JP2018115170A - Factor fviii-polymer conjugates - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide Factor FVIII-polymer conjugates.SOLUTION: The invention relates to a proteinaceous construct comprising a Factor VIII molecule conjugated to an aqueous polymer through a carbohydrate moiety of the Factor VIII, and a method for preparing the same. In a first embodiment of the invention, the aqueous polymer is selected from polyethylene glycol (PEG), polysialic acid (PSA) or dextran. The invention relates to a proteinaceous construct comprising coagulation Factor VIII (FVIII) bound to at least one aqueous polymer including poly(alkylene oxide) such as polyethylene glycol. Further the invention relates to a method for extending in vivo half life of FVIII in the blood of a mammal having hemorrhagic disease associated with functional defect or deficit of FVIII.SELECTED DRAWING: None

Description

(Field of Invention)
This application is a continuation-in-part of US patent application Ser. No. 11 / 729,625, filed Mar. 29, 2007, which is filed on June 6, 2006. Claims priority to US Provisional Patent Application No. 60 / 790,239, filed on March 31, 2006, and US Provisional Patent Application No. 60 / 787,968, filed March 31, 2006.

  The present invention relates to proteinaceous constructs comprising coagulation factor VIII (FVIII) bound to at least one water soluble polymer, including poly (alkylene oxides) such as polyethylene glycol. Furthermore, the present invention relates to a method for extending the in vivo half-life of FVIII in the blood of a mammal having a bleeding disorder associated with a functional defect or deficiency of FVIII.

(Background of the Invention)
Coagulation factor VIII (FVIII) circulates in plasma at very low concentrations and binds non-covalently to von Willebrand factor (VWF). During hemostasis, FVIII separates from VWF and activates factor X (FX) mediated by activated factor IX (FIXa) by increasing the activation rate in the presence of calcium and phospholipids or cell membranes Acts as a cofactor for

  FVIII is synthesized as a single chain precursor of about 270-330 kD with domain structure A1-A2-B-A3-C1-C2. When purified from plasma (eg, “plasma derived” or “plasma”), FVIII is composed of a heavy chain (A1-A2-B) and a light chain (A3-C1-C2). The light chain has a molecular weight of 80 kD, but the heavy chain ranges from 90 to 220 kD due to proteolysis within the B domain.

  FVIII is also synthesized as a recombinant protein for treatment in bleeding disorders. Various in vitro assays have been devised to determine the potential efficacy of recombinant FVIII (rFVIII) as a therapeutic agent. These assays mimic the in vivo action of endogenous FVIII. When FVIII is thrombin treated in vitro, its procoagulant activity rapidly increases and subsequently decreases, as measured by an in vitro assay. This activation and inactivation occurs simultaneously with specific proteolytic hydrolysis in both heavy and light chains, altering the availability of different binding epitopes in FVIII, eg, FVIII dissociates from VWF and becomes phospholipid surface Or the ability to bind to a particular monoclonal antibody is altered.

  FVIII deficiency or dysfunction is associated with hemophilia A, the most common bleeding disorder. The treatment of choice for management of hemophilia A is plasma-derived FVIII concentrate or rFVIII concentrate replacement therapy. Severe hemophilia A patients with FVIII levels less than 1% generally receive prophylactic therapy aimed at maintaining FVIII between doses higher than 1%. Considering the average half-life of the various FVIII products in circulation, this can usually be achieved by administering FVIII 2-3 times / week.

  A number of concentrates are commercially available for the treatment of hemophilia A. One of these concentrates is the recombinant product Advert (R). It is produced in CHO cells and manufactured by Baxter Healthcare Corporation. In the cell culture process, purification or final formulation of this product, no human or animal plasma protein or albumin is added.

  The goal of many manufacturers of FVIII concentrates and therapeutic polypeptide drugs is to develop next generation products with improved pharmacodynamic and pharmacokinetic properties while maintaining all other product properties. is there.

  Therapeutic polypeptide drugs are rapidly degraded by proteolytic enzymes and neutralized by antibodies. This shortens its half-life and circulation time, thereby limiting its therapeutic effectiveness. It has been found that adding soluble polymers or carbohydrates to a polypeptide inhibits degradation and extends the half-life of the polypeptide. For example, PEGylation of a polypeptide drug protects it and improves its pharmacodynamic and pharmacokinetic profile (1). The PEGylation process adds polyethylene glycol (PEG) repeat units to a polypeptide drug. PEGylation of the molecule increases the drug's resistance to enzymatic degradation, extends in vivo half-life, reduces the number of doses, decreases immunogenicity, increases physical and thermal stability, Solubility can be increased, liquid stability can be increased, and aggregation can be suppressed.

  Therefore, the addition of soluble polymers, such as by PEGylation, is one way to improve the properties of the FVIII product. The current state of the art is documented by various patents and patent applications.

  U.S. Patent No. 6,057,031 describes poly (alkylene oxide) -FVIII or FIX conjugates. This protein is covalently bonded to poly (alkylene oxide) via the carbonyl group of FVIII.

  U.S. Patent No. 6,099,056 describes a method for preparing a conjugate of FVIII and a biocompatible polymer. This patent was supplemented by a publication by Roestin et al. This conjugate comprises B domain deleted recombinant FVIII modified with monomethoxypolyethylene glycol. This conjugate had reduced FVIII function and blood coagulation activity decreased rapidly with the degree of modification.

  U.S. Patent No. 6,057,031 describes polymer-FVIII molecular conjugates that include multiple conjugates. Each conjugate has 1 to 3 water soluble polymers covalently linked to the FVIII molecule. The FVIII molecule lacks the B domain.

  U.S. Patent No. 6,057,031 describes modified FVIII in which an infusible conjugate comprising a protein having FVIII activity is covalently bound to a non-antigenic ligand.

  Patent Document 5 describes a conjugate of a polypeptide and a biocompatible polymer.

  U.S. Patent No. 6,057,031 describes FVIII bound to polyethylene glycol having a preferred molecular weight of 5,000 daltons or less.

  FVIII to which a soluble polymer that extends the half-life of FVIII in vivo, for example, 10,000 daltons, which has an increased half-life in vivo while maintaining functional activity compared to non-PEGylated FVIII. There remains a need for PEGylated FVIII, such as full length FVIII conjugated with more PEG.

US Pat. No. 6,037,452 European Patent No. 1258497 International Publication No. 04/075923 US Pat. No. 4,970,300 US Pat. No. 6,048,720 International Publication No. 94/15625

Harris JM et Chess RB, Nat Rev Drug Discov 2003; 2: 214-21 Rostin et al., Bioconj Chem 2000; 11: 387-96.

(Summary of the Invention)
The present invention relates to a proteinaceous construct comprising a Factor VIII molecule conjugated with a water-soluble polymer via the carbohydrate moiety of Factor VIII and a method for preparing the same.

In one embodiment of the present invention, a method of conjugating a water soluble polymer and an oxidized carbohydrate moiety of FVIII comprising contacting the oxidized carbohydrate moiety with an activated water soluble polymer under conditions that allow conjugation. I will provide a. In a related embodiment, the water soluble polymer is selected from the group consisting of PEG, PSA and dextran. In yet another embodiment, the activated water soluble polymer is selected from the group consisting of PEG-hydrazide, PSA-hydrazine and aldehyde activated dextran. In another aspect of the present invention, the carbohydrate moiety is oxidized by incubation in buffer containing NaIO _4. In yet another aspect of the invention, the oxidized carbohydrate portion of FVIII is present in the B domain of FVIII.

  In another embodiment of the invention, there is provided a modified FVIII produced by a method according to any of the above methods. In yet another embodiment, the method comprises: (a) a Factor VIII molecule; and (b) at least one water soluble polymer bound to said Factor VIII molecule, wherein the water soluble polymer is a B domain of Factor VIII. Proteinaceous constructs are provided that are linked to Factor VIII via one or more carbohydrate moieties present in In a related aspect of the invention, the water soluble polymer is selected from the group consisting of PEG, PSA and dextran.

FIG. 1 shows the spread and mass increase of rFVIII after conjugation with PEG as measured by SDS-PAGE followed by immunoblotting. FIG. 2 is a graph showing the pharmacokinetics of PEG-rFVIII conjugates compared to unconjugated FVIII in hemophilia mice. Open squares: PEGrFVIII, FVIII dose 200 IU / kg. Filled diamonds: native rFVIII, FVIII dose 200 IU / kg. FIG. 3 is a diagram showing a detailed analysis of PEGylation sites by SDS-PAGE using various anti-FVIII antibodies. FIG. 4 is a graph showing activation and inactivation of native rFVIII and pegylated rFVIII induced by thrombin. FIG. 5 is a diagram showing bands indicating native rFVIII and PEGylated rFVIII domains. FIG. 6 is a graph showing the degree of PEGylation of various domains of native rFVIII and PEGylated rFVIII. FIG. 7 is a graph showing the thrombin inactivation rate of native rFVIII and PEGylated rFVIII.

(Detailed description of the invention)
The present invention is a proteinaceous construct comprising an FVIII molecule having at least a portion of an intact B domain attached to a water soluble polymer. Examples of the water-soluble polymer include polyalkylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, polyoxazoline, polyacryloylmorpholine, or carbohydrates such as polysialic acid (PSA) and dextran. In one embodiment of the invention, the water soluble polymer is a polyethylene glycol molecule having a molecular weight greater than 10,000 daltons. In another embodiment, the water soluble polymer has a molecular weight of greater than 10,000 Da to about 125,000 Da, about 15,000 Da to 20,000 Da, or about 18,000 Da to about 25,000 Da. In one embodiment, the construct retains the full functional activity of a standard therapeutic FVIII product and provides a longer in vivo half-life than a standard therapeutic FVIII product. In another embodiment, the construct is at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 relative to native factor VIII. , 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 (%) of biological activity. In a related embodiment, the biological activity of the construct and native factor VIII is determined by the ratio of chromogenic activity to FVIII antigen value (FVIII: Chr: FVIII: Ag). In yet another embodiment of the invention, the half-life of the construct is 0.5, 0.6, 0.7, 0.8, 0.9, relative to the in vivo half-life of native factor VIII. 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times decrease or increase.

  The starting material of the present invention is FVIII. FVIII can be obtained from human plasma or US Pat. No. 4,757,006, US Pat. No. 5,733,873, US Pat. No. 5,198,349, US Pat. No. 5,250,421. , US Pat. No. 5,919,766 and European Patent No. 306968.

  As used herein, the term “Factor VIII” or “FVIII” refers to any FVIII molecule that has at least a portion of the complete B domain and exhibits biological activity associated with native FVIII. In one embodiment of the invention, the FVIII molecule is full length factor VIII. An FVIII molecule is a protein encoded by a DNA sequence capable of hybridizing to DNA encoding Factor VIII: C. Such proteins may contain amino acid deletions at various sites between or within domains A1-A2-B-A3-C1-C2 (US Pat. No. 4,868,112). An FVIII molecule can also be an analog of native FVIII in which one or more amino acid residues have been replaced by site-directed mutagenesis.

  FVIII molecules useful in the present invention include full-length proteins, precursors of the proteins, biologically active or functional subunits or fragments of the proteins, and functional derivatives thereof, and The variants described below are mentioned. The notation FVIII is intended to include all potential forms of such proteins. Here, each form of FVIII has at least some or all of the complete native B domain sequence.

  The term “recombinant factor VIII” (rFVIII) according to the present invention may include any heterologous or naturally occurring rFVIII obtained by recombinant DNA technology, or a biologically active derivative thereof. In certain embodiments, the term encompasses the above proteins and nucleic acids encoding the rFVIII of the present invention. Such nucleic acids include, but are not limited to, for example, genes, pre-mRNAs, mRNAs, polymorphic variants, alleles, synthetic and naturally occurring variants. Proteins encompassed by the term rFVIII include, for example, the proteins and polypeptides described herein above, the proteins encoded by the nucleic acids, (1) (up to the full length sequence of 406 amino acids of the mature native protein) ) About 60% relative to 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. About 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96% An amino acid sequence having about 97%, about 98%, or about 99% or more amino acid sequence homology, and / or (2) an immunological sequence comprising a reference amino acid sequence described herein. Interspecies homologues and other polypeptides that specifically bind to antibodies, eg, polyclonal or monoclonal antibodies, generated against the antigen, immunogenic fragments thereof, and / or conservatively modified variants thereof But not limited to.

  The polynucleotide encoding rFVIII of the present invention includes (1) specific nucleic acids encoding the reference amino acid sequences described herein and conservatively modified variants thereof under stringent hybridization conditions. (2) at least about 25, about 50, about 100, about 150, about 200, about 250, about 500, about 1000 or more nucleotides (up to the full length sequence of 1218 nucleotides of the mature protein) Having a nucleic acid sequence having greater than about 95%, about 96%, about 97%, about 98%, about 99% or more nucleotide sequence homology to a reference nucleic acid sequence described herein over a region of Examples include, but are not limited to polynucleotides.

  As used herein, “endogenous FVIII” includes FVIII derived from a mammal to be treated. The term also includes FVIII transcribed from a transgene or any other foreign DNA present in the mammal. As used herein, “exogenous FVIII” includes FVIII not derived from said mammal.

  Variant (or analog) polypeptides include insertional variants in which one or more amino acid residues are added to the FVIII amino acid sequence of the invention. The insertion can be located at one or both ends of the protein and / or within an internal region of the FVIII amino acid sequence. Insertion variants having additional residues at one or both ends include, for example, fusion proteins, and proteins that contain an amino acid tag or another amino acid tag. In one aspect, the FVIII molecule, particularly if the molecule is E. coli. When expressed recombinantly in bacterial cells such as E. coli, an N-terminal Met may optionally be included.

  In deletion mutants, one or more amino acid residues of the FVIII polypeptide described herein are removed. Deletions can be caused at one or both ends of the FVIII polypeptide and / or by removal of one or more residues within the FVIII amino acid sequence. Thus, deletion mutants include all fragments of the FVIII polypeptide sequence.

  In substitutional variants, one or more amino acid residues of the FVIII polypeptide are removed and replaced with alternative residues. In one aspect, the substitution is inherently conservative, and this type of conservative substitution is well known in the art. Alternatively, the invention includes substitutions that are also non-conservative. Exemplary conservative substitutions are described in Lehninger, [Biochemistry, 2nd Edition; Worth Publishers, Inc. , New York (1975), pp. 71-77] and is described immediately below.

あるいは、例示的な保存的置換をすぐ下に述べる。

Alternatively, exemplary conservative substitutions are described immediately below.

「天然に存在する」ポリヌクレオチド又はポリペプチド配列は、典型的には、霊長類、例えばヒト；げっ歯類、例えばラット、マウス、ハムスター；ウシ、ブタ、ウマ、ヒツジ又は任意のほ乳動物を含むが、ただしそれだけに限定されないほ乳動物に由来する。本発明の核酸及びタンパク質は、（例えば、非相同で野生型配列若しくはその変異体をコードする、又は天然に存在しない）組換え分子とすることができる。基準ポリヌクレオチド及びポリペプチド配列としては、例えば、ＵｎｉＰｒｏｔＫＢ／Ｓｗｉｓｓ−Ｐｒｏｔ Ｐ００４５１（ＦＡ８＿ＨＵＭＡＮ）、Ｇｉｔｓｃｈｉｅｒ Ｊ ｅｔ ａｌ．，Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ｔｈｅ ｈｕｍａｎ Ｆａｃｔｏｒ ＶＩＩＩ ｇｅｎｅ，Ｎａｔｕｒｅ，３１２（５９９２）：３２６−３０（１９８４）、Ｖｅｈａｒ ＧＨ ｅｔ ａｌ．，Ｓｔｒｕｃｔｕｒｅ ｏｆ ｈｕｍａｎ Ｆａｃｔｏｒ ＶＩＩＩ，Ｎａｔｕｒｅ，３１２（５９９２）：３３７−４２（１９８４）、及びＴｈｏｍｐｓｏｎ ＡＲ．Ｓｔｒｕｃｔｕｒｅ ａｎｄ Ｆｕｎｃｔｉｏｎ ｏｆ ｔｈｅ Ｆａｃｔｏｒ
ＶＩＩＩ ｇｅｎｅ ａｎｄ ｐｒｏｔｅｉｎ，Ｓｅｍｉｎ Ｔｈｒｏｍｂ Ｈｅｍｏｓｔ，２００３：２９；１１−２９（２００２）が挙げられる（参照によりその全体を本明細書に援用する）。

A “naturally occurring” polynucleotide or polypeptide sequence typically includes a primate, such as a human; a rodent, such as a rat, mouse, hamster; cow, pig, horse, sheep or any mammal However, it is derived from a mammal that is not so limited. The nucleic acids and proteins of the invention can be recombinant molecules (eg, non-homologous, encoding wild type sequences or variants thereof, or non-naturally occurring). Reference polynucleotide and polypeptide sequences include, for example, UniProtKB / Swiss-Prot P00451 (FA8_HUMAN), Gitschier J et al. , Characterization of the human Factor VIII gene, Nature, 312 (5992): 326-30 (1984), Vehar GH et al. , Structure of human Factor VIII, Nature, 312 (5992): 337-42 (1984), and Thompson AR. Structure and Function of the Factor
VIII gene and protein, Semin Thromb Hemost, 2003: 29; 11-29 (2002), which is incorporated herein by reference in its entirety.

  As used herein, “biologically active derivative” or “biologically active variant” refers to substantially the same functional and / or biological properties of a molecule, such as binding, and / or peptide backbone, or basic polymerization. Includes any derivative or variant of the molecule having the same structural basis, such as units.

  As used herein, “plasma-derived FVIII” or “plasma” includes all forms of proteins found in blood obtained from mammals that have the property of activating the coagulation pathway.

  In various embodiments, the production of rFVIII includes (i) the production of recombinant DNA by genetic engineering, (ii) but is not limited to, for example, recombinant DNA is transformed into prokaryotic cells by transfection, electroporation or microinjection. Or (iii) culturing the transformed cell, (iv) expressing rFVIII, eg, constitutively or by induction, and (vi) obtaining purified rFVIII ( v) Any method known in the art of isolating said rFVIII, eg, from the medium or by collecting transformed cells.

  In another aspect, rFVIII is produced by expression in a suitable prokaryotic or eukaryotic host system characterized by producing a pharmacologically acceptable rFVIII molecule. Examples of eukaryotic cells are mammalian cells such as CHO, COS, HEK293, BHK, SK-Hep, HepG2.

  In yet another aspect, a wide variety of vectors are used for the preparation of rFVIII and are selected from eukaryotic and prokaryotic expression vectors. Examples of prokaryotic expression vectors include plasmids such as, but not limited to, pRSET, pET, and pBAD. Here, promoters used in prokaryotic expression vectors include, but are not limited to, one or more lac, trc, trp, recA or araBAD. Examples of eukaryotic expression vectors include (i) for expression in yeast, such as, but not limited to, AOX1, GAP, GAL1, or AUG1, such as pAO, pPIC, pYES, or pMET, Vectors not limited thereto, (ii) for expression in insect cells, such as PH, p10, MT, Ac5, OpIE2, gp64, polh, but not limited to promoters such as pMT, pAc5, pIB, pMIB, or pBAC But not limited to vectors, and (iii) for expression in mammalian cells, use promoters such as, but not limited to, CMV, SV40, EF-1, UbC, RSV, ADV, BPV, β-actin Derived from viral systems such as, but not limited to, vaccinia virus, adeno-associated virus, herpes virus, retrovirus and the like, in one embodiment, such as, but not limited to, pSVL, pCMV, pRc / RSV, pcDNA3, or pBPV Vector.

  In certain embodiments, the FVIII 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, FVIII is PEGylated by conjugation of PEG with the free amino group of the protein using N-hydroxysuccinimide (NHS) ester. In another embodiment, a water soluble polymer, such as PEG, is coupled with a free SH group by maleimide chemistry or by coupling of PEG hydrazide or PEG amine after pre-oxidation with the carbohydrate moiety of FVIII.

  In another embodiment, FVIII is conjugated with another water soluble polymer. The water-soluble polymer is, for example, a carbohydrate or polysaccharide such as polyalkylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, polyoxazoline, polyacryloylmorpholine, polysialic acid (PSA) or dextran. Coupling of the water soluble polymer can be performed by direct coupling with the protein or via a linker molecule. An example of a chemical linker is MBPH (4- [4-N-maleimidophenyl] butyric hydrazide) containing a carbohydrate-selective hydrazide and a sulfhydryl-reactive maleimide group (Chamow et al., J Biol Chem 1992; 267: 15916). -22).

  Conjugation can be performed by direct coupling of a water-soluble polymer and Factor VIII (or coupling through a linker system) under the formation of a stable bond. Furthermore, degradable, free or hydrolysable linker systems can be used in the present invention (Tsubery et al. J Biol Chem 2004; 279: 38118-24, Greenwald et al., J Med Chem 1999; 42 : 3657-67, Zhao et al., Bioconj Chem 2006; 17: 341-51, WO 2006/138572 A2, U.S. Pat. No. 7,259,224 B2, U.S. Pat. No. 7,060,259 B2).

As discussed herein, an embodiment of the present invention is the coupling of an activated soluble polymer with the oxidized carbohydrate portion of FVIII. The term “activated water-soluble polymer” is used herein to refer to FVIII having an active functional group that allows chemical conjugation of the water-soluble polymer to a linker or directly to FVIII (including an active aldehyde group). Used to refer to a water-soluble polymer used in coupling with. As used herein, the term “oxidized carbohydrate moiety” refers to FVIII that contains a free aldehyde group produced by an oxidizing agent such as NaIO ₄ . In one aspect of the invention, an aldehyde activated dextran (containing an active aldehyde group) is coupled to the aldehyde group of FVIII via a dihydrazide linker.

  According to the glycosylation pattern of FVIII (Lenting et al; Blood, 92: 3983-96 (1998)), conjugation of FVII via the carbohydrate moiety should likely occur at the B domain of FVIII. . Since the B domain does not play a role in the activity of FVIII, it is desirable to target the B domain for such conjugation reactions. Enzymatic glycoconjugation is described in US Patent Application Publication No. 2008/00700275.

  In one embodiment of the invention, FVIII removes lysine residues by using a polyethylene glycol derivative comprising an active N-hydroxysuccinimide ester (NHS) such as succinimidyl succinate, succinimidyl glutarate, or succinimidyl propionate. Modified through. These derivatives react with the lysine residue of FVIII under mild conditions by forming a stable amide bond. In one embodiment of the invention, the chain length of the PEG derivative is 5,000 Da. 500 to 2,000 Da, 2,000 to 5,000 Da, more than 5,000 to 10,000 Da, or more than 10,000 to 20,000 Da, including linear and branched structures, or 20 Other PEG derivatives having chain lengths greater than 1 000 and up to 150,000 Da are used in various embodiments.

  Another method for PEGylation of amino groups is chemical conjugation with PEG carbonate by forming urethane linkages or reaction with aldehydes or ketones by reductive amination to form secondary amide linkages.

  In the present invention, FVIII molecules are chemically modified using commercially available PEG derivatives. These PEG derivatives can have a linear structure or a branched structure. Examples of PEG derivatives containing NHS groups are shown below.

  The following PEG derivatives are examples of those commercially available from Nektar Therapeutics (see Huntsville, Ala., Www.nektar.com/PEG reagent catalog; Nektar Advanced PEGylation, price list, 6-6).

分枝構造を有するこの試薬は、Ｋｏｚｌｏｗｓｋｉ等によってより詳細に記述されている（ＢｉｏＤｒｕｇｓ ２００１；５：４１９−２９）。

This reagent with a branched structure is described in more detail by Kozlowski et al. (BioDrugs 2001; 5: 419-29).

  Another example of a PEG derivative is commercially available from NOF Corporation (Tokyo, Japan; see www.nof.co.jp/english: Catalog 2005).

これらのプロパン誘導体は、１，２置換パターンを有するグリセロール骨格を示す。本発明では、１，３置換を有するグリセロール構造、又は米国特許出願公開第２００３／０１４３５９６号Ａ１に記載の別の分枝構造に基づく分枝ＰＥＧ誘導体を使用することもできる。

These propane derivatives exhibit a glycerol skeleton with a 1,2 substitution pattern. In the present invention, a branched PEG derivative based on a glycerol structure having 1,3 substitution or another branched structure described in US 2003/0143596 A1 can also be used.

  Use in the present invention PEG derivatives having degradability (eg, hydrolyzable linkers) as described by Tsubery et al. (J Biol Chem 2004; 279: 38118-24) and Shechter et al. (WO 04089280 A3). You can also.

  Remarkably, the PEGylated FVIII of the present invention exhibits sufficient functional activity along with a long FVIII half-life in vivo. Furthermore, PEGylated rFVIII is believed to be more resistant to thrombin inactivation. This has been demonstrated by various in vitro and in vivo methods. And it is illustrated in the following examples.

  As used herein, a “sialic acid moiety” is soluble in an aqueous solution or suspension and has little or no adverse effect on mammals, such as side effects, upon administration of a pharmaceutically effective amount of a PSA-FVIII conjugate. Includes sialic acid monomers or polymers ("polysaccharides") that do not extend. There are no particular restrictions on the sialic acid units used in accordance with the present invention. The polymer, in one aspect, is characterized by having 1 to 4 units. In certain embodiments, different sialic acid units are attached to the chain.

  In various aspects of the invention, the sialic acid moiety is coupled to FVIII, for example, by the methods described in US Pat. No. 4,356,170, incorporated herein by reference. In various embodiments of the present invention, the polysaccharide compound is a naturally occurring polysaccharide, a naturally occurring polysaccharide derivative, or a naturally occurring polysaccharide derivative. In general, all of the sugar residues in the compound are sialic acid residues.

  Other techniques for coupling PSA and polypeptides are also known. For example, US Patent Application Publication No. 2007/0282096 describes conjugating an amine or hydrazide derivative of, for example, PSA with a protein. In addition, US Patent Application Publication No. 2007/0191597 describes PSA derivatives that contain an aldehyde group at the reducing end for reaction with a substrate (eg, protein).

  In one embodiment of the invention, the polysialic acid moiety of the polysaccharide compound is extremely hydrophilic, and in another embodiment, the entire compound is very hydrophilic. Hydrophilicity is mainly imparted by pendant carboxyl groups of sialic acid units as well as hydroxyl groups. The sugar unit may contain another functional group such as an amine, hydroxyl, or sulfate group, or a combination thereof. These groups can be present in naturally occurring sugar compounds or can be introduced into derivative polysaccharide compounds.

  The specific use polysaccharide compounds of the present invention, in one aspect, are those produced by bacteria. Some of these naturally occurring polysaccharides are known as glycolipids. In one embodiment, the polysaccharide compound is substantially free of terminal galactose units.

  In one embodiment of the invention, the in vivo half-life of the proteinaceous construct is increased. In related embodiments, the in vivo half-life of the proteinaceous construct is increased by at least 2-fold compared to FVIII that is not conjugated to a water-soluble polymer, and in another embodiment, the in-vivo half-life is extended by at least 3-fold. Is done.

  In one embodiment, the proteinaceous constructs of the invention can be administered by injection, such as intravenous, intramuscular, intraperitoneal injection.

  In order to administer a composition comprising a proteinaceous construct of the invention to a human or test animal, in one aspect, the composition comprises one or more pharmaceutically acceptable carriers. The terms “pharmaceutically” or “pharmacologically acceptable” are stable, inhibit aggregation and proteolysis such as degradation products, and are well known in the art, as shown below. Molecular entities and compositions that do not cause allergic reactions or other adverse reactions when administered by route. “Pharmaceutically acceptable carrier” includes any and all clinically useful solvents, dispersion media, coatings, antibacterial agents, and antifungal agents, isotonic agents, including the agents disclosed above And absorption retardants.

  As used herein, an “effective amount” includes a dose suitable for the treatment of a mammal having a bleeding disorder as outlined above.

  The composition can be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal injection or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retroocular, intrapulmonary injection, or surgical implantation at a specific site is also contemplated. In general, the composition is essentially free of pyrogens and other impurities that may be harmful to the recipient.

  Single or multiple administrations of the compositions can be carried out, the dose level and pattern being selected by the treating physician. In the case of prevention or treatment of a disease, the appropriate dosage is, as described above, 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 treatment, It depends on the patient's medical history and response to the drug, and at the discretion of the attending physician.

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

  As an additional aspect, the invention includes a kit comprising a composition of the invention packaged to facilitate use for administration to a subject. In one embodiment, such a kit is a compound or composition as described herein (eg, a composition comprising a proteinaceous construct) packaged in a container such as a sealed bottle or vessel. And a label describing the use of the compound or composition in carrying out the method is affixed to the container or included in the package. In one embodiment, the kit includes a first container comprising a composition comprising a proteinaceous construct, and a second container comprising a physiologically acceptable reconstitution solution for the composition of the first container. including. In one aspect, the compound or composition is packaged as a unit dosage form. The kit can further include a device suitable for administering the composition according to a particular route of administration. Preferably, the kit includes a label describing the use of the therapeutic protein or peptide composition.

Example 1
PEGylation of lysine residues in rFVIII using mPEG succinimidyl succinate A solution of bulk rFVIII from the Advert manufacturing process (3,400 U / ml) was added to 20 mM Hepes buffer containing 0.5% sucrose and 0.1% polysorbate 80 Gel filtration using an Econo-Pac 10DG column (Bio-Rad) with 150 mM NaCl, pH 7.4. Next, 5,000 sda mPEG succinimidyl succinate (Abuchowski et al. Cancer Biochim Biophys 1984; 7: 175-86) (PEG-SS5000) (PEG-SS5000) was added to this solution with gentle stirring (PEG-SS 5 mg / s). mg protein) and 0.5 M NaOH were added dropwise to adjust the pH value to 7.4. The PEGylation was then carried out for 1 hour at room temperature with gentle stirring.

  Subsequently, an ion exchange chromatography resin (Fractogel EMD TMAE650M / Pharmacia XK-10 column, bed height 15 equilibrated with 20 mM Hepes buffer containing 0.5% sucrose and 0.1% polysorbate 80, 150 mM NaCl, pH 7.4. (0.0 cm) was applied to the reaction mixture. The column is then washed with 20 CV equilibration buffer to remove excess reagent and PEGylated rFVIII is eluted with elution buffer (20 mM Hepes, 1.0 M NaCl, 0.5% sucrose, 0.1% polysorbate 80, pH 7. Elute in 4). Using a membrane with a molecular weight cut-off of 30 kD made of regenerated cellulose and using a buffer system consisting of 20 mM Hepes, 150 mM NaCl, 0.5% sucrose, pH 7.4, the eluate is obtained by ultrafiltration / diafiltration. Concentrated.

(Example 2)
In vitro biochemical characterization of PEGylated rFVIII rFVIII from the Advate manufacturing process was PEGylated according to Example 1 and the PEGylated FVIII product was biochemically characterized. The functional activity of PEG-rFVIII was determined using the FVIII chromogenic assay (Rosen S, Scand J Haematol 1984; 33 (Suppl40): 139-45). This method is described in Ph. Eur. 5th edition (5.05) 2.7.4 Based on Assay of Blood Coagulation Factor VIII.

  A sample containing Factor VIII (FVIII: C) is mixed with thrombin, activated Factor IX (FIXa), phospholipid and Factor X (FX) in a buffer containing calcium. FVIII is activated by thrombin and subsequently forms a conjugate with phospholipids, FIXa and calcium ions. This conjugate activates factor X to factor Xa, which then cleaves the chromogenic substrate FXa-1 (AcOH * CH3OCO-D-CHA-Gly-Arg-pNA). The time course of liberated para-nitroaniline (pNA) is measured at 405 nm with a microplate reader. The slope of the reaction is proportional to the factor VIII concentration in the sample. FVIII antigen levels were measured using a commercial ELISA system (Cedarlane, Hornby, Ontario, Canada) with minor modifications. From these values, the FVIII plastid / FVIII antigen ratio was calculated. The protein content in the preparation was determined by measuring the optical density at 280 nm. From these data, the protein content was calculated (Hoyer LW in: Human Protein Data. Installations 1-6; Heberli Ed .; Wiley VCH, Weinheim, Germany, 1998) and expressed in mg / ml.

表１のデータによれば、ＰＥＧ化ｒＦＶＩＩＩ調製物においては、（ＦＶＩＩＩ色素形成活性とＦＶＩＩＩ抗原の比で表される）生物活性は、未変性ｒＦＶＩＩＩの生物活性（１００％）に比べて９０％を超えるまで回収される。

According to the data in Table 1, in the PEGylated rFVIII preparation, the biological activity (expressed as the ratio of FVIII chromogenic activity to FVIII antigen) is 90% compared to the biological activity of native rFVIII (100%). It is collected until it exceeds.

(Example 3)
Characterization of PEGylated rFVIII by SDS-PAGE and immunoblotting techniques 4-12% polyacrylamide gradient gel obtained from Invitrogen (Carlsbad, Calif., USA) was used and SDS PAGE under reducing conditions according to the manufacturer's instructions. Was used to analyze the properties of native rFVIII. As a molecular weight marker (MW), a Precision Plus marker (10 kD to 250 kD) obtained from Bio-Rad (Hercules, Calif., USA) was used. The protein was then transferred by electroblotting onto a PVDF membrane obtained from Bio-Rad (Hercules, Calif., USA), followed by a polyclonal sheep anti-human FVIII: C antibody obtained from Cedarlane (Hornby, Ontario, Canada). Incubated with. The final stage of the immunostaining procedure consisted of incubation with alkaline phosphatase (ALP) conjugated anti-sheep antibody obtained from Accurate (Westbury, NY, USA), followed by ALP substrate kit (Bio-Rad, Hercules). , Calif., USA). The results are summarized in FIG. This blot shows the domain structure of native rFVIII and PEGylated rFVIII. PEGylated rFVIII was found to have a broader band and higher molecular weight than the native recombinant protein.

Example 4
Pharmacokinetics of PEGylated rFVIII in FVIII-deficient knockout mouse model FVIII-deficient mice detailed by Bi et al. (Nat Genet 1995; 10: 119-21) were used as a model for severe human hemophilia A. Groups of 5 mice were injected bolus (10 ml) with PEG-rFVIII (PEG-SS, 5K) or native rFVIII prepared according to Example 1 at a dose of FVIII 200 IU / kg body weight via the tail vein. / Kg). Citrate plasma by cardiac puncture after anesthesia was prepared from each group at 5 minutes, 3, 6, 9, and 24 hours after injection. FVIII activity levels in plasma samples were measured. The results of this experiment are summarized in FIG. Average half-life increased from 1.9 hours (for native rFVIII) to 4.9 hours (for PEGylated rFVIII) and area under the curve (AUC) increased from 13.0 to 25.2 hours * IU / ml did. Half-life calculations were performed using MicroMath Scientist's model 1 (MicroMath, Saint Louis, Mo., USA) from the pharmacokinetic library.

(Example 5)
Detailed analysis of PEGylation of rFVIII by SDS-PAGE and immunoblot techniques Digestion of native and PEGylated rFVIII with 1 nM thrombin at 60 ° C. for 60 minutes resulted in specific cleavage of the FVIII molecule, clearly defined A degradation product was produced. These heavy and light chain fragments were separated by SDS-PAGE followed by electroblotting as described in Example 3. To visualize the cleaved fragments, polyclonal and monoclonal antibodies against heavy chain A1 and A2 domains, B domain and light chain N-terminal A3 domain were applied.

  As shown in FIG. 3, all three domains were PEGylated to varying degrees. The B domain was strongly PEGylated. Both the A1 and A2 domains of the heavy chain were partially PEGylated. Various degrees of PEGylation (mono-, di-, tri -...) could be observed in the light chain A3 domain. Consistent with Example 6, PEGylated FVIII was considered more resistant to thrombin.

(Example 6)
Thrombin resistance of PEGylated rFVIII When FVIII is treated with thrombin in vitro, its procoagulant activity rapidly increases and subsequently decreases. Activation and inactivation rates depended on thrombin concentration and FVIII integrity and were monitored by the FIXa cofactor assay as follows.

FVIII was incubated at 37 ° C. with 0.5 or 1 nM thrombin. Subsamples are withdrawn at time intervals between 0.5 and 40 minutes and added to a mixture of FIXa, FX, PL vesicles and CaCl ₂ that also contains certain thrombin inhibitors to further thrombin-mediated reactions. Was stopped and incubated for 3 minutes. A secondary sample was added to the chromogenic substrate. The chromogenic substrate contained EDTA that was selectively cleaved by FXa to stop further Xa activation. After 15 minutes incubation, the reaction was terminated with acetic acid. Absorbance (A405) values proportional to FXa concentration were measured by an ELISA reader and converted to FXa concentration using a purified FXa reference curve. The resulting FXa concentration was plotted against the incubation time with thrombin.

  The decay portion of the curve was fitted with a single exponential fit to determine the quasi-first-order inactivation rate of FVIII.

図４及び表２に示すように、ＰＥＧ化ｒＦＶＩＩＩは、適用した両方のトロンビン濃度においてより遅い不活性化速度を示した。

As shown in FIG. 4 and Table 2, PEGylated rFVIII showed slower inactivation rates at both applied thrombin concentrations.

(Example 7)
PEGylation of lysine residues in rFVIII with branched 2,3-bis (methylpolyoxyethylene-oxy) -1- (1,5-dioxo-5-succinimidyloxy, pentyloxy) propane 150 mM NaCl A solution of rFVIII in 20 mM Hepes buffer pH 7.4 containing 0.5% sucrose and 0.1% polysorbate 80 was prepared from the bulk material from the Advate manufacturing process containing FVIII489 IU / ml. Branched PEG succinimidyl PEG glutarate (PEG-SG) reagent (2,3-bis (methylpolyoxyethylene-oxy) -1- (1,5-dioxo-5) obtained from NOF Corporation (Tokyo, Japan) -Succinimidyloxy, pentyloxy) propane) was added to 153 ml of this solution with gentle stirring (5 mg / mg protein reagent) and after 10 minutes 0.5 M NaOH was added dropwise to bring the pH value to 7.4. Adjusted. PEGylation of rFVIII was then performed for 1 hour at room temperature with gentle stirring.

  Subsequently, an ion exchange chromatography resin (Fractogel EMD TMAE650M / Pharmacia XK-50 column, bed height 14 equilibrated in 20 mM Hepes buffer containing 0.5% sucrose and 0.1% polysorbate 80, 150 mM NaCl, pH 7.4. The reaction mixture was applied at a linear flow rate of 1 cm / min. The column was washed with 25 CV equilibration buffer to remove excess reagent (linear flow rate 2 cm / min) and PEGylated rFVIII was eluted with buffer (20 mM Hepes, 1.0 M NaCl, 0.5% sucrose, 0.1% Polysorbate 80, pH 7.4) was used for elution at a linear flow rate of 0.5 cm / min. The eluate was then concentrated by ultrafiltration / diafiltration using a membrane with a molecular weight cutoff of 30 kD made of regenerated cellulose using a buffer system consisting of 20 mM Hepes, 150 mM NaCl, 0.5% sucrose, pH 7.4. .

(Example 8)
In-vitro characterization of rFVIII PEGylated with branched PEG-SG 20 kD rFVIII from the Advate manufacturing process is PEGylated via a lysine residue using a branched PEG-SG reagent according to Example 7 to produce PEGylated rFVIII The material was biochemically characterized as described in Example 2.

表３のデータによれば、ＰＥＧ化ｒＦＶＩＩＩ調製物においては、（ＦＶＩＩＩ色素形成活性とＦＶＩＩＩ抗原の比で表される）生物活性は、未変性ｒＦＶＩＩＩの生物活性（１００％）に比べて完全に回収された。

According to the data in Table 3, in the PEGylated rFVIII preparation, the biological activity (expressed as the ratio of FVIII chromogenic activity to FVIII antigen) is completely greater than that of native rFVIII (100%). It was recovered.

  PEGylated rFVIII was characterized by SDS-PAGE and immunoblot techniques under reducing conditions using 4-12% polyacrylamide gradient gels as described in Example 3. The results are summarized in FIG. This blot shows the domain structure of native and PEGylated rFVIII. PEGylated rFVIII was found to have a broader band and higher molecular weight than the native recombinant protein.

  For a more detailed analysis of PEGylation of rFVIII preparations by SDS-PAGE and immunoblotting techniques, digest native and PEGylated rFVIII with 1 nM thrombin for 60 min at 60 ° as described in Example 5. This resulted in specific cleavage of the FVIII molecule, producing a well-defined degradation product. The fragments were separated by SDS-PAGE followed by electroblotting and visualized with different anti-FVIII antibodies. As shown in FIG. 6, all domains were PEGylated to some extent. The B domain was strongly PEGylated. Various degrees of PEGylation (mono-, di-, tri-PEGylation) could be observed in the light chain A3 domain. From the results, PEGylated rFVIII was considered to be more resistant to thrombin.

  As described in Example 6, the rate of activation and inactivation by thrombin was monitored by the FIXa cofactor assay. The decay portion of the curve was fitted with a single exponential fit to determine the quasi-first-order inactivation rate of FVIII.

図７及び表４に示すように、ＰＥＧ化ｒＦＶＩＩＩは、適用した両方のトロンビン濃度においてより遅い不活性化速度を示した。

As shown in FIG. 7 and Table 4, PEGylated rFVIII showed slower inactivation rates at both applied thrombin concentrations.

Example 9
PEGylation of rFVIII via a carbohydrate moiety For the preparation of PEG-rFVIII conjugates via a carbohydrate residue, an rFVIII solution (final concentration 1.2 mg / ml) is prepared with 25 mM phosphate buffer, pH 6.7. NaIO ₄ is added for oxidation of carbohydrate residues (final concentration 0.3 mM) (Roberts et al .; Advanced Drug Del Rev .; 54: 459-76 (2002); Meir and Wilchek; Meth Enzymol; 138 : 429-42 (1987)). Glycerol was added at a final concentration of 10% to quench the reaction, and centrifugation was repeated using an Amicon Micron-10 apparatus (Amicon, Billerica, Mass.) To separate excess reagent. PEG-hydrazide (MW3300 Da / Nektar, Huntsville, Alabama) was added to obtain a reagent with a final concentration of 1.5 mM. PEGylation was then performed at room temperature for 2 hours. Subsequently, the resulting conjugate and excess reagent were separated by repeated centrifugation with an Amicon Micron-10 apparatus using 25 mM phosphate buffer, pH 6.7.

(Example 10)
Polysialylation of rFVIII using PSA-hydrazine For the preparation of PSA-rFVIII conjugates via carbohydrate residues, an rFVIII solution (final concentration 1 mg / ml) is prepared with 20 mM sodium acetate buffer, pH 6.0. For the oxidation of carbohydrate residues, NaIO ₄ is added (final concentration 0.25 mM). Oxidation is performed at 4 ° C. in the dark for 60 minutes. Sodium bisulfite (final concentration 25 mM) is added to stop the reaction. Excess sodium periodate is separated by gel filtration on a DG-10 column (Bio-Rad). Subsequently, PSA-hydrazine with a chain length of 20 kD (prepared according to WO 2006/016168) is added (final concentration 10 mM). The polysialylation procedure is performed at room temperature for 2 hours. Polysialylated rFVIII is purified by HIC from Butyl-Sepharose (GE-Healthcare). A 5M NaCl solution is added to the mixture to a final concentration of 3M NaCl. This mixture is applied to a Butyl-Sepharose (GE-Healthcare) packed column and the rFVIII-PSA conjugate is eluted using 50 mM Hepes buffer, pH 7.4 containing 6.7 mM CaCl ₂ . After elution of the conjugate, the pH is adjusted to pH 6.9.

(Example 11)
Purification and derivatization of polysialic acid Polysialic acid was purified by anion exchange chromatography on Q-Sepharose FF as described in WO06016161 A1. 5 grams of PSA was dissolved in 50 mL of 10 mM triethanolamine buffer, pH 7.4 (= starting buffer) containing 25 mM NaCl. This solution was applied to a Pharmacia XK50 column packed with Q-Sepharose FF (GE Healthcare, Munich, Germany) equilibrated with starting buffer. The column was then washed with 8 column volumes (CV) of starting buffer and bound PSA was eluted stepwise with 3 CV of 200 mM NaCl, 350 mM NaCl and 500 mM NaCl in the starting buffer. The fraction eluted with 350 mM NaCl had a molecular weight of 20 kDa as shown by SDS gel electrophoresis. This fraction was concentrated by ultrafiltration using a 5 kD membrane (Millipore, Billerica, Mass.) Made of regenerated cellulose, followed by diafiltration with 50 mM phosphate buffer, pH 7.2. PSA was oxidized with NaIO ₄ and terminal primary amino groups were introduced by reductive amination as described in WO05016973 A1. For reductive amination, 11 mL of 2M NH ₄ Cl solution was added to 20 mL of a solution containing 58 mg / ml oxidized PSA in 50 mM phosphate buffer, pH 7.2. A 5M NaCNBH ₃ solution in 1M NaOH was then added to a final concentration of 75 mM. The reaction was carried out at room temperature and pH 8.0 for 5 days.

The mixture was then dialyzed against (NH ₄ ) ₂ CO ₃ solution (50 mg / L) containing 10 mM NaCl followed by 50 mM phosphate buffer containing 5 mM EDTA, pH 8.0. A sulfhydryl group was then introduced by reaction of the terminal primary amino group with 2-iminothiolane (Traut reagent / Pierce, Rockford, IL). The reaction was carried out at room temperature for 1 hour using a 20-fold molar excess of reagent in 50 mM phosphate buffer containing 5 mM EDTA, pH 8.0. Finally, the PSA solution containing terminal free SH groups was subjected to ultrafiltration / diafiltration using a cut-off 5 kD membrane (Millipore, Billerica, MA) made of regenerated cellulose.

(Example 12)
Polysialylation of rFVIII using a heterobifunctional crosslinker For coupling of PSA-SH to rFVIII, a heterobifunctional crosslinker MBPH (4- [4- N-maleimidophenyl] butyric acid hydrazide HCl / Pierce, Rockford, IL) was used (Chamow et al., J Biol Chem; 267: 15916-22 (1992)). PSA-SH containing an active sulfhydryl group was prepared according to Example 11.

2 ml rFVIII (638 mg, 3.856 mg / ml protein concentration) was transferred to an oxidation buffer (50 mM sodium acetate, pH 6) using a desalting column (Bio-Rad Econopac 10DG) according to the manufacturer's instructions. The protein was then oxidized with 0.25 mM NaIO ₄ (Merck) (1 hour in the dark at 4 ° C.). The oxidation reaction was quenched with glycerol at a final concentration of 10%. Glycerol and NaIO ₄ were removed and the protein was transferred to reaction buffer (50 mM sodium phosphate pH 6.5) using a desalting column (Bio-Rad Econopac 10DG) according to the manufacturer's instructions. A mixture containing 1 mg / mg MBPH protein and PSA-SH (200-fold molar excess over protein) was then incubated at room temperature for 2 hours at pH 6.5. Excess linker was removed using a desalting column (Bio-Rad Econopac 10DG) according to the manufacturer's instructions and the linker-PSA conjugate was transferred into the reaction buffer.

MPBH-PSA conjugate was added to oxidized rFVIII (0.105 mg / ml protein) and the reaction mixture was incubated for 2 hours at room temperature with gentle shaking. The rFVIII-PSA conjugate was purified by HIC using a pre-packed Butyl Sepharose column (GE Healthcare, Butyl HiTrap FF 5 ml). To hydrophobically interact the conjugate with Butyl Sepharose, the sample was cooled to 2-8 ° C. and a buffer solution containing 5 M NaCl (50 mM Hepes, 5 M NaCl, 6.7 mM CaCl ₂ , 0.01% Tween). PH 6.9) was added to increase the ionic strength of the reaction mixture to a conductivity of about 185 mS / cm. The reaction mixture was loaded at a flow rate of 1.2 cm / min onto a column equilibrated with equilibration buffer pH 6.9 (containing 50 mM Hepes, 3M NaCl, 6.7 mM CaCl ₂ , 0.01% Tween 80). Unbound sample was washed away using 10 column volumes (CV) of equilibration buffer. The conjugate was eluted at a flow rate of 1.2 cm / min using a low ionic strength buffer, pH 7.4 (50 mM Hepes, 6.7 mM CaCl ₂ ). Samples and buffer were cooled by an ice bath during the chromatography step. Finally, the pH of the eluate was adjusted to 6.9.

(Example 13)
Conjugation of rFVIII and dextran For conjugation of rFVIII and dextran, 2 ml of rFVIII (638 mg, 3.4 mg / ml protein) was used using a desalting column (Bio-Rad Econopac 10DG). To an oxidation buffer (50 mM sodium acetate, pH 6). The protein was then oxidized with 0.25 mM NaIO ₄ (1 hour in the dark at 4 ° C.). The oxidized protein was first concentrated using a MWCO 30 kDa vivaspin ultrafiltration spin column (Sartorius Stedim Biotech GmbH) according to the manufacturer's instructions. The sample was then dialyzed overnight at 4 ° C. against reaction buffer (50 mM sodium phosphate pH 7).

  After dialysis, 26.58 mg of adipic acid dihydrazide (ADH) (Sigma) was added (500-fold molar excess) and the reaction mixture was incubated at room temperature for 2 hours with gentle shaking. ADH was removed using a desalting column (Bio-Rad Econopac 10DG) according to the manufacturer's instructions. 10 mg of aldehyde activated dextran (Pierce) was added (17-fold molar excess over protein) and the mixture was incubated at room temperature, pH 7, for 2 hours.

  The conjugate was purified by IEX chromatography on Q-Sepharose HP (GE-Healthcare). The sample was loaded at a flow rate of 0.5 ml / min onto a column (6.4 mm × 3 cm, V = 1 ml) equilibrated with buffer A (50 mM sodium phosphate pH 6.8). Unbound samples were washed away with 5 CV buffer A. Finally, the conjugate was eluted with a linear salt gradient (10 CV 0-100% buffer B [50 mM sodium phosphate pH 6.8 + 1 M NaCl]) at a flow rate of 0.5 ml / min.

Claims (8)

  A method of conjugating a water-soluble polymer and an oxidized carbohydrate moiety of Factor VIII comprising contacting the oxidized carbohydrate moiety with an activated water-soluble polymer under conditions that allow conjugation. .   The method of claim 1, wherein the water soluble polymer is selected from the group consisting of PEG, PSA, and dextran.   2. The method of claim 1, wherein the activated water soluble polymer is selected from the group consisting of PEG-hydrazide, PSA-hydrazine and aldehyde activated dextran. The method of claim 1, wherein the carbohydrate moiety is oxidized by incubation in a buffer comprising NaIO ₄ .   2. The method of claim 1, wherein the oxidized carbohydrate moiety of FVIII is present in the B domain of Factor VIII.   Modified Factor VIII produced by the method according to any one of claims 1-5. A proteinaceous construct comprising (a) a Factor VIII molecule, and (b) at least one water-soluble polymer bound to said Factor VIII molecule, wherein said water-soluble polymer is in the B domain of said Factor VIII Bound to the factor VIII via one or more carbohydrate moieties present;
Proteinaceous construct.   8. A proteinaceous construct according to claim 7, wherein the water soluble polymer is selected from the group consisting of PEG, PSA and dextran.

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