Conjugates of factor VIII moieties having oxime-containing linkages

Abstract

The present disclosure provides conjugates comprising a Factor VIII moiety covalently attached to a water-soluble polymer, e.g., a polyethylene glycol polymer, via an oxime-containing linkage. Methods for preparing and administering such conjugates are also provided, including, among other things, water-soluble polymeric oxyamine reagents useful for preparing the subject conjugates. The present disclosure generally relates to conjugates of a water-soluble polymer and a Factor VIII moiety, wherein the linkage between the Factor VIII moiety and the water-soluble polymer comprises an oxime (-CH=NO-).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 365,836, filed July 22, 2016, which is incorporated herein by reference in its entirety.

[0002] The present disclosure generally relates to conjugates of water-soluble polymers and Factor VIII moieties, in which the linkage between the Factor VIII moiety and the water-soluble polymer comprises an oxime (~CH=NO~). More specifically, a water-soluble polymer, such as polyethylene glycol, is covalently attached to an oxidized carbohydrate moiety of a Factor VIII molecule via an oxime linkage. Additionally provided herein are methods for synthesizing the conjugates, methods for purifying the conjugates, and novel polymer-oxyamine reagents, among others. [Background technology]

[0003] Scientists and clinicians face many challenges in their attempts to develop active agents into a form suitable for delivery to patients. For example, active agents that are polypeptides are often delivered via injection rather than orally. In this way, the polypeptide is introduced into the systemic circulation without being exposed to the proteolytic environment of the stomach. However, injection of polypeptides has several drawbacks. For example, many polypeptides have a relatively short half-life, necessitating repeated injections, which are often inconvenient and painful. Furthermore, some polypeptides may induce one or more immune responses, which results in the patient's immune system attempting to destroy or neutralize the immunogenic polypeptide. Of course, once the polypeptide is destroyed or neutralized, the polypeptide cannot exert its intended pharmacodynamic activity. Therefore, delivery of active agents such as polypeptides is often problematic, even when these agents are administered by injection.

[0004] The problem of delivering active agents via injection has been addressed with some success. Several techniques have been used to extend the half-life of proteins, including genetic fusion with albumin (Schulte, S. (2011), Thrombosis Research 128 Suppl 1, S9-12) or the Fc portion of antibodies (Schulte, S. (2011), Thrombosis Research 128 Suppl 1, S9-12; Powell, JS, et al., (2012), Blood 119, 3031-3037), non-covalent association with liposomes (Powell, JS, et al., (2008) J Thromb Haemost 6, 277-283), as well as polyethylene glycol (Turecek, PL, et al., Hamostaseologie 32 Suppl 1, S29-38; Turecek, PL, et al. al., (2016), J.Pharm.Sci.105,460-475), sialic acid (Bader, RA, and Wardwell, PR (2014) Therapeutic delivery 5,235-237), dextran (Caliceti, P., et al., (2003) Bioconjugate Chemistry 14,899-908) and hydroxyethyl starch (Baldwin, JE, et al., (1981) Tetrahedron 37,1723-1726), polyvinylpyrrolidone (PVP) (Kaneda, Y., et al., (2004) Biomaterials 25,3259-3266). For example, conjugation of active agents to water-soluble polymers has resulted in polymer-active agent conjugates with reduced immunogenicity and antigenicity. In addition, these polymer-active agent conjugates often have a much longer half-life compared to their non-conjugated counterparts due to reduced clearance through the kidney and / or reduced enzymatic degradation in the systemic circulation. As a result of having a longer half-life, the polymer-active agent conjugates require less frequent administration, thus reducing the overall number of painful injections and inconvenient visits to a medical professional.Furthermore, active agents that are only slightly soluble can have their water solubility significantly increased when conjugated to a water soluble polymer.

[0005] Polyethylene glycol has been conjugated to active agents due to its proven safety and FDA approval for both topical and oral administration. When an active agent is conjugated to a polymer of polyethylene glycol, or "PEG," the conjugated active agent is commonly referred to as "PEGylated." The commercial success of PEGylated macromolecules such as PEGASYS® PEGylated interferon alpha-2a (Hoffmann-La Roche, Nutley, NJ), PEG-INTRON® PEGylated interferon alpha-2b (Schering Corp., Kennilworth, NJ), and NEULASTA™ PEG-filgrastim (Amgen Inc., Thousand Oaks, CA) has demonstrated that administration of conjugated forms of active agents may have significant advantages over their non-conjugated counterparts. PEGylated small molecules such as MOVANATIK™ (naloxegol) (AstraZeneca Pharmaceuticals LP, Wilmington, DE) have also been commercially successful. Harris et al. provide a review of some of the effects of PEGylation on pharmaceuticals. Harris et al. (2003) Nat. Rev. Drug Discov. 2(3):214-221.

[0006] Despite these successes, it is often difficult to conjugate polymers to active agents to produce commercially suitable drugs. For example, conjugation may result in polymers attached to or near sites on active agents required for pharmacological activity (e.g., at or near binding sites). For example, random conjugation at lysines of the amino acid backbone may result in significant loss of biological activity. In such cases, the corresponding prolonged circulation time may or may not be sufficient to compensate for the loss of activity. In addition, conjugates with increased half-life may be further improved by combined longer half-life or by a beneficial combination of extended half-life and therapeutic activity. In the field of bleeding disorders, proteins (such as, for example, Factor VIII) may be administered to patients to combat or ameliorate bleeding disorders. Various approaches have been described for PEGylation of Factor VIII, which (among other things) has the advantage of a longer half-life compared to its non-conjugated counterpart. Although conjugation of Factor VIII has been successful, it may be possible to improve upon previous success by providing additional conjugates of Factor VIII (for example). Thus, the present disclosure seeks to address this and other needs. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Schulte, S. (2011), Thrombosis Research 128 Suppl 1, S9-12 [Non-Patent Document 2] Powell,JS,et al.,(2012),Blood 119,3031-3037 [Non-Patent Document 3] Powell,JS,et al.,(2008)J Thromb Haemost 6,277-283 [Non-Patent Document 4] Turecek,PL,et al.,Hamostaseologie 32 Suppl 1,S29-38 [Non-Patent Document 5] Turecek,PL,et al.,(2016),J.Pharm.Sci.105,460-475 [Non-Patent Document 6] Bader,RA,and Wardwell,PR(2014)Therapeutic delivery 5,235-237 [Non-Patent Document 7] Caliceti,P.,et al.,(2003)Bioconjugate Chemistry 14,899-908 [Non-Patent Document 8] Baldwin, JE, et al., (1981) Tetrahedron 37, 1723-1726 [Non-Patent Document 9] Kaneda, Y., et al., (2004) Biomaterials 25, 3259-3266 [Non-Patent Document 10] Harris et al.(2003)Nat.Rev.Drug Discov.2(3):214-221 Summary of the Invention [Means for solving the problem]

[0008] In one or more embodiments, a conjugate is provided that comprises a Factor VIII moiety covalently attached to a water-soluble polymer via an oxime-containing linkage. More particularly, provided herein is a conjugate that comprises one or more water-soluble polymers covalently attached to one or more oxidized carbohydrates of the Factor VIII moiety.

[0009] For example, in some embodiments, the conjugate has the structure POLY-XON=CH-FVIII, where POLY is a water-soluble polymer, X is any spacer containing one or more atoms, FVIII is a Factor VIII moiety, and ∼CH-FVIII is the residue of a carbonyl group of an oxidized carbohydrate of the Factor VIII moiety.

[0010] Regardless of the spacer, in some embodiments, the spacer X is hydrolytically stable. In some further embodiments related to the above, X comprises at least one heteroatom selected from O, S, and NH. In some still further related embodiments of each and all of the foregoing, X has an atomic length of about 5 atoms to about 25 atoms.

[0011] In some preferred embodiments of the conjugates provided herein, the water-soluble polymer is a poly(alkylene oxide).

[0012] In yet another embodiment of the water-soluble polymer, the water-soluble polymer has a weight average molecular weight of from 2,000 Daltons to 85,000 Daltons.

[0013] In some particular embodiments, the conjugate has the structure: CH3O-CH2CH2O-(CH2CH2O) m’ -CH2CH2-O-NH=CH-(FVIII) where (m') ranges from 0 to about 4,000, FVIII is a factor VIII moiety, and -CH-(FVIII) is the residue of a carbonyl group of an oxidized carbohydrate of the factor VIII moiety.

[0014] In one or more still further embodiments, the water soluble polymer is branched. In one or more related embodiments, the conjugate comprises: [ka] wherein (m') or (n) for each of the above branched structures independently ranges from about 1 to about 4,000, and X is as described above in any one or more of the illustrative embodiments. In some embodiments of the above branched structures, each of (m') or (n) is about 227.

[0015] Illustrative factor VIII portions for each and every embodiment provided herein include, for example, human recombinant B-domain deleted factor VIII, and human recombinant full-length factor VIII, among others.

[0016] Also provided is a composition comprising a Factor VIII moiety covalently attached to a water-soluble polymer via an oxime-containing linkage according to any one or more of the exemplary embodiments, and a pharma- ceutically acceptable excipient.

[0017] In yet another aspect, provided herein is a method for treating a blood disorder, such as hemophilia A, comprising administering to a patient a composition as described above.

[0018] In yet another embodiment, the compound has the structure: [ka] where X is a spacer containing one or more atoms, and each (n) ranges from about 1 to about 4,000.

[0019] In some embodiments of the oxyamine reagent (or the corresponding conjugate), X is -NH(CH) 1~6 In some particular embodiments, X is or includes -NH(CH2)2-. In some still further embodiments, X is -NH(CH2) 1~6 In some particular embodiments, X is -NH(CH)NHC(O)CH-.

[0020] In one or more further embodiments, methods for preparing such conjugates are provided.

[0021] In one or more related embodiments, a pharmaceutical formulation comprising the conjugate is provided.

[0022] In yet another one or more additional aspects provided herein, there are provided methods for preparing water-soluble polymers having an oxime reactive group or a reactive water-soluble polymer having a reactive functional group capable of forming an oxime-containing linkage after reaction with a Factor VIII moiety.

[0023] In yet a further aspect, a method of administering the conjugate is provided.

[0024] Additional aspects and embodiments are set forth in the following description and claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] In describing and claiming particular features of the present disclosure, the following terminology will be used in accordance with the definitions set out below unless otherwise indicated.

[0026] The present disclosure is not limited to particular polymers, synthesis techniques, active agents, etc., as these may vary.

[0027] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a "polymer" includes a single polymer and two or more of the same or different polymers, a reference to a "conjugate" includes a single conjugate and two or more of the same or different conjugates, a reference to an "excipient" includes a single excipient and two or more of the same or different excipients, etc.

[0028] As used herein, "PEG," "polyethylene glycol," and "poly(ethylene glycol)" are intended to encompass any water-soluble poly(ethylene oxide). Typically, PEGs for use according to the present invention have the following structure: -O(CH2CH2O) m -" where (m) is 2 to 4000. As used herein, PEG can be any of the following: -CH2CH2-O(CH2CH2O) depending on whether the terminal oxygen has been removed or not, e.g., during synthetic transformation. m -CH2CH2-" and "-(CH2CH2O) m -". When PEG further comprises a spacer moiety (described in more detail below), the atoms constituting the spacer moiety do not result in the formation of an oxygen-oxygen bond (i.e., "-OO-", i.e., peroxide linkage) when covalently attached to a water-soluble polymer segment. It should be recalled that throughout the specification and claims, the term "PEG" includes structures having various terminal or "end-capping" groups, etc. The term "PEG" also refers to polymers that include a majority, i.e., greater than 50%, of -CH2CH2O- monomer subunits. Generally, substantially all (and preferably all) of the monomer subunits are ethylene oxide subunits, although polyethylene glycol polymers may include different end-capping moieties or functional groups, e.g., for conjugation. With regard to specific configurations, PEG can have any number of different molecular weights and structures or geometries, such as "branched", "linear", "branched", "multifunctional", etc. (described in more detail below).

[0029] The terms "end capped" or "terminally capped" are used interchangeably herein to refer to the end or terminus of a polymer having an end capping moiety. Typically, but not necessarily, the end capping moiety is a hydroxy or C 1~20Including alkoxy groups. Thus, examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxy) and benzyloxy. Additional end-capping moieties include aryl, heteroaryl, cyclo, heterocyclo, etc. Saturated, unsaturated, substituted and unsubstituted forms of each of the foregoing are envisioned.

[0030] The end-capping group may be a silane. The end-capping group may also advantageously comprise a detectable label. When the polymer has an end-capping group that comprises a detectable label, the amount or location of the polymer and / or the moiety of interest (e.g., active agent) to which the polymer is linked can be determined by using a suitable detector. Such labels include, but are not limited to, fluorescers, chemiluminescers, moieties used in enzyme labeling, color-producing (e.g., dyes), metal ions, radioactive moieties, and the like. Suitable detectors include photometers, films, spectrometers, and the like.

[0031] "Non-naturally occurring" in reference to a polymer or water-soluble polymer means a polymer that, as a whole, is not found in nature. However, a non-naturally occurring polymer or water-soluble polymer can contain one or more subunits or portions of subunits that are naturally occurring, so long as the entire polymer structure is not found in nature.

[0032] The term "water-soluble polymer" refers to any polymer that is soluble in water at room temperature. Typically, the water-soluble polymer will transmit at least about 75%, more preferably at least about 95%, of the light transmitted by the same solution after filtration. On a weight basis, the water-soluble polymer will preferably be at least about 35% (by weight) soluble in water, more preferably at least about 50% (by weight) soluble in water, even more preferably about 70% (by weight) soluble in water, and even more preferably about 85% (by weight) soluble in water. However, even more preferably, the water-soluble polymer will be about 95% (by weight) soluble in water, and most preferably, the water-soluble polymer will be completely soluble in water.

[0033] Molecular weight in the context of the water-soluble polymers of the present invention, such as PEG, can be expressed as either number average molecular weight or weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to weight average molecular weight. Both number average and weight average molecular weight determinations can be measured using gel permeation chromatography or other liquid chromatography techniques. Other methods for measuring molecular weight values ​​can also be used, such as using end group analysis or measurements of colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure) to determine number average molecular weight, or using light scattering techniques, ultracentrifugation, or viscometry to determine weight average molecular weight. The polymers described herein are typically polydisperse (i.e., the number average molecular weight and weight average molecular weight of the polymer are not equal), and preferably have a low polymer dispersity of less than about 1.2, more preferably less than about 1.15, even more preferably less than about 1.10, even more preferably less than about 1.05, and most preferably less than about 1.03.

[0034] The term "spacer" or "spacer moiety" is used herein to refer to an atom or collection of atoms that optionally occurs between one moiety and another moiety.

[0035] As used herein, an "organic group" includes, for example, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl.

[0036] "Alkyl" refers to a hydrocarbon chain, typically ranging from about 1 to 20 atoms in length. Such hydrocarbon chains may be branched or straight-chained, although straight-chained is generally preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like. As used herein, "alkyl" includes lower alkyl.

[0037] "Lower alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms, which may be straight or branched, as exemplified by methyl, ethyl, n-butyl, iso-butyl, and tert-butyl.

[0038] "Cycloalkyl" refers to a saturated cyclic hydrocarbon chain, including bridged, fused, or spirocyclic compounds, preferably composed of 3 to about 12 carbon atoms, more preferably 3 to about 8 carbon atoms.

[0039] "Non-interfering substituents" are those groups that, when present in a molecule, are typically non-reactive with other functional groups contained within the molecule.

[0040] The term "substituted," as in, for example, "substituted alkyl," refers to a moiety (e.g., an alkyl group) in which one or more hydrogen atoms have been replaced with one or more non-interfering substituents, such as, but not limited to: C3-C8 cycloalkyl, such as cyclopropyl, cyclobutyl, etc.; halo, such as fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl; substituted phenyl; etc. A "substituted aryl" is an aryl having one or more non-interfering groups as a substituent. In the case of substitution on the phenyl ring, the substituents can be in any orientation (i.e., ortho, meta, or para). A "substituted ammonium" is an ammonium having one or more non-interfering groups (e.g., organic groups) as a substituent.

[0041] "Alkoxy" refers to the group -OR, where R is alkyl or substituted alkyl, preferably C1-C 20 It is preferably alkyl (eg, methoxy, ethoxy, propyloxy, benzyl, etc.), more preferably C1 to C7 alkyl.

[0042] As used herein, "alkenyl" refers to a branched or unbranched hydrocarbon group of 2 to 15 atoms in length containing at least one double bond. Exemplary alkenyls include (but are not limited to) ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, and the like.

[0043] The term "alkynyl" as used herein refers to a branched or unbranched hydrocarbon group of 2 to 15 atoms in length containing at least one triple bond. Exemplary alkynyls include (but are not limited to) ethynyl, n-butynyl, iso-pentynyl, octynyl, decynyl, and the like.

[0044] "Aryl" means one or more aromatic rings of 5 or 6 core carbon atoms each. Aryl includes multiple aryl rings, fused, as in naphthyl, or unfused, as in biphenyl. The aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings. As used herein, "aryl" includes heteroaryl. Aromatic-containing moieties (e.g., Ar 1 , Ar 2 etc.) refer to structures containing aryl.

[0045] "Heteroaryl" is an aryl group containing one to four heteroatoms, preferably N, O, or S, or a combination thereof. Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings.

[0046] "Heterocycle" or "heterocyclic" means one or more rings of 5 to 12 atoms, preferably 5 to 7 atoms, with or without unsaturation or aromatic character and with at least one ring atom that is not carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.

[0047] A "substituted heteroaryl" is a heteroaryl having one or more non-interfering groups as substituents.

[0048] A "substituted heterocycle" is a heterocycle having one or more side chains formed from non-interfering substituents.

[0049] "Electrophile" refers to an ion, or an atom or collection of atoms which may be ionic, having an electrophilic center, i.e., an electrophilic center, capable of reacting with a nucleophile.

[0050] "Nucleophile" refers to an ion, or an atom or collection of atoms which may be ionic, having a nucleophilic center, i.e., a nucleophilic center, or with an electrophile.

[0051] A "hydrolytically stable" linkage or bond refers to a chemical bond, typically a covalent bond, that is substantially stable in water, i.e., does not undergo hydrolysis to any appreciable extent under physiological conditions over an extended period of time. Examples of hydrolytically stable linkages include, but are not limited to, carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, and the like. In general, hydrolytically stable linkages are those that exhibit a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks. It should be noted that some linkages can be hydrolytically stable or hydrolytically stable depending (for example) on adjacent and proximal atoms and on the ambient conditions. One of skill in the art can determine whether a given linkage or bond is hydrolytically stable or hydrolytically stable under a given set of circumstances, for example, by subjecting the linkage-containing molecule of interest to the conditions of interest and testing for evidence of hydrolysis (e.g., the presence and amount of two molecules resulting from the cleavage of a single molecule). Other techniques known to those of skill in the art for determining whether a given linkage or bond is hydrolytically stable or hydrolytically stable can also be used.

[0052] The terms "active agent", "biologically active agent" and "pharmacologically active agent" are used interchangeably herein and are defined to include any agent, drug, compound, composition or mixture of substances that provides some pharmacological, often beneficial, effect that can be demonstrated in vivo or in vitro. This includes supplements, nutrients, nutriceuticals, drugs, proteins, vaccines, antibodies, vitamins, and other beneficial agents. As used herein, these terms further include any physiologically or pharmacologically active substance that produces a local or systemic effect in a patient. A preferred biologically active agent is a factor VIII moiety.

[0053] A "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to an excipient that may be included in a composition of the present invention and that causes no significant adverse toxicological effects to a patient.

[0054] "Pharmacologically effective amount," "physiologically effective amount," and "therapeutically effective amount" are used interchangeably herein to refer to the amount of polymer-active agent conjugate (typically present in a pharmaceutical formulation) necessary to provide a desired level of active agent and / or conjugate in the bloodstream or target tissue. The exact amount will depend on many factors, such as the particular active agent, components and physical properties of the pharmaceutical formulation, the intended patient population, patient considerations, etc., and can be readily determined by one of skill in the art based on the information provided herein and available in the relevant literature.

[0055] Certain compounds described herein may exist in particular geometric or stereoisomeric forms, and the present disclosure contemplates all such compounds, including, where applicable, cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic and other mixtures thereof, that are within the scope of the present disclosure.

[0056] In addition, certain compounds, polymers, conjugates, etc. may contain basic functional groups and thus may form pharma- ceutically acceptable salts with pharma-ceutically acceptable acids. In this context, the term "pharma-ceutically acceptable salts" refers to the relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention. These salts may be prepared in situ during the administration vehicle or dosage form manufacturing process, or by separately reacting the free base form of the compound with a suitable organic or inorganic acid and isolating the salt thus formed during subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate, etc. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0057] Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the compounds, derived, for example, from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothioic acid, and the like.

[0058] In other cases, compounds, polymers, conjugates, etc. may contain one or more acidic functional groups and thus can form pharma- ceutically acceptable salts with pharma-ceutically acceptable bases. The term "pharma-ceutically acceptable salts" in this case refers to the relatively non-toxic inorganic and organic base addition salts of such compounds, polymers, or conjugates. These salts can be prepared in situ during the administration vehicle or dosage form manufacturing process, or by separately reacting the purified free acid form of the compound with a suitable base, such as a hydroxide, carbonate, or bicarbonate of a pharma-ceutically acceptable metal cation, with ammonia, or with a pharma-ceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, etc. Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc. (see, for example, Berge et al., supra).

[0059] "Multifunctional" in the context of a polymer means a polymer that contains three or more functional groups, where the functional groups can be the same or different. A multifunctional polymer will typically contain about 3-100 functional groups, or 3-50 functional groups, or 3-25 functional groups, or 3-15 functional groups, or 3-10 functional groups, or 3, 4, 5, 6, 7, 8, 9, or 10 functional groups in the polymer. A "bifunctional" polymer means a polymer that contains two functional groups that are the same (i.e., homobifunctional) or different (i.e., heterobifunctional).

[0060] "Branched" in reference to the overall shape or structure of a polymer refers to a polymer having two or more polymer "arms". A branched polymer may have two polymer arms, three polymer arms, four polymer arms, six polymer arms, eight polymer arms or more. One particular type of highly branched polymer is a dendritic polymer or dendrimer, which for purposes of the present invention is considered to have a structure distinct from that of a branched polymer.

[0061] "Dendrimers" or dendritic polymers are globular, size-monodisperse polymers with all bonds radiating from a central focal point, or core, with a regular branching pattern and repeating units each contributing a branch point. Dendrimers exhibit certain dendritic state properties, such as core encapsulation, that make them unique from other types of polymers.

[0062] "Substantially" or "essentially" means nearly entirely or completely, e.g., 95% or more of a given amount.

[0063] Basic or acidic reactants described herein include their neutral, charged, and any corresponding salt forms.

[0064] The term "patient" or "subject" refers to an organism suffering from or susceptible to a condition that can be prevented or treated by administration of a conjugate or composition provided herein, and includes both humans and animals. Subjects include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and are preferably humans.

[0065] "Optional" and "optionally" mean that the subsequently described situation may or may not occur, so that the description includes cases where the situation occurs and cases where it does not occur.

[0066] As used herein, the "halo" designators (e.g., fluoro, chloro, iodo, bromo) are generally used when the halogen is attached to a molecule, while the suffix "chloride" (e.g., fluoride, chloride, iodide, bromide, etc.) is used when the halogen is present in its separate ionic form (e.g., when a leaving group leaves the molecule, etc.).

[0067] It should be recognized that, in the context of this disclosure, a definition of a variable provided in the context of one structure or formula may also apply to the same variable repeated in a different structure, unless the context dictates otherwise.

[0068] Overview Random PEGylation of blood factors such as factor VIII has been described previously and can result in therapeutically useful products.See, for example, U.S. Patent No. 7,199,223.However, due to the large number of lysines in proteins such as factor VIII, random PEGylation can present difficulties, such as reproducible preparation of conjugate mixtures, characterization of products, and formation of products that maintain sufficient bioactivity so that the products are therapeutically useful.

[0069] In the case of proteins such as Factor VIII that contain carbohydrate groups, PEGylation at the carbohydrate offers the possibility of placing the PEG at a position less likely to interfere with biological activity. Carbohydrate PEGylation using enzymes to facilitate the conjugation of PEG to carbohydrate moieties of proteins has been previously described for the PEGylation of the major blood factors, FVIIa, FIX and FVIII (Ostergaard, H., et al., (2011) Blood 118, 2333-2341; Stennicke, HR, et al., (2008), Thrombosis and haemostasis 100, 920-928; Tiede, A., et al., (2013); J Thromb Haemost 11, 670-678; Agerso, H., et al., (2012) Haemophilia 18, 941-947; Stennicke, HR, et al., (2013), Blood 121, 2108-2116). However, this approach requires the additional enzymatic GMP generation to produce the resulting PEGylated protein. Although chemical oxidation approaches (i.e., non-enzymatic) for the formation of reactive groups on sugars of O- or N-linked glycans provide a more direct approach when compared to enzymatic oxidation, the harsh reaction conditions commonly used are not considered suitable for the oxidation of carbohydrates contained in large blood factor molecules such as factor VIII due to the possibility of significant protein inactivation. Although such approaches may be postulated for factor VIII, the inventors are not aware of any such oxidation and subsequent conjugation being successfully performed with the concomitant maintenance of significant activity of the factor VIII moiety. Surprisingly, the inventors have found conditions using classical periodate oxidation of glycols and carbohydrates that allow the conjugation of FVIII without the need for additional enzyme or protein manipulation and without destroying the activity of the protein. Chemical PEGylation of protein-bound carbohydrates typically requires the oxidation of unreactive sugar hydroxyls to reactive aldehydes followed by coupling to a suitable PEG reagent to achieve a stable linked conjugate.

[0070] Mild oxidation (~1 mM) conditions using sodium periodate can selectively oxidize the vicinal hydroxyl groups of N-acetyl-D-neuraminic acid (sialic acid), the terminal sugar found in glycoprotein carbohydrate trees. High concentrations of periodate can cleave adjacent hydroxyl-bearing carbon bonds in other sugars, resulting in undesired ring opening. Careful control of periodate oxidation allows successful selective tritium labeling of carbohydrate residues for radiolabeled biodistribution studies without incorporating tritium into the non-sialic acid portions of proteins. [ka]

[0071] Quenching excess periodate, for example with a large excess of ethylene glycol, followed by rapid removal of formaldehyde, minimizes back reactions with protein amines (Wolfe, CA, and Hage, DS (1995), Anal Biochem 231, 123-130).

[0072] As described herein, the preferred coupling for carbohydrate PEGylation involves the use of water-soluble polymeric oxyamine reagents, which may optionally have a linker between the PEG and the oxyamine group. In the illustrative reaction scheme below, no linker is present. Oxime linkages are preferred because they have higher stability than linkages obtained using hydrazide-functionalized polyethylene glycol reagents. [ka]

[0073] This approach is particularly advantageous for PEGylation of FVIII based on conjugation to carbohydrate moieties due to the fact that there are far fewer carbohydrate moieties (23 chains) in full-length FVIII than lysines (159) (Lenting, PJ, van Mourik, JA, and Mertens, K. (1998) Blood 92, 3983-3996). Details surrounding the provision of water-soluble polymeric oxime-linked conjugates of Factor VIII, the conjugates themselves, related polymeric reagents and related methods are described in the following sections.

[0074] Water-soluble polymers Prior to describing exemplary conjugates, the characteristics of the water-soluble polymer are detailed below. Each of the various embodiments of the water-soluble polymer applies to the reactive water-soluble polymers, conjugates, compositions, related methods, etc. that are the subject of this disclosure.

[0075] With respect to a given water-soluble polymer, each water-soluble polymer (e.g., POLY, POLY 1 and POLY 2) can include any polymer, so long as the polymer is water-soluble and non-peptidic. The water-soluble polymer for use herein is preferably poly(ethylene glycol), but may also be other water-soluble polymers, such as other poly(alkylene glycols) [also referred to as "poly(alkylene oxides)], such as poly(propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol, poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines, poly(N-acryloylmorpholines), as described in U.S. Pat. No. 5,629,384. The water-soluble polymer may be any homopolymer, copolymer, terpolymer, non-random block polymer, and random block polymer of the foregoing. In addition, the water-soluble polymer may be linear, but may also be other morphologies (e.g., branched, bifurcated, etc.), as described in more detail below. When present within the overall structure, the water-soluble polymer has from 1 to about 300 termini.

[0076] When a polymeric reagent comprises more than one water-soluble polymer, each water-soluble polymer in the overall structure may be the same or different. However, it is preferred that all water-soluble polymers in the overall structure are of the same type. For example, it is preferred that all water-soluble polymers in a given structure are poly(ethylene glycol) polymers.

[0077] While the weight average molecular weight of any individual water-soluble polymer can vary, the weight average molecular weight of any given water-soluble polymer will typically be within the following ranges: 100 Daltons to about 150,000 Daltons. However, exemplary ranges include weight average molecular weights within the following ranges: about 880 Daltons to about 5,000 Daltons; greater than 5,000 Daltons to about 100,000 Daltons; about 6,000 Daltons to about 90,000 Daltons; about 10,000 Daltons to about 85,000 Daltons; greater than 10,000 Daltons to about 85,000 Daltons; about 20,000 Daltons to about 85,000 Daltons; about 53,000 Daltons. and about 40,000 daltons to about 120,000 daltons. For any given water-soluble polymer, PEG having a molecular weight within one or more of these ranges is preferred.

[0078] Exemplary weight average molecular weights for water soluble polymers include about 100 daltons, about 200 daltons, about 300 daltons, about 400 daltons, about 440 daltons, about 500 daltons, about 600 daltons, about 700 daltons, about 750 daltons, about 800 daltons, about 900 daltons, about 1,000 daltons, about 1,500 daltons, about 2,000 daltons, about 2,200 daltons, about 2,500 daltons, about 3,000 daltons, about 4,000 daltons, about 4,400 daltons, about 4,500 daltons, about 5,000 daltons, about 5,500 daltons, about 6,000 daltons, about 7,000 daltons, about 7,500 daltons, about 8,000 daltons, about 9,000 daltons, about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, about 16,000 daltons, about 17,000 daltons, about 18,000 daltons, about 19,000 daltons, about 21,000 daltons, about 22,000 daltons, about 23,000 daltons, about 24,000 daltons, about 25,000 dalton ,000 daltons, about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, about 16,000 daltons, about 17,000 daltons, about 18,000 daltons, about 19,000 daltons, about 20,000 daltons, about 22,500 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons, and about 75,000 daltons. Branched versions of water-soluble polymers having any of the foregoing total weight average molecular weights can also be used (eg, a branched 40,000 dalton water-soluble polymer composed of two 220,000 dalton polymers).

[0079] When PEG is the polymeric reagent, it typically comprises multiple (OCH2CH2) monomers [or (CH2CH2O) monomers, depending on how PEG is defined]. As used throughout the specification, the number of repeating units is referred to as "(OCH2CH2) n". Thus, the value of (n) typically falls within one or more of the following ranges: from 2 to about 3400, from about 4 to about 1500, from about 100 to about 2300, from about 100 to about 2270, from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to about 1,900. For any given polymer whose molecular weight is known, the number of repeating units (i.e., "n") can be determined by dividing the total weight average molecular weight of the polymer by the molecular weight of the repeating monomer.

[0080] Each water-soluble polymer is typically biocompatible and non-immunogenic. In relation to biocompatibility, a material is considered biocompatible if the beneficial effects associated with its use (e.g., administration to a patient) in association with living tissue, alone or with another material (e.g., an active agent), outweigh any adverse effects, as assessed by a clinician, e.g., a physician. In relation to non-immunogenicity, a material is considered non-immunogenic if its use in association with living tissue, alone or with another material, does not result in an immune response (e.g., formation of antibodies), or if an immune response does result, such a response is not considered clinically significant or important, as assessed by a clinician. It is particularly preferred that the water-soluble polymers and conjugates of active agents and polymers described herein are biocompatible and non-immunogenic.

[0081] In one form, free or unconjugated PEG is a linear polymer terminated at each end with a hydroxyl group: HO-CH2CH2O-(CH2CH2O) m’ -CH2CH2-OH wherein (m') typically ranges from 0 to about 4,000, preferably from about 20 to about 1,000. The aforementioned PEGs, also referred to as PEG diols, can be used as starting materials to provide PEG reactants suitable for reaction with a Factor VIII moiety, such as a carboxyl group of a Factor VIII moiety. In some cases, the number of repeating units in a polyethylene glycol polymer can be designated herein by (n) rather than (m'). In such cases, the illustrative values ​​and ranges of (m') provided herein also apply to (n). The aforementioned polymer, α-,ω-dihydroxypoly(ethylene glycol), as described above, can be represented in shorthand form as HO-PEG-OH, where the -PEG- symbol represents the following structural unit: -CH2CH2O-(CH2CH2O) m’ -CH2CH2- where (m') is as defined above.

[0082] Another type of free or unbound PEG useful for preparing, for example, reactive PEG reagents or corresponding Factor VIII moiety conjugates with oxime-containing linkers is methoxy-PEG-OH, or mPEG for short, in which one terminus is a relatively inert methoxy group and the other terminus is a hydroxyl group. The structure of mPEG is shown below: CH3O-CH2CH2O-(CH2CH2O) m’ -CH2CH2- (In the formula, (m') is as defined above.)

[0083] Multi-arm or branched PEG molecules, such as those described in U.S. Patent No. 5,932,462, can also be used as the PEG polymer. For example, PEG can be represented by the structure: [ka] may have: (In the formula: Poly a and poly bare PEGs (same or different), such as methoxypoly(ethylene glycol); R″ is a non-reactive moiety such as H, methyl or a PEG backbone; P and Q are non-reactive linkages. In a preferred embodiment, the branched PEG polymer is a methoxypoly(ethylene glycol) disubstituted lysine, which upon reaction with the Factor VIII moiety forms a conjugate having an oxime-containing linkage.

[0084] In addition, PEG can include branched PEG. An example of a free or unbound branched PEG is represented by the following formula: [ka] where X is a spacer moiety and each Z is an oxyamine linked to CH by a chain of atoms of a defined length. The chain of atoms linking the Z functionality to the branching carbon atom serves as a linking group and can include, for example, alkyl chains, ether chains, ester chains, amide chains and combinations thereof. U.S. Pat. No. 6,362,254 discloses various branched PEG structures that can be used to provide the Factor VIII moiety conjugates disclosed herein.

[0085] Additionally, the PEG can have a branched form, as follows, and the water-soluble polymer includes the following branched structure: [ka] In the formula, each (m') is independently an integer having a value of 2 to 4000, or about 1 to about 3,000, or 1 to about 2,000, preferably about 20 to about 1,000.

[0086] The PEG polymer can include pendant PEG molecules having reactive groups, such as oxyamines, covalently attached along the length of the PEG rather than at the ends of the PEG chains. The pendant reactive groups can be attached to the PEG directly or through a spacer moiety, such as an alkylene group.

[0087] It will be understood by those of skill in the art that the term poly(ethylene glycol) or PEG refers to or includes all of the above forms of PEG.

[0088] Those skilled in the art will recognize that the foregoing detailed description of substantially water-soluble polymers is by no means exhaustive, but merely illustrative, and that all polymeric materials having the above-mentioned properties are contemplated. As used herein, the term "water-soluble polymer" refers to both molecules and water-soluble polymer moieties attached to other moieties, such as Factor VIII moieties. The following description of water-soluble polymers is applicable not only to the polymeric reagents but also to the corresponding conjugates formed using the described polymeric reagents.

[0089] The functional group of the water-soluble polymer-containing polymeric reagents used to form the conjugates described herein is an oxyamine, "~O-NH2" (also referred to as an amino-oxypolymer). Thus, the reactive PEG can, in certain cases, optionally include one or more additional atoms, i.e., spacers, interposed between the PEG portion of the molecule and the reactive oxyamine, as described below. PEG-XO-NH2

[0090] The oxyamine-terminated polymeric reagent can react with a carbonyl group of a Factor VIII moiety (e.g., a carbohydrate) to provide an oxime linkage between the Factor VIII moiety and a water-soluble polymer, such as polyethylene glycol, as described above. Typically, but not necessarily, the carbonyl group is part of an aldehyde in the Factor VIII moiety (e.g., generated after oxidation of the Factor VIII moiety). In addition, however, the carbonyl group may be part of a ketone in the Factor VIII moiety. In general, such reactive PEGs are encompassed by the following formula shown above: PEG-XO-NH2, where PEG can have any of the forms described herein, e.g., linear, branched, unbranched, etc., and X is an optional spacer of one or more atoms, preferably one that is hydrolytically stable, if present.

[0091] Typically, X contains at least one heteroatom, such as O, or S, or NH. Illustrative examples are provided below. Generally, X has an atomic length of 1 to about 50 atoms, or about 4 atoms to about 50 atoms, or about 5 atoms to about 25 atoms, or even more preferably about 5 atoms to about 20 atoms, or 3 to about 15 atoms. Exemplary spacers include, -C(O)-NH-, -NH-C(O)-NH-, -OC(O)-NH-, -C(S)-, -CH-, -CH-CH-, -CH-CH-CH-, -CH-CH-CH-CH-, -CH-CH-CH-CH-CH-, -O-CH-, -CH-O-, -O-CH-CH-, -CH-CH-O-CH-, -O-CH-CH-CH-, -CH-O-CH-, -CH-CH-O-CH-, -CH-CH- CH2-O-, -O-CH2-CH2-CH2-CH2-, -CH2-O-CH2-CH2-CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-CH2-CH2-O-CH2-, -CH2-CH2-CH2-CH2-O- , -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -CH2-C(O)-NH-CH2-, -CH2-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-CH2-, -CH2-C(O)-NH-CH 2-CH2-, -CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-CH2-, -C H2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-C(O)-NH-, -C(O)-O-CH2-, -CH2-C(O)-O-CH2-, -CH2-CH2-C(O)-O-CH2-, -NH-C(O)-CH2-, -CH2-NH-C(O)-CH2-, -CH2-CH2-NH-C(O)-CH2-, - NH-C(O)-CH2-CH2-, -CH2-NH-C(O)-CH2-CH2-, -CH2-CH2-NH-C(O)-CH2-CH2-, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -NH-CH2-,-NH-CH2-CH2-, -CH2-NH-CH2-, -CH2-CH2-NH-CH2-, -C(O)-CH2-, -C(O)-CH2-CH2-, -CH2-C(O)-CH2-, -C H2-CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-CH2-, -CH2-CH2-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH- , -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-, -(CH2)3C(O)NH(CH2)2NHC(O)CH2-, -CH2-CH2-CH2-C(O) -NH-CH2-CH2-NH-C(O)-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-CH2-, -OC(O)-NH-[CH2], 0~6 -(OCH2CH2) 0-2 -, -C(O)-NH-(CH2) 1~6 -NH-C(O)-, -NH-C(O)-NH-(CH2) 1~6 Examples of such segments include those independently selected from the group consisting of -NH-C(O)-. However, for purposes of this disclosure, a series of atoms is not a spacer if it is directly adjacent to a water-soluble polymer segment, the series of atoms being merely another monomer, and thus the proposed spacer moiety represents a mere extension of the polymer chain. The spacers or linkers described herein may also include combinations of any two or more of the above groups, in any orientation. Illustrative PEG-oxyamines are shown in the table below:

[0092] [Table 1]

[0093] [Table 2]

[0094] [Table 3]

[0095] [Table 4]

[0096] With respect to the PEG reagents provided in Table 1, it should be understood that the foregoing structures also include pharma- ceutically acceptable salt forms thereof, such as the hydrochloride salt. With respect to the structures in Table 1, X is a spacer comprising one or more atoms as previously described herein. In one or more particular embodiments applicable to each of the foregoing structures comprising the variable X, the spacer X is 〜NH(CH2) 1~6 The spacer X is equal to or comprises ~NH(CH2)2~, where the number of methylenes is selected from 1, 2, 3, 4, 5 and 6. More particularly, the spacer X is equal to or comprises ~NH(CH2)2~. Another preferred spacer applicable to each of the above structures where appropriate is ~NH(CH2)2NHC(O)CH2~, which comprises the segment ~NH(CH2)2~ with an additional carboxamidomethyl group. More generally, the additional specific spacer is, for example, ~NH(CH2) 1~6 NHC(O)CH2~, where the number of intervening methylene groups inserted between the amino groups is selected from 1, 2, 3, 4, 5, and 6. That is, in some embodiments, the number of intervening methylenes is 1; in yet some further embodiments, the number is 2; in yet some further embodiments, the number is 3; in yet some further embodiments, the number is 4; in yet some further embodiments, the number is 5; and in some additional embodiments, the number is 6. Furthermore, although not expressly shown, Table 1 above is also intended to expressly describe, for each of structures I-XXII, the corresponding PEG-Factor VIII moiety (also sometimes referred to herein as FVIII and F8) conjugates, where for each of structures I-XXII, ~ONH2 is replaced with ~ONH=CH-(FVIII) and the attachment to the Factor VIII moiety is via one or more oxidized carbohydrates of the Factor VIII moiety.

[0097] Exemplary polymeric reagents include, for example, the following structures: [ka] where X is a spacer as generally described above.

[0098] The carbonyl group of the Factor VIII moiety may be present as part of the molecule or may be obtained through modification of a protein having Factor VIII activity. In one approach to modifying a protein having Factor VIII activity, the protein (having at least one glycosylation modification) is subjected to oxidizing conditions (e.g., exposed to periodate or any other suitable oxidizing agent) to oxidize one or more hydroxy groups contained in the Factor VIII moiety, resulting in a carbonyl-containing (e.g., aldehyde-containing) Factor VIII moiety, which carbonyl group is suitable for reaction with a water-soluble polymeric oxyamine, such as PEG oxyamine. It has been found that periodate can be used to oxidize vicinal diols in carbohydrates present in the Factor VIII moiety without significantly impairing the activity of the protein. Conjugates formed by covalent attachment via oxime linkage to carbonyl groups (e.g., aldehydes) present in the sugar or carbohydrate moiety of the Factor VIII moiety, resulting from oxidation, for example, may sometimes be referred to as glycoconjugates. As mentioned above, due to the significantly fewer carbohydrates (23 chains) in full-length factor VIII (i.e., only 14% using the techniques described herein) when compared to lysine (159), more polymer-oriented modified factor VIII moieties can be accessed in which water-soluble polymers are covalently attached to known regions of the factor VIII molecule via oxime-containing linkages. See, for example, FIG. 1 in Lenting, PJ, et al., Haemophilia (2010), 16, 6-15, which illustrates the domain structure and post-translational modifications of factor VIII (FVIII). Mature FVIII consists of 2332 amino acids arranged in distinct domain structures: A1 (residues 1-336), A2 (373-710), B (741-1648), A3 (1690-2019), C1 (2020-2172) and C2 (2173-2332). The A domain is bounded by the acidic regions a1 (337-372), a2 (711-740) and a3 (1649-1689). Most of the Cys-residues are involved in disulfide bridges, but some are present as free cysteines.FVIII is highly glycosylated, with most of the carbohydrate residues present in the B domain. The B domain contains approximately seven O-linked glycans (the exact locations of which are unknown) and 16 sites for N-linked glycosylation. Five N-linked glycans are found in the other domains, two of which are mannose-type glycans. Thus, the factor VIII polymer modification approach described herein is effective in concentrating the majority of PEG attachment within the B domain, particularly when full-length factor VIII is used, due to the B domain being highly glycosylated.

[0099] Additional exemplary polymeric reagents are discussed in further detail below. Although stereochemistry has not been specifically depicted in any formula or structure (whether for the polymeric reagents, conjugates, or any other formula or structure), it should be remembered that the formulas and structures provided contemplate both enantiomers, as well as compositions containing equal amounts of each enantiomer (i.e., racemic mixtures) and unequal amounts of each enantiomer.

[0100] An exemplary linear polymeric reagent has the following structure: CH3O-CH2CH2O-(CH2CH2O) m’ -CH2CH2-O-NH2 In the formula, (m') typically ranges from 0 to about 4,000, or from about 0 to about 3,000, or from about 0 to about 2,000, preferably from about 20 to about 1,000. Specific illustrative linear PEG oxyamines having the structures shown above will have molecular weights of, for example, about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, or about 30,000 daltons. The foregoing linear PEG oxyamines do not include a spacer; in this PEG reagent, polyethylene glycol is covalently attached to the oxyamine group. Polymeric reagents encompassed by this formula can be synthesized according to procedures described in the literature or obtained commercially (e.g., from JenKem Technology USA, Plano, TX). For example, PEG oxyamines can be prepared from methoxy-PEG (i.e., conversion of the terminal hydroxyl group) by O-alkylation of tert-butyl N-hydroxycarbamate with PEG-O-methanesulfonate followed by acidic N-deprotection as described in S. Albrecht, et al., Synthesis, 2006, 1635-1638. Representative Factor VIII moiety conjugates prepared using such polymeric reagents are described in the supporting examples.

[0101] Additional illustrative branched PEG oxyamine reagents include the following: [ka]

[0102] Another exemplary PEG oxyamine reagent has the following branched structure: [ka] wherein (m') or (n) for each structure provided herein typically ranges from about 1 to about 4,000, or from about 1 to about 3,000, or from 1 to about 2,000, preferably from about 20 to about 1,000. This branched PEG reagent is sometimes referred to herein as RU-PEG2-oxyamine. Certain branched PEGs having the above structure have two PEG "arms" each having a molecular weight of about 10,000 daltons, such that (m') is about 227 for each PEG arm. Additional illustrative branched PEG oxyamines according to the above structures have two PEG "arms" or branches each having a molecular weight of about 5,000 daltons, or 10,000 daltons (as described above), or 20,000 daltons, etc. In the above branched structures, the two PEG arms, CH3-(OCH2CH2O) m’ extends from an organic core which is 2-(4-((2-(2-(aminooxy)acetamido)ethyl)amino)-4-oxobutoxy)propane-1,3-diyl dicarbamate. In the branched structure shown above, the intervening segment or spacer between the branched PEG and the oxyamine group is ~(CH2)3C(O)NH(CH2)2NHC(O)CH2~, although any suitable spacer as described above can be used.

[0103] In relation to the factor VIII moiety (e.g., for reaction with a water-soluble polymeric oxyamine such as PEG oxyamine), the factor VIII moiety useful in the present invention includes any protein that has the same activity (although not necessarily to the same extent) as native human factor VIII and is glycosylated (i.e., contains sugar residues such as glycosidically linked monosaccharides). Possible factor VIII moieties include native human factor VIII, which is a single-chain glycoprotein of 2,351 amino acids, structurally organized as A1-A2-B-A3-C1-C2. However, when the expressed polypeptide is translocated into the lumen of the endoplasmic reticulum, the 19 amino acid signal sequence is cleaved, resulting in a second sequence. This second sequence provided herein lacks the leading 19 amino acids. The factor VIII moiety for use herein is recognized. It will be appreciated that the factor VIII moiety is not limited to merely the "active" form of factor VIII (e.g., factor VIIIa), but that the term "factor VIII moiety" encompasses the "precursor" form, as well as other substances that have similar procoagulant effects.

[0104] For any given portion, it can be determined whether the portion has factor VIII activity. For example, some animal strains have been purposely bred with a genetic mutation for hemophilia so that animals derived from such strains have very low and insufficient levels of factor VIII. Such strains can be obtained from a variety of sources, including, but not limited to, the Division of Laboratories and Research, New York Department of Public Health, Albany, NY, and the Department of Pathology, University of North Carolina, Chapel Hill, NC. Both of these sources provide, for example, dogs suffering from canine hemophilia A. To test the factor VIII activity of any given portion in question, the portion is injected into a diseased animal, a small cut is made, and the bleeding time is compared to that of an untreated diseased animal as a control. Another method useful for determining factor VIII activity is to determine cofactor and procoagulant activity. See, for example, Mertens et al. (1993) Brit. J. Haematol. 85:133-42. Other methods known to those of skill in the art can also be used to determine whether a given moiety has Factor VIII activity. Such methods are useful for determining the Factor VIII activity of both the moiety itself (and thus can be used as a "Factor VIII moiety"), and the corresponding polymer-moiety conjugate having an oxime-containing linkage.

[0105] Non-limiting examples of factor VIII moieties include: factor VIII; factor VIIIa; factor VIII:C; factor VIII:vWF; B-domain deleted factor VIII (and other truncated forms of factor VIII); hybrid proteins such as those described in U.S. Pat. No. 6,158,888; glycosylated proteins with factor VIII activity such as those described in U.S. Patent Application Publication No. 2003 / 0077752; and peptidomimetics with factor VIII activity. A preferred truncated factor VIII (encompassed by the term "B-domain deleted factor VIII") is a factor VIII having the amino acid sequence represented by the amino acid sequence of Arg. 759 ~Ser 1709 More preferably, the deletion corresponds to at least 581 amino acids in the region Pro 1000 ~Asp 1582 , area Thr 778 ~Pro 1659 , and the domain Thr 778 ~Glu 1694 The present invention relates to a protein having an amino acid sequence of human factor VIII that corresponds to one of the following:

[0106] In relation to the Factor VIII moiety, any biologically active fragment, deletion variant, substitution variant or addition variant of the foregoing that maintains at least some Factor VIII activity can also be used.

[0107] The Factor VIII moiety can be advantageously modified to include one or more amino acid residues, such as, for example, lysine, cysteine, and / or arginine, to provide for facile attachment of the polymer to atoms in the side chains of the amino acids, so long as the linkage is an oxime-containing linkage. Techniques for adding amino acid residues are well known to those of skill in the art.

[0108] Additionally, the active agent can be obtained from blood-derived sources. For example, factor VIII can be fractionated from human plasma using precipitation and centrifugation techniques known to those skilled in the art. See, for example, Wickerhauser (1976) Transfusion 16(4):345-350 and Slichter et al. (1976) Transfusion 16(6):616-626. Factor VIII can also be isolated from human granulocytes. See, for example, Szmitkoski et al. (1977) Haematologia (Budap.) 11(1-2):177-187.

[0109] In addition, active agents can be obtained by recombinant methods. In summary, recombinant methods include constructing a nucleic acid encoding a desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., bacteria, yeast, or mammalian cells such as Chinese hamster ovary cells or baby hamster kidney cells), and expressing the nucleic acid to produce the desired polypeptide or fragment. Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic and eukaryotic host cells are known to those skilled in the art. See, for example, U.S. Pat. No. 4,868,122.

[0110] The above exemplary Factor VIII moieties are intended to encompass, where applicable, analogs, agonists, antagonists, inhibitors, isomers, and pharma- ceutically acceptable salt forms thereof. In relation to peptides and proteins, the present disclosure is intended to encompass synthetic, recombinant, native, glycosylated, and non-glycosylated forms, as well as biologically active fragments thereof. In addition, the term "active agent" in relation to a Factor VIII moiety is intended to encompass the active agent prior to conjugation, and the "residue" of the active agent following conjugation.

[0111] A general outline of exemplary conjugation reactions for the Factor VIII moieties provided herein is provided below: [ka]

[0112] An exemplary conjugate has the formula: [ka] where PEG includes any PEG moiety, particularly those described herein, and X is any spacer moiety, as described in detail above. Suitable and exemplary molecular weight values ​​for the PEG moiety attached to the Factor VIII moiety via an oxime-containing linkage are described above. For example, one illustrative Factor VIII moiety conjugate is generally described below, where the spacer X is not present in the general formula: CH3O-CH2CH2O-(CH2CH2O) m’ -CH2CH2-O-NH=CH-(FVIII) where (m') typically ranges from 0 to about 4,000 and F8 is the residue of a carbonyl-containing Factor VIII moiety, i.e., the "CH" of the oxime represents the carbon atom of the carbonyl group of the Factor VIII moiety prior to reaction with the PEG-oxyamine reagent.

[0113] Further illustrative Factor VIII moiety conjugates include, for example: [ka] wherein (m') or (n) of each of the above branched structures is typically in the range of about 1 to about 4,000, or about 1 to about 3,000, or 1 to about 2,000, preferably about 20 to about 1,000. In some embodiments, each PEG "arm" has a molecular weight of about 10,000 daltons, such that (m') is about 227 in each PEG arm.

[0114] Also provided herein is a method for preparing branched polyethylene glycol oxyamines, as described in detail in Examples 1-3. In this method, a branched inverse urethane PEG reagent, typically activated as an active ester such as an NHS-ester (although any suitable active ester can be used), is reacted with, for example, H2N(CH2) 1~6 Reaction with a diamine such as NH2 provides the corresponding inverse urethane PEG carboxamide-amine intermediate, see general reaction scheme provided below. [ka]

[0115] The reaction is typically carried out in the presence of a base, such as triethylamine, in an organic solvent, such as dichloromethane or chloroform. Preferably, the reaction is carried out under an inert atmosphere using a dry (i.e., anhydrous) solvent. Typical reaction times include about 1 hour to about 10 hours, or about 1 hour to about 8 hours, and the reaction is generally carried out at a temperature ranging from 0°C to about 60°C.

[0116] Preferably, the reaction is carried out at room temperature (about 20° C. to about 25° C.). The intermediate RU-PEG2-carboxamidoamine intermediate is then recovered, for example, by precipitation with an alcohol (e.g., isopropyl alcohol) and suitably dried. For additional details, see Example 1, in which the reaction is carried out with ethylenediamine. The RU-PEG2-carboxamidoamine intermediate is then reacted with a carboxy-protected 2-(carbonylaminooxy)acetic acid using conventional coupling chemistry and conditions (e.g., in the presence of a coupling agent such as N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride in the presence of N,N-dimethylpyridine) to provide the corresponding protected oxyamine product, where any suitable protecting group can be used. An illustrative protected RU-PEG2-oxyamine product, including a t-Boc protecting group, is provided below. [ka] The protecting group is then removed, for example, with trifluoroacetic acid in the case of a t-Boc protecting group, to provide the desired product, which is typically recovered by precipitation with an alcohol, such as isopropyl alcohol, as described above, and has the following structure (including its salt forms): [ka]

[0117] Also provided herein is a method for preparing a Factor VIII moiety conjugate, which comprises contacting a polymeric reagent bearing an oxyamine group with a Factor VIII moiety under conditions suitable for forming a covalent oxime bond between the polymer, e.g., PEG moiety, and the biologically active agent, i.e., Factor VIII. Typically, the polymeric reactant, i.e., the polymeric oxyamine, is reacted with the Factor VIII moiety in equimolar amounts (relative to the desired number of groups suitable for reaction with the reactive group) or in molar excess. See, e.g., the supporting examples. For example, the polymeric reagent may be added to the target active agent in a molar ratio of about 1:1 (polymeric reagent:active agent), 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, 10:1, 20:1, 30:1, 40:1, or even greater. The conjugation reaction is allowed to proceed until the desired degree of polymer attachment occurs or until substantially no further conjugation occurs (which can generally be determined by monitoring the progress of the reaction over time). The progress of the reaction can be monitored by withdrawing aliquots from the reaction mixture at various time points and analyzing the reaction mixture by SDS-PAGE or MALDI-TOF mass spectrometry or any other suitable analytical method. The reaction is considered complete once a plateau is reached in relation to the amount of conjugate formed or unconjugated polymer remaining. Typically, the conjugation reaction takes anywhere from a few minutes to several hours (e.g., 5 minutes to 24 hours or more). The resulting product mixture is preferably, but not necessarily, purified to separate excess reagents, unconjugated reaction participants (e.g., active agent), undesired multi-conjugated species, and free or unreacted polymer. The resulting conjugates can then be further characterized using analytical methods such as MALDI, capillary electrophoresis, gel electrophoresis, and / or chromatography.

[0118] For example, as described in representative Examples 4, 5, and 6, Factor VIII is generally activated for reaction with a water-soluble polymeric oxyamine reagent, e.g., PEG-oxyamine, by first incubating the Factor VIII moiety with a chemical oxidizing agent, such as a periodate (e.g., sodium periodate), thereby oxidizing one or more carbohydrates of Factor VIII. For example, the oxidizing agent is added to a solution of the Factor VIII moiety in a suitable buffer, typically at a pH of about 6. Exemplary concentrations of the Factor VIII moiety range from about 1.0 mg / mL to about 7 mg / mL, or from about 2.0 mg / mL to about 5 mg / mL, or from about 3 to 4 mg / mL, in a suitable buffer (e.g., including HEPES, sodium chloride, calcium chloride, and Tween 80). Oxidation is preferably performed in the absence of light, e.g., in the dark. The reaction is then allowed to proceed for a suitable period of time sufficient to oxidize the carbohydrate groups in the protein but not result in significant loss of activity, for example, from about 10 minutes to about 2 hours, preferably less than about 1 hour, or from about 30 to about 45 minutes. The oxidation is typically carried out at a temperature ranging from about 5° C. to about 35° C., or from about 5° C. to about 25° C. The oxidized Factor VIII, i.e., Factor VIII having one or more carbohydrates oxidized to provide reactive aldehyde groups suitable for reaction with PEG-oxyamine, is then recovered and a suitable bioactivity assay, such as measuring clotting activity, is used to confirm that its bioactivity has been maintained.

[0119] The oxidized Factor VIII thus prepared is then reacted with a water-soluble polymeric oxyamine, such as a PEG oxyamine, as previously described herein. For example, the oxidized Factor VIII is generally, but not necessarily, in molar excess relative to the oxidized Factor VIII moiety (e.g., about equimolar to a 250 molar excess, such as 5-fold, 10-fold, 30-fold, 50-fold, 70-fold, 100-fold, 120-fold, 150-fold, 200-fold, and 300-fold molar excess (expressly including the range reached by a combination of any two of the foregoing). The oxime-coupling reaction is generally carried out at a temperature of about 2° C. to about 35° C. (e.g., room temperature of about 20-25° C.) for a period of about 0.5 hours to about 24 hours, or about 1 hour to about 8 hours.

[0120] In some embodiments, the coupling reaction between the water-soluble polymeric oxyamine reagent and the Factor VIII moiety is carried out in the absence of a catalyst selected from o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid, sulfanilic acid, o-aminobenzamide, o-toluidine, m-toluidine, p-toluidine, o-anisidine, m-anisidine, and p-anisidine. In some particular embodiments, the coupling reaction between the water-soluble polymeric oxyamine reagent and the Factor VIII moiety is carried out in the absence of toluidine. In some other embodiments, the reaction is carried out in the absence of aniline.

[0121] Any unreacted aldehyde groups remaining on the oxidized Factor VIII moiety are then desirably capped (i.e., rendered unreactive) by the addition of a suitable capping agent, such as an alkoxyamine, such as methoxylamine hydrochloride (e.g., in sodium hydroxide, such as 1N sodium hydroxide). The resulting reaction mixture is then allowed to react for an additional period of time, such as from about 10 minutes to about 5 hours, or from about 15 minutes to 2 hours, or from about 15 minutes to 1 hour. After the capping reaction, the pH of the reaction mixture may be adjusted to about pH 7 by the addition of a suitable buffer. The resulting PEG-oxime-Factor VIII conjugate mixture may then be further purified by any of a number of suitable methods. Suitable purification methods include, for example, chromatography, filtration, and precipitation. Illustrative chromatographic methods include, for example, hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC), size exclusion chromatography (SEC), affinity chromatography, and reverse phase chromatography, with a preferred purification method being anion exchange chromatography.

[0122] As noted above, the degree of conjugation (i.e., the degree of PEGylation relative to the PEGoxyamine reagent) can be adjusted, if desired, by (for example) adjusting the reaction conditions. For example, different numbered polymer (i.e., PEG)-to-FVIII ratios (e.g., 1-mer, 2-mer, 3-mer, etc., where "1-mer" indicates one polymer per Factor VIII moiety, "2-mer" indicates two polymers per Factor VIII moiety, etc.) can be obtained by stopping the conjugation reaction before it reaches completion, by varying the molar ratio of PEGoxyamine to Factor moiety, etc. The conjugation reaction can be stopped in a variety of ways, including, for example, the addition of a quenching agent (e.g., quenching with an alkoxyamine such as methoxyamine).

[0123] It is possible to characterize the degree of polkymer attachment of the conjugate (i.e., the number of polymeric reagents attached to the carbohydrate moiety of Factor VIII by reaction of one or more oxidized carbohydrates (e.g., aldehydes) of Factor VIII with PEGoxyamine, often expressed in terms of the average number in the context of the conjugate composition). Analytical techniques such as SDS-PAGE, SEC, IEC, MALDI-TOF, etc. can be used to determine the average number of water-soluble polymer molecules covalently attached to the Factor VIII moiety conjugate. Exemplary techniques for determining the degree of PEGylation are described in Example 4. In the method used, the PEG-oxime-Factor VIII conjugate composition is desirably digested with a protease such as pronase to exhaustively digest Factor VIII, thereby releasing the PEG moieties that were covalently attached to Factor VIII. The amount of released water-soluble polymer, e.g., PEG, is then quantified using an appropriate method such as SEC-HPLC, or RP-HPLC.

[0124] The methods provided herein are effective to provide Factor VIII-PEG oxime conjugates, in which PEG (or more generally, a water-soluble polymer) is covalently attached to the carbohydrate of the Factor VIII moiety via an oxime-containing linkage. As is evident from the supporting examples, illustrative conjugates (and conjugate compositions) have been prepared having both relatively high and low average degrees of PEGylation, e.g., ranging from about 1 to about 20. In the case of Factor VIII-linear PEG oxime carbohydrate-based conjugates, the average degree of PEGylation of the composition is within the range of about 4 to about 20. Preferred compositions of Factor VIII-linear PEG oxime carbohydrate-based conjugates have an average degree of PEGylation of about 3 to about 8, or more preferably about 4 to about 7. That is, in some embodiments, preferred compositions have the following structure: [CHO-CHCHO-(CHCHO) m’ -CH2CH2-O-NH=CH] 3~8 -(FVIII), where m' is as previously described. In some further embodiments, the preferred compositions include a Factor VIII-linear PEG oxime carbohydrate-based conjugate having the following structure: [CHO-CHCHO-(CHCHO) m’ -CH2CH2-O-NH=CH] 4~7 -(FVIII)-linear PEG oxime carbohydrate-based conjugates, where m' is as previously described. In some additional embodiments of the foregoing, the average degree of PEGylation is about 6. Illustrative compositions include conjugates according to the foregoing structures, where polyethylene glycol chains are covalently attached to carbohydrate moieties of Factor VIII, each having an average molecular weight of about 20,000 daltons.

[0125] Turning now to the illustrative branched RU-PEG-2 oxime-Factor VIII conjugates and related compositions, the average degree of PEGylation of the compositions is somewhat lower, for example, within the range of about 0.5 to about 4, or about 1 to about 3.5. In some embodiments, the molecular weight of each PEG chain in the structure below is about 10,000 daltons. [ka]

[0126] The Factor VIII conjugates and compositions provided herein surprisingly retain significant biological activity when compared to the unmodified Factor VIII moiety based on any suitable bioassay that measures the biological activity of Factor VIII. Preferred PEG-oxime-Factor VIII conjugates retain about 45% to about 75% or more, or 50% to 75% or more, biological activity when compared to the unmodified Factor VIII moiety based on any suitable bioassay, such as, for example, a one-stage APTT-based clotting assay.

[0127] A challenge in providing therapeutically useful conjugates of Factor VIII is reaching a therapeutically useful balance between half-life and bioactivity. Particularly preferred conjugates and related compositions have an average number of polymer attachment sites per Factor VIII moiety, e.g., 3-8, or 4-7, or about 6, as described above for linear PEG oximes, and retain about 40% to about 75% or more of the biological activity, as compared to the unmodified Factor VIII moiety, based on any suitable assay, such as a one-stage APTT-based clotting assay. Additional preferred conjugates and compositions have an average number of branched polymer attachment sites per Factor VIII moiety, e.g., 0.5 to about 4, or about 1 to 3.5, using, for example, the branched inverse urethane PEG oxyamine reagents described above, and retain about 60% or more, e.g., about 60% to about 80% of the biological activity, as compared to the unmodified Factor VIII moiety, based on any suitable assay, such as a one-stage APTT-based clotting assay.

[0128] In the context of Factor VIII-PEG oxime-containing conjugates, the conjugates can be purified (e.g., by ion exchange using resins designed for purification of polymer-conjugated drugs, such as the MACROCAP brand of ion exchangers available from GE Healthcare Bio-Sciences AB, Uppsala, Sweden) to obtain / isolate the different conjugated species. Alternatively, and more preferably, for smaller molecular weight polymers (e.g., less than about 20 kilodaltons, more preferably less than about 10 kilodaltons), the product mixture can be purified to obtain a distribution of water-soluble polymer segments per Factor VIII. For example, the product mixture can be purified to obtain an average of anywhere between 1-5 PEGs per active agent (e.g., Factor VIII polypeptide). The strategy for purification of the final conjugate reaction mixture will depend on many factors, including, for example, the molecular weight of the polymer used, the particular active agent, the desired administration regimen, and the residual activity and in vivo properties of the individual conjugates.

[0129] If desired, conjugates with different molecular weights can be isolated using gel filtration chromatography (this technique works best when the polymer size is relatively close to the active agent size). That is, gel filtration chromatography is used to fractionate the different numbered polymer-to-active agent ratios (e.g., 1-mer, 2-mer, 3-mer, etc.) based on their different molecular weights (where the difference essentially corresponds to the average molecular weight of the water-soluble polymer segment). For example, in an exemplary reaction in which a 100 kDa protein is randomly conjugated to a polymeric reagent with a molecular weight of about 20 kDa, the resulting reaction mixture may include unmodified protein (MW 100 kDa), 1-PEGylated protein (MW 120 kDa), 2-PEGylated protein (MW 140 kDa), etc. Although this technique can be used to separate PEG and other polymer conjugates with different molecular weights, this technique is generally ineffective for separating positional isomers with different polymer attachment sites within the protein. For example, gel filtration chromatography can be used to separate mixtures of PEG 1-mers, 2-mers, 3-mers, etc. from one another, with each recovered PEG-mer composition containing PEGs attached to different reactive amino groups (e.g., lysine residues) within the active agent.

[0130] Suitable gel filtration columns for carrying out this type of separation include Superdex™ and Sephadex™ columns available from Amersham Biosciences (Piscataway, NJ). The choice of a particular column will depend on the desired fractionation range desired. Elution is generally carried out using a suitable buffer, such as phosphate, acetate, etc. The collected fractions can be analyzed by a number of different methods, including (i) optical density (OD) at 280 nm for protein content, (ii) dye-based methods (e.g., BCA) using BSA as a standard to determine protein content, (iii) iodine test for PEG content [Sims et al. (1980) Anal. Biochem, 107:60-63], and (iv) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) followed by staining with barium iodide.

[0131] In addition, ion exchange chromatography can be used to separate the different numbered polymer-to-active agent ratios (e.g., 1-mer, 2-mer, 3-mer, etc.) To separate the different numbered polymer-to-protein ratios using ion exchange chromatography, the conjugate-containing composition can be eluted through a column, with unconjugated (i.e., unreacted) polymer eluting first, followed by the higher multi-conjugated species, then progressively lower multi-conjugated species, and then completing with unconjugated protein.

[0132] Separation of positional isomers is accomplished by reverse phase chromatography using a reverse phase-high performance liquid chromatography (RP-HPLC) C18 column (Amersham Biosciences or Vydac) or by ion exchange chromatography using an ion exchange column, such as a Sepharose™ ion exchange column available from Amersham Biosciences. Either approach can be used to separate polymer-active agent isomers that have the same molecular weight (positional isomers). In addition to other techniques, peptide mapping can be used to help determine the location of polymer attachment of the active agent.

[0133] Factor VIII-water-soluble polymer conjugates that include oxime linkage to the carbohydrate of the Factor VIII moiety exhibit increased in vivo half-life compared to their corresponding unconjugated forms. Increased in vivo half-life can be assessed by measuring the pharmacokinetics of the conjugate and the Factor VIII moiety prior to conjugation in Factor VIII-deficient mice, as described in the literature. In summary, Factor VIII-deficient mice can be treated with a bolus injection of Factor VIII or Factor VIII-water-soluble polymer conjugate, and Factor VIII antigen levels in plasma samples can be measured at various time points. Factor VIII antigen can be measured by ELISA assay. Preferred conjugates and compositions will have a significant measure of Factor VIII activity, e.g., retention of at least about 30% or more, preferably at least about 40%, of the unmodified Factor VIII moiety, and a circulating half-life that is extended by at least about 1.2 to about 1.5 times the circulating half-life of unmodified Factor VIII.

[0134] In some embodiments, when using full-length factor VIII or non-B domain deleted factor VIII, the corresponding PEG-oxime-Factor VIII conjugate will have a substantial number of PEG oxime attachment sites in the B domain based on the number of glycosylation sites present in the B domain of factor VIII. See, e.g., Lenting, PJ, et al., Haemophilia (2010), 16, 6-15. For example, in some embodiments, all PEG oxime attachments in the conjugate composition are located in the B domain of factor VIII. In yet other embodiments, on average, 90% or more of the PEG oxime attachments are in the B domain. In yet some other embodiments, on average, 80% or more of the PEG oxime attachments are in the B domain.

[0135] The present disclosure also includes pharmaceutical formulations comprising a conjugate or composition provided herein in combination with one or more pharma- ceutically acceptable excipients. Generally, the conjugate itself is a solid (e.g., precipitate, lyophilizate, etc.), which can be combined with a suitable pharmaceutical excipient, which can be either solid or liquid.

[0136] Exemplary excipients include, but are not limited to, those selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.

[0137] Carbohydrates such as sugars, derivatized sugars such as alditols, aldonic acids, esterified sugars and / or sugar polymers can be present as excipients. Specific carbohydrate excipients include, for example: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myo-inositol, etc.

[0138] The excipient may also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium dihydrogen phosphate, disodium phosphate, and combinations thereof.

[0139] The formulation may also include an antimicrobial agent to prevent or inhibit microbial growth. Non-limiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof.

[0140] Antioxidants may also be present in the formulation. Antioxidants are used to prevent oxidation and, thereby, to prevent deterioration of the conjugate or other components of the formulation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0141] Surfactants can be present as excipients. Exemplary surfactants include: polysorbates such as "Tween 20" and "Tween 80" and pluronics such as F68 and F88 (both available from BASF, Mount Olive, New Jersey); sorbitan esters; lipids such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (but preferably not in liposomal form), fatty acids and fatty acid esters; and steroids such as cholesterol.

[0142] Acids or bases may be present as excipients in the formulation. Non-limiting examples of acids that can be used include acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloro acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, but are not limited to, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof.

[0143] Pharmaceutical formulations include all types of formulations, particularly those suitable for injection, such as powders that can be reconstituted, as well as suspensions and solutions. The amount of water-soluble polymer oxime-Factor VIII conjugate (i.e., the conjugate formed between the active agent and the polymer described herein) in the composition will vary depending on many factors, but will be a therapeutically effective dose when the composition is stored in a unit dose container (e.g., a vial). In addition, the pharmaceutical formulation may be contained in a syringe. The therapeutically effective dose can be determined empirically by repeatedly administering increasing amounts of the conjugate to determine which amount produces the clinically desired endpoint.

[0144] The amount of any individual excipient in the composition will vary depending on the activity of the excipient and the specific needs of the composition. Typically, the optimal amount of any individual excipient is determined by routine experimentation, i.e., by preparing compositions containing various amounts of the excipient (from low to high amounts) and testing the stability and other parameters, and then determining the range in which optimal performance is achieved without significant adverse effects.

[0145] Generally, however, the excipient will be present in the composition in an amount of from about 1% to about 99% by weight, preferably from about 5% to 98% by weight, more preferably from about 15 to 95% by weight of the excipient, with concentrations of less than 30% by weight being most preferred.

[0146] These aforementioned pharmaceutical excipients, together with other excipients, are listed in "Remington: The Science & Practice of Pharmacy, 19 th ed., Williams & Williams, (1995), the "Physician's Desk Reference", 52 nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, AH, Handbook of Pharmaceutical Excipients, 3 rd Edition, American Pharmaceutical Association, Washington, DC, 2000.

[0147] The pharmaceutical formulations of the present invention are typically, though not necessarily, administered via injection and therefore are generally liquid solutions or suspensions immediately prior to administration. Pharmaceutical formulations can also take other forms, such as syrups, creams, ointments, tablets, powders, etc. Other modes of administration, such as pulmonary, rectal, transdermal, transmucosal, oral, intrathecal, subcutaneous, intraarterial, etc., are also included.

[0148] As previously mentioned, the conjugates can be administered parenterally by intravenous injection, or less preferably by intramuscular or subcutaneous injection. Suitable formulation types for parenteral administration include, among others, solutions ready for injection, dry powders that are combined with a solvent before use, suspensions ready for injection, dry insoluble compositions that are combined with a vehicle before use, and emulsions and liquid concentrates that are diluted before administration.

[0149] The present invention also provides a method for administering a water-soluble polymer oxime-Factor VIII conjugate provided herein to a patient suffering from a condition responsive to treatment with the conjugate, such as hemophilia A. The method generally involves administering a therapeutically effective amount of the conjugate (preferably provided as part of a pharmaceutical formulation) via injection. This method of administration can be used to treat any condition that can be treated or prevented by administration of a particular conjugate. Those of skill in the art will recognize conditions that a particular conjugate can effectively treat. The actual dose administered will vary depending on the age, weight, and general condition of the subject, as well as the severity of the condition being treated, the judgment of the medical professional, and the conjugate being administered. Therapeutically effective amounts are known to those of skill in the art and / or described in the relevant reference documents and literature. In general, a therapeutically effective amount ranges from about 0.001 mg to 100 mg, preferably from 0.01 mg / day to 75 mg / day in doses, and more preferably from 0.10 mg / day to 50 mg / day in doses. For example, exemplary dosing regimens include administering a PEG-oxime Factor VIII conjugate provided herein at a dose of about 10-90 IU / kg, or about 15-80 IU / kg, or about 20-75 IU / kg, where for each dose, the conjugate is administered, for example, once every month, once every two weeks, once every week, or even twice weekly.

[0150] This method of administration can be used to treat any condition that can be treated or prevented by administration of the conjugate. Those skilled in the art will recognize the conditions that a particular conjugate can effectively treat. For example, the conjugate can be used to treat individuals with hemophilia A. In addition, the conjugate is suitable for use as a prophylactic against uncontrolled bleeding in patients with or without hemophilia. Thus, for example, the conjugate can be administered to patients at risk of uncontrolled bleeding before surgery.

[0151] The unit dosage of any given conjugate (again, preferably provided as part of a pharmaceutical formulation) can be administered in a variety of dosing schedules, depending on the clinician's judgment, the patient's needs, etc. The particular dosing schedule will be known to those skilled in the art or can be determined empirically using routine methods. Exemplary dosing schedules include, but are not limited to, administration 5 times a day, 4 times a day, 3 times a day, 2 times a day, once a day, 3 times a week, 2 times a week, once a week, twice a month, once a month, and any combination thereof. Once the clinical endpoint is reached, administration of the composition is stopped.

[0152] While the present invention has been described in connection with certain preferred embodiments thereof, it should be understood that the foregoing description and the experiments which follow are intended to be illustrative of the invention and not limiting of the scope thereof. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0153] All articles, books, patents, patent publications, and other publications referenced herein are incorporated by reference in their entirety. EXAMPLES

[0154] The practice of the present invention employs, unless otherwise indicated, conventional techniques of protein chemistry and the like, as understood by those of skill in the art and described in the literature. In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation must be accounted for. Temperatures are in degrees Celsius and pressures are at or near atmospheric pressure at sea level unless otherwise indicated. All reagents were obtained commercially unless otherwise indicated.

[0155] The Factor VIII used in the following illustrative PEGylation reactions was commercially available full-length Factor VIII (ADVATE, Shire) at 3.57 mg / ml in 20 mM HEPES, 0.75 M NaCl, 2 mM CaCl2, 0.1% Tween 80, pH 7.1. This composition is hereafter referred to as "FVIII Solution."

[0156] Protein concentrations were determined using the BCA protein assay (Pierce) with BSA as the standard.

[0157] The following abbreviations are used:

[0158] Example 1 Preparation of inverse urethane (RU)-PEG2-amine, 20 kD [ka] To a solution of ethylenediamine (1.20 g, 0.0200 mol) and triethylamine (1.01 g, 0.0200 mol) in anhydrous dichloromethane (200 mL) was added solid inverse urethane PEG2-NHS 20K (leftmost in the reaction scheme, 20.0 g, 0.0020 mol) for 30 min, and the mixture was stirred overnight under nitrogen atmosphere. The mixture was then filtered, concentrated under reduced pressure, and added to 600 mL of isopropyl alcohol. The precipitated product was filtered off and dried under vacuum. Yield 19.2 g.

[0159] NMR analysis confirmed the structure of the reverse-urethane PEG2-amine-20 kD product.

[0160] Example 2 Preparation of t-Boc protected inverse urethane PEG2-Oxymine, 20 kD [ka] To a solution of RU-PEG2-amine 20K (19.2 g, 0.0019 mol) in anhydrous dichloromethane (500 mL) was added 2-(tert-butoxycarbonylaminooxy)acetic acid (0.367 g, 0.0019 mol), followed by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.368 g, 0.0019 mol) and N,N-dimethylpyridine (0.059 g, 0.0048 mol). The mixture was stirred overnight under nitrogen atmosphere, then it was filtered, concentrated under reduced pressure, and added to 500 mL of isopropyl alcohol. The precipitated product was filtered off and dried under vacuum. Yield 19.2 g.

[0161] NMR analysis confirmed the structure of the compound obtained.

[0162] Example 3 Preparation of inverse urethane PEG2-oxyamine, 20 kD [ka] A solution of t-Boc protected inverse urethane PEG2-Oxymine 20K (19.2 g, 0.0019 mol) in a mixture of anhydrous dichloromethane (100 mL) and trifluoroacetic acid (100 mL) was stirred at room temperature overnight. The solution was then concentrated under reduced pressure and added to 500 mL of isopropyl alcohol. The precipitated product was filtered off and dried under vacuum overnight. Yield 19.2 g. (white powder, Mn=21946, polydispersity 1.004).

[0163] NMR analysis confirmed the structure of the compound obtained.

[0164] Example 4 Linear mPEG 20kD Carbohydrate PEGylation of full-length rFVIII using -oxyamine The Factor VIII used in the exemplary PEGylation reactions was commercially available full-length Factor VIII (ADVATE, Shire) at 3.57 mg / ml in 20 mM HEPES, 0.75 M NaCl, 2 mM CaCl2, 0.1% Tween 80, pH 7.1. This composition is hereafter referred to as the "FVIII solution." Prior to reaction with the illustrative reactive aminooxy-PEG, Factor VIII was oxidized to convert the vicinal diol of the carbohydrate to an aldehyde suitable for reaction with the oxyamine PEG reagent.

[0165] Factor VIII oxidation: Prior to periodate oxidation, the pH of the FVIII solution was adjusted from pH 7.1 to 6.0 with 500 mM MES, pH 5.5 solution. 0.0245 mL of a freshly prepared 0.1 M solution of sodium periodate solution was transferred to 3.27 mL (3.27 mg / mL) of pH 6.0 FVIII solution. The reaction mixture was stirred for 30 minutes at room temperature in the dark. After periodate oxidation, the oxidized FVIII sample was buffer exchanged into 20 mM MES, 0.15 M NaCl, 2 mM CaCl2, pH 6.0 buffer using a 10 mL Zeba™ spin desalting column according to the manufacturer's instructions. Briefly, the column was centrifuged to remove the storage liquid, followed by the addition of 5 mL 20 mM MES, 0.15 M NaCl, 2 mM CaCl2, pH 6.0 buffer in three cycles and centrifugation to equilibrate the column. The oxidized FVIII sample was applied to a spin column, and the column was centrifuged again to recover oxidized FVIII in 20 mM MES, 0.15 M NaCl, 2 mM CaCl2, pH 6.0 buffer. After buffer exchange, the FVIII concentration was determined to be 3.17 mg / mL.

[0166] PEGylation and subsequent capping of unreacted aldehyde groups with methoxyamine: 0.23 mL of 100 mg / mL mPEG-20K methoxyamine (CHO-CHCHO-(CHCHO) m’A solution of 0.23 mL of 100 mg / ml methoxylamine hydrochloride (in 1N NaOH) was added to the PEGylation reaction mixture for capping any unreacted aldehyde groups of the oxidized factor VIII and further incubated for 15 min at room temperature. Finally, the pH of the reaction mixture was adjusted to 7.1 with 1 M Tris, pH 9.0 and diluted with 20 mM Tris, 2 mM CaCl2 until the conductivity was less than 4.2 ms / cm.

[0167] Purification: PEGylated FVIII conjugates were purified using an anion exchange chromatography column (HiTrap Q HP, GE Healthcare, 5 mL, Cat. No.: 17-1154-01) with an AKTA system. Elution buffers were 20 mM Tris, 2 mM CaCl2, pH 7.1 (Buffer A) and 20 mM Tris, 2 mM CaCl2, 1 M NaCl, pH 7.1 (Buffer B). After sample loading, the column was washed with 5 CV of Buffer A, followed by a gradient elution of 0-100% B for 10 CV. PEGylated FVIII was pooled and 1 mL of purified PEGylated FVIII was set aside for PEGylation degree analysis. Finally, Tween 80 was added to the remainder of the purified PEGylated FVIII to a final concentration of 0.1%.

[0168] The general procedure described above was also used to prepare Factor VIII conjugates using PEG-5K-oxyamine. Conjugates with different degrees of PEGylation were prepared by varying the molar ratio of PEG-oxyamine to Factor VIII. In general, a higher ratio of PEG to Factor VIII resulted in PEG-oxime-Factor VIII conjugates with a higher degree of PEGylation.

[0169] Analysis of PEGylation Degree (DP): The definition of PEGylation degree is the average number of PEG-20K or PEG-5K molecules per FVIII molecule in the conjugate. That is, PEGylation degree describes the number of PEG molecules covalently attached to the factor VIII moiety via oxime-containing linkages (i.e., oxime linkages) after PEGylation reaction with PEGoxyamine and purification. FVIII-PEG-20K or FVIII-PEG-5K conjugates were exhaustively digested with a protease (pronase) to digest the protein and thereby release PEG (and potentially peptide residues). Released PEG was quantified by HPLC using the PEG reagent as a standard. Briefly, 5 μL of 3 mg / ml pronase (Calbiochem, Cat. No.: 53072) was added to 145 μL of 0.61 mg / ml (protein equivalent) PEGylated FVIII in 20 mM Tris, 2 mM CaCl2, pH 7.1. After 24 hours of incubation at 37°C, the PEG released from the conjugates was quantified using SEC-HPLC coupled with a refractive index detector for the 20K PEG FVIII conjugates or RP-HPLC coupled with an MS detector for the 5K PEG FVIII conjugates, using the PEG reagent as a standard. Using this technique, it was possible to determine the degree of PEGylation.

[0170] Multiple lots of Factor VIII-oxime-PEG-20kD and Factor VIII-oxime-PEG-5dD were prepared and analyzed; conjugates with higher degrees of PEGylation were generally prepared by increasing the molar ratio of PEG oxyamine reagent to Factor VIII. PEG-oxime-20kD-Factor VIII conjugates were prepared and determined to have degrees of PEGylation within the range of about 4-20.

[0171] Clotting activity was determined for the conjugates (one-stage APTT-based clotting assay; U / mg); the PEG-oxime-20 kD-Factor VIII conjugates retained about 50 percent to about 75% of the clotting activity relative to unmodified Factor VIII.

[0172] Example 5 Linear MPEG 5kD Carbohydrate PEGylation of full-length rFVIII using -oxyamine The linear PEG-5 kD oxyamine was reacted with the carbohydrates on Factor VIII (i.e., oxidation of Factor VIII, PEGylation, and capping of unreacted aldehyde functional groups) to prepare the corresponding PEG-5 kD-oxime-Factor VIII conjugates as described above in Example 4. The amounts of specific reactants were varied during the PEGylation reaction (e.g., ratio of PEG reagent to Factor VIII) to prepare conjugates with different degrees of PEGylation.

[0173] [Table 5]

[0174] After the PEGylation reaction, the pH of the reaction mixture was adjusted to 7.1 with 1 M Tris, pH 9.0, and the reaction mixture was further diluted with FPLC buffer A to a conductivity of <4.0 ms / cm (40–60 mL). FPLC purification was performed using the following elution system: column QHP prepacked, 5 mL. Buffer A: 20 mM Tris, 2 mM CaCl2, pH 7.1 / HCl. Buffer B: 20 ​​mM Tris, 2 mM CaCl2, 1 M NaCl, pH 7.1 / HCl. Fractions were collected and analyzed to determine the degree of PEGylation.

[0175] [Table 6]

[0176] Example 6 Carbohydrate PEGylation of full-length rFVIII using 20kD-RU-PEG-oxyamine Factor VIII oxidation: Prior to periodate oxidation, the pH of the FVIII solution was adjusted from pH 7.1 to 6.0 with 500 mM MES, pH 5.5 solution. 0.0592 mL of a freshly prepared 0.1 M solution of sodium periodate solution was transferred to 7.9 mL (3.27 mg / mL) of pH 6.0 FVIII solution. The reaction mixture was stirred for 30 minutes at room temperature in the dark. After periodate oxidation, the oxidized FVIII sample was buffer exchanged into 20 mM MES, 0.15 M NaCl, 2 mM CaCl2, pH 6.0 buffer using a 10 mL Zeba™ spin desalting column according to the manufacturer's instructions. Briefly, the column was centrifuged to remove the storage liquid, followed by three cycles of adding 5 mL of 20 mM MES, 0.15 M NaCl, 2 mM CaCl2, pH 6.0 buffer and centrifuging to equilibrate the column. The oxidized FVIII sample was applied to a spin column, and the column was centrifuged again to recover oxidized FVIII in 20 mM MES, 0.15 M NaCl, 2 mM CaCl2, pH 6.0 buffer. After buffer exchange, the FVIII concentration was determined to be 3.17 mg / mL.

[0177] PEGylation and subsequent capping of unreacted aldehyde groups of oxidized FVIII with methoxyamine: A solution of inverse urethane PEG2-Oxymine 20K was added to the solution of oxidized FVIII (pH 6.0). The reaction mixture was stirred at room temperature for about 2 hours. To cap any unreacted aldehyde groups of oxidized Factor VIII, methoxylamine hydrochloride (in 1N NaOH) was added to the PEGylation reaction mixture and further incubated at room temperature for 15 minutes. Finally, the pH of the reaction mixture was adjusted to 7.1 with 1M Tris, pH 9.0, and diluted with 20mM Tris, 2mM CaCl2 until the conductivity was less than 4.2ms / cm. The amounts of reagents and reaction conditions for RU-PEG2-Oxymine-20kD PEGylation reactions A-D are summarized in the following table.

[0178] [Table 7]

[0179] Purification: PEGylated FVIII conjugates were purified using an anion exchange chromatography column (HiTrap Q HP, GE Healthcare, 5 mL, Cat. No.: 17-1154-01) with an AKTA system. Elution buffers were 20 mM Tris, 2 mM CaCl2, pH 7.1 (Buffer A) and 20 mM Tris, 2 mM CaCl2, 1 M NaCl, pH 7.1 (Buffer B). After sample loading, the column was washed with 5 CV of Buffer A, followed by a gradient elution of 0-100% B for 10 CV. PEGylated FVIII was pooled and 1 mL of purified PEGylated FVIII was set aside for PEGylation degree analysis. Finally, Tween 80 was added to the remainder of the purified PEGylated FVIII to a final concentration of 0.1%.

[0180] Clotting activity was determined for the conjugates (one-stage APTT-based clotting assay; U / mg); the RU-PEG2-oxime-20kD Factor VIII conjugate retained approximately 70% more clotting activity relative to unmodified Factor VIII. According to a preferred embodiment of the present invention, for example, the following is provided: (Section 1) A conjugate comprising a water-soluble polymer covalently attached to an oxidized carbohydrate of a Factor VIII moiety via an oxime-containing linkage. (Section 2) Item 2. The conjugate according to item 1, having the structure POLY-XON=CH-FVIII, where POLY is a water-soluble polymer, X is any spacer containing one or more atoms, FVIII is a factor VIII moiety, and 〜CH-FVIII is a residue of a carbonyl group of an oxidized carbohydrate of said factor VIII moiety. (Section 3) The conjugate of paragraph 2 above, wherein X is hydrolytically stable. (Section 4) 4. The conjugate according to claim 2 or 3, wherein X comprises at least one heteroatom selected from O, S and NH. (Section 5) 5. The conjugate according to any one of items 2 to 4, wherein X has an atomic length of about 5 atoms to about 25 atoms. (Section 6) 6. The conjugate according to any one of items 1 to 5, wherein the water-soluble polymer is a poly(alkylene oxide). (Section 7) 7. The conjugate according to any one of items 1 to 6, wherein the water-soluble polymer has a weight-average molecular weight of 2,000 daltons to 85,000 daltons. (Section 8) structure: CH 3 O-CH 2 CH 2 O-(CH 2 CH 2 O) m’ -CH 2 CH 2 -O-NH=CH-(FVIII) wherein (m') ranges from 0 to about 4,000, FVIII is a factor VIII moiety, and -CH-(FVIII) is a residue of a carbonyl group of an oxidized carbohydrate of the factor VIII moiety. (Section 9)

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Claims

1. 1. A conjugate comprising a water-soluble polymer covalently attached to an oxidized carbohydrate of a Factor VIII moiety via an oxime-containing linkage, The conjugate has the structure: POLY-X-ON=CH-FVIII having where POLY is the structure: 【Chemistry 1A】 is a branched poly(ethylene glycol) polymer having the formula: X is a spacer, -NH(CH ２ ) １～６ NHC(O)CH ２ - and each (n) independently ranges from 1 to 4,000; and FVIII is a human recombinant glycosylated Factor VIII moiety, where 〜CH-(FVIII) is the residue of a carbonyl group of an oxidized carbohydrate of said Factor VIII moiety. Conjugate.

2. 2. The conjugate of claim 1, wherein the entire poly(ethylene glycol) polymer portion of the conjugate has a weight average molecular weight of from 2,000 Daltons to 85,000 Daltons.

3. 2. The conjugate of claim 1, wherein each (n) is independently an integer having a value from 20 to 1,000.

4. The CH ３ (O-CH ２ CH ２ ) ｎ 2. The conjugate of claim 1, wherein each of the chains has a molecular weight selected from 5,000 daltons, 10,000 daltons or 20,000 daltons.

5. 【Chemical 1A】 having a structure selected from The conjugate of any one of claims 1 to 4, wherein (n) for each of said branched structures independently ranges from 1 to 4,000.

6. The conjugate of claim 5, wherein (n) is 227.

7. The conjugate according to any one of claims 1 to 6, wherein the human recombinant glycosylated Factor VIII moiety is human recombinant glycosylated B domain-deleted Factor VIII.

8. The conjugate according to any one of claims 1 to 6, wherein the human recombinant glycosylated Factor VIII moiety is human recombinant glycosylated full-length Factor VIII.

9. 8. The conjugate according to any one of claims 1 to 7, wherein the conjugate retains from 45% to 75% or more, or from 50% to 75% or more, of Factor VIII biological activity when compared to the unmodified Factor VIII moiety.

10. 8. The conjugate of any one of claims 1 to 7, wherein the conjugate retains 60% to 80% of the Factor VIII biological activity when compared to the unmodified Factor VIII moiety and has an average number of branched polymers attached to the Factor VIII moiety of 1 to 3.

5.

11. A composition comprising the conjugate of any one of claims 1 to 10 and a pharma- ceutically acceptable excipient.

12. The composition of claim 11 for the treatment of hemophilia A in a patient.

13. structure: 【Chemistry 2】 (Wherein, X is —NH(CH ２ ) １～６ NHC(O)CH ２ - and each (n) ranges from 1 to 4,000.

14. X is -NH(CH ２ ) ２ NHC(O)CH ２ The polymeric oxyamine reagent of claim 13, wherein

15. 12. The composition of claim 11 for use in treating a blood disorder such as hemophilia A.

16. 1. A method for preparing a branched polyethylene glycol oxyamine reagent, comprising the steps of: The method comprises: (i) Structure: 【Chemistry 19-1A】 wherein (m') is independently in the range of 20 to 1,000, is reacted with a branched polyethylene glycol active ester reagent in an organic solvent in the presence of a base to form a branched polyethylene glycol carboxamide-amine intermediate. 【Chemistry 19-1B】 under suitable reaction conditions to form the structure, NH ２ (CH ２ ) １～６ NH ２ with a diamine having the formula (ii) recovering the branched polyethylene glycol carboxamide-amine intermediate. Step, (iii) reacting the recovered branched polyethylene glycol carboxamide-amine intermediate with a carboxy-protected 2-(carbonylaminooxy)acetic acid in the presence of a coupling agent to give a protected branched polyethylene glycol oxyamine reagent; 【Chemistry 19-3】 where PG is a protecting group; and (iv) deprotecting the protected branched polyethylene glycol oxyamine reagent to obtain a compound of the structure: 【Chemistry 19-4】 providing a branched polyethylene glycol oxyamine reagent having A method comprising:

17. The activated ester from step (i) has the structure 【Chemistry 20】 The method of claim 16, wherein the NHS-ester has the formula:

18. 17. The method of claim 16, wherein step (i) is carried out under an inert atmosphere in an organic solvent selected from anhydrous dichloromethane and anhydrous chloroform.

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