HK1229229A - Blood coagulation protein conjugates - Google Patents

Description

Blood coagulation protein conjugates

the present application is a divisional application of the invention patent application having application number 201080037002.8, application date 26/7/2010, entitled "blood coagulation protein conjugate".

The present application claims benefit of U.S. provisional application No. 61/347,136 filed on 21/5/2010 and U.S. provisional application No. 61/228,828 filed on 27/7/2009, all of which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to materials and methods for conjugating water soluble polymers to blood coagulation proteins.

Background

Therapeutic polypeptides such as coagulation proteins (including Factor ix (fix), Factor viii (fviii), Factor viia (fviia), Von Willebrand Factor (VWF), Factor fv (fv), Factor x (fx), Factor xi (fxi), Factor xii (fxii), thrombin (FII), protein C, protein S, tPA, PAI-1, Tissue Factor (TF) and ADAMTS 13 protease) are rapidly degraded by proteolytic enzymes and neutralized by antibodies. This shortens its half-life and circulation time, limiting its therapeutic effectiveness. Relatively high doses and frequent administration are necessary to achieve and maintain the desired therapeutic or prophylactic effect of these coagulation proteins. Thus, proper dose adjustment is difficult to achieve and the need for frequent intravenous administration places restrictions on the lifestyle of the patient.

Pegylation of polypeptide drugs protects the Drug in the circulation and improves its pharmacodynamic and pharmacokinetic properties (Harris and Chess, Nat Rev Drug Discov. 2003: 2: 214-21). The pegylation process attaches ethylene glycol repeat units (polyethylene glycol (PEG)) to the polypeptide drug. PEG molecules have a large hydrodynamic volume (5-10 times the size of globular proteins), are highly water soluble and hydrated, non-toxic, non-immunogenic, and rapidly clear themselves in vivo. Pegylation of the molecule can enhance resistance of the drug to enzymatic degradation, increase half-life in vivo, reduce dosing frequency, reduce immunogenicity, enhance physical and thermal stability, enhance solubility, enhance liquid stability, and reduce aggregation. The first pegylated drug received FDA approval early in the 90's of the 20 th century. Since then, several pegylated drugs have been approved by the FDA for oral, injectable, and topical administration.

Polysialic acid (PSA), also known as Colominic Acid (CA), is a naturally occurring polysaccharide. It is a homopolymer containing an alpha (2 → 8) ketoglycoside-bonded N-acetylneuraminic acid and contains a vicinal diol group at its non-reducing end. It is negatively charged and is a natural component of the human body. It can be easily manufactured by bacteria in large quantities and with predetermined physiological characteristics (us patent No. 5,846,951). Since PSA produced by bacteria is chemically and immunologically identical to PSA produced by humans, bacterial PSA is non-immunogenic even if coupled to proteins. Unlike certain polymers, polysialic acid is biodegradable. Covalent coupling of colominic acid to catalase and asparaginase has been shown to enhance enzyme stability in the presence of proteolytic enzymes or plasma. In vivo comparison studies with polysialized asparaginase and unmodified asparaginase revealed that polysialization increased the half-life of the enzyme (Femandes and Gregoriadis, Int Biochimica Biophysica Acta 1341:26-34, 1997).

The preparation of conjugates by forming covalent linkages between water-soluble polymers and therapeutic proteins can be carried out using a variety of chemical methods. For example, Roberts et al (Adv Drug Deliv Rev 2002: 54:459-76) reviewed conjugation of PEG derivatives to peptides or proteins. One method of coupling water-soluble polymers to therapeutic proteins is to conjugate the polymer via the carbohydrate moiety of the protein. Sodium periodate (NaIO) can be easily used₄) The ortho hydroxyl groups (OH) of carbohydrates in proteins are oxidized to form reactive aldehyde groups (Rothfus and Smith, J Biol Chem 1963: 238: 1402-10; van Lenten and Ashweli, J Biol Chem 1971: 246:1889-94). Next, the polymer can be coupled to the aldehyde group of the carbohydrate by using a reagent containing, for example, a reactive hydrazide group (Wilchek M and Bayer EA, methods enzymol 1987: 138: 429-42). A more recent technique is the use of reagents containing aminooxy groups which react with aldehydes to form oxime linkages (WO 96/40662, WO 2008/025856).

Other examples describing the conjugation of water-soluble polymers to therapeutic proteins are described in the following documents: WO 06/071801, teaches oxidation of the carbohydrate moiety in von willebrand factor and subsequent coupling to PEG using hydrazide chemistry; U.S. publication No. 2009/0076237, teaches oxidizing rFVIII and then coupling with PEG and other water-soluble polymers (e.g., PSA, HES, dextran) using hydrazide chemistry; WO2008/025856, which teaches the oxidation of different coagulation factors (e.g. rFIX, FVIII and FVIIa) and then coupling with e.g. PEG via oxime linkage formation using aminoxy chemistry; and U.S. patent No. 5,621,039, which teaches oxidizing FIX and then coupling to PEG using hydrazide chemistry.

More recently, an improved process has been described which involves mild oxidation of sialic acid with periodate to produce an aldehyde followed by reaction with an aminooxy-containing reagent in the presence of catalytic amounts of aniline (Dirksen A and Dawson PE, Bioconjugate chem.2008: 19, 2543-8; and Zeng Y et al, Nature Methods 2009: 6: 207-9). Aniline catalysis significantly accelerates oxime ligation, allowing the use of very low concentrations of the reagents.

Despite the existence of methods available for conjugating water-soluble polymers to therapeutic proteins, there remains a need to develop materials and methods for conjugating water-soluble polymers to proteins that improve the pharmacodynamic and/or pharmacokinetic properties of the proteins while minimizing the costs associated with various agents.

Summary of The Invention

The present invention provides materials and methods for conjugating polymers to proteins that improve the pharmacodynamic and/or pharmacokinetic properties of the proteins while minimizing the costs associated with various agents.

In one embodiment of the invention, a method of conjugating a water soluble polymer to an oxidized carbohydrate moiety of a blood coagulation protein comprises contacting the oxidized carbohydrate moiety with an activated water soluble polymer under conditions that allow conjugation; the coagulation protein is selected from the group consisting of: factor ix (fix), factor viii (fviii), factor viia (fviia), Von Willebrand Factor (VWF), factor fv (fv), factor x (fx), factor xi (fxi), factor xii (fxii), thrombin (FII), protein C, protein S, tPA, PAI-1, Tissue Factor (TF), and ADAMTS 13 protease or biologically active fragments, derivatives, or variants thereof; the water soluble polymer contains an active aminooxy group and is selected from the group consisting of: polyethylene glycol (PEG), branched PEG, polysialic acid (PSA), carbohydrates, polysaccharides, pullulan (pulullan), chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG)Alcohol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, poly (1-hydroxymethylethylene-hydroxymethylformal) (PHF), 2-methacryloyloxy-2' -ethyltrimethylammonium phosphate (MPC); and the carbohydrate moiety is oxidized by incubation with a buffer comprising an oxidizing agent selected from the group consisting of: sodium periodate (NaIO)₄) Lead tetraacetate (Pb (OAc)₄) And potassium perruthenate (KRuO)₄) (ii) a Wherein an oxime linkage is formed between the oxidized carbohydrate moiety and the active aminooxy group on the water soluble polymer.

In another embodiment of the present invention, the water-soluble polymer according to the above process is a PSA. In related embodiments, the PSA comprises about 5-500 or 10-300 sialic acid units. In a further embodiment, the coagulation protein according to the above method is FIX. In another embodiment, the coagulation protein according to the above method is FVIIa. In a further embodiment, the coagulation protein according to the above method is FVIII. In yet another embodiment, there is provided a process as described above, wherein the oxidizing agent is sodium periodate (NaIO)₄). In another embodiment, the oxidized carbohydrate moiety of the blood coagulation protein according to the above method is located in an activating peptide of the blood coagulation protein.

In yet another embodiment of the present invention, there is provided the above process, wherein the PSA is prepared by reacting an activated aminooxy linking group with an oxidized PSA;

wherein the aminooxy linking group is selected from the group consisting of:

a 3-oxa-pentane-1, 5-dihydroxyamine linking group of the formula:

and

a3, 6, 9-trioxa-undecane-1, 11-dihydroxyamine linking group of the formula:

wherein the PSA is oxidized by incubation with an oxidizing agent to form a terminal aldehyde group at the non-reducing end of the PSA. In yet another embodiment, a method as described above is provided, wherein the activated aminooxy linking group comprises 1 to 50 ethylene glycol units.

In yet another embodiment, there is provided a method as described above, wherein the aminooxy linking group is 3-oxa-pentane-1, 5-dihydroxyamine. In a related embodiment, the oxidizing agent is NaIO₄。

In another embodiment of the present invention, there is provided the above method, wherein the contacting of the oxidized carbohydrate moiety with the activated water-soluble polymer is performed in a buffer comprising a nucleophilic catalyst selected from the group consisting of aniline and aniline derivatives.

In yet another embodiment of the present invention, there is provided the above method further comprising the step of reducing oxime linkages in the conjugated blood coagulation protein by incubating the conjugated blood coagulation protein in a buffer comprising a reducing compound selected from the group consisting of sodium cyanoborohydride (NaCNBH3) and ascorbic acid (vitamin C). In a related embodiment, the reducing compound is sodium cyanoborohydride (NaCNBH 3).

In another embodiment of the present invention, there is provided a modified blood coagulation protein produced by the above method.

In yet another embodiment of the present invention, there is provided a modified FIX comprising a FIX molecule or a biologically active fragment, derivative or variant thereof; and at least one aminoxy PSA bound to a FIX molecule, wherein the aminoxy PSA is linked to FIX via one or more carbohydrate moieties.

In another embodiment of the invention, there is provided a modified FVIIa comprising a FVIIa molecule, or a biologically active fragment, derivative or variant thereof; and at least one aminoxy PSA bound to the FVIIa molecule, wherein the aminoxy PSA is linked to FVIIa via one or more carbohydrate moieties.

In yet another embodiment of the present invention, there is provided a modified FVIII comprising a FVIII molecule or a biologically active fragment, derivative or variant thereof; and at least one aminoxy PSA bound to a FVIII molecule, wherein the aminoxy PSA is linked to FVIII via one or more carbohydrate moieties.

In yet another embodiment of the present invention, there is provided a modified FIX comprising a FIX molecule or a biologically active fragment, derivative or variant thereof; and at least one aminoxy PEG conjugated to FIX molecule, wherein the aminoxy PEG is linked to FIX via one or more carbohydrate moieties.

In another embodiment of the invention, there is provided a modified FVIIa comprising a FVIIa molecule, or a biologically active fragment, derivative or variant thereof; and at least one aminoxy PEG conjugated to a FVIIa molecule, wherein said aminoxy PEG is linked to FVIIa via one or more carbohydrate moieties.

In yet another embodiment of the present invention, there is provided a modified FVIII comprising a FVIII molecule or a biologically active fragment, derivative or variant thereof; and at least one aminoxy PEG conjugated to a FVIII molecule, wherein said aminoxy PEG is attached to FVIII via one or more carbohydrate moieties.

In yet another embodiment, a water-soluble polymer comprising a reactive aminooxy linking group is provided; the water soluble polymer is selected from the group consisting of: polyethylene glycol (PEG), branched PEG, polysialic acid (PSA), carbohydrates, polysaccharides, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, poly (1-hydroxymethylethylene-hydroxymethylformal) (PHF), 2-methacryloyloxy-2' -ethyltrimethylammonium phosphate (MPC); the reactive aminooxy linking group is selected from the group consisting of: a 3-oxa-pentane-1, 5-dihydroxyamine linking group of the formula:

and

a3, 6, 9-trioxa-undecane-1, 11-dihydroxyamine linking group of the formula:

in yet another embodiment, a method as described above is provided, wherein the activated aminooxy linking group comprises 1 to 50 ethylene glycol units.

Drawings

Figure 1 shows the primary structure of coagulation factor IX.

FIG. 2 shows the coupling of oxidized rFIX to aminoxy-PSA.

FIG. 3 shows the synthesis of the water-soluble diamino-oxy linking group 3-oxa-pentane-1, 5-dihydroxy amine and 3,6, 9-trioxa-undecane-1, 11-dihydroxy amine.

FIG. 4 shows the preparation of aminoxy-PSA.

FIG. 5 shows the analytical characterization of PSA-rFIX conjugates using SDS-PAGE and Coomassie blue staining.

FIG. 6 shows the analytical characterization of PSA-rFIX conjugates using detection with anti-FIX and anti-PSA antibodies.

Figure 7 shows the activity of native rFIX and PSA-rFIX conjugates relative to time post infusion.

Figure 8 shows the content of PSA-rFVIII and Advate versus time post infusion.

Detailed Description

The pharmacological and immunological properties of therapeutic proteins can be improved by chemical modification and conjugation with polymeric compounds, the polymeric compounds are, for example, polyethylene glycol (PEG), branched PEG, polysialic acid (PSA), carbohydrates, polysaccharides, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, poly (1-hydroxymethylethylene-hydroxymethylformal) (PHF), 2-methacryloyloxy-2' -ethyltrimethylammonium phosphate (MPC). The properties of the resulting conjugates are generally highly dependent on the structure and size of the polymer. Thus, in the art, polymers having a defined and narrow size distribution are generally preferred. Synthetic polymers such as PEG can be readily made with narrow size distributions, while PSA can be purified in a manner that results in a final PSA formulation with a narrow size distribution. In addition, pegylation reagents with defined polymer chains and narrow size distribution are commercially available and can be purchased at a reasonable price.

Adding soluble polymers, for example by polysialylation, is a method of improving the properties of blood coagulation proteins such as FIX and other blood coagulation proteins such as VWF, FVIIa (see e.g. US 2008/0221032a1, incorporated herein by reference) and FVIII.

Blood coagulation proteins

As described herein, the present invention encompasses coagulation proteins including, but not limited to, factor ix (fix), factor viii (fviii), factor viia (fviia), Von Willebrand Factor (VWF), factor fv (fv), factor x (fx), factor XI, factor xii (fxii), thrombin (FII), protein C, protein S, tPA, PAI-1, Tissue Factor (TF), and ADAMTS 13 protease. The term "blood coagulation protein" as used herein refers to any factor ix (fix), factor viii (fviii), factor viia (fviia), Von Willebrand Factor (VWF), factor fv (fv), factor x (fx), factor xii (fxii), thrombin (FII), protein C, protein S, tPA, PAI-1, Tissue Factor (TF), and ADAMTS 13 protease that exhibit biological activities associated with a particular native blood coagulation protein.

The coagulation cascade is divided into three distinct parts: endogenous, exogenous, and common pathways (Schenon et al, Curr Opin Hematol.2004: 11: 272-7). The cascade involves a series of serine proteases (zymogens) and protein cofactors. If desired, the inactive zymogen precursor is converted to the active form, thereby converting the subsequent enzyme in the cascade.

The intrinsic pathway requires coagulation factors VIII, IX, X, XI, and XII. Initiation of the endogenous pathway occurs when current kallikrein, high molecular weight kininogen, factor xi (fxi) and factor xii (fxii) are exposed to negatively charged surfaces. Calcium ions and phospholipids secreted from platelets are also required.

Exogenous pathways are initiated when the vascular lumen of a blood vessel is damaged. The membrane glycoprotein tissue factor is exposed and subsequently bound to circulating factor vii (fvii) and a small amount of its pre-existing activated form FVIIa. This binding favours the complete conversion of FVII to FVIIa and the subsequent conversion of Factor IX (FIX) to factor IXa (FIXa) and Factor X (FX) to factor Xa (FXa) in the presence of calcium and phospholipids. Association of FVIIa with tissue factor enhances proteolytic activity by bringing the matrix (FIX and FX) binding sites of FVII closer and inducing conformational changes that enhance the enzymatic activity of FVIIa.

Activation of FX is common to both pathways. Factors va (fva) and Xa together with phospholipids and calcium convert prothrombin to thrombin (prothrombinase complex), which then cleaves fibrinogen to form fibrin monomers. The monomers polymerize to form fibrin strands. Factor xiiia (fxiiia) covalently bonds these chains to each other to form a rigid network.

The conversion of FVII to FVIIa is also catalyzed by several proteases including thrombin, FIXa, FXa, factor xia (fxia) and factor xiia (fxiia). To inhibit the early stages of the cascade, tissue factor pathway inhibitors target the FVIIa/tissue factor/FXa product complex.

A. Polypeptides

In one aspect, the starting material of the present invention is a blood coagulation protein that can be derived from human plasma or prepared by recombinant engineering techniques as described in U.S. patent No. 4,757,006; U.S. patent No. 5,733,873; U.S. patent No. 5,198,349; U.S. patent No. 5,250,421; U.S. patent No. 5,919,766; and EP 306968. As used herein, the term coagulation protein refers to any coagulation protein molecule that exhibits biological activity associated with a native coagulation protein. In one embodiment of the invention, the coagulation protein molecule is a full length coagulation protein.

Contemplated coagulation protein molecules include full-length proteins, precursors of full-length proteins, biologically active subunits or fragments of full-length proteins, and biologically active derivatives and variants of any of these coagulation protein forms. Thus, blood coagulation proteins include those that satisfy the following conditions: (1) an amino acid sequence having greater than about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identity to the amino acid sequence of a polypeptide encoded by a reference nucleic acid or amino acid sequence described herein over a region of at least about 25, about 50, about 100, about 200, about 300, about 400, or more amino acids; and/or (2) specifically binds to an antibody, e.g., a polyclonal or monoclonal antibody, raised against an immunogen comprising a reference amino acid sequence as described herein, an immunogenic fragment thereof, and/or conservatively modified variants thereof.

According to the present invention, the term "recombinant coagulation protein" includes any coagulation protein obtained via recombinant DNA technology. In certain embodiments, the term encompasses a protein as described herein.

As used herein, "intrinsic coagulation protein" includes coagulation proteins originating from a mammal intended to receive treatment. The term also includes coagulation proteins transcribed from transgenes or any other foreign DNA present in the mammal. As used herein, "extrinsic coagulation proteins" include coagulation proteins that do not originate from the mammal intended to receive treatment.

As used herein, "plasma-derived coagulation protein" or "plasmatic" includes all forms of proteins found in blood obtained from a mammal that have properties involved in the coagulation pathway.

As used herein, "biologically active derivative" or "biologically active variant" includes any derivative or variant of a molecule that has substantially the same functional and/or biological properties (e.g., binding properties) and/or the same structural basis (e.g., peptide backbone or underlying polymeric unit) as the molecule.

An "analog," "variant," or "derivative" is a compound that is substantially similar in structure and has the same biological activity as a naturally occurring molecule, but in some cases to a different degree. For example, a polypeptide variant refers to a polypeptide that shares a substantially similar structure and has the same biological activity as a reference polypeptide. A variant or analog differs in amino acid sequence composition as compared to the naturally-occurring polypeptide from which it is derived based on one or more mutations that involve (i) deletion of one or more amino acid residues at one or more termini of the polypeptide and/or in one or more internal regions (e.g., fragments) of the naturally-occurring polypeptide sequence, (ii) insertion or addition of one or more amino acids at one or more termini of the polypeptide (typically "addition" or "fusion") and/or in one or more internal regions of the naturally-occurring polypeptide sequence (typically "insertion"), or (iii) substitution of one or more amino acids in the naturally-occurring polypeptide sequence with other amino acids. For example, a "derivative" refers to a polypeptide that has been modified (e.g., chemically modified) to share the same or substantially similar structure as a reference polypeptide.

Variant or analog polypeptides include insertion variants in which one or more amino acid residues are added to the amino acid sequence of the blood coagulation protein of the invention. Insertions may be located at either or both ends of the protein, and/or may be located in internal regions of the amino acid sequence of the coagulation protein. Insertional variants with additional residues at either or both termini include, for example, fusion proteins and proteins that include amino acid tags or other amino acid labels. In one aspect, the coagulation protein molecule optionally contains an N-terminal Met, particularly when the molecule is recombinantly expressed in a bacterial cell such as e.

In a deletion variant, one or more amino acid residues are removed in a blood coagulation protein polypeptide as described herein. Deletions may be effected at one or both termini of the coagulation protein polypeptide, and/or removal of one or more residues within the coagulation protein amino acid sequence. Thus, deletion variants include fragments of the coagulation protein polypeptide sequence.

In substitution variants, one or more amino acid residues of the blood coagulation protein polypeptide are removed and replaced with a replacement residue. In one aspect, the substitutions are conservative in nature, and such types of conservative substitutions are well known in the art. Alternatively, the invention encompasses substitutions that are also non-conservative. Exemplary conservative substitutions are described in Lehninger [ Biochemistry, 2 nd edition; worth Publishers, New York (1975), pages 71-77 ], and is set forth immediately below.

Conservative substitutions

Alternatively, exemplary conservative substitutions are set forth immediately below.

Conservative substitutions II

B. Polynucleotide

The blood coagulation protein-encoding nucleic acids of the present invention include, for example, but are not limited to, genes, pre-mRNA, cDNA, polymorphic variants, alleles, synthetic and naturally occurring mutants.

The coagulation protein-encoding polynucleotide of the present invention also includes (but is not limited to) those satisfying the following conditions: (1) specifically hybridizes under stringent hybridization conditions to a nucleic acid encoding a reference amino acid sequence described herein and conservatively modified variants thereof; (2) a nucleic acid sequence having greater than about 95%, about 96%, about 97%, about 98%, about 99% or more nucleotide sequence identity to a reference nucleic acid sequence described herein over a region of at least about 25, about 50, about 100, about 150, about 200, about 250, about 500, about 1000 or more nucleotides (up to the full length of 1218 nucleotides of the mature protein). Exemplary "stringent hybridization" conditions include hybridization at 42 ℃ in 50% formamide, 5 XSSC, 20mM Na. PO4, pH 6.8; and washed with 1 XSSC at 55 ℃ for 30 minutes. It will be appreciated that variations of these exemplary conditions can be made based on the length of the sequence to be hybridized and the GC nucleotide content. Standard formulas in the art are suitable for determining appropriate hybridization conditions. See Sambrook et al, Molecular Cloning: A Laboratory Manual (2 nd edition, Cold Spring harbor Laboratory Press, 1989) § 9.47-9.51.

A "naturally-occurring" polynucleotide or polypeptide sequence is typically from a mammal, including (but not limited to) a primate, such as a human; rodents, such as rats, mice, hamsters; bovine, porcine, equine, ovine or any mammal. The nucleic acids and proteins of the invention may be recombinant molecules (e.g., heterologous and encoding a wild-type sequence or variant thereof, or non-naturally occurring).

In certain embodiments of the invention, the polypeptides and polynucleotides described above are, for example, the following coagulation proteins.

Factor VIIa

FVII (also known as a stabilizing factor or a prenvertin) is a vitamin K-dependent serine protease glycoprotein with key roles in hemostasis and coagulation (Eigenbrot, Curr Protein Pept Sci.2002: 3: 287-99).

FVII is synthesized in the liver and secreted as a 48kD single chain glycoprotein. FVII shares a similar protein domain structure with all vitamin K-dependent serine protease glycoproteins consisting of: an amino-terminal gamma-carboxyglutamic acid (Gla) domain with 9-12 residues responsible for protein interaction with lipid membranes, a carboxy-terminal serine protease domain (catalytic domain), and two epidermal growth factor-like domains containing calcium ion binding sites that mediate tissue factor interaction. Gamma-glutamyl carboxylase catalyzes the carboxylation of Gla residues in the amino terminal part of the molecule. The action of carboxylase is dependent on the reduced form of vitamin K, which is oxidized to the epoxide form. Vitamin K epoxide reductase is required to convert the epoxide form of vitamin K to a reduced form.

A large proportion of FVII circulates in plasma in zymogen form, and activation of this form results in cleavage of the peptide bond between arginine 152 and isoleucine 153. The resulting activated FVIIa is composed of NH linked via a single disulfide bond (Cys 135 and Cys 262)₂A derivatized light chain (20kD) and a COOH-terminally derivatized heavy chain (30 kD). The light chain contains a membrane-bound Gla domain, while the heavy chain contains a catalytic domain.

Genetic and environmental factors determine FVII plasma concentrations of about 0.5mg/mL (Pinotti et al, blood.2000: 95: 3423-8). The average FVII content of different FVII genotypes may differ by several fold. Plasma FVII levels in healthy women rise during pregnancy and plasma FVII levels increase with age and are higher in women and in those with hypertriglyceridemia. FVII has the shortest half-life among all procoagulant factors (3-6 h). The average FVIIa plasma concentration of healthy individuals is 3.6ng/mL and the circulating half-life of FVIIa is relatively long compared to other coagulation factors (2.5 h).

Hereditary FVII deficiency is a rare autosomal recessive bleeding disorder, and the incidence of the total population is estimated to be 1 per 500,000 individuals (Acharya et al, J Thromb Haemost.2004: 2248-56). Acquired FVII deficiency by inhibitors is also extremely rare. Cases have also been reported in which the occurrence of defects is associated with drugs such as cephalosporins, penicillins and oral anticoagulants. In addition, acquired FVII deficiency has been reported to occur spontaneously, or with other conditions (e.g., myeloma, sepsis, aplastic anemia), with interleukin-2, and anti-thymocyte globulin therapy.

Reference polynucleotide and polypeptide sequences include, for example, GenBank accession No. J02933 (genomic sequence), M13232(cDNA) (Hagen et al, PNAS 1986: 83:2412-6), and P08709 (polypeptide sequence) (incorporated herein in its entirety). Various polymorphisms of FVII have been described, see, for example, Sabater-Lleal et al (Hum Genet.2006: 118:741-51) (the entire contents of which are incorporated herein).

Factor IX

FIX is a vitamin K-dependent plasma protein involved in the coagulation intrinsic pathway by converting FX into its active form in the presence of calcium ions, phospholipids and FVIIIa. The main catalytic ability of FIX is as a serine protease specific for a particular arginine-isoleucine bond within FX. Activation of FIX occurs via FXIa, which causes cleavage of the activating peptide from FIX, resulting in an activated FIX molecule comprising two chains held together via one or more disulfide bonds. A deficiency in FIX is the cause of recessive X-linked hemophilia B.

Hemophilia a and B are genetic diseases characterized by deficiencies in FVIII and FIX polypeptides, respectively. The underlying cause of the defect is often the result of mutations in the FVIII and FIX genes, both located on the X chromosome. Traditional therapies for hemophilia typically involve intravenous administration of pooled plasma or semi-purified coagulation proteins from normal individuals. These agents may be contaminated with pathogens or viruses such as infectious prions, HIV, parvoviruses, hepatitis a and hepatitis C. Thus, there is an urgent need for therapeutic agents that do not require the use of human serum.

The degree of decrease in FIX activity is directly proportional to the severity of hemophilia B. Current treatments for hemophilia B consist of: replacement of defective proteins with plasma-derived FIX or recombinant FIX (so-called FIX replacement or replacement therapy or therapy).

The polynucleotide and polypeptide sequences of FIX can be found, for example, in UniProtKB/Swiss-Prot accession number P00740, U.S. Pat. No. 6,531,298 and FIG. 1.

Factor VIII

Coagulation factor viii (fviii) circulates in plasma at very low concentrations and is non-covalently bound to Von Willebrand Factor (VWF). During hemostasis, FVIII separates from VWF and acts as a cofactor for FX activation mediated by activated factor ix (fixa) by enhancing the rate of activation in the presence of calcium and phospholipids or cell membranes.

FVIII can be synthesized as a single chain precursor of approximately 270-330kD with the domain structure A1-A2-B-A3-C1-C2. When purified from plasma (e.g., "plasma-derived" or "plasmatic"), FVIII consists of heavy chains (A1-A2-B) and light chains (A3-C1-C2). The molecular mass of the light chain is 80kD, while that of the heavy chain is in the range of 90-220kD due to the presence of proteolysis in the B domain.

FVIII is also synthesized as a recombinant protein for therapeutic use in hemostatic conditions. Various in vitro assays have been designed to determine the potential efficacy of recombinant fviii (rfviii) as a therapeutic drug. These assays mimic the in vivo effects of endogenous FVIII. In vitro thrombin treatment of FVIII results in a rapid increase in procoagulant activity followed by a decrease as measured by in vivo assays. This activation and inactivation is consistent with specific limited proteolysis in the heavy and light chains, which alters the availability of different binding epitopes in FVIII, for example allowing FVIII to dissociate from VWF and bind to phospholipid surfaces or altering the binding capacity to certain monoclonal antibodies.

Deficiencies or dysfunctions of FVIII result in the most common bleeding disorder, haemophilia a. The treatment of choice for managing haemophilia a is replacement therapy with plasma sources or rFVIII concentrates, and patients with severe haemophilia a who have FVIII levels below 1% typically use prophylactic therapy with the aim of maintaining FVIII above 1% between doses. Given the average half-life of various FVIII products in the circulation, this result can typically be achieved by administering FVIII two to three times per week.

Reference polynucleotide and polypeptide sequences include, for example, UniProtKB/Swiss-Prot P00451(FA8_ HUMAN); gitschier J et al, Characterisation of the human Factor VIII gene, Nature, 312 (5992: 326-30 (1984)); vehar GH et al, Structure of human Factor VIII, Nature, 312(5992):337-42 (1984); structure and Function of the Factor VIIIgene and protein, Semin Thromb Hemost, 2003: 29: 11-29(2002).

Von Willebrand factor

Von Willebrand Factor (VWF) is a glycoprotein that circulates in plasma in a series of multimers ranging in size from about 500kD to 20,000 kD. The multimeric form of VWF consists of 250kD polypeptide subunits bonded together via disulfide bonds. VWF mediates the adhesion of the original platelets to the subendothelial layer of the damaged vessel wall. Only the larger multimer exhibited hemostatic activity. It is assumed that endothelial cells secrete large multimeric forms of VWF, and those forms of VWF with low molecular weight (low molecular weight VWF) are produced by proteolytic cleavage. Polymers with large molecular masses are stored in the weibull-Palladie body of endothelial cells and released upon stimulation.

VWF is synthesized by endothelial cells and megakaryocytes as a pro-VWF precursor that is largely composed of repetitive domains. Upon cleavage of the signal peptide, the pro-VWF dimerizes via disulfide bonding in its C-terminal region. The homodimers act as multimers, which are controlled by disulfide bonding between the free terminal ends. After assembly into multimers, the propeptide sequence is removed by proteolysis (Leyte et al, biochem. J.274(1991), 257-261).

The primary translational product predicted from the cloned cDNA of VWF is a 2813-residue-containing precursor polypeptide (pro-VWF precursor). The pro-VWF precursor consists of a signal peptide with 22 amino acids and a propeptide with 741 amino acids, wherein mature VWF comprises 2050 amino acids (Ruggeri z.a. and Ware, j., faeb j., 308-316 (1993)).

The deficiency of VWF is the cause of Von Willebrand Disease (VWD) characterized by a markedly different hemorrhagic phenotype. VWD type 3 is the most severe form, in which VWF is completely defective, and VWD type 1 is associated with quantitative loss of VWF and its phenotype may be extremely mild. VWD type 2 is associated with qualitative defects in VWF and may be as severe as VWD type 3. VWD type 2 has many sub-forms, some of which are associated with the loss or reduction of high molecular weight multimers. Von Willebrand disease type 2A (VWD-2A) is characterized by the loss of both meso-and large-scale multimers. VWD-2B is characterized by the loss of the highest molecular weight multimers. Other diseases and disorders associated with VWF are known in the art.

The polynucleotide and amino acid sequences of the pro-VWF precursor are available in GenBank accession nos. NM _000552 and NP _000543, respectively.

Other coagulation proteins according to the invention are described in the art, e.g. Mann KG, thrombomb Haemost, 1999: 82:165-74.

C. Preparation of blood coagulation proteins

The preparation of the coagulation protein includes any method known in the art for: (i) preparing a recombinant DNA by genetic engineering, (ii) introducing the recombinant DNA into a prokaryotic or eukaryotic cell by, for example but not limited to, transfection, electroporation or microinjection, (iii) incubating the transformed cell, (iv) expressing the coagulation protein, e.g. constitutively or after induction, and (v) isolating the coagulation protein, e.g. from the culture medium or by harvesting the transformed cells to obtain a purified coagulation protein.

In other aspects, the coagulation protein is prepared by expression in a suitable prokaryotic or eukaryotic host system characterized by the production of a pharmacologically acceptable coagulation protein molecule. Examples of eukaryotic cells are mammalian cells, such as CHO, COS, HEK 293, BHK, SK-Hep and HepG 2.

A variety of vectors are available for the preparation of blood coagulation proteins and are selected from eukaryotic and prokaryotic expression vectors. Examples of vectors for prokaryotic expression include plastids such as (but not limited to) pRSET, pET, and pBAD, where promoters for prokaryotic expression vectors include (but are not limited to) one or more of the following: lac, trc, trp, recA or araBAD. Examples of vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as, but not limited to, pAO, pPIC, pYES or pMET, and promoters such as, but not limited to, AOX1, GAP, GAL1 or AUG 1; (ii) for expression in insect cells, vectors such as, but not limited to, pMT, pAc5, pIB, pMIB or pBAC, promoters such as, but not limited to, PH, p10, MX, Ac5, OpIE2, gp64 or polh are used, and (iii) for expression in mammalian cells, vectors such as, but not limited to, pSVL, pCMV, pRc/RSV, pcDNA3 or pBPV, and in one aspect, vectors derived from viral systems such as, but not limited to, vaccinia virus, adeno-associated virus, herpes virus or retrovirus, promoters such as, but not limited to, CMV, SV40, EF-1, UbC, RSV, ADV, BPV and β -actin are used.

D. Administration of

In one embodiment, the conjugated blood coagulation proteins of the present invention may be administered by injection (e.g., intravenous, intramuscular, or intraperitoneal injection).

Administering to a human or test animal a composition comprising a conjugated blood coagulation protein of the present invention, in one aspect, the composition comprises one or more pharmaceutically acceptable carriers. The terms "pharmaceutically" or "pharmacologically acceptable" refer to molecular entities and compositions that are stable, inhibit protein degradation (e.g., aggregation and cleavage products), and otherwise do not produce allergic or other untoward reactions when administered as described below using routes well known in the art. "pharmaceutically acceptable carrier" includes any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, including those disclosed above.

As used herein, "effective amount" includes a dosage suitable for treating a mammal having a bleeding disorder as described herein.

The composition may be applied as follows: oral, topical, transdermal, parenteral, by inhalation spray, vaginal, rectal or by intracranial injection. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal injection or infusion techniques. The following administration is also contemplated: intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection, and/or surgical implantation at a specific site. Generally, the compositions are substantially free of pyrogens and other impurities that may be harmful to the recipient.

Single or multiple administrations of the composition are carried out with dose levels and pattern being selected by the treating physician. For the prevention or treatment of disease, the appropriate dosage will depend on the following factors: the type of disease to be treated, the severity and course of the disease, whether the drug is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the drug, as described above, and the discretion of the attending physician.

The present invention also relates to a pharmaceutical composition comprising an effective amount of a conjugated blood coagulation protein as defined herein. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, salt, buffer or excipient. The pharmaceutical composition may be used for the treatment of a bleeding disorder as defined above. The pharmaceutical composition of the present invention may be a solution or a lyophilized product. The solution of the pharmaceutical composition may be subjected to any suitable lyophilization process.

As another aspect, the invention includes a kit comprising a composition of the invention packaged in a manner that facilitates use of the composition of the invention for administration to an individual. In one embodiment, the kit comprises a compound or composition described herein (e.g., a composition comprising a conjugated blood coagulation protein) packaged in a container (e.g., a sealed bottle or container), and a label affixed to the container or included in the package describing the use of the compound or composition in practicing the method. In one embodiment, the kit contains a first container having a composition comprising a conjugated coagulation protein and a second container having a physiologically acceptable reconstitution solution for the composition in the first container. In one aspect, the compound or composition is packaged in unit dosage form. The kit may further comprise a device suitable for administering the composition according to a particular route of administration. Preferably, the kit contains a label describing the use of the therapeutic protein or peptide composition.

Water-soluble polymers

In one aspect, provided coagulation protein derivative (i.e., conjugated coagulation protein) molecules are bound to a water-soluble polymer, including, but not limited to, polyethylene glycol (PEG), branched PEG, polysialic acid (PSA), carbohydrates, polysaccharides, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, poly (1-hydroxymethylethylene-hydroxymethylformal) (PHF), 2-methacryloyloxy-2' -ethyltrimethylammonium phosphate (MPC). In one embodiment of the invention, the water-soluble polymer consists of sialic acid molecules with a molecular weight in the following range: 350 to 120,000Da, 500 to 100,000Da, 1000 to 80,000Da, 1500 to 60,000Da, 2,000 to 45,000Da, 3,000 to 35,000Da, and 5,000 to 25,000 Da. The coupling of the water-soluble polymer can be carried out by direct coupling to the protein or via a linker molecule. An example of a chemical linking group is MBPH (4- [ 4-N-maleimidophenyl ] butanoic acid hydrazide) containing a carbohydrate selective hydrazide and a sulfhydryl-reactive maleimido group (Chamow et al, J Biol Chem 1992: 267: 15916-22). Other exemplary and preferred linking groups are described below.

In one embodiment, the derivative retains the full functional activity of the native therapeutic coagulation protein product and provides an extended half-life in vivo as compared to the native therapeutic coagulation protein product. In another embodiment, the derivative retains at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, or 150 percent (%) biological activity relative to the native blood coagulation protein. In a related aspect, the biological activity of the derivative and native coagulation protein is determined as the ratio of chromogenic activity to coagulation factor antigen value (coagulation factor Chr: coagulation factor Ag). In yet another embodiment of the invention, the half-life of the construct is decreased or increased to 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold relative to the in vivo half-life of the native blood coagulation protein.

A. Sialic acid and PSA

As used herein, "sialic acid moiety" includes sialic acid monomers or polymers ("polysaccharides") that are soluble in aqueous solutions or suspensions and that have little negative impact (e.g., side effects) on a mammal after administration of a pharmaceutically effective amount of a PSA-coagulation protein conjugate. In one aspect, the polymer is characterized as having 1,2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 sialic acid units. In certain aspects, different sialic acid units are combined in one chain.

In one embodiment of the invention, the sialic acid portion of the polysaccharide compound is highly hydrophilic, and in another embodiment, the entire compound is highly hydrophilic. Hydrophilicity is mainly conferred by the pendant carboxyl groups of the sialic acid units as well as by the hydroxyl groups. The saccharide units may contain other functional groups such as amine, hydroxyl or sulfate groups or combinations thereof. These groups may be present on naturally occurring sugar compounds or incorporated into derivatized polysaccharide compounds.

Naturally occurring polymeric PSAs are available in polydispersed formulations that exhibit broad size distributions (e.g., Sigma C-5762) and high Polydispersity (PD). Since polysaccharides are usually produced in bacteria with an inherent risk of co-purifying endotoxins, purification of long sialic acid polymer chains can increase the chance of increased endotoxin content. Short PSA molecules with 1-4 sialic acid units can also be prepared synthetically (Kang SH et al, Chem Commun.2000: 227-8; Res DK and Linhardt RJ, Current Organic Synthesis.2004: 1:31-46) in order to minimize the risk of high endotoxin levels. However, PSA preparations with narrow size distribution and low polydispersity and also free of endotoxins can now be manufactured. In one aspect, the polysaccharide compounds particularly useful in the present invention are polysaccharide compounds produced by bacteria. Some of these naturally occurring polysaccharides are called glycolipids. In one embodiment, the polysaccharide compound is substantially free of terminal galactose units.

B. Polyethylene glycol (PEG) and pegylation

In certain aspects, a coagulation factor (e.g., FVIII, FVIIa, FIX, or other coagulation factor molecule) is conjugated to a water-soluble polymer by any of a variety of chemical methods (Roberts JM et al, Advan Drug Delivery Rev 2002: 54: 459-76). For example, in one embodiment, the protein is modified by conjugating PEG to the free amino group of FVIII, FVIIa or FIX using N-hydroxysuccinimide (NHS) ester. In another embodiment, the water soluble polymer (e.g., PEG) is coupled to the free SH group using maleimide chemistry or coupling of PEG hydrazide or PEG amine to the carbohydrate moiety of FVIII, FVIIa or FIX after prior oxidation.

In one aspect, conjugation is performed by coupling a water-soluble polymer directly (or via a linking system) to a coagulation factor (e.g., FVIII, FVIIa, or FIX) under formation of a stable bond. In addition, in certain aspects of the invention, degradable, releasable or hydrolyzable linkage systems are used (Tsubbery et al, J Biol Chem 2004: 279:38118-24/Greenwald et al, J Med Chem 1999: 42:3657-67/ZHao et al, Bioconj Chem 2006: 17:341-51/WO2006/138572A2/US7259224B2/US7060259B 2).

In one embodiment of the invention, a coagulation factor (e.g. FVIII, FVIIa or FIX) is modified via a lysine residue by using a polyethylene glycol derivative containing an active N-hydroxysuccinimide ester (NHS) (e.g. succinimidyl succinate, succinimidyl glutarate or succinimidyl propionate). These derivatives react with lysine residues of FVIII, FVIIa or FIX under mild conditions by forming stable amide bonds. In one embodiment of the invention, the PEG derivative has a chain length of 5,000 Da. Other PEG derivatives having chain lengths of 500 to 2,000Da, 2,000 to 5,000Da, 5,000 up to 10,000Da, or 10,000 up to 20,000Da, or 20,000 up to 150,000Da are used in various embodiments, including linear and branched structures.

Alternative methods of aminopolylation are, but are not limited to, chemical conjugation to PEG carbonates through formation of urethane linkages, or reaction with aldehydes or ketones through reductive amination to form secondary amide linkages.

In one embodiment of the invention, commercially available PEG derivatives are used to chemically modify a coagulation factor (e.g., FVIII, FVIIa, FIX, or other coagulation factors) molecule. In alternative aspects, these PEG derivatives have a linear or branched structure. Examples of PEG derivatives containing NHS groups are listed below.

The following PEG derivatives are available from Nektar Therapeutics (Huntsville, Ala.; seewww.nektar.com/PEGA list of reagents; nektar Advanced PEGylation, price List 2005-2006) non-limiting examples of PEG derivatives available:

mPEG-succinimidyl propionate (mPEG-SPA)

mPEG-alpha-methylbutyrate succinimide ester (mPEG-SMB)

mPEG-CM-HBA-NHS (CM ═ carboxymethyl; HBA ═ hydroxybutyric acid)

Structure of branched PEG derivatives (Nektar Therapeutics):

branched PEGN-hydroxysuccinimide (mPEG2-NHS)

Kozlowski et al (BioDrugs 2001: 5:419-29) describe in detail such reagents with a branched structure.

Can be obtained from NOF Corp (Tokyo, Japan; seewww.nof.co.jp/english: catalog 2005) Other non-limiting examples of commercially available PEG derivatives.

General structure of linear PEG derivatives (NOF Corp.):

x ═ carboxymethyl

X ═ carboxypentyl

x ═ succinate group

x ═ glutarate radical

Structure of branched PEG derivatives (NOF Corp.): 2, 3-bis (methylpolyoxyethylene-oxy) -1- (1, 5-dioxo-5-succinimidyloxypentoxy) propane

2, 3-bis (methylpolyoxyethylene-oxy) -1- (succinimidylcarboxypentyloxy) propane

These propane derivatives show a1, 2 substituted glycerol backbone. In the present invention, branched PEG derivatives based on 1,3 substituted glycerol structures or other branched structures described in US2003/0143596A1 are also contemplated.

Also contemplated are PEG derivatives containing degradable (e.g., hydrolyzable) linking groups as described by Tsubery et al (J Biol Chem 2004: 279:38118-24) and Shechter et al (WO04089280A 3).

Surprisingly, the pegylated FVIII, FVIIa, FIX or other coagulation factors of the invention exhibit functional activity as well as an extended half-life in vivo. In addition, pegylated rFVIII, FVIIa, FIX, or other coagulation factors appear to be more resistant to thrombin inactivation.

C. Connection method

The blood coagulation protein may be covalently linked to the polysaccharide compound via any of a variety of techniques known to those skilled in the art. In various aspects of the invention, the sialic acid moiety can be bound to a blood coagulation protein (e.g., FIX, FVIII, FVIIa, or VWF), for example, by methods described in U.S. Pat. No. 4,356,170, which is incorporated herein by reference.

Other techniques for coupling PSA to polypeptides are also known and encompassed by the present invention. For example, U.S. publication No. 2007/0282096 describes conjugating amine or hydrazide derivatives, such as PSA, to proteins. In addition, U.S. publication No. 2007/0191597 describes PSA derivatives containing aldehyde groups for reaction with the reducing end of a substrate (e.g., a protein). These references are incorporated herein by reference in their entirety.

U.S. patent No. 5,846,951 (which is incorporated herein by reference in its entirety) column 7, column 15 through column 8, column 5, discloses various methods. Exemplary techniques include bonding via a peptide bond between a carboxyl group on either the blood coagulation protein or polysaccharide and an amine group of the blood coagulation protein or polysaccharide, or an ester bond between a carboxyl group of the blood coagulation protein or polysaccharide and a hydroxyl group of the blood coagulation protein or polysaccharide. Another bond of the covalent bonding of the coagulation protein to the polysaccharide compound is the Schiff base formed via the reaction between the free amino group on the coagulation protein and the aldehyde group formed at the non-reducing end of the polysaccharide by periodate oxidation (JenningsHJ and Lugowski C, J Immunol.1981: 127: 1011-8; Fernandes AI and Gregoriadis G, BiochimBiochimBiophys acta.1997: 1341: 26-34). In one aspect, the generated schiff base is stabilized via specific reduction with NaCNBH3 to form a secondary amine. An alternative approach is to generate terminal free amino groups in the PSA by reductive amination with NH4Cl after prior oxidation. Bifunctional reagents can be used to link two amino groups or two hydroxyl groups. For example, amino-containing PSA is coupled to amino groups of proteins with reagents such as BS3 (bis (sulfosuccinimidyl) suberate/Pierce, Rockford, IL). In addition, heterobifunctional crosslinking reagents such as sulfo-EMCS (N-maleimidocaproyloxy) sulfosuccinimidyl ester/Pierce) are used, for example, to link amino groups to thiol groups.

In another method, PSA hydrazides are prepared and coupled to the carbohydrate portion of the protein after prior oxidation and generation of aldehyde functional groups.

As described above, the free amino group of the therapeutic protein reacts with the 1-carboxyl group of the sialic acid residue to form a peptide bond, or an ester bond between the 1-carboxylic acid group and a hydroxyl or other suitable reactive group on the blood coagulation protein. Alternatively, the carboxyl group forms a peptide bond with the deacetylated 5-amino group, or the aldehyde group of the coagulation protein molecule forms a schiff base with the N-deacetylated 5-amino group of the sialic acid residue.

Alternatively, the polysaccharide compound is associated with the blood coagulation protein in a non-covalent manner. For example, in one aspect, the polysaccharide compound and the pharmaceutically active compound are linked via a hydrophobic interaction. Other non-covalent associations include electrostatic interactions in which oppositely charged ions attract each other.

In various embodiments, the coagulation protein and polysaccharide compound are linked or associated in stoichiometric amounts (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, etc.). In various embodiments, 1-6, 7-12, or 13-20 polysaccharides are linked to the blood coagulation protein. In other embodiments, 1,2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more polysaccharides are linked to a blood coagulation protein.

In various embodiments, the coagulation protein is modified to introduce glycosylation sites (i.e., sites other than the native glycosylation site). Such modifications can be accomplished using standard molecular biology techniques known in the art. In addition, the blood coagulation proteins may be glycosylated in vivo or in vitro prior to conjugation to the water soluble polymer via one or more carbohydrate moieties. These glycosylation sites can serve as targets for conjugating the protein to a water soluble polymer (U.S. patent application No. 20090028822, U.S. patent application No. 2009/0093399, U.S. patent application No. 2009/0081188, U.S. patent application No. 2007/0254836, U.S. patent application No. 2006/0111279, and frees s. et al, Glycobiology, 2006, 16, 9, 833-43).

D. Aminooxy linkage

In one embodiment of the invention, the blood coagulation protein conjugates are prepared using the reaction of hydroxylamine or a hydroxylamine derivative with an aldehyde (e.g., an aldehyde on a carbohydrate moiety after oxidation by sodium periodate) to form an oxime group. For example, first an oxidizing agent (e.g., sodium periodate (NaIO) is used₄) Oxidized glycoproteins (e.g., the clotting proteins of the invention) (Rothfus JA and smithhel., J Biol Chem 1963, 238, 1402-10; and Van Lenten L and Ashwell G., J Biol Chem 1971, 246, 1889-94). Periodate oxidation of glycoproteins is based on the classical Malaprade reaction (Malaprade reaction) described in 1928, i.e., the oxidation of vicinal diols with periodate to form reactive aldehyde groups (Malaprade l., Analytical application, fill Soc Chim France, 1928, 43, 683-96). Other examples of such oxidants are lead tetraacetate (Pb (OAc)₄) Manganese acetate (MnO (Ac))₃) Cobalt acetate (Co (OAc)₂) Thallium acetate (TlOAc), cerium sulfate (Ce (SO)₄)₂) (US 4,367,309) or potassium perruthenate (KRuO)₄) (Marko et al, J Am Chem Soc 1997, 119, 12661-2). "oxidizing agent" refers to a mild oxidizing agent capable of oxidizing a vicinal diol in a carbohydrate under physiological reaction conditions to produce an active aldehyde group.

The second step is to couple the aminooxy-containing polymer with the oxidized carbohydrate moiety to form an oxime linkage. In one embodiment of the invention, this step may be carried out in the presence of a catalytic amount of the nucleophilic catalyst aniline or aniline derivative (Dirksen A and Dawson PE, Bioconjugate Chem.2008; Zeng Y et al, Nature Methods 2009: 6: 207-9). Aniline catalysis significantly accelerates oxime ligation, allowing the use of very low concentrations of reagents. In another embodiment of the invention, the oxime linkage may be stabilized by the formation of an alkoxyamine linkage upon reduction with NaCNBH3 (fig. 2).

In one embodiment of the invention, the reaction steps of conjugating the water-soluble polymer to the blood coagulation protein are performed separately and sequentially (i.e., the starting material (e.g., blood coagulation protein, water-soluble polymer, etc.), reagents (e.g., oxidizing agent, aniline, etc.), and reaction products (e.g., oxidized carbohydrate on blood coagulation protein, activated aminoxy water-soluble polymer, etc.) are separated between the individual reaction steps).

Additional information on aminooxy technology can be found in the following references, each of which is incorporated herein in its entirety: EP1681303a1 (hased erythropoietin); WO 2005/014024 (conjugate where polymer and protein are linked via oxime linker); WO96/40662 (amino-containing linking compounds and their use in conjugates); WO2008/025856 (modified protein); peri F et al, Tetrahedron 1998, 54, 12269-78: Kubler-Kielb J and Pozsgay V., J Org Chem 2005, 70, 6887-90; lees A et al, Vaccine 2006, 24(6), 716-29; and Heredia KL et al, macromolecules 2007, 40(14), 4772-9.

In various embodiments of the invention, water-soluble polymers attached to the oxidized carbohydrate moiety of a blood coagulation protein (e.g., FVIII, FVIIa, or FIX) according to the aminooxy technique described herein include, but are not limited to, polyethylene glycol (PEG), branched PEG, polysialic acid (PSA), carbohydrates, polysaccharides, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, poly (1-hydroxymethylethylidene-hydroxymethylformal) (PHF), 2-methacryloyloxy-2' -ethyltrimethylammonium phosphate (MPC).

The following examples are not intended to be limiting, but merely illustrate specific embodiments of the invention.

Examples

Example 1

Preparation of homobifunctional linking group NH ₂ [OCH ₂ CH ₂ ] ₂ ONH ₂

Synthesis of the homobifunctional linking group NH containing two reactive aminoxy groups according to Boturyn et al (Tetrahedron 1997: 53:5485-92) in a two-step organic reaction using a modified Cabereli Synthesis of a Primary amine (Gabriel-Synthesis)₂[OCH₂CH₂]₂ONH₂

(3-oxa-pentane-1, 5-dihydroxy amine) (FIG. 3). In a first step, one molecule of 2, 2-chlorodiethyl ether is reacted with two molecules of endo-N-hydroxy-5-norbornene-2, 3-dicarboximide in Dimethylformamide (DMF). The desired homobifunctional product was prepared from the resulting intermediate by hydrazinolysis in ethanol.

Example 2

Preparation of homobifunctional linking group NH ₂ [OCH ₂ CH ₂ ] ₄ ONH ₂

The homobifunctional linking group NH containing two active aminoxy groups was synthesized according to Boturyn et al (Tetrahedron 1997: 53:5485-92) in a two-step organic reaction using a modified Cabery synthesis of primary amines₂[OCH₂CH₂]₄ONH₂

(3,6, 9-trioxa-undecane-1, 11-dihydroxy amine) (FIG. 3). In a first step, one molecule of bis- (2- (2-chloroethoxy) -ethyl) -ether is reacted with two molecules of endo-N-hydroxy-5-norbornene-2, 3-dicarboximide in DMF. The desired homobifunctional product was prepared from the resulting intermediate by hydrazinolysis in ethanol.

Example 3

Preparation of aminoxy-PSA

500mg of oxidized PSA (MW 18.8kD) obtained from the Indian Serum Institute of India (Pu, India) was dissolved in 8ml of 50mM sodium acetate buffer (pH 5.5). Next, 100mg of 3-oxa-pentane-1, 5-dihydroxyamine was added. After 2 hours shaking at room temperature, 44mg of sodium cyanoborohydride was added. After shaking at 4 ℃ for 4 hours, the reaction mixture was loaded into Slide-A-Lyzer (Pierce, Rockford, IL) dialysis cassettes (3.5kD membrane, regenerated cellulose) and dialyzed against PBS (pH7.2) for 4 days. The product was frozen at-80 ℃. FIG. 4 illustrates the preparation of aminoxy-PSA according to this procedure.

Alternative procedure for the preparation of aminoxy PSA

1000mg of oxidized PSA (MW 20kD) obtained from the Indian serum institute (Pune, India) was dissolved in 16ml of 50mM phosphate buffer (pH 6.0). Subsequently, 170mg of 3-oxa-pentane-1, 5-dihydroxyamine were added to the reaction mixture. After 2 hours of shaking at room temperature, 78.5mg of sodium cyanoborohydride was added and the reaction was allowed to proceed for 18 hours overnight. The reaction mixture was then subjected to an ultrafiltration/diafiltration procedure (UF/DF) using a 5kD cut-off membrane (Millipore) made of regenerated cellulose.

Example 4

Coupling aminoxy-PSA with rFIX and purifying the conjugate

To 12.6mg rFIX dissolved in 6.3ml of 50mM sodium acetate buffer (pH 6.0) was added 289. mu.l of an aqueous sodium periodate solution (10 mM). The mixture was shaken in the dark at 4 ℃ for 1 hour and quenched by the addition of 6.5. mu.l of 1M glycerol at room temperature for 15 minutes. Low molecular weight contaminants were removed by ultrafiltration/diafiltration (UF/DF) using a Vivaspin (Sartorius, Goettingen, Germany) concentrator (30kD membrane, regenerated cellulose). Next, 43mg of aminoxy-PSA was added to the UF/DF retentate and the mixture was shaken at 4 ℃ for 18 hours. Excess PSA reagent was removed using Hydrophobic Interaction Chromatography (HIC). The conductivity of the cooled reaction mixture was raised to 180mS/cm and loaded onto a 5ml HiTrap Butyl FF (GE Healthcare, Fairfield, CT) HIC column (1.6X 2.5cm) pre-equilibrated with 50mM HEPES, 3M sodium chloride, 6.7mM calcium chloride, 0.01% Tween80(pH 6.9). The conjugate was eluted with 2.4 Column Volumes (CV) of 50mM HEPES, 6.7mM calcium chloride, 0.005% Tween80 (pH7.4) at a flow rate of 5 ml/min. The preparation was characterized analytically by measuring total protein (BCA) and FIX chromogenic activity. For the PSA-rFIX conjugate, the specific activity per mg of protein was determined to be 80.2IU (56.4% compared to native rFIX). The results are summarized in table 1.

TABLE 1

FIG. 5 illustrates the analytical characterization of PSA-rFIX conjugates by SDS-PAGE using Coomassie Brilliant blue staining. FIG. 6 shows SDS-PAGE followed by Western blotting using anti-FIX and anti-PSA antibodies.

Example 5

Coupling of aminoxy-PSA with rFIX in the Presence of Aniline as nucleophilic catalyst

To 3.0mg rFIX dissolved in 1.4ml of 50mM sodium acetate buffer (pH 6.0) was added 14.1. mu.l of an aqueous sodium periodate solution (10 mM). The mixture was shaken in the dark at 4 ℃ for 1 hour and quenched by the addition of 1.5. mu.l of 1M glycerol at room temperature for 15 minutes. Low molecular weight contaminants were removed by Size Exclusion Chromatography (SEC) using a PD-10 desalting column (GE Healthcare, Fairfield, CT). 1.2mg of oxidized rFIX dissolved in 1.33ml of 50mM sodium acetate buffer (pH 6.0) was mixed with 70. mu.l of aniline (200mM stock solution) and shaken at room temperature for 45 minutes. Next, 4.0mg of aminooxy-PSA was added, and the mixture was shaken at room temperature for 2 hours and then at 4 ℃ for 16 hours. Samples were taken after 1 hour, after 2 hours and at the end of the reaction after 18 hours. Next, excess PSA reagent and free rFIX were removed by means of HIC. The conductivity of the cooled reaction mixture was raised to 180mS/cm and loaded onto a 5ml HiTrap Butyl FF (GE Healthcare, Fairfield, CT) HIC column (1.6X 2.5cm) pre-equilibrated with 50mM HEPES, 3M sodium chloride, 6.7mM calcium chloride, 0.01% Tween80(pH 6.9). The conjugate was eluted with a linear gradient of 20CV to 50mM HEPES, 6.7mM calcium chloride, 0.005% Tween80 (pH7.4) at a flow rate of 5 ml/min.

Example 6

Aminoxy-PSA with rFIX and Using NaCNBH ₃ Reduction of

To 10.5mg rFIX dissolved in 5.25ml of 50mM sodium acetate buffer (pH 6.0) was added 53. mu.l of an aqueous sodium periodate solution (10 mM). The mixture was shaken in the dark at 4 ℃ for 1 hour and quenched by the addition of 5.3. mu.l of 1M glycerol at room temperature for 15 minutes. Low molecular weight contaminants were removed by means of UF/DF using a Vivaspin (Sartorius, Goettingen, Germany) concentrator (30kD membrane, regenerated cellulose). Next, 35.9mg of aminoxy-PSA was added to the UF/DF retentate and the mixture was shaken at room temperature for 2 hours. Subsequently 53. mu.l aqueous sodium cyanoborohydride (5M) was added and the reaction was carried out for a further 16 hours. Subsequently, excess PSA reagent was removed by means of HIC. The conductivity of the cooled reaction mixture was raised to 180mS/cm and loaded on a 5ml HiTrap Butyl FF HIC (GE Healthcare, Fairfield, CT) column (1.6X 2.5cm) pre-equilibrated with 50mM HEPES, 3M sodium chloride, 6.7mM calcium chloride, 0.01% Tween80(pH 6.9). The conjugate was eluted with 2.4CV of 50mM HEPES, 6.7mM calcium chloride, 0.005% Tween80 (pH7.4) at a flow rate of 5 ml/min.

Example 7

Reacting aminooxy-PSA (linking group: NH) ₂ [OCH ₂ CH ₂ ] ₄ ONH ₂ ) Coupling with rFIX and purification of the conjugate

To 5.6mg rFIX dissolved in 2.8ml of 50mM sodium acetate buffer (pH 6.0) was added 102. mu.l of an aqueous sodium periodate solution (10 mM). The mixture was shaken in the dark at 4 ℃ for 1 hour and quenched by the addition of 2.9. mu.l of 1M glycerol at room temperature for 15 minutes. Low molecular weight contaminants were removed by means of UF/DF using a Vivaspin (Sartorius, Goettingen, Germany) concentrator (30kD membrane, regenerated cellulose). Subsequently, 19mg of aminoxy-PSA was added to the UF/DF retentate and the mixture was shaken at 4 ℃ for 18 hours. Excess PSA reagent was removed by means of HIC. The conductivity of the cooled reaction mixture was raised to 180mS/cm and loaded on a 5ml HiTrap Butyl FF (GE Healthcare, Fairfield, CT) HIC column (1.6X 2.5cm) pre-equilibrated with 50mM HEPES, 3M sodium chloride, 6.7mM calcium chloride, 0.01% Tween80(pH 6.9). The conjugate was eluted with 2.4CV of 50mM HEPES, 6.7mM calcium chloride, 0.005% Tween80 (pH7.4) at a flow rate of 5 ml/min.

Example 8

Coupling aminooxy-PSA with rFVIII

To the solution was dissolved in 11ml of Hepes buffer (pH 6) (50mM Hepes, 5mM CaCl₂150mM NaCl, 0.01% Tween) to 11mg rFVIII 57. mu.l of 10mM sodium periodate was added. The mixture was shaken in the dark at 4 ℃ for 30 minutes and quenched by the addition of 107. mu.l of 1M aqueous glycerol solution at 4 ℃ for 30 minutes. Subsequently, 19.8mg aminooxy-PSA (18.8kD) was added and the mixture was shaken overnight at 4 ℃. By adding a buffer containing 8M ammonium acetate (8M ammonium acetate, 50mM hepes, 5mM CaCl)₂350mM NaCl, 0.01% Tween80, pH6.9) to increase the ionic strength to give a final concentration of 2.5M ammonium acetate. Next, the reaction mixture was loaded into equilibration buffer (2.5M ammonium acetate, 50mM hepes, 5mM CaCl)₂350mM NaCl, 0.01% Tween80, pH6.9) on a HiTrap Butyl FF (GE Healthcare, Fairfield, CT) column. With elution buffer (50mM Hepes, 5mM CaCl)₂0.01% Tween80, pH7.4) and the eluate was concentrated by centrifugation using a Vivaspin (Sartorius, Goettingen, Germany) apparatus with a MWCO of 30,000.

Example 9

PK study in hemophilia mice

FIX-deficient mice were injected with a 10ml volume dose of rFIX or PSA-rFIX (prepared according to example 4) in a formulation buffer (10mM histidine, 260mM glycine, 29mM sucrose, 0.005% Tween80, pH 6.8) per kg body weight. Each group of 6 mice were sacrificed at 5 minutes, 3 hours, 9 hours, 16 hours, 24 hours, and 48 hours after substance injection, and blood was collected by cardiac puncture. Plasma containing citrate was prepared and stored frozen until FIX activity was analyzed.

FIX activity was measured by chromogenic FIX assay (Biophen FIX assay, Hyphen Biomed, Neuville-sur-Oise, France) and an elimination curve was constructed (FIG. 7). The actual FIX active dose of PSA-rFIX was 123IU FIX/kg, and the actual FIX active dose of rFIX was 143IU FIX/kg. Pharmacokinetic parameters were calculated using program R (The R Foundation for statistical computing, 2008). The in vivo recovery of rFIX was 13% and the in vivo recovery of PSA-rFIX was 29%. Dose-adjusted AUC for PSA-rFIX increased 6.4-fold relative to rFIX, terminal half-life increased 1.2-fold, and MRT for PSA-rFIX was 1.7-fold longer compared to rFIX (table 2).

TABLE 2

Example 10

Polysialylating blood coagulation proteins

Polysialylation as described herein may be extended to other coagulation proteins. For example, in various aspects of the invention, coagulation proteins such as FVIII, FVIIa and VWF are repeatedly polysialylated with aminoxy-PSA as described in examples 5,6 and 9 above.

Example 11

Preparation of homobifunctional linking group NH ₂ [OCH ₂ CH ₂ ] ₆ ONH ₂

Synthesis of homo-bis-containing two active aminoxy groups according to Boturyn et al (Tetrahedron 1997: 53:5485-92) in a two-step organic reaction using a modified Caberley synthesis of primary aminesFunctional linking group NH₂[OCH₂CH₂]₆ONH₂

(3,6,9,12, 15-pentaoxa-heptadecane-1, 17-dihydroxyamine). In a first step, one molecule of hexaethylene glycol dichloride (hexaethylene glycol dichloride) is reacted with two molecules of endo-N-hydroxy-5-norbornene-2, 3-dicarboximide in DMF. The desired homobifunctional product was prepared from the resulting intermediate by hydrazinolysis in ethanol.

Example 12

Polysialylation of rFIX Using a maleimido/aminoxy linking System

A. Preparation of modifying reagents

The aminoxy-PSA reagent was prepared by using the maleimido/aminoxy ligation system (Toyokuni et al, Bioconjugate Chem 2003: 14, 1253-9). PSA-SH (20kD) containing free terminal SH-groups was prepared using a two-step procedure: a) by using NH according to WO05016973A1₄Reductive amination of oxidized PSA with Cl to produce PSA-NH₂And b) introduction of sulfhydryl groups by reacting the terminal primary amino group with 2-iminothiolane (yurt's reagent)/Pierce, Rockford, IL) as described in US 7645860. The PSA-SH is coupled to the maleimido group of the linker in PBS buffer at pH 7.5 using a 10-fold molar excess of the linker and a PSA-SH concentration of 50 mg/ml. The reaction mixture was incubated at room temperature for 2 hours with gentle shaking. Subsequently, excess linking reagent was removed and the aminoxy-PSA buffer was replaced by diafiltration into an oxidation buffer (50mM sodium phosphate, pH 6.0). The regenerated cellulose membrane (Millipore, Billerica, MA) was buffer exchanged 25 times with Pellicon XL5 kD.

B. In use with NaIO₄Pre-oxidizedPost-modified rFIX

rFIX was oxidized in 50mM sodium phosphate buffer (pH 6.0) with 100. mu.M sodium periodate in this buffer. The mixture was shaken in the dark at 4 ℃ for 1 hour and quenched at room temperature by the addition of glycerol to a final concentration of 5mM for 15 minutes. Low molecular weight contaminants were removed by Size Exclusion Chromatography (SEC) using a PD-10 desalting column (GE Healthcare, Fairfield, CT). Subsequently, aniline was incorporated into the oxidized rFIX to obtain a final concentration of 10mM and mixed with aminooxy-PSA reagent to achieve a 5-fold molar excess of PSA. The reaction mixture was incubated for 2 hours at room temperature in the dark with gentle shaking.

C. Purification of conjugates

Excess PSA reagent and free rFIX were removed by HIC. The conductivity of the reaction mixture was raised to 180mS/cm and loaded onto a column pre-equilibrated with 50mM HEPES, 3M sodium chloride, 6.7mM calcium chloride, 0.01% Tween80 (pH6.9) filled with 48ml butyl-agarose FF (GE Healthcare, Fairfield, CT). The conjugate was then eluted with a linear gradient of 40CV of 60% elution buffer (50mM Hepes, 6.7mM calcium chloride, pH 7.4). Finally, fractions containing PSA-rFIX were collected and UF/DF was performed using a 30kD membrane (Millipore) made of regenerated cellulose. The preparation was characterized analytically by measuring total protein (BCA) and FIX chromogenic activity. For PSA-rFIX conjugates prepared from both variants, specific activity was determined to be greater than 50% of native rFIX.

Example 13

Preparation of aminoxy-PSA reagent

aminoxy-PSA reagent was prepared according to example 3. The final product was diafiltered with buffer (pH7.2) (50mM Hepes) using a 5kD membrane (regenerated cellulose, Millipore), frozen at-80 ℃ and lyophilized. After lyophilization, the reagents are dissolved in an appropriate volume of water and used to prepare PSA-protein conjugates via carbohydrate modification.

Example 14

Pharmacokinetics of polysialylated rFVIII in FVIII-deficient knockout mouse models

PSA-FVIII conjugates were prepared according to example 8. The conjugate showed a specific activity of 6237IU/mg (FVIII activity determined by chromogenic assay; total protein determined by Bradford assay) and a degree of polysialylation of 6.7 (moles PSA per mole FVIII) as measured by the resorcinol assay (Svennerholm L, Biochim Biophys Acta 1957: 24: 604-11).

FVIII-deficient mice, described in detail by Bi et al (Nat Genet 1995: 10:119-21), were used as models of severe human hemophilia A. Each group (6 mice per group) received a bolus injection (200IU FVIII/kg) of PSA-rFVIII or native rFVIII (advanced Healthcare) prepared according to example 8 at a dose of 200IU FVIII per kg body weight via the tail vein. Citrate-containing plasma was prepared from individual groups by cardiac puncture after anesthesia 5 minutes, 3 hours, 6 hours, 9 hours, 16 hours, 24 hours, 32 hours and 42 hours after injection. The extent of FVIII activity in plasma samples was measured by using a chromogenic assay. The results of this experiment are summarized in table 3 and illustrated in fig. 8. Version 2.10.1R (a Statistical calculation for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria was used.http://www.R-project.org.)All calculations were performed. As a result, the Mean Residence Time (MRT) increased from 5.4 hours (advante control) to 11.1 hours (PSA-rFVIII conjugate).

Table 3:

example 15

Detailed Synthesis of aminoxy-PSA reagents

3-oxa-pentane-1, 5-dihydroxyamine was synthesized according to Botyryn et al (Tetrahedron 1997: 53:5485-92) in a two-step organic synthesis as outlined in example 1.

Step 1:

to a solution of endo-N-hydroxy-5-norbornene-2, 3-dicarboximide (59.0 g; 1.00 eq) in 700ml of anhydrous N, N-dimethylformamide was added anhydrous K₂CO₃(45.51 g; 1.00 eq.) and 2, 2-dichlorodiethyl ether (15.84 ml; 0.41 eq.). The reaction mixture was stirred at 50 ℃ for 22 hours. The mixture was evaporated to dryness under reduced pressure. The residue was suspended in 2L of dichloromethane and extracted twice with saturated aqueous NaCl solution (1L each). The dichloromethane layer was washed with Na₂SO₄Drying, followed by evaporation to dryness under reduced pressure and drying under high vacuum, gave 64.5g of 3-oxapentane-1, 5-dioxy-endo-2 ', 3' -dicarboxyidiimide norbornene (intermediate 1) as a yellow-white solid.

Step 2:

to a solution of intermediate 1(64.25 g; 1.00 eq) in 800ml of absolute ethanol was added 31.0ml of hydrazine hydrate (4.26 eq). The reaction mixture was then refluxed for 2 hours. The mixture was concentrated to half of the initial volume by evaporation of the solvent under reduced pressure. The precipitate that appeared was filtered off. The remaining ethanol layer was evaporated to dryness under reduced pressure. The residue containing the crude 3-oxa-pentane-1, 5-dihydroxyamine was dried in vacuo to give 46.3 g. The crude product was further purified by column chromatography (silica gel 60; isocratic elution with dichloromethane/methanol mixture 9+ 1) to yield 11.7g of pure final product 3-oxa-pentane-1, 5-dihydroxyamine.

Example 16

Polysialylation of rFIX Using PSA hydrazide

rFIX was polysialylated using PSA hydrazide reagent prepared by reacting oxidized PSA with adipic Acid Dihydrazide (ADH).

Step 1: preparation of PSA hydrazides

500mg of oxidized PSA (MW 20kD) obtained from the Indian serum institute (Pune, India) was dissolved in 8ml of 50mM sodium acetate buffer (pH 5.5). 100mg of adipic Acid Dihydrazide (ADH) was then added. The solution was gently shaken for 2 hours. 44mg of sodium cyanoborohydride are subsequently added. After incubating the reaction at 4 ℃ for 4 hours, the reaction mixture was loaded into Slide-A-Lyzer (Pierce, Rockford, IL) dialysis cassettes (3.5kD membrane, regenerated cellulose) and dialyzed against PBS (pH7.2) for 4 days. The product was frozen at-80 ℃.

Step 2: reaction of PSA hydrazide with rFIX and purification of conjugate

rFIX was polysialylated by using PSA hydrazide reagent as described in step 1. With NaIO in the dark at 4 ℃ with gentle shaking₄(concentration: 80. mu.M) rFIX was oxidized (concentration: 1mg/ml) for 1 hour. The reaction was stopped by the addition of glycerol and the oxidized FIX was UF/DF using a 30kD membrane made of regenerated cellulose (Vivaspin). The oxidized rFIX was subsequently polysialylated at pH 6.5 using a 200-fold molar excess of reagent and a protein concentration of 1 mg/ml. rFIX and polysialylating reagents were incubated for 2 hours at room temperature in the dark with gentle shaking. Finally, the PSA-rFIX conjugate was purified using HIC. The conductivity of the reaction mixture was raised to 130mS/cm by adding ammonium acetate containing buffer (50mM Hepes, 350mM NaCl, 5mM calcium chloride, 8M ammonium acetate, 0.01% Tween80, pH6.9) and loaded on a HiTrap Butyl FF column (5ml, GE Healthcare, Fairfield, CT) pre-equilibrated with 50mM Hepes, 2.5M ammonium acetate, 350mM sodium chloride, 5mM calcium chloride, 0.01% Tween80(pH 6.9). Next, the conjugate was eluted with 50mM Hepes, 5mM calcium chloride, 0.01% Tween80(pH 7.4). Finally, fractions containing PSA-rFIX were collected and UF/DF was performed using a 30kD membrane (Vivaspin) made of regenerated cellulose. For the PEG-rFIX conjugate, specific activity was determined to be greater than 50% of native rFIX (chromogenic assay).

Example 17

Polysialylation of rFIX Using PSA hydrazide in the Presence of Aniline as nucleophilic catalyst

123mg of rFIX were dissolved in 60ml of phosphate buffer (50mM NaPO)₄pH 6.5) buffer. 1.2ml of aqueous sodium periodate (10mM) are subsequently added and the mixture is incubated for 1 hour at 4 ℃ in the dark with gentle stirring. Next, the reaction was quenched by the addition of 600. mu.l of 1M aqueous glycerol solution at room temperature for 15 minutes. The mixture was then UF/DF using a Pellicon XLUltracel 30kD membrane.

The UF/DF retentate (63.4ml) containing oxidized rFIX was further diluted with 59.6ml of phosphate buffer (50mM NaPCU, pH6.0) and mixed with 6.5ml of aqueous aniline (200mM) and incubated at room temperature for 30 minutes. 12.3ml of PSA-hydrazide reagent (prepared according to example 16) was then added, giving a 5-fold molar excess of reagent. The mixture was incubated at room temperature in the dark for 2 hours with gentle shaking.

Excess PSA-hydrazide reagent and free rFIX were removed by HIC. The conductivity of the reaction mixture was raised to 180mS/cm and loaded onto a column pre-equilibrated with 50mM HEPES, 3M sodium chloride, 6.7mM calcium chloride, 0.01% Tween80 (pH6.9) filled with 48ml butyl-agarose FF (GE Healthcare, Fairfield, CT). Next, the conjugate was eluted with 50mM hepes, 5mM calcium chloride, 0.01% Tween80(pH 7.4). Finally, fractions containing PSA-rFIX were collected and UF/DF was performed using a 30kD membrane (Millipore) made of regenerated cellulose. The preparation was characterized analytically by measuring total protein (BCA) and FIX chromogenic activity. For PSA-rFIX conjugates, specific activity was determined to be greater than 50% of native rFIX.

Example 18

Polysialylation of rFIX and purification using a two-step procedure

140mg of rFIX were dissolved in 62ml of phosphate buffer (50mM NaPO)₄pH6.0) buffer. Followed by addition of1.92ml of aqueous sodium periodate (10mM) and the mixture is incubated for 1 h at 4 ℃ in the dark with gentle stirring and quenched at room temperature for 15 min by the addition of 64. mu.l of 1M aqueous glycerol. The mixture was then UF/DF using a Pellicon XL Ultracel 30kD membrane.

The UF/DF retentate (69.4ml) containing oxidized rFIX was further diluted with 73.8ml of phosphate buffer (50mM NaPCU, pH6.0), mixed with 8.2ml of aqueous aniline (200mM) and incubated at room temperature for 30 minutes. 12.3ml of aminooxy reagent (prepared according to example 3) were then added, giving a 2.5-fold molar excess of reagent. The mixture was incubated at room temperature in the dark with gentle shaking for 2.5 hours.

Free rFIX was removed by means of anion exchange chromatography (AIEC). With 20ml of buffer A (50mM Hepes, 5mM CaCl)₂pH 7.5) and loaded on a Q-Sepharose FF 26/10 column (GEHealthcare, Fairfield, CT) pre-equilibrated with buffer A. Followed by buffer B (50mM Hepes, 1M NaCl, 5mM CaCl)₂pH 7.5) elution column. Free rFIX elutes at a conductivity between 12-25mS/cm, and the conjugate elutes at a conductivity between 27-45 mS/cm. Followed by addition of buffer C (50mM Hepes, 5M NaCl, 5mM CaCl)₂pH6.9) the conductivity of the conjugate-containing eluate was raised to 190mS/cm and loaded in buffered solution D (50mM Hepes, 3M NaCl, 5mM CaCl)₂pH6.9) on a pre-equilibrated butyl sepharose FF 26/10 column (GE Healthcare, Fairfield, CT). Free PSA-reagent was washed off with 5CV buffer D. Next, 100% buffer E (50mM Hepes, 5mM CaCl) was used₂pH7.4) eluting the conjugate. Use of a 10kD membrane (88 cm) made of regenerated cellulose²Cut-off 10kD/Millipore) was run on UF/DF to concentrate the conjugate-containing fractions. With a solution containing 150mM NaCl and 5mM CaCl₂The final diafiltration step was performed with histidine buffer (pH 7.2). The preparation was characterized analytically by measuring total protein (BCA) and FIX chromogenic activity. For PSA-rFIX conjugates, specific activity was determined to be greater than 50% of native rFIX.

Example 19

Coupling aminoxy-PSA with rFVIIa and purifying the conjugate

Mix a solution of 10mg rFVIIa in 5ml reaction buffer (50mM Hepes, 150mM sodium chloride, 5mM calcium chloride, pH6.0) with NaIO₄Aqueous solution (final concentration: 100. mu.M), and incubated at 4 ℃ in the dark for 1 hour with gentle stirring, and quenched by addition of aqueous cysteine solution (final concentration: 1mM) for 15 minutes. Next, the reaction mixture was subjected to UF/DF. To the retentate (10ml) was added a 30-fold molar excess of aminoxy reagent (prepared according to example 1). The coupling reaction was carried out in the dark for 2 hours at room temperature with gentle shaking. Excess aminoxy reagent was removed by HIC. The conductivity of the reaction mixture was raised to 130mS/cm by adding ammonium acetate containing buffer (50mM Hepes, 350mM NaCl, 5mM calcium chloride, 8M ammonium acetate, 0.01% Tween80, pH6.9) and loaded on a HiTrap Butyl FF column (5ml, GEHealthcare, Fairfield, CT) pre-equilibrated with 50mM Hepes, 2.5M ammonium acetate, 350mM sodium chloride, 5mM calcium chloride, 0.01% Tween80(pH 6.9). The conjugate was then eluted with a linear gradient of 50mM Hepes, 5mM calcium chloride, 0.01% Tween80 (pH7.4) through 20CV of 100% elution buffer. Finally, fractions containing PSA-rFVIIa were collected and UF/DF was performed using a 30kD membrane made of regenerated cellulose (Vivaspin). The formulations were characterized analytically by measuring total protein (BCA) and FVIIa chromogenic activity (Staclot assay, diagnostic Stago, assieres, France) and showed specific activities of more than 20% of rFVIIa starting material.

Example 20

Coupling of aminoxy-PSA with rFVIIa in the presence of aniline as nucleophilic catalyst

To 3.0mg of rFVIIa dissolved in 1.4ml of 50mM sodium acetate buffer (pH 6.0) was added 14.1. mu.l of an aqueous solution of sodium periodate (10 mM). The mixture was shaken in the dark at 4 ℃ for 1 hour and quenched by the addition of 1.5. mu.l of 1M glycerol at room temperature for 15 minutes. Low molecular weight contaminants were removed by Size Exclusion Chromatography (SEC) using a PD-10 desalting column (GE Healthcare, Fairfield, CT). 3mg of rFVIIa oxide dissolved in 3ml of 50mM sodium acetate buffer (pH 6.0) was mixed with aniline (a nucleophilic catalyst, final concentration: 10mM) and shaken at room temperature for 30 minutes. Next, aminoxy-PSA was added to give a 5-fold molar excess, and the mixture was shaken at room temperature for 2 hours. Next, excess PSA reagent and free rFIX were removed by means of HIC. The conductivity of the cooled reaction mixture was raised to 180mS/cm and loaded on a 5ml HiTrap Butyl FF (GEHealthcare, Fairfield, CT) HIC column (1.6X 2.5cm) pre-equilibrated with 50mM HEPES, 3M sodium chloride, 6.7mM calcium chloride, 0.01% Tween80(pH 6.9). The conjugate was eluted with a linear gradient of 20CV of 50mM HEPES, 6.7mM calcium chloride, 0.005% Tween80 (pH7.4) at a flow rate of 5 ml/min.

Example 21

Preparation of aminoxy-PEG reagent

A branched PEG-aldehyde (MW 40kD) was used to couple to the diamino linking group prepared as described in example 1. This PEG-aldehyde reagent can be obtained from NOF (NOF corp., Tokyo, Japan). 500mg of PEG-aldehyde were dissolved in 8ml of 50mM sodium acetate buffer (pH 5.5). Subsequently, 100mg of 3-oxa-pentane-1, 5-dihydroxyamine were added. After 2 hours shaking at room temperature, 44mg of sodium cyanoborohydride was added. After shaking at 4 ℃ for 4 hours, the reaction mixture was loaded into Slide-A-Lyzer (Pierce, Rockford, IL) dialysis cassettes (3.5kD membrane, regenerated cellulose) and dialyzed against PBS (pH7.2) for 4 days. The product was frozen at-80 ℃.

Example 22

Pegylation of rFIX with aminoxy PEG reagent

rFIX was pegylated by using an aminooxy-containing linear 20kD pegylation reagent. Examples of agents of this type are those from NOFCA series (NOF Corp., Tokyo, Japan). Using NaIO at a protein concentration of 2mg/ml in reaction buffer (50mM Hepes, 150mM sodium chloride, 5mM calcium chloride, pH6.0) in the dark at 4 ℃ with gentle shaking₄rFIX was oxidized (final concentration: 100. mu.M) for 1 hour and quenched by addition of an aqueous glycerol solution (final concentration: 1mM) for 15 minutes. Next, the reaction mixture was subjected to UF/DF. To the retentate was added a 3-fold molar excess of aminooxy reagent and aniline (nucleophilic catalyst, final concentration: 10 mM). The coupling reaction was carried out in the dark for 2 hours at room temperature with gentle shaking. Finally, the PEG-rFIX conjugate was purified by ion exchange chromatography on Q-sepharose FF. 1.5mg protein per ml gel was loaded onto a column pre-equilibrated with 50mM Tris (pH 8.0). The conjugate was eluted with 20CV 50mM Tris and 1M sodium chloride (pH 8.0) and subsequently UF/DF was performed using a 30kD membrane. The preparation was characterized analytically by measuring total protein (BCA) and FIX chromogenic activity. For the PEG-rFIX conjugate, specific activity was determined to be greater than 75% of native rFIX.

Example 23

PEGylation of rFVIII with aminoxy PEG reagent

rFVIII was pegylated by using an aminooxy-containing linear 20kD pegylation reagent. Examples of agents of this type are those from NOFCA series (NOF Corp., Tokyo, Japan). Using NaIO at a protein concentration of 1mg/ml in reaction buffer (50mM Hepes, 150mM sodium chloride, 5mM calcium chloride, pH6.0) in the dark at 4 ℃ with gentle shaking₄rFVIII was oxidized (final concentration: 100. mu.M) for 1 hour and quenched by addition of aqueous cysteine solution (final concentration: 1mM) for 15 minutes. Next, the reaction mixture was subjected to UF/DF. Adding a 20-fold molar excess of aminoxy groups to the retentateAgent and aniline (nucleophilic catalyst, final concentration: 10 mM). The coupling reaction was carried out in the dark for 2 hours at room temperature with gentle shaking. Finally, the PEG-rFVIII conjugate was purified by ion exchange chromatography on Q-sepharose FF. 1.5mg protein per ml gel was loaded with 5mM CaCl₂50mM Hepes buffer (pH 7.4). With a medium containing 5mM CaCl₂And 500mM sodium chloride in 50mM Hepes buffer (pH7.4) and subsequent UF/DF using a 30kD membrane. Analytical characterization of the conjugates by FVIII chromogenic assay and assay total protein (BCA assay) showed specific activity greater than 60% of rFVIII starting material.

Example 24

PEGylation of rFVIIa with aminoxy PEG reagent

rFVIIa was pegylated by using an aminooxy-containing linear 20kD pegylation reagent. Examples of agents of this type are those from NOFCA series (NOF Corp., Tokyo, Japan). Using NaIO at a protein concentration of 2mg/ml in reaction buffer (50mM Hepes, 150mM sodium chloride, 5mM calcium chloride, pH6.0) in the dark at 4 ℃ with gentle shaking₄rFVIIa was oxidized (final concentration: 100. mu.M) for 1 hour and quenched by addition of aqueous glycerol (final concentration: 1mM) for 15 minutes. Next, the reaction mixture was subjected to UF/DF. To the retentate was added a 5-fold molar excess of aminooxy reagent and aniline (nucleophilic catalyst, final concentration: 10 mM). The coupling reaction was carried out in the dark for 2 hours at room temperature with gentle shaking. Finally, the PEG-rFVIIa conjugate was purified by ion exchange chromatography on Q-sepharose FF. 1.5mg protein per ml gel was loaded with 1mM CaCl₂20mM Hepes buffer (pH 7.4). With a 1mM CaCl content₂And 500mM sodium chloride in 20mM Hepes buffer (pH7.4) and subsequent UF/DF using a 30kD membrane. By measuring FVIIanalytical characterization of the conjugates for a activity (Staclot assay, diagnostic Stago, Asnieres, France) and total protein (BCA assay) showed specific activities greater than 25% of the rFVIIa starting material.

Example 25

PEGylation of rFIX with PEG-hydrazide reagent

rFIX was pegylated by using a linear 20kD pegylation reagent containing a hydrazide group. Examples of agents of this type are those from NOFHZ series (NOF Corp., Tokyo, Japan). Using NaIO at a protein concentration of 2mg/ml in reaction buffer (50mM Hepes, 150mM sodium chloride, 5mM calcium chloride, pH6.0) in the dark at 4 ℃ with gentle shaking₄rFIX was oxidized (final concentration: 100. mu.M) for 1 hour and quenched by addition of an aqueous glycerol solution (final concentration: 1mM) for 15 minutes. Next, the reaction mixture was subjected to UF/DF. To the retentate was added a 50-fold molar excess of hydrazide reagent and aniline (nucleophilic catalyst, final concentration: 10 mM). The coupling reaction was carried out in the dark for 2 hours at room temperature with gentle shaking. Finally, the PEG-rFIX conjugate was purified by ion exchange chromatography on Q-sepharose FF. The reaction mixture was loaded onto a column (1.5 mg protein per ml gel) pre-equilibrated with 50mM Tris buffer (pH 8.0). The conjugate was eluted with 20CV Tris buffer (pH 8.0) (50mM Tris, 1M NaCl) and subsequently UF/DF using a 30kD membrane. The preparation was characterized analytically by measuring total protein (BCA) and FIX chromogenic activity. For the PEG-rFIX conjugate, specific activity was determined to be greater than 50% of native rFIX (chromogenic assay).

Example 26

Polysialylation of rFVIII in the presence of 2mM aniline

Transfer of rFVIII toReaction buffer (50mM Hepes, 350mM sodium chloride, 5mM calcium chloride, 0.01% Tween80, pH 6), diluted to a protein concentration of 1mg/ml and treated with NaIO in reaction buffer (50mM Hepes, 150mM sodium chloride, 5mM calcium chloride, pH6.0) at 4 ℃ in the dark with gentle shaking₄(final concentration: 100. mu.M) for 1 hour, and quenched by addition of aqueous cysteine solution (final concentration: 1mM) for 15 minutes. Next, the reaction mixture was subjected to UF/DF. To the retentate was added a 20-fold molar excess of aminooxy reagent and aniline (nucleophilic catalyst, final concentration: 2 mM). The coupling reaction was carried out in the dark for 2 hours at room temperature with gentle shaking. Excess aminoxy reagent was removed by means of HIC. The conductivity of the reaction mixture was raised to 130mS/cm by adding ammonium acetate containing buffer (50mM Hepes, 350mM sodium chloride, 5mM calcium chloride, 8M ammonium acetate, 0.01% Tween80, pH6.9) and loaded onto a column pre-equilibrated with 50mM Hepes, 2.5M ammonium acetate, 350mM sodium chloride, 5mM calcium chloride, 0.01% Tween80 (pH6.9) filled with 53ml butyl-Sepharose FF (GE Healthcare, Fairfield, CT). Next, the conjugate was eluted with 50mM Hepes, 5mM calcium chloride, 0.01% Tween80(pH 7.4). Finally, fractions containing PSA-rFIX were collected and UF/DF was performed using a 30kD membrane (Millipore, Billerica, MA) made of regenerated cellulose. The preparations were characterized analytically by measuring total protein (BCA) and FVIII chromogenic activity. For PSA-rFVIII conjugates, specific activity was determined to be 80% of native rFVIII.

Example 27

Polysialylation of rFVIII in the presence of 10mM aniline

rFVIII was transferred to reaction buffer (50mM Hepes, 350mM sodium chloride, 5mM calcium chloride, 0.01% Tween80, pH 6), diluted to a protein concentration of 1mg/ml and treated with NaIO in reaction buffer (50mM Hepes, 150mM sodium chloride, 5mM calcium chloride, pH6.0) in the dark with gentle shaking at 4 ℃₄(final concentration: 100. mu.M) for 1 hour, and quenched by addition of aqueous cysteine solution (final concentration: 1mM) for 15 minutesA clock. Next, the reaction mixture was subjected to UF/DF. To the retentate was added a 20-fold molar excess of aminooxy reagent and aniline (nucleophilic catalyst, final concentration: 10 mM). The coupling reaction was carried out in the dark for 2 hours at room temperature with gentle shaking. Excess aminoxy reagent was removed by means of HIC. The conductivity of the reaction mixture was raised to 130mS/cm by adding ammonium acetate containing buffer (50mM Hepes, 350mM sodium chloride, 5mM calcium chloride, 8M ammonium acetate, 0.01% Tween80, pH6.9) and loaded onto a column pre-equilibrated with 50mM Hepes, 2.5M ammonium acetate, 350mM sodium chloride, 5mM calcium chloride, 0.01% Tween80 (pH6.9) filled with 53ml butyl-Sepharose FF (GE Healthcare, Fairfield, CT). Next, the conjugate was eluted with 50mM Hepes, 5mM calcium chloride, 0.01% Tween80(pH 7.4). Finally, fractions containing PSA-rFIX were collected and UF/DF was performed using a 30kD membrane (Millipore, Billerica, MA) made of regenerated cellulose. The preparations were characterized analytically by measuring total protein (BCA) and FVIII chromogenic activity. For PSA-rFVIII conjugates, specific activity was determined to be 80% of native rFVIII.

Example 28

PEGylation of blood coagulation proteins using branched PEGs

Pegylation of coagulation proteins (e.g., FIX, FVIII, and FVIIa as described in examples 22-25) can be extended to branched or linear pegylation reagents made from aldehydes and suitable linking groups containing an active aminooxy group as described in example 21.

Claims (22)

1. A method of conjugating a water soluble polymer to an oxidized carbohydrate moiety of a blood coagulation protein, the method comprising contacting the oxidized carbohydrate moiety with an activated water soluble polymer under conditions that allow conjugation;

the coagulation protein is selected from the group consisting of: factor ix (fix), factor viii (fviii), factor viia (fviia), Von Willebrand Factor (VWF), factor fv (fv), factor x (fx), factor xi (fxi), factor xii (fxii), thrombin (FII), protein C, protein S, tPA, PAI-1, Tissue Factor (TF), and ADAMTS 13 protease or biologically active fragments, derivatives, or variants thereof;

the water soluble polymer contains an active aminooxy group and is selected from the group consisting of: polyethylene glycol (PEG), branched PEG, polysialic acid (PSA), carbohydrates, polysaccharides, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, poly (1-hydroxymethylethylene-hydroxymethylformal) (PHF), 2-methacryloyloxy-2' -ethyltrimethylammonium phosphate (MPC); and is

The carbohydrate moiety is oxidized by incubation with a buffer comprising an oxidizing agent selected from the group consisting of: sodium periodate (NaIO)₄) Lead tetraacetate (Pb (OAc)₄) And potassium perruthenate (KRuO)₄) (ii) a Wherein an oxime linkage is formed between the oxidized carbohydrate moiety and the active aminooxy group on the water soluble polymer.

2. The method of claim 1, wherein the water soluble polymer is PSA.

3. The method of claim 2, wherein the PSA comprises about 10-300 sialic acid units.

4. The method of any one of claims 1 to 3, wherein the coagulation protein is FIX.

5. The method according to any one of claims 1 to 3, wherein the coagulation protein is FVIIa.

6. The method of any one of claims 1-3, wherein the coagulation protein is FVIII.

7. The method of any one of claims 1 to 6, wherein the oxidizing agent is sodium periodate (NaIO)₄)。

8. The method according to any one of claims 4 to 7, wherein the oxidized carbohydrate moiety of the blood coagulation protein is located in an activating peptide of the blood coagulation protein.

9. The method of claim 2, wherein the PSA is prepared by reacting an activated aminooxy linker with oxidized PSA;

wherein the aminooxy linking group is selected from the group consisting of:

a) a 3-oxa-pentane-1, 5-dihydroxyamine linking group of the formula:

and

b) a3, 6, 9-trioxa-undecane-1, 11-dihydroxyamine linking group of the formula:

wherein the PSA is oxidized by incubation with an oxidizing agent to form a terminal aldehyde group at the non-reducing end of the PSA.

10. The method of claim 7, wherein the aminooxy linking group is 3-oxa-pentane-1, 5-dihydroxyamine.

11. The method of claim 7, wherein the oxidizing agent is NaIO₄。

12. The method of any one of claims 1 to 9, wherein contacting the oxidized carbohydrate moiety with the activated water-soluble polymer is performed in a buffer comprising a nucleophilic catalyst selected from the group consisting of aniline and aniline derivatives.

13. The method of claim 2, further comprising treating the cell with a composition comprising a compound selected from the group consisting of sodium cyanoborohydride (NaCNBH)₃) And ascorbic acid (vitamin C) in a buffer of a reducing compound to reduce oxime linkages in the conjugated blood coagulation protein.

14. The method of claim 11, wherein the reducing compound is sodium cyanoborohydride (NaCNBH)₃)。

15. A modified blood coagulation protein produced using the method according to any one of claims 1 to 12.

16. A modified FIX, comprising:

(a) FIX molecule or biologically active fragment, derivative or variant thereof; and

(b) at least one aminoxy PSA bound to the FIX molecule of (a), wherein the aminoxy PSA is linked to the FIX via one or more carbohydrate moieties.

17. A modified FVIIa, comprising:

(a) a FVIIa molecule or a biologically active fragment, derivative or variant thereof; and

(b) at least one aminoxy PSA bound to the FVIIa molecule of (a), wherein said aminoxy PSA is linked to said FVIIa via one or more carbohydrate moieties.

18. A modified FVIII comprising:

(a) a FVIII molecule or a biologically active fragment, derivative or variant thereof; and

(b) at least one aminoxy PSA bound to the FVIII molecule of (a), wherein said aminoxy PSA is linked to said FVIII via one or more carbohydrate moieties.

19. A modified FIX, comprising:

(a) FIX molecule or biologically active fragment, derivative or variant thereof; and

(b) at least one aminoxy PEG conjugated to the FIX molecule of (a), wherein the aminoxy PEG is attached to the FIX via one or more carbohydrate moieties.

20. A modified FVIIa, comprising:

(a) a FVIIa molecule or a biologically active fragment, derivative or variant thereof; and

(b) at least one aminoxy PEG conjugated to the FVIIa molecule of (a), wherein said aminoxy PEG is linked to said FVIIa via one or more carbohydrate moieties.

21. A modified FVIII comprising:

(a) a FVIII molecule or a biologically active fragment, derivative or variant thereof; and

(b) at least one aminooxy PEG conjugated to the FVIII molecule of (a), wherein said aminooxy PEG is linked to said FVIII via one or more carbohydrate moieties.

22. A water-soluble polymer comprising a reactive aminooxy linking group; the water soluble polymer is selected from the group consisting of: polyethylene glycol (PEG), branched PEG, polysialic acid (PSA), carbohydrates, polysaccharides, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, poly (1-hydroxymethylethylene-hydroxymethylformal) (PHF), 2-methacryloyloxy-2' -ethyltrimethylammonium phosphate (MPC); the reactive aminooxy linking group is selected from the group consisting of:

a) a 3-oxa-pentane-1, 5-dihydroxyamine linking group of the formula:

and

b) a3, 6, 9-trioxa-undecane-1, 11-dihydroxyamine linking group of the formula: