyoxyethylene blocks polymkeric substance (if poloxamer is as Pluronic f68, poloxamer 188 and 407, Triton X-100), polyoxyethylene sorbitan aliphatic ester, polyoxyethylene and polyoxyethylene deriv are as alkylation and alkoxy derivative (tween, as tween 20, Tween-40, tween-80 and Brij-35), monoglyceride or its ethoxylated derivative, triglyceride or its polyoxyethylene deriv, alcohols, glycerine, Sugar receptors and phospholipid are (as phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositols, diphosphatidylglycerol and, lipid sphyngomyelin), phospholipid derivative (as two palmityl palmityls) and lysophospholipid are (as palmityl lysophospholipid-Serine and thanomin, choline, Serine or Threonine 1-acyl group-sn-glycerol-3-phosphate ester) and the alkyl of lysophospholipid and GranulestinLecithin, alkoxyl group (alkyl ester), alkoxyl group (alkyl oxide) derivative, as lysophosphatidylcholine, the lauroyl of dipalmitoyl phosphatidylcholine and mnyristoyl derivative and polar head group modifier, i.e. choline, thanomin, phosphatidic acid, Serine, Threonine, glycerine, inositol, DODAC with lotus positive electricity, DOTMA, DCP, BISHOP, lysophosphoglyceride and lysophospholipid Threonine and glyceryl phosphatide (as kephalin), glyceroglycolipid (as galactopyranoside), sphingoglycolipid is (as ceramide, ganglioside), lauryl phosphorylcholine, chicken lysolecithin, fusidic acid derivatives (as taurine dihydrofusin sodium etc.), C ₆-C ₁₂the N of longer chain fatty acid and its salt (as oleic acid and sad), acylcarnitines and derivative, Methionin, arginine or Histidine ^αthe N of the dipeptides of-acylated derivatives or Methionin or arginic side chain acylated derivatives, the Methionin that contains any associating, arginine or Histidine and neutral amino acids ^αthe N of-acylated derivatives, the neutral amino acids that contains any associating and two charged amino acid whose tripeptides ^α-acylated derivatives, DSS (Docusate Sodium, CAS registration number [577-11-7]), dioctyl calcium sulfosuccinate, CAS registration number [128-49-4]), docusate potassium, CAS registration number [7491-09-0]), SDS (Sodium Lauryl Sulphate BP/USP or sodium lauryl sulphate), Sodium octoate, cholic acid or derivatives thereof, bile acide and its salt and glycine or taurine conjugate, ursodesoxycholic acid, Glycocholate sodium, sodium deoxycholate, Taurocholic acid sodium salt, NaGC, N-hexadecyl-N, N-dimethyl-3-ammonium-1-propyl sulphonate, negatively charged ion (alkyl-aryl-sulfonic acid ester) schedule of rates surface-active agent, zwitterionics (as N-alkyl-N, N-dimethylammonio-1-propane sulfonic acid ester, 3-alcohol amido-1-propyl-dimethyl ammonium-1-propane sulfonic acid ester, cats product (quaternary ammonium hydroxide) is (as CETRIMIDE POWDER, cetylpyridinium chloride), nonionogenic tenside (as dodecyl β-D-glucopyranoside), poloxamines (as Tetronic TR 913R), it is derived from the four function group block copolymers that add continuously propylene oxide and oxyethane in aminophylline, maybe can be selected from tensio-active agent or its mixture of imidazolidine derivatives.Each concrete tensio-active agent has formed the selectable embodiment of the present invention.

In pharmaceutical composition, using tensio-active agent is that those skilled in the art of the present technique are known.The reference being applicable to is Remington:The Science and Practice of Pharmacy, 20 ^thedition, 2000.

In pharmaceutical composition of the present invention, also can add other compositions.The composition of described interpolation comprises wetting agent, emulsifying agent, antioxidant, weighting agent, tonicity contributor, sequestrant, metal ion, oiliness carrier, protein (as human serum albumin, gelatin or protein) and zwitter-ion (if amino acid is as trimethyl-glycine, taurine, arginine, glycine, Methionin and Histidine).This added ingredients should be unable to have a negative impact to the resistance to overturning of pharmaceutical composition of the present invention certainly.

The pharmaceutical composition that contains modified proteins of the present invention can be administered to the patient that need to treat at different sites, for example outside, as skin and mucous membrane, the position that bypass absorbs, as artery, vein, heart administration, the position of absorption, as intracutaneous, subcutaneous, intramuscular or intraperitoneal administration.

Using of pharmaceutical composition of the present invention can be by different route of administration, as by tongue, hypogloeeis, cheek, mouthful in, oral cavity, stomach and intestine, nose, lung, for example by bronchiole and alvei or its both, epidermis, corium, through skin, vagina, rectum, eye, for example by conjunctiva, ureter and parenteral, to patient, use this treatment.

The present composition can be with different dosage form administration, solution for example, suspension, emulsion, micro emulsion, multiple emulsion, foam, paste, paste, plaster, ointment, tablet, coated tablet, lotion, capsule, for example hard gelatin capsule and soft gelatin capsule, suppository, rectal capsule, drops, gelifying agent, sprays, pulvis, gaseous solvents, inhalation, eye drops, eye ointment, eye wass, vaginal suppository, pesseulum, vagina ointment, injection liquid, converted in-situ is solution, for example in-situ gelling effect, original position is adjusted, in-situ precipitate, in-situ crystallization, infusion solution and implant.

Composition of the present invention can be further by covalency, hydrophobic and electrostatic interaction is mixed or medication carrier, drug delivery system and advanced drug delivery system with the stability that further strengthens, improve bioavailability, increase solvability, reduce side effect, reach the known chronotherapy of those skilled in the art of the present technique, strengthen patient's compliance or its combination.Carrier, the example of drug delivery system and advanced drug delivery system includes but not limited to polymkeric substance, as Mierocrystalline cellulose and derivative, polysaccharide, as dextrin and derivative, starch and derivative, poly-(vinyl alcohol), acrylate and methacrylic acid copolymer, poly(lactic acid) and polyglycolic acid and its segmented copolymer, polyoxyethylene glycol, carrier proteins, albumin for example, gel, heat-sensitive gel system for example, block copolymerization system well-known in the art for example, meagre profit, liposome, microballoon, nano particle, liquid crystal and its dispersion, L2 phase and its dispersion, phase behavior in fat water system well-known in the art, polymer particulate, multiple emulsion, self-emulsifying, self-emulsifying microemulsion, cyclodextrin and its derivative, and dendritic macromole.

The present composition carrys out pulmonary administration modified proteins for solid, semisolid, powder, liquid composite, uses for example metered dose inhaler, powder inhaler and atomizer, and all devices are that those skilled in the art of the present technique are known.

The present composition is specifically for controlled release, sustained release, prolongation release, slowbreak and Atrigel.More specifically but be not limited to, those skilled in the art of the present technique are known, and composition is for parenteral controlled release and sustained release system (all systems cause administration number of times to reduce many times).More concrete, controlled release and sustained release system subcutaneous administration.Be not subject to the restriction of the scope of the invention, effectively controlled release system and composition are hydrogel, oleogel, liquid crystal, polymer particulate, microballoon and nano particle.

The method for present composition controlled release system of producing includes but not limited to crystallization, condensation, cocrystallization, precipitation, co-precipitation, emulsification, dispersion, high pressure homogenize, encapsulated, spraying is dry, micro encapsulation, condense, be separated, evaporating solvent becomes microballoon, extrude and SCF process.Common reference drug controlled release handbook (Wise, D.L., ed.Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol.99:ProteinComposition and Delivery (MacNally, E.J., ed.Marcel Dekker, New York, 2000).

Parenteral admistration can be passed through syringe, and optional pen-type injection device is through subcutaneous, intramuscular, intraperitoneal or passages through which vital energy circulates injection.Selectable, parenteral admistration can be passed through infusion pump.Further select is that solution or suspension composite are used modified proteins with the form of nose or lung spraying.Further select, the pharmaceutical composition that contains modified proteins of the present invention also can percutaneous dosing, as by the separated syringe of syringe needle or by patch, and optional iontophoresis patch or through mucous membrane, as cheek administration.

Term " stable composition " refers to that composition has the physics and chemistry stability of the physical stability of increase, the chemical stability of increase or increase.

" physical stability " of term protein composition refer to due to albumen is exposed to thermal-mechanical stress and/or with interface and surface interaction lose stable, as hydrophobic interface and surface, the lifeless matter actives of Protein formation protein and/or do not allow aggregate.The physical stability of moisture protein composition is exposed under machinery/physical stress (as stirred) by visual inspection or turbidity inspection and evaluation in different temperature and different time after composition being loaded on to appropriate containers (as cartridge case or phial).The visual inspection of composition carries out in the focused light of black background.The turbidity of composition represents by vision scoring, show muddy degree as scope be 0 to 3 (composition without muddiness corresponding to vision scoring 0, composition under daylight vision muddiness corresponding to vision scoring 3).When composition occurs that under daylight vision is muddy, composition is classified as the physical instability relevant with protein aggregation.Selectable, the turbidity of composition can be used the known simple turbidity measuring method assessment of those skilled in the art of the present technique.The physical stability of moisture protein composition also can be assessed with reagents for spectrometry or by the configuration states of probe survey albumen.The preferred small molecules of probe is preferentially bonded to the non-natural configurational isomer of albumen.The example of the small molecules spectral probe of protein structure is thioflavin T.Thioflavin T is the fluorescence dye that is widely used in detecting amyloid fibril.When fibril or other albumen configurations exist, thioflavin T, when when fibril albumen form is combined, causes new excitation maximum at 450nm, causes the transmitting value of enhancing at about 482nm.Unconjugated thioflavin T substantially at this wavelength without fluorescence.The small molecules that other protein structures change between natural and non-natural state can be used as probe.For example " hydrophobic macular area " probe is preferentially bonded to the hydrophobic macular area that albumen exposes.Hydrophobic macular area is imbedded the tertiary structure of native state albumen conventionally, but comes out when expansion or sex change when albumen.These micromolecular examples, spectral probe is aromatic series hydrophobic dye, as anthracene, acridine, ferrosin etc.Other spectral probe is metal-aminoacid inner complex, and the cobalt metallo-chelate of hydrophobic amino acid for example, as phenylalanine, leucine, Isoleucine and α-amino-isovaleric acid etc.

The chemical covalency variation of " chemical stability " finger protein structure of term protein composition causes forming chemical degradation product, and this product may reduce biological activity and/or may increase immunogenicity than native protein structure.The difference of chemical degradation product depends on the environment of native protein kind and character and exposure thereof.As known in those skilled in the art of the present technique, the elimination thing of chemical degradation can not be avoided completely, and the increasing of chemical degradation product quantity appears at that protein composition stores and between the usage period conventionally.Most protein is easy to desamidation, and in this process, the hydrolysis of the amide side chain group of glutamyl or asparaginyl residue forms free carboxy acid.Other degradation pathway cause the formation of high molecular products of metamorphism, wherein two or more protein moleculars cause forming covalently bound dimer, oligopolymer and polymer degradation products (Stability of Protein Pharmaceuticals by transmidation and/or the mutual covalent attachment of disulphide effect, Ahern.T.J. & Manning M.C., Plenum Press, New York 1992).Oxidation (for example methionine residue) is another kind of chemical degradation.The chemical stability of protein composition can be assessed by being exposed to the rear quantity of measuring different time points chemical degradation product of varying environment condition (temperature that the formation of degraded product can for example be raise is conventionally accelerated).Use different chromatographic technique (as SEC-HPLC and/or RP-HPLC) thus according to molecular weight and/or charge measurement, distinguish the quantity that degraded product is measured various degraded products.

Therefore, as mentioned above, " stable composition " refers to have the composition of the physics and chemistry stability of the physical stability of raising, the chemical stability of raising or raising.Conventionally composition is used and between the shelf lives, should stablize (in accordance with use and the condition of storage of recommendation) before validity period arrives.The stable use of pharmaceutical composition of containing modified proteins in embodiments of the present invention is longer than 6 weeks, and stably stored is longer than 3 years.

Another embodiment Chinese traditional medicine composition stable of the present invention is used and is longer than 4 weeks, and stably stored is longer than 3 years.

The further embodiment Chinese traditional medicine of the present invention composition stable is used and is longer than 4 weeks, and stably stored is longer than 2 years.

The present invention further embodiment Chinese traditional medicine composition stable use is longer than 2 weeks, and stably stored is longer than 2 years.

All references, comprise that publication, patent application and patent are incorporated herein its full content as a reference, if each reference is that independent and concrete demonstration is incorporated herein by reference for same degree, at this, illustrate its all (at utmost allowed by law), no matter the concrete paragraph of proposition of any separation is introduced in other places.

The term of describing in content of the present invention " one " and " this " and similarly refer to be interpreted as comprising single and a plurality of, unless context is otherwise noted or clearly oppose.

Except as otherwise noted, in the corresponding approximation of these all definite value representations (as all routine value providing about specific factor or measurement can be considered to provide corresponding approximate measure, suitable being revised as " approximately ").

The term that any part of the present invention or embodiment are used in this description as " comprising ", " having " or " containing " refer to key element or composition for the similar part of the present invention that provides support or embodiment " by ... form ", " in essence by ... form ", " substantially containing " this key element or composition, unless context is otherwise noted or clearly oppose (composition contains element-specific and should be understood to describe by this elementary composition composition as described in this, unless context is otherwise noted or clearly oppose).

All titles and subtitle be only for convenient as used herein, and be not used as the restriction to any mode of the present invention.

Use at these any and all embodiment or the language (for example, as " ") of giving an example is intended to better describe the present invention, rather than limitation of the scope of the invention, Unless Otherwise Requested.In specification sheets, all language should be interpreted as showing to realize the element of the requisite any requirement of the present invention.

At this, quote as proof with referenced patents document and be only used to convenient and do not reflect any this patent documentation validity, patentability and/or enforceable viewpoint.

Embodiment

Abbreviation:

SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis

EDTA: ethylenediamine tetraacetic acid (EDTA)

The FVIIa:VIIa factor

The FVIII:VIII factor

The FIX:IX factor

Material, instrument and method

Purifying:

Protein purification carries out on ion exchange column (HiTrap Q HP, Amersham Bioscience) or size exclusion chromatography post (XK26/60 HiLoad Superdex 200, Amersham Bioscience), uses fPLC and FC-950 fraction collector (Amersham Bioscience).The equal constant temperature of elution buffer, post and fraction collector is in 5 ℃.

SDS-PAGE：

SDS-PAGE is used Invitrogen ^tMx cell Surelock ^tMmini cell system and the pre-solidifying 4-12% bisacrylamide-tromethane gel, NuPAGE MES SDS electrophoretic buffer, Mark 12 standard models and the NUPAGELDS sample buffer that according to Invitrogen standard test, design.According to Invitrogen experimental design, gel SimpleBlue ^tMdyeing.

Buffered soln:

GlyGly damping fluid: 25mM Gly-Gly, 50mM NaCl, 25mM CaCl ₂, pH6.0CaCl ₂free GlyGly damping fluid: 25mM Gly-Gly, 50mM NaCl, pH6.0Hepes damping fluid: 50mM hydroxyethyl piperazine ethanesulfonic acid, 100mM NaCl, 5mM CaCl ₂, 0,01% tween 80, pH=7.4

Embodiment

NaIO ₄process FVIIa and S2288 active

Correlative study NaIO ₄with the obvious dependency of the peptidolytic activity → nothing of residue.

Test I

The decomposition peptide of measuring FVIIa is active:

Use comes from Chromogenix, the chromogenic substrate S-2288 of Sweden (H-D-Ile-Pro-Arg-p-N-methyl-p-nitroaniline) detects peptidolytic activity.By following experimental design, undertaken: the S2288 stoste of preparation 10mM in Hepes damping fluid.Similarly prepare the tissue factor stoste of 200nM in Hepes damping fluid.The stoste of 200nM experiment product and related compound (as wild-type FVIIa) are prepared with Hepes damping fluid.Compound is tested with ELISA hole by three parts.In typical experiment, 10ul 200nM tissue factor stoste and 135ul Hepes damping fluid add in hand-hole.By adding the initiation reaction of 5ul 200nM S2288 stoste.With ELISA-reader, in room temperature (absorbancy is in 405nm), analyze continuously hole 15 minutes.By the rate that starts, calculate relative reactivity, contrast with the ratio of wild-type FVIIa.The activity of modification FVIIa analogue is expressed as the per-cent of wild-type FVIIa activity.

Embodiment 1

10K PEG-ONH ₂: 10K-SMB-PEG (1,00g; 0.1mmol; Nektar Inc) be dissolved in DCM (10ml).Add 4-(N-tertbutyloxycarbonyl aminooxy) butylamine (0.20g, 1mmol, by WO 2005014049A2 preparation), mixture stirs 16h in room temperature.Add ether (90ml), leach white precipitate.Repeat by DCM (10ml) dissolution precipitation and the step that adds ether (90ml).Then use DCM (6ml) redissolution sedimentable matter, add Amberlyst15 ion exchange resin (2,0g; First with the DCM solution of DCM and 10%EtOH, wash).Mixture, in stirring at room 30 minutes, then leaches resin, with DCM, fully washs.Merge DCM solution and be concentrated into small volume with Rotary Evaporators, then add ether (90ml) that product is separated out.DCM for product (6ml) dissolves, and adds TFA (6ml).Mixture was in stirring at room 1/2 hour.With ether (100ml), product is separated out, leach.DCM for product (5ml) redissolves, and adds triethylamine (1ml).Mixture stirs 5 minutes, and then with ether (100ml), product is separated out.Leach precipitation, with ether, wash twice, vacuum drying oven dried overnight.Yield: 475mg (46%). ¹h-NMR (CDCl ₃; Select peak):

δ1.25ppm(d，3H)；1.50-1.75(m，6H)；1.82(m，1H)；3.38(s，3H)；3,45-3,90(m，ca.900H).

Embodiment 2

Single 10K-PEG-FVIIa (0128-0000-1018-1A):

Be dissolved in 10ml 25mM Gly-Gly, 50mM NaCl, 25mM CaCl ₂, pH6.0 (GlyGly damping fluid) Factor VIIa (14mg, 0.28umol) add 100ul CaCl ₂20mM NaIO in free GlyGly damping fluid ₄10K-PEG-ONH in solution and 1000ul GlyGly damping fluid ₂solution (embodiment Isosorbide-5-Nitrae 2mg; 4.2umol; And shake once in a while 15 equivalents of the VIIa factor): mixture is placed in ice bath 2h.Then in reaction mixture, add the 500ul MeONH in GlyGly damping fluid (1M NaOH adjusts pH to 6.0) ₂.HCl solution (17mg; 0,21mmol).Mixture is placed in ice bath 10min again.Then to add in reaction mixture the 100mM EDTA cold soln in bis-generations (4,5ml), maintain pH lower than 9.0.Then with 100ul 1N HCl solution, adjust pH to 8.0.

Ion-exchange chromatography:

With ion-exchange chromatography, remove unnecessary PEG-ONH ₂.Cooling reaction mixture is loaded on 5ml HiTrap Q ion exchange column (Amersham Bioscience), and this exchange column is used 25mM GlyGly, 50mM NaCl, pH8.0 balance in advance.With 25mM GlyGly, 50mM NaCl, pH8.0 (buffer A) wash-out, surpass 10cv; Then use 25mM GlyGly, 50mM NaCl, 25mM CaCl ₂, pH8.0 (buffer B) wash-out surpasses 15cv, constant current (1ml/min) constant temperature (5 ℃).Effluent is monitored at 280nm by absorbancy.

Size exclusion chromatography:

In 5 ℃, on Superdex 200, use fPLC and Frac-950 (AmershamBioscience) remove the not FVIIa of PEGization (Pegylated) from the Glycopegylated FVIIa variant reclaiming in HiTrap Q mixed fraction by size exclusion chromatography.XK26/60HiLoadSuperdex 200 posts (320ml cv) pre-balances (10cv), use 25mM GlyGly, 50mM NaCl, 25mM CaCl after dress post ₂, pH6.0 is with flow velocity 2.5ml/min wash-out.Effluent liquid 280nm absorbance detection.Collect 10ml cut.According to the description of Invitrogen, SDS-PAGE for cut (4-12%Bis-Tris NuPAGE gels) analyzes, with Simple Blue dyeing.Merge the cut that contains pure mono-pegylated FVIIa, in 4000rpm, 10 ℃ are passed through AmiconUltra ^tM-15 (10K MWCO) centrifugal filter filtering and concentrating 13.5 minutes, final volume is 4.1ml.

UV measuring method for protein concentration (is used E _280nm=1.32ml/mg/cm) be determined as 0.29mg/ml, it equals 0.35mg/ml FVIIa-10K PEG.Peptidolytic activity (S2288 test) is 79% with respect to wt FVIIa.

Embodiment 3

High (HS) and low replacement (LS) 10K-PEG-FVIIa of replacing of preparation

Be dissolved in 10ml 25mM Gly-Gly, 50mM NaCl, 25mM CaCl ₂, pH6.0 (GlyGly damping fluid) factor VIIa (14mg, 0.28umol) in add 100ul CaCl ₂20mM NaIO in free GlyGly damping fluid ₄10K-PEG-ONH in solution and 1000ul GlyGly damping fluid ₂solution (embodiment Isosorbide-5-Nitrae 2mg; 4.2umol; 15 equivalents of the VIIa factor): mixture is placed in 4 ℃ of 24h, and shakes once in a while.Then in reaction mixture, add the MeONH in 500ul GlyGly damping fluid (1M NaOH adjusts pH to 6.0) ₂.HCl solution (17mg; 0,21mmol).Mixture is placed in ice bath 10min.Then to add in reaction mixture 100mM bis-generations EDTA cold soln (4,5ml), maintain pH lower than 9.0.Then with 60ul 1N HCl solution, adjust pH to 8.0.

Ion-exchange chromatography:

With ion-exchange chromatography, remove unnecessary PEG-ONH ₂.Cooling reaction mixture is loaded on 5ml HiTrap Q ion exchange column (Amersham Bioscience), and this exchange column is used 25mM GlyGly, 50mM NaCl, pH8.0 balance in advance.With 25mM GlyGly, 50mM NaCl, pH8.0 (buffer A) wash-out, surpass 10cv; Then use 25mM GlyGly, 50mM NaCl, 25mM CaCl ₂, pH8.0 (buffer B) wash-out surpasses 15cv, constant current (1ml/min) constant temperature (5 ℃).Effluent is monitored by absorbancy at 280nm.

Size exclusion chromatography:

In 5 ℃ with fPLC and Frac-950 (Amersham Bioscience) remove the not FVIIa of PEGization by size exclusion chromatography on Superdex 200 from the Glycopegylated FVIIa variant reclaiming in HiTrap Q mixed fraction.XK26/60HiLoad Superdex200 post (320ml cv) pre-balance (10cv), uses 25mM GlyGly, 50mMNaCl, 25mM CaCl after dress post ₂, pH6.0 is with flow velocity 2.5ml/min wash-out.Effluent liquid is in 280nm absorbance detection.Collect 10ml cut.According to the description of Invitrogen, SDS-PAGE for cut (4-12%Bis-Tris NuPAGE gels) analyzes, with Simple Blue dyeing.According to PEG substitution level mixed fraction, in 4000rpm, 10 ℃ by Amicon Ultra ^tM-15 (10K MWCO) centrifugal filter filtering and concentrating 13.5 minutes.

HS (tri/tetra 10K PEG) FVIIa:0.92mg albumen (UV _280nm)/ml, cumulative volume=1.6ml.

HS (di/tri 10K PEG) FVIIa:0.63mg albumen (UV _280nm)/ml, cumulative volume=1.4ml.

LS (mono/di 10K PEG) FVIIa:1.2mg albumen (UV _280nm)/ml, cumulative volume=1.4ml.

Single 10K PEG FVIIa:0.7mg albumen (UV _280nm)/ml, cumulative volume=2.3ml.

Embodiment 4

High (HS) and low replacement (LS) 40K-PEG-FVIIa of replacing of preparation

Be dissolved in 10ml 25mM Gly-Gly, 50mM NaCl, 25mM CaCl ₂, pH6.0 (GlyGly damping fluid) the VIIa factor (14mg, 0.28umol) in add 100ul CaCl ₂20mM NaIO in free GlyGly damping fluid ₄40K-PEG-ONH in solution and 1500ul GlyGly damping fluid ₂solution ((20k PEG) OCH ₂(20K PEG) OCHCH ₂oC (=O) O-CH ₂cH ₂oNH ₂; WO 2006042847,112mg; 2.8umol; Be equivalent to the VIIa factor 10 equivalents).Mixture is placed in 4 ℃ of 48h, and shakes once in a while.In reaction mixture, add the MeONH in 500ul GlyGly damping fluid ₂.HCl solution (17mg; 0,21mmol), then with 1N NaOH, adjust pH to 6.0.Mixture is placed in ice bath 10min.Then to add in reaction mixture 100mM bis-generations EDTA cold soln (4,5ml), maintain pH lower than 9.0.Then with 60ul 1N HCl solution, adjust pH to 8.0.

Ion-exchange chromatography:

With ion-exchange chromatography, remove unnecessary PEG-ONH ₂.Cooling reaction mixture is loaded on 5ml HiTrap Q ion exchange column (Amersham Bioscience), and this exchange column is used 25mM GlyGly, 50mM NaCl, pH8.0 balance in advance.With 25mM GlyGly, 50mM NaCl, pH8.0 (buffer A) wash-out, surpass 10cv; Then use 25mM GlyGly, 50mM NaCl, 25mM CaCl ₂, pH8.0 (buffer B) wash-out surpasses 15cv, constant current (1ml/min) constant temperature (5 ℃).Effluent is monitored by absorbancy at 280nm.Mix effluent, with 1N HCl, adjust pH to 6.0.

Size exclusion chromatography:

In 5 ℃ with fPLC and Frac-950 (Amersham Bioscience) remove the not FVIIa of PEGization by size exclusion chromatography on Superdex 200 from the Glycopegylated FVIIa variant reclaiming in HiTrap Q mixed fraction.XK26/60 HiLoad Superdex200 post (320ml cv) pre-balance (10cv), uses 25mM GlyGly, 50mMNaCl, 25mM CaCl after dress post ₂, pH6.0 is with flow velocity 2.5ml/min wash-out.Effluent liquid is in 280nm absorbance detection.Collect 10ml cut.According to the description of Invitrogen, the SDS-PAGE for cut (4-12%Bis-Tris NuPAGE gels) with UV activity (280nm) analyzes, with Simple Blue dyeing.According to PEG substitution level mixed fraction, in 4000rpm, 10 ℃ by Amicon Ultra ^tM-15 (10K MWCO) centrifugal filter filtering and concentrating.

HS (di/tri 40K PEG) FVIIa:0.46mg albumen (UV _280nm)/ml, cumulative volume=3.0ml.

LS (mono/di 40K PEG) FVIIa:0.75mg albumen (UV _280nm)/ml, cumulative volume=3.7ml.

Mono 40K-PEGFVIIa:0.53mg albumen (UV _280nm)/ml, cumulative volume=4.5ml.

Embodiment 5

High (HS) and low replacement (LS) 5K-PEG-FVIIa of replacing of preparation

Impact on Periodic acid or periodate concentration and the final peptidolytic activity of compound:

Prepare following stoste:

2.8mM PEG stoste: by 5K-PEG-ONH ₂(4.03mg; 0.8umol, is prepared by 5K-PEG-NHS ester by described in embodiment 1) be dissolved in 281ul CaCl ₂free GlyGly damping fluid (25mM Gly-Gly, 50mM NaCl, pH6.0).20mM NaIO ₄stoste: NaIO ₄(426mg; 100ml CaCl 2mmol) ₂free GlyGly damping fluid (25mM Gly-Gly, 50mM NaCl, pH6.0) solution.100mM EDTA solution: EDETATE DISODIUM (292mg) is dissolved in 10ml water.1M CaCl ₂solution: CaCl ₂(1.12g) the 10ml aqueous solution (by 0.45um membrane filtration).12.8uMVIIa factor solution: (1.4mg, 0.028umol) is dissolved in 1.0ml 25mM Gly-Gly, 50mM NaCl, pH6.0 (GlyGly damping fluid), adds 95ul 100mMEDTA solution, then adds 1.095ml CaCl ₂free GlyGly damping fluid (final FVIIa concentration=0.64mg/ml).Stoste is mixed according to following table:

Entry	12.8uM FVIIa	Not containing CaCl ₂-GlyGly damping fluid	20mM NaIO ₄	2.8mM PEG5000-ONH ₂	Cumulative volume
						1	50ul	40ul	0ul	10ul	100ul
2	50ul	20ul	20ul	10ul	100ul
						3	50ul	30ul	10ul	10ul	100ul
4	50ul	38ul	2ul	10ul	100ul
						5	50ul	39,5ul	0,5ul	10ul	100ul

Under all reaction mixture room temperatures, gentleness is shaken 2h.In each phial, add 25ul 1MCaCl ₂solution.Then sample is by analyzing peptidolytic activity described in part of detecting.

Entry	NaIO ₄Ultimate density	FVIIa∶NaIO ₄Ratio	Sialic acid: NaIO ₄Ratio ^*	Relative peptidolytic activity
					1	0.0mM	1∶0	1∶0	100
2	4.0mM	1∶625	1∶138	3,6
					3	2.0mM	～1∶312	～1∶70	15
4	0.4mM	～1∶63	～1∶14	47
					5	0.1mM	～1∶16	～1∶3.5	82

* the upper sialic average number of FVIIa equals 4.5, by neuraminidase-semi-lactosi oxidation test described in following examples 8, measures.

In all cases, obtain single-, two-and the equal mixture of three-PEGization product, as described in Invitrogen, in order to SDS-PAGE (the 4-12%Bis-Tris NuPAGE gel) analysis of SilverQuest dyeing.

Embodiment 6

High (HS) and low replacement (LS) 10K-PEG-FIX of replacing of preparation:

Experimental technique preparation described in these materials'uses embodiment 3.

Embodiment 7

High (HS) and low replacement (LS) 40K-PEG-FIX of replacing of preparation:

The IX factor (Benefix) is purifying from auxiliary material according to the following steps: lyophilized substance (1000IU) is redissolved with sterilized water (4ml), add EDTA (200ul, the 0.25M aqueous solution, pH6).After 5 ℃ of 10min, on sample, use the 1ml Resource Q post (AmershamBioscience) of 25mM GlyGly, 100mM NaCl, pH6.0 pre-balance.Pillar flow velocity is 1ml/min.For pillar, 25mM GlyGly, 100mMNaCl, pH8.0 (buffer A) wash-out surpass 30cv; Then use 25mM GlyGly, 100mM NaCl, 10mM CaCl ₂, pH8.0 (buffer B) wash-out surpasses 15cv, constant current (1ml/min) constant temperature (5 ℃).Effluent is monitored by absorbancy at 280nm.Collect cut 4.2ml, absorbancy for protein concentration (1.32/mg/ml) is determined as 0.52mg/ml.

PEGization (Pegylation) reaction:

The 25mM GlyGly of purification of Recombinant DNA source plasma thromboplastin component, 100mM NaCl, pH6.0 solution (2ml; 1.04mg; 18.54mmol) add 80ul (1mM NaIO4,80nmol are equivalent to sialic 1 equivalent in the plasma thromboplastin component of purification of Recombinant DNA source).Mixture stirring at room 2h, then adds 40K-PEG-ONH ₂((20K PEG) OCH ₂(20KPEG) OCHCH ₂oC (=O) O-CH ₂cH ₂oNH ₂; WO 2006042847; 926ul; 500uM; 25mM GlyGly 25x), 100mM NaCl, pH6.0 solution.Mixture is placed in room temperature 48h.Then add methionine(Met) (100ul, 5mM in 25mM GlyGly, 100mMNaCl, pH6.0), then add MeONH ₂(100ul, the 25mM GlyGly of 5mM, 100mM NaCl, pH6.0 solution).Mixture is placed in room temperature 30min, is then refrigerated to-80 ℃ until be further purified.

Ion-exchange chromatography

With 1ml Resource Q post with fPLC and Frac-950 operation (all in Amersham Bioscience) are removed unnecessary PEG-ONH by ion-exchange chromatography from Glycopegylated FIX variant ₂the FIX of reagent and not PEGization.Effluent is monitored by optical density at 280nm.Collect 1000ul cut, in process, flow velocity keeps 1ml/min.Freezing reaction mixture slowly melts, add EDTA (400ul, the 0.25M aqueous solution, pH6).Add water to regulate the electric conductivity of mixture to 9.3mS.Then mixture is loaded on to the Resource Q post by the 10mM Histidine of 20cv, 50mM NaCl, 10mM EDTA, 0.01% tween 80, pH6.0 pre-balance.Pillar is washed with 10mM Histidine, 50mM NaCl, 10mMEDTA, 0.01% tween 80, the pH6.0 of 15cv, then uses 10mM Histidine, 50mMNaCl, 0.01% tween 80, the pH6.0 (buffer A) of 15cv to wash.Pillar is with increasing gradient (0-80%) 10mM Histidine, 50mM MgCl ₂, 0.01% tween 80, pH6.0 (buffer B), surpasses 15cv wash-out, then by 100% buffer B over 20cv wash-out.Cut, according to analyzing with SDS-PAGE (4-12%Bis-Tris NuPAGE gel) described in Invitrogen, dyes with Simple Blue.According to PEG substitution level, merge cut.

LS (mono/di 40K PEG) FIX:34.3ug albumen (UV _280nm)/ml, cumulative volume=3ml.

Mono 40K-PEG-FIX:70.7ug albumen (UV _280nm)/ml, cumulative volume=3ml.

Embodiment 8

The sialic acid concentration of FVIIa quantitatively:

As described in WO 2005014035A2, use is purchased from Molecular Probes Inc. (29851 Willow Creek Road, Eugene, OR 97402) the red neuraminidase of reodorant (sialidase) measure test kit (A-22178) and measure sialic acid concentration.

Embodiment 9

The FVIII 40K-PEGization of Periodic acid or periodate mediation

The VIII factor (Refacto; Wyeth, 2000IE) be dissolved in 1ml and disperse damping fluid to contain: NaCl (36mg/ml); Sucrose (12mg/ml); L-Histidine (6mg/ml); KCl (1mg/ml); Polysorbate 80 (0,4mg/ml).Then 400IE (200ul tissue buffer solution) is used AmiconUltra 15,10K MWCO (3 * 12min, 13.000rpm) by spin-flip buffer exchange, be 20mM GlyGly, 0.15M NaCl; 10mM CaCl ₂; 0.02% tween 80, pH7.3.In this FVIII solution of 30ul, add 10ul 500uM (SunBright GL2-400CA; 20mM GlyGly 20mg/ml), 0.15M NaCl; 10mM CaCl ₂; 0.02% tween 80, pH7.3, then add the moisture NaIO4 of 3ul 100uM and 12ul 20mM GlyGly, 0.15M NaCl; 10mM CaCl ₂; 0.02% tween 80, pH7.3 solution.React on 4 ℃ of hatching 16h, then, according to analyzing with SDS-Gel (7% tromethane acetate gel 150V/70min) described in Invitrogen, take SilverQuest dyeing as silvery white.

Illustrative embodiments of the present invention and feature

In order better to understand invention described herein, provide some nonrestrictive illustrative embodiments of the present invention and features:

Joint

1. a method of preparing the modified proteins with following general formula:

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I)

Wherein M and optional M ' are the polymeric part that increases modified proteins molecular weight independently, wherein L and L ' represent divalence joint independently, wherein P represents glycoprotein, the one or more oxidized dextran end that described glycoprotein comprises glycoprotein, O, N, C and H represent respectively oxygen, nitrogen, carbon and hydrogen atom, n is 1-10, and m is 0-50, and the method comprises the following steps:

A) with Periodic acid or periodate ion, be oxidized at least one and have glycoprotein P ^*on glycan end, P wherein ^*represent a plurality of sugared types, to obtain the glycoprotein P-(CHO) that contains one or more aldehyde groups _n+m, and

2. according to the method described in embodiment 1, wherein said glycoprotein is that N-glycosylation and/or O-are glycosylated and/or contain sialic acid residues.

3. according to the method described in aforementioned any embodiment, the method comprises that this modified proteins of further confirmation has the step of the pharmacological properties of improvement with respect to initial glycoprotein.

4. according to the method described in aforementioned any embodiment, the pharmacological properties of wherein said improvement is selected from the body of the function Half-life in vivo of the bioavailability of raising, prolongation, prolongation plasma half-life, the immunogenicity of reduction, the function Half-life in vivo of the albumin avidity of the protease resistant of raising, raising, the receptor affinity of improvement, the stability in storage of raising, shortening, plasma half-life in the body of shortening.

5. according to the method described in embodiment 4, the transformation period wherein extending is reduced as the group that increases molecular weight or is eliminated kidney removing and/or realize as the group of sheltering the binding partners (partner) of liver receptor by M by M and/or M '.

6. according to the method described in embodiment 4, the immunogenicity wherein reducing by M and/or M ' as blocking antibody with cause the group realization of being combined in immune site.

7. according to the method described in embodiment 4, wherein improve albumin avidity and realize with the group that albumin has high affinity by M and/or M ' conduct.

8. according to the method described in embodiment 4, wherein improve receptor affinity and realize as the group with target cell surface receptor specific binding by M and/or M '.

9. according to the method described in aforementioned any embodiment, wherein M and/or M ' are selected from: the organic charged groups of lower molecular weight, and it can contain one or more carboxylic acids, amine, sulfonic acid, phosphonic acids or its combination; Lower molecular weight neutral hydrophilic molecule, for example Polyethylene Chain of cyclodextrin or optional branching; Low-molecular-weight lipophilic molecule, as lipid acid or cholic acid or derivatives thereof; Molecular-weight average is the polyoxyethylene glycol of 2-40kDa; Well-defined accurate polymkeric substance, as the definite molecular weight ranges dendritic macromole (dendrimer) that is 700Da to 20kDa; Substantially the polypeptide of non-immunogenic, as albumin, antibody or optionally contain the part of the antibody of Fc-structural domain; With high molecular organic polymer.

10. according to the method described in aforementioned any embodiment, wherein M and/or M ' are selected from: dendritic macromole, polyoxyalkylene (PAO), comprise polyalkylene glycol (PAG), for example polyoxyethylene glycol (PEG) and polypropylene glycol (PPG), branching PEG, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyethylene-copolymerization-maleic anhydride, polystyrene-copolymerization-maleic anhydride, dextran, Sensor Chip CM 5, HES, MPC and PHF.

11. according to the method described in embodiment 1-9 any one, wherein M and/or M ' are selected from serum protein binding partner and contain the organic molecule that changes the part of charge property under physiological condition, the structure that suppresses glycan and receptors bind, and the middle substituent of prevention glycan specific recognition.

12. according to the method described in aforementioned any embodiment, and wherein P is selected from FVII, FVIII, FIX, FX, FII, FV, PROTEIN C, Protein S, tPA, PAI-1, tissue factor, FXI, FXII, FXIII and sequence variants thereof, immunoglobulin (Ig), cytokine is as interleukin, α-, β-and gamma-interferon, G CFS, comprise granulocytosis colony stimulative factor, fibroblast growth factor, Thr6 PDGF BB, Phospholipid hydrolase activator (PUP), Regular Insulin, vegetable-protein is as lectin and ricin, tumour necrosis factor and relevant allelotrope, the Tumor Necrosis Factor Receptors of solubilized form, the interleukin-2-receptor of interleukin-2-receptor and solubilized form, somatomedin is tissue growth factor for example, as TGFa ' s or TGFps and Urogastron, hormone, somatomedin, erythropoietin, pigment hormone, hypothalamic releasing factor, vassopressin, prolactin, chorionic-gonadotropin hormone, follicle stimulating hormone, thyrotropic hormone, tissue plasminogen activator, with immunoglobulin (Ig) as IgG, IgE, IgM, IgA and IgD and fragment thereof, or any fusion rotein that contains any aforementioned albumen or its fragment.

13. according to the method described in embodiment 1-12 any one, and wherein glycoprotein is VII factor polypeptide.

14. according to the method described in embodiment 1-12 any one, the aminoacid sequence that wherein glycoprotein contains the wild-type people VII factor.

15. according to the method described in embodiment 13-14 any one, the specific activity that wherein modified proteins shows in conjunction with test at one or more CAs described in specification sheets of the present invention, proteolysis effects test or TF be unmodified VII factor polypeptide specific activity at least about 10%, as at least about 20%, as at least about 40%, as at least about 60%, as at least about 80%, as at least about 100%.

16. according to the method described in embodiment 1-15 any one, the bioavailability that wherein modified proteins shows be unmodified glycoprotein bioavailability at least about 110%, be for example unmodified glycoprotein bioavailability at least about 120%, approximately 130% or at least about 140%.

17. according to the method described in embodiment 1-15 any one, the serum half-life that wherein modified proteins shows be unmodified sugared egg serum half-life at least about 125%, according to appointment 150%, approximately 200% or be unmodified sugared egg serum half-life at least about 250%.

18. according to the method described in embodiment 1-17 any one, wherein, with respect to the quantity of not reducing glycan end existing on glycoprotein, Periodic acid or periodate ion to be less than the amount of 20 equivalents, for example, are less than 10 equivalents, for example be less than 5 equivalents, the amount that is for example less than 1 equivalent exists, for example, and with respect to the quantity of not reducing glycan end existing on glycoprotein, with 0.1-20 equivalent, 0.1-10 equivalent for example, 0.1-5 equivalent for example, for example the amount of 0.1-1 equivalent exists.

19. have the modified proteins of following general formula

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I)

Modified proteins described in 20. embodiments 19, wherein said glycoprotein is that N-glycosylation and/or O-are glycosylated and/or contain sialic acid residues.

21. according to the modified proteins described in embodiment 19-20 any one, and the pharmacological properties wherein improving is selected from the bioavailability of raising, plasma half-life in plasma half-life, the immunogenicity of reduction, the function Half-life in vivo of the albumin avidity of the protease resistant of raising, raising, the receptor affinity of improvement, the stability in storage of raising, shortening, the body of shortening in the body of the function Half-life in vivo of prolongation, prolongation.

22. according to the modified proteins described in embodiment 21, and wherein the transformation period of prolongation reduces as the group that increases molecular weight by M and/or M ' or eliminates kidney removing and/or realize as the group of sheltering the binding partners of liver receptor by M and/or M '.

23. according to the modified proteins described in embodiment 21, the immunogenicity wherein reducing by M and/or M ' as blocking antibody with cause the group realization of being combined in immune site.

24. according to the modified proteins described in embodiment 21, wherein improves albumin avidity and realizes with the group that albumin has high affinity by M and/or M ' conduct.

25. according to the modified proteins described in embodiment 21, wherein improves receptor affinity and realizes as the group with target cell surface receptor specific binding by M and/or M '.

26. according to the modified proteins described in embodiment 19-25 any one, and wherein M and/or M ' are selected from: the organic charged groups of lower molecular weight, and it can contain one or more carboxylic acids, amine, sulfonic acid, phosphonic acids or its combination; Lower molecular weight neutral hydrophilic molecule, for example Polyethylene Chain of cyclodextrin or optional branching; Low-molecular-weight lipophilic molecule, as lipid acid or cholic acid or derivatives thereof; Molecular-weight average is the polyoxyethylene glycol of 2-40kDa; Well-defined accurate polymkeric substance, as the definite molecular weight ranges dendritic macromole that is 700Da to 20kDa; Substantially the polypeptide of non-immunogenic, as albumin, antibody or optionally contain the part of the antibody of Fc-structural domain; With high molecular organic polymer.

27. according to the modified proteins described in embodiment 19-26 any one, wherein M and/or M ' are selected from: dendritic macromole, polyoxyalkylene (PAO), comprise polyalkylene glycol (PAG), for example polyoxyethylene glycol (PEG) and polypropylene glycol (PPG), branching PEG, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyethylene-copolymerization-maleic anhydride, polystyrene-copolymerization-maleic anhydride, dextran, Sensor Chip CM 5, HES, MPC and PHF.

28. according to the modified proteins described in embodiment 19-26 any one, wherein M and/or M ' are selected from serum protein binding partner and contain the organic molecule that changes the part of charge property under physiological condition, the structure that suppresses glycan and receptors bind, and the middle substituent of prevention glycan specific recognition.

29. according to the modified proteins described in embodiment 19-28 any one, and wherein P is selected from variant FVII, FVIII, FIX, FX, FII, FV, PROTEIN C, Protein S, tPA, PAI-1, tissue factor, FXI, FXII, FXIII and sequence variants thereof, immunoglobulin (Ig), cytokine is as interleukin, α-, β-and gamma-interferon, G CFS, comprise granulocytosis colony stimulative factor, fibroblast growth factor, Thr6 PDGF BB, Phospholipid hydrolase activator (PUP), Regular Insulin, vegetable-protein is as lectin and ricin, tumour necrosis factor and relevant allelotrope, the Tumor Necrosis Factor Receptors of solubilized form, the interleukin-2-receptor of interleukin-2-receptor and solubilized form, somatomedin is tissue growth factor for example, as TGFa ' s or TGFps and Urogastron, hormone, somatomedin, erythropoietin, pigment hormone, hypothalamic releasing factor, vassopressin, prolactin, chorionic-gonadotropin hormone, follicle stimulating hormone, thyrotropic hormone, tissue plasminogen activator, with immunoglobulin (Ig) as IgG, IgE, IgM, IgA and IgD and fragment thereof, or any fusion rotein that contains any aforementioned albumen or its fragment.

30. according to the modified proteins described in embodiment 19-29 any one, and wherein glycoprotein is VII factor polypeptide.

31. according to the modified proteins described in embodiment 19-29 any one, the aminoacid sequence that wherein glycoprotein contains the wild-type people VII factor.

32. according to the modified proteins described in embodiment 30-31 any one, the specific activity that wherein modified proteins shows in conjunction with test at one or more CAs described in specification sheets of the present invention, proteolysis effects test or TF be unmodified VII factor polypeptide specific activity at least about 10%, as at least about 20%, as at least about 40%, as at least about 60%, as at least about 80%, as at least about 100%.

33. according to the modified proteins described in embodiment 19-32 any one, the bioavailability that wherein modified proteins shows be unmodified glycoprotein bioavailability at least about 110%, be for example unmodified glycoprotein bioavailability at least about 120%, approximately 130% or at least about 140%.

34. according to the modified proteins described in embodiment 19-33 any one, the serum half-life that wherein modified proteins shows be unmodified sugared egg serum half-life at least about 125%, according to appointment 150%, approximately 200% or be unmodified sugared egg serum half-life at least about 250%.

35. have the modified proteins of following general formula

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I)

Joint wherein M and optional M ' independently for increasing the polymeric part of modified proteins molecular weight, wherein L and L ' represent divalence joint independently, wherein P represents glycoprotein, the one or more oxidized dextran end that described glycoprotein comprises glycoprotein, O, N, C and H represent respectively oxygen, nitrogen, carbon and hydrogen atom, n is 1-10, and m is 0-50, and wherein said modified proteins is with respect to initial glycoprotein P ^*have the pharmacological properties of improvement and keep its functionally active, described modified proteins is to obtain by the method described in any one in embodiment 1-12.

36. comprise according to the preparation of the multiple modified proteins of embodiment 19-35 any one.

37. pharmaceutical compositions containing the modified proteins of with good grounds embodiment 19-36 any one and the pharmaceutically acceptable carrier, thinner, auxiliary material or the vehicle that mix with it.

38. be used for the treatment of according to the modified proteins of embodiment 19-36 any one.

Claims

1. a method of preparing the modified proteins with following general formula:

(M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _m(formula I)

Wherein M and optional M ' are the polyoxyethylene glycol (PEG) that increases modified proteins molecular weight independently, wherein L and L ' represent divalence joint independently, wherein P represents glycoprotein, the one or more oxidized dextran end that described glycoprotein comprises glycoprotein, O, N, C and H represent respectively oxygen, nitrogen, carbon and hydrogen atom, n is 1-10, and m is 0-50, and the method comprises the following steps:

A) with Periodic acid or periodate ion, be oxidized at least one and have the glycan end on glycoprotein P*, wherein P* represents a plurality of sugared types, to obtain the glycoprotein P-(CHO) that contains one or more aldehyde groups _n+m, and

C) will there is structure (M-L-O-N=CH) _n-P-(CHO) _mglycoprotein in any responseless aldehyde group and M '-L '-O-NH ₂reaction, to obtain having structure (M-L-O-N=CH) _n-P-(CH=N-O-L '-M ') _mmodified proteins,

Wherein with respect to the quantity of not reducing glycan end existing on glycoprotein, described Periodic acid or periodate ion exist with the amount of 0.1-5 equivalent, and wherein said modified proteins has the pharmacological properties of improvement and keeps its functionally active with respect to initial glycoprotein P*, and wherein P is selected from FVII, FVIII and FIX.

2. method according to claim 1, the molecular-weight average of wherein said polyoxyethylene glycol is 2-40kDa.

3. according to the method described in any one in claim 1-2, wherein glycoprotein is VII factor polypeptide.

4. method according to claim 1 and 2, the bioavailability that wherein modified proteins shows is at least 110% of unmodified glycoprotein bioavailability.

5. method according to claim 4, the bioavailability that wherein said modified proteins shows is at least 120% of unmodified glycoprotein bioavailability.

6. method according to claim 4, the bioavailability that wherein said modified proteins shows is at least 130% of unmodified glycoprotein bioavailability.

7. method according to claim 4, the bioavailability that wherein said modified proteins shows is at least 140% of unmodified glycoprotein bioavailability.

8. method according to claim 1 and 2, wherein, with respect to the quantity of not reducing glycan end existing on glycoprotein, described Periodic acid or periodate ion exist with the amount of 0.1-1 equivalent.