MXPA06006023A - Glycopegylated factor ix. - Google Patents

Abstract

The present invention provides conjugates between Factor IX and PEG moieties. The conjugates are linked via an intact glycosyl linking group interposed between and covalently attached to the peptide and the modifying group. The conjugates are formed from glycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar moiety onto a glycosyl residue on the peptide. Also provided are methods for preparing the conjugates, methods for treating various disease conditions with the conjugates, and pharmaceutical formulations including the conjugates.

Description

FACTOR IX GLICOPEGILADO BACKGROUND OF THE INVENTION Vitamin K dependent proteins (eg, factor IX) contain 9 to 13 gamma-carboxyglutamic acid (Gla) residues at their 45 amino terminal residues. Gla residues are produced by enzymes in the liver that use vitamin K to carboxylate the side chains of glutamic acid residues in protein precursors. Vitamin K dependent proteins are involved in a number of biological processes, of which the best described are blood coagulation (reviewed in Nelsestuen, Vi tamin, Horm 58: 355-389 (2000)). Vitamin K dependent proteins include Z protein, S protein, prothrombin (factor II), factor X, factor IX, protein C, factor VII, Gas6 and matrix GLA protein. Factors VII, IX, X and II work in procoagulation processes while protein C, protein S and protein Z serve in anticoagulation roles. Gas6 is a growth-arrest hormone encoded by gene 6 specific for growth arrest (gas6) and is related to protein S.

See, Manfioletti et al., Mol. Cell. Biol. 13: 4976-4985 (1993). The matrix GLA protein is normally found in bone and is critical for the prevention of calcification of soft tissues in the circulation. Luo et al. Nature 386: REF .: 172384 78-81 (1997). The regulation of blood coagulation is a process that presents a number of major health problems, including both failure to form blood clots as well as thrombosis, the formation of unwanted blood clots. Agents that prevent unwanted clots are used in many situations and a variety of agents are available. Unfortunately, the most current therapies have unpleasant side effects. Orally administered anticoagulants such as warfarin act by inhibiting the action of vitamin K in the liver, thus preventing a complete carboxylation of glutamic acid residues in vitamin K-dependent proteins, resulting in a reduced concentration of active proteins in the system circulatory and reduced capacity to form clots. Warfarin therapy is complicated by the competitive nature of the drug with its goal. Fluctuations in dietary vitamin K may result in an overdose or sub-dose of warfarin. Fluctuations in clotting activity are an undesirable result of this therapy. Injected substances such as heparin, including low molecular weight heparin, are also commonly used anticoagulants. Again, these compounds are subject to overdose and should be carefully monitored. A newer category of anticoagulants includes the active site-modified vitamin K-dependent coagulation factors such as factor Vlla and IXa.The active sites are blocked by serine protease inhibitors such as chloromethyl ketone derivatives of amino acids or short peptides. modified in active site they retain the ability to form complexes with their respective cofactors, but they are inactive, thus producing no enzymatic activity and avoiding the complexation of the cofactor with the respective active enzymes.In short, these proteins seem to offer the benefits of anticoagulation therapy without Adverse side effects of other anticoagulants Factor X modified in active site is another possible anticoagulant in this group Its cofactor protein is factor Va. Activated protein modified in active site (APC) can also form an effective inhibitor of coagulation See, Sorensen et al. J. Biol. Chem. 272: 11863-11868 (1997). Active modified APC binds to factor Va and prevents factor Xa from binding. A major inhibition for the use of vitamin K-dependent coagulation factors is cost. The biosynthesis of vitamin K-dependent proteins depends on a carboxylation system of intact glutamic acid, which is present in a small number of animal cell types. The overproduction of these proteins is limited by this system of enzymes. Moreover, the effective dose of these proteins is high. A common dose is 1,000 μg peptide / kg body weight. See, Harker et al. 1997, cited above. Other phenomena that hinder the use of therapeutic peptides are the well-known aspect of protein glycosylation and the relatively short in vivo average life exhibited by these peptides. Above all, the short in vivo average life problem means that therapeutic glycopeptides should be administered frequently in high doses, which ultimately results in health care costs higher than would be necessary if one more method were available. efficient to make therapeutic glycoproteins longer lasting and more effective. The Vlla factor, for example, illustrates this problem. Factor VII and Vlla have average circulation lives of about 2-4 hours in humans. That is, within 2-4 hours, the concentration of the peptide in the serum is reduced by half. When the factor Vlla is used as a procoagulant to treat certain forms of hemophilia, the standard protocol is to inject Vlla every two hours at high doses (45 to 90 μg / kg of body weight). See, Hedner et al., Transfus. Med. Rev. 7: 78-83 (1993)). Thus, the use of these proteins as procoagulants or anticoagulants (in the case of factor Vlla) requires that the proteins be administered at frequent intervals and at high doses. One solution to the problem of providing inexpensive glycopeptide therapeutics has been to provide peptides with longer in vivo half-lives. For example, glycopeptide therapeutics with improved pharmacokinetic properties have been produced by linking synthetic polymers to the base structure of the peptide. An exemplary polymer that has been conjugated to peptides is poly (ethylene glycol) ("PEG"). The use of PEG to derive therapeutic peptides has been shown to reduce the immunogenicity of the peptides. For example, the patent of E.U.A. No. 4,179,337 (Davis et al.) Discloses non-immunogenic polypeptides such as enzymes and peptide hormones coupled to poly (ethylene glycol) (PEG) or polypropylene glycol. In addition to reduced immunogenicity, the elimination time in the circulation is prolonged due to the increased size of the PEG conjugate of the polypeptides in question. The main way of fixing the PEG, and. its derivatives, to peptides is a non-specific binding through an amino acid residue peptide (see, for example, US Patent No. 4,088,538, US Patent No. 4,496,689, US Patent No. 4,414,147, US Patent No. 4,055,635 and PCT WO 87/00056). Another way to fix PEG to peptides is through the non-specific oxidation of glycosyl residues in a glycopeptide (see, for example, WO 94/05332). In these non-specific methods, poly (ethylene glycol) is added in a random and non-specific manner to reactivate residues on a base structure of the peptide. Of course, the random addition of PEG molecules has its disadvantages, including a lack of homogeneity of the final product, and the possibility of the reduction in the biological or enzymatic activity of the peptide. Therefore, for the production of therapeutic peptides, a derivation strategy that results in the formation of a specially marked, easily characterized and essentially homogeneous product is superior. These methods have been developed. Therapeutic homogenous and specifically labeled peptides can be produced in vi tro through an action of enzymes. Unlike the typical non-specific methods to join. a synthetic polymer or another marker to a peptide, the enzyme-based syntheses have the advantages of regioselectivity and stereoselectivity. Two major classes of enzymes for use in the synthesis of labeled peptides are glycosyltransferases (e.g., sialyltransferases, oligosaccharyltransferases, N-acetylglucosaminyltransferases) and glucosidases. These enzymes can be used for the specific binding of sugars which can subsequently be modified to comprise a therapeutic moiety. Alternatively, modified glycosyltransferases and glycosidases can be used to directly transfer modified sugars to a base structure of a peptide (see, e.g., U.S. Patent 6,399,336 and U.S. Patent Application Publications 20030040037, 20040132640, 20040137557, 20040126838 and 20040142856, each of which is incorporated by reference in the present). Methods that combine both chemical and enzymatic synthetic elements are also known (see, for example, Yamamoto et al., Carbohydr Res. 305: 415-422 (1998) and publication of the US patent application 20040137557 which is incorporated in the present as a reference). Factor IX is an extremely valuable therapeutic peptide. Although the commercially available forms of factor IX are currently in use, these peptides can be improved by modifications that increase the pharmacokinetics of the resulting isolated glycoprotein product. Thus, the need in the technique of factor IX peptides of longer duration with improved effectiveness and better pharmacokinetics remains. Even more, to be effective for the largest number of individuals, it should be possible to produce, on an industrial scale, a factor IX peptide with improved therapeutic pharmacokinetics that has a predictable and essentially homogeneous structure and can be easily reproduced again and again. Fortunately, factor IX peptides with improved pharmacokinetics and methods for making them have now been discovered. In addition to factor IX peptides with improved pharmacokinetics, the invention also provides industrially practical and economical methods for the production of these factor IX peptides. The factor IX peptides of the invention comprise modifying groups such as PEG portions, therapeutic portions, biomolecules and the like. The present invention thus satisfies the need for factor IX peptides with improved therapeutic effectiveness and improved pharmacokinetics for the treatment of disease conditions in which factor IX provides effective therapy.

BRIEF DESCRIPTION OF THE INVENTION It has now been discovered that controlled modification of factor IX with one or more portions of poly (ethylene glycol) produces novel factor IX derivatives with improved pharmacokinetic properties. Moreover, economic methods for reliable production of the modified factor IX peptides of the invention have been discovered and developed.

In one aspect, the present invention provides a factor IX peptide that includes the portion: In the above formula, D is -OH or R1-L-HN-. The symbol G represents R1-L- or -C (0) (C_-C6 alkyl). R1 is a portion comprising a straight or branched chain poly (ethylene glycol) residue; and L is a linker that is a member selected from a bond, substituted or unsubstituted alkyl or unsubstituted heteroalkyl. Generally, when D is OH, G is Rx-L-, and when G is -C (0) (C? -C6 alkyl), D is RYL-NH-. As will be appreciated by those skilled in the art, in the sialic acid analogs described herein, COOH also represents COO "or a salt thereof In another aspect, the invention provides a method for making a PEGylated factor IX comprising the The method of the invention includes (a) contacting a substrate factor IX peptide with a PEG-sialic acid donor and an enzyme that transfers PEG-sialic acid onto an amino acid or glycosyl residue of the peptide of factor IX, under conditions suitable for transfer A donor portion of exemplary PEG-sialic acid has the formula: In one embodiment, the host is a mammalian cell. In other embodiments, the host cell is an insect cell, plant cell, bacteria or fungus. In another aspect, the invention provides a method for treating a condition in a subject that requires it, wherein the condition is characterized by compromised coagulation in the subject. The method comprises the step of administering to a subject an amount of the factor IX peptide conjugate of the invention effective to reduce the condition in the subject. An exemplary disease treatable by this method is hemophilia. In another aspect, the invention provides a pharmaceutical formulation comprising the factor IX peptide of the invention and a pharmaceutically acceptable carrier. Other objects and advantages of the invention will be apparent to those skilled in the art from the following detailed description.

Description of the Figures Figure 1 is the structure of factor IX, which shows the presence and location of potential glycosylation sites in Asn 157, Asn 167; Ser 53, Ser 61, Thr 159, Thr 169 and Thr 172. Figures 2A-2F are a scheme showing an exemplary embodiment of the invention in which a carbohydrate residue in a factor IX peptide is remodeled and glycopegylated: ( A) portions of sialic acid are removed by sialidase and the resulting galactose residues are glycopeglylated with the sialic acid derivative of Figure 5; (B) a mannose residue is glycopeglylated with the PEG of sialic acid; (C) a sialic acid portion of an N-glycan is glycopegylated with the PEG of sialic acid; (D) a sialic acid portion of an O-glycan is glycopeglylated with the PEG of sialic acid; (E) SDS-PAGE gel factor IX of 2 (A); (F) SDS-PAGE gel of factor IX of the reaction producing 2 (C) and 2 (D). Figure 3 is a graph comparing the lifetimes of in vivo residence of non-glycosylated factor IX and enzymatically glycopegylated factor IX. Figure 4 is a table comparing the activities of the species shown in Figure 3. Figure 5 is the amino acid sequence of factor IX.

Figure 6 is a graphical presentation of the pharmacokinetic properties of several glycopegylated factor IX molecules compared to a non-pegylated factor IX. Figure 7 is a table of representative modified sugar species for use in the present invention. Figure 8 is a table of representative modified sugar species for use in the present invention. Figures 9A-9N are a table of sialyltransferases useful for transferring on a receptor a modified sialic acid moiety, such as those shown herein and unmodified sialic acid moieties.

Detailed description of the invention Abbreviations PEG, poly (ethylene glycol); PPG, poly (propylene glycol); Ara, arabinosyl; Fru, fructosyl; It was, fucosilo; Gal, galactosyl; GalNAc, N-acetylgalactosaminyl; Glc, glucosyl; GlcNAc, N-acetylglucosaminyl; Man, manosilo; ManAc, manosaminyl acetate; Xyl, xylosyl and NeuAc, sialyl (N-acetylneuraminyl); M6P, mannose-6-phosphate; Sia, sialic acid, N-acetylneuraminyl and derivatives and analogs thereof.

Definitions Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one skilled in the art to which this invention pertains. In general, the nomenclature used herein and the laboratory procedures of cell cultures, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well known and commonly employed in the art. Standard techniques are used for the synthesis of nucleic acid and peptides. The techniques and procedures are generally carried out according to conventional methods in the art and several general references (see generally, Sambrook et al.) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference), which are provided throughout this document. The nomenclature used herein and the analytical chemistry laboratory procedures, and the organic synthetic materials described below are those well known and commonly used in the art. Standard techniques, or modifications thereof, are used for chemical synthesis and chemical analysis. All oligosaccharides described herein are described by the name or abbreviation for the non-reducing saccharide (ie, Gal), followed by the configuration of the glycosidic bond (a or β), the ring bond (1 or 2), the position in the ring of the reducing saccharide involved in the bond (2, 3, 4, 6 or 8) and then the name or abbreviation of the reducing saccharide (i.e., GlcNAc). Each saccharide is preferably a pyranose. For a review of the standard glycobiology nomenclature, see, Essentials of Glycobiology Varki et al. eds. CSHL Press (1999). The oligosaccharides are considered to have a reducing end and a non-reducing end, whether or not the saccharide at the non-reducing end is in fact a reducing sugar. According to the accepted nomenclature, the oligosaccharides are illustrated herein with the non-reducing end on the left and the reducing end on the right. The term "sialic acid" refers to any member of the nine-carbon carboxylated sugar family. The most common member of the sialic acid family is N-acetylneuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactonomulopyrans-l -onic acid (commonly abbreviated as Neu5Ac, NeuAc or NANA)). A second member of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc is hydroxylated. A third member of the sialic acid family is 2-keto-3- acid. deoxy-nonulosonic (KDN) (Nadano et al 198 &) J. Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265: 21811-21819 (1990)). Also included are 9-substituted sialic acids such as 9-0-C1-C6-acyl-Neu5Ac such as 9-0-lactyl-Neu5Ac or 9-0-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For a review of the sialic acid family, see, for example, Varki, Glycobiology 2: 25-40 (1992); Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. (Springer-Verlag, New York (1992)). The synthesis and use of sialic acid compounds in a sialylation process are described in international application WO 92/16640, published on October 1, 1992. "Peptide" refers to a polymer in which the monomers are amino acids and are united together through amide bonds, alternatively referred to as a polypeptide. In addition, non-natural amino acids, for example, β-alanine, phenylglycine and homoarginine are also included. Amino acids that are not encoded by genes can also be used in the present invention. In addition, amino acids that have been modified to include reactive groups, glycosylation sites, polymers, therapeutic moieties, biomolecules, and the like can also be used in the invention. All of the amino acids used in the present invention can be either the d or 1 isomer. Isomer 1 is generally preferred. In addition, other peptidomimetics are also useful in the present invention. As used herein, "peptide" refers to both glycosylated and non-glycosylated peptides. Peptides that are incompletely glycosylated by a system that expresses the peptide are also included. For a general review, see, Spatola, A. F., in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983). The term "peptide conjugate" refers to species of the invention in which a peptide is conjugated to a modified sugar shown herein. The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as to amino acid analogs and amino acid mimetics that function in a manner similar to that of naturally occurring amino acids. The naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are subsequently modified, for example, hydroxyproline, β-carboxyglutamate and O-phosphoserine. "Amino acid analogues" refers to compounds that have the same basic chemical structure as the naturally occurring amino acid, ie, a carbon atom that is attached to a hydrogen, a carboxyl group, an amino group and an R group, for example , homoserin, norleucine, methionine sulfoxide, methionine methionine. These analogs have modified R groups (e.g., norleucine) or modified peptide base structures, but retain the same basic chemical structure as that of the naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to that of a naturally occurring amino acid. As used herein, "amino acid", whether it is a linker or a component of a peptide sequence, refers to both isomer d and 1 of the amino acid as well as mixtures of these two isomers. As used herein, the term "modified sugar" refers to a naturally occurring or non-naturally occurring carbohydrate which is added enzymatically to an amino acid or a glycosyl residue of a peptide in a process of the invention. The modified sugar is selected from a number of enzyme substrates including, but not limited to sugar nucleotides (mono-, di- and tri-phosphates), activated sugars (for example, glycosyl halides, glycosyl mesylates) and sugars that are neither activated nor nucleotides. The "modified sugar" is functionalized covalently with a "modifying group". Useful modifying groups include, but are not limited to, PEG portions, therapeutic portions, diagnostic portions, biomolecules, and the like. The preferred modifier group is not a naturally occurring carbohydrate or an unmodified carbohydrate. The functionalization locus with the modifier group is selected such that it prevents the "modified sugar" from being added enzymatically to a peptide. The term "water soluble" refers to portions that have a high degree of detectable water solubility. Methods for detecting and / or quantifying solubility in water are well known in the art. Exemplary water soluble polymers include peptides, saccharides, poly (ethers), poly (amines), poly (carboxylic acids), and the like. The peptides may have mixed sequences that are composed of a single amino acid, for example, poly (lysine). An exemplary polysaccharide is poly (sialic acid). An exemplary poly (ether) is poly (ethylene glycol). Poly (ethyleneimine) is an exemplary polyamine and poly (acrylic acid) is a representative polycarboxylic acid. The polymeric base structure of the water soluble polymer can be poly (ethylene glycol) (ie, PEG). However, it should be understood that other related polymers are also suitable for use in the practice of this invention and that the use of the term PEG or poly (ethylene glycol) is intended to include and not exclude this respect. The term PEG includes poly (ethylene glycol) in any of its forms, including PEG alkoxy, difunctional PEG, multi-armed PEG, hairpin PEG, branched PEG, pendant PEG (ie, PEG or related polymers having one or more pendant functional groups in the base structure of the polymer), or PEG with degradable linkages therein. The base structure of the polymer can be linear or branched. Branched polymer base structures are generally known in the art. Typically, a branched polymer has a central portion of the central branch and a plurality of linear polymer chains linked to the central branch core. PEG is commonly used in branched forms which can be prepared by the addition of ethylene oxide to various polyols, such as glycerol, pentaerythritol and sorbitol. The central branch portion can also be derived from several amino acids, such as lysine, the branched poly (ethylene glycol) can be represented in general form as R- (-PEG-OH) m in which R represents the central portion, such as glycerol or pentaerythritol, and m represents the number of arms. The multi-arm PEG molecules, such as those described in the U.S.A. No. 5,932,462, which is incorporated by reference in its entirety, can also be used as the polymeric base structure. Many other polymers are also suitable for the invention. Polymer base structures that are non-peptidic and water soluble, with from about 2 to about 300 terms, are particularly useful in the invention. Examples of suitable polymers include, but are not limited to, other poly (alkylene glycols), such as poly (propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol and the like, polyol (polyethoxylated), poly (olefinic) alcohol, poly (vinylpyrrolidone), poly (hydroxypropylmetracrylamide), poly (α-hydroxy acid), poly (vinyl) alcohol, polyphosphazene, polyoxazoline, poly (N-acryloylmorpholine), such as those described in the US patent No. 5,629,384, which is incorporated by reference in its entirety, and copolymers, terpolymers and mixtures thereof. Although the molecular weight of each chain of the base structure of the polymer may vary, typically on the scale of about 100 Da to about 100,000 Da, commonly about 6,000 Da to about 80,000 Da. The "area under the curve" or "AUC", as used herein in the context of administering a peptide drug to a patient, is defined as the total area under the curve that describes the concentration of drug in the systemic circulation in the patient. patient as a function of time from zero to infinity. The term "average life" or "t5_" as used herein in the context of administering a peptide drug to the patient, is defined as the time required for the plasma concentration of a drug in a patient to be reduced by half. . There may be more than one average life associated with the drug peptide of different elimination mechanisms, redistribution and other mechanisms well known in the art. Normally, the average alpha and beta lives are defined in such a way that the alpha phase is associated with the redistribution and the beta phase is associated with the elimination. However, with protein drugs that are, for the most part, confined to the bloodstream, there can be at least two average elimination lives. For some glycosylated peptides, rapid elimination in the beta phase can be mediated by receptors or macrophages, or endothelial cells that recognize terminal galactose, N-acetylgalactosamine, N-acetylgucosamine, mannose or fucose. Slower beta-phase elimination can occur through renal glomerular filtration for molecules with an effective radius <; 2 nm (approximately 68 kD) and / or specific or non-specific absorption and metabolism in tissues. The glycopegilation can be terminal blocking sugars (for example, galactose or N-acetylgalactosamine) and in this way block the rapid elimination in alpha phase by means of receptors that recognize these sugars. It can also confer a larger effective radius and therefore reduce the volume of distribution and absorption in tissues, thus prolonging the subsequent beta phase. In this way, the precise impact of glycopegilation in the high phase and in the average lives of alpha phase and beta phase will vary depending on size, glycosylation status and other parameters, as is well known in the art. An additional explanation of the "average life" is found in Pharmaceutical Biotechnoiogy (1997, DFA Crommelin and RD Sindelar, eds., Harwood Publishers, Amsterdam, pp. 101-120). The term "glycoconjugation" as used herein, refers to the enzymatically mediated conjugation of a modified sugar species to an amino acid or glycosyl residue of a polypeptide, for example, a factor IX peptide substrate. A sub-genus of "glycoconjugation" is "glycol-pegylation", in which the modified sugar modifier group is poly (ethylene glycol), and the alkyl derivative (eg, m-PEG) or reactive derivative (eg H2N-PEG, HOOC-PEG) thereof. The terms "large scale" and "industrial scale" are used interchangeably and refer to a reaction cycle that produces at least 250 mg, preferably at least about 500 mg and most preferably at least about 1 gram of glycoconjugate at the conclusion of a single reaction cycle. The term "glycosyl linking group", as used herein, refers to a glycosyl residue to which a modifying group (eg, PEG portion, therapeutic moiety, biomolecule) is covalently linked; the glycosyl linking group binds the modifying group to the rest of the conjugate. In the methods of the invention, the "glycosyl linkage group" is covalently linked to a glycosylated or unglycosylated peptide, thereby binding the agent to an amino acid and / or glycosyl residue in the peptide. A "glycosyl linkage group" is generally derived from a "modified sugar" by the enzymatic attachment of the "modified sugar" to an amino acid and / or glycosyl residue of the peptide. The glycosyl linkage group can be a structure derived from saccharides that is degraded during the formation of a modified sugar cassette by modifying groups (eg, oxidation - »Schiff-reduction formation), or the glycosyl linking group can be intact An "intact glycosyl linkage group" refers to a linking group that is derived from a glycosyl moiety in which the saccharide monomer that binds the modifier group to the remainder of the conjugate is not degraded, eg, oxidized, for example, by sodium metaperiodate. The "intact glycosyl linkage groups" of the invention can be derived from an oligosaccharide that occurs naturally by the addition of glycosyl units or the removal of one or more glycosyl unit from a saccharide structure of origin. The term "address portion" as used herein, refers to species that will selectively be located in a particular tissue or region of the body. The location is mediated by specific recognition of molecular determinants, molecular size of the targeting agent or conjugate, ionic interactions, hydrophobic interactions and the like. Other mechanisms of directing an agent to a particular tissue or region are known to those skilled in the art. Exemplary targeting moieties include antibodies, antibody fragments, transferrin, HS-glycoprotein, coagulation factors, serum proteins, β-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO, whey proteins (e.g. , factors VII, Vlla, VIII, IX and X) and similar. As used herein, "therapeutic moiety" means any agent useful for therapy including, but not limited to, antibiotics, anti-inflammatory agents, anti-tumor drugs, cytotoxins, and radioactive agents. "Therapeutic portion" includes prodrugs of bioactive agents, constructions in which more than one therapeutic moiety is attached to a vehicle, for example multivalent agents. A therapeutic moiety also includes proteins and constructs including proteins. Exemplary proteins include, but are not limited to, Granulocyte Colony Stimulator Factor (GCSF), Granulocyte-Macrophage Colony Stimulating Factor (GMCSF), Interferon (e.g., Interferon-a, -β, -?), Interleukin (for example, Interleukin II), whey proteins (for example, factors VII, Vlla, VIII, IX and X), Gonadotropin Human Chorionic (HCG), Follicle Stimulating Hormone (FSH) and Lutenizing Hormone (LH) as well as antibody fusion proteins (e.g., domain fusion protein) (TNFR) / Fc of Tumor Necrosis Factor Receptor). As used herein, "pharmaceutically acceptable carrier" includes any material, which when combined with the conjugate retains the activity of the conjugate and is not reactive with the subjects' immune systems. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as oil in water emulsion, and various types of wetting agents. Other vehicles may also include sterile solutions, tablets including coated tablets and capsules. Typically, these vehicles contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable oils or fats, gums, glycols or other known excipients. These vehicles may also include flavor and color additives or other ingredients. The compositions comprising these vehicles are formulated by conventional well-known methods. As used herein, "administering" means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, or the implantation of a slow release device, for example, a miniosmotic pump to the subject. Administration is by any route including parenteral and transmucosal (eg, oral, nasal, vaginal, rectal or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial administration. Moreover, when the injection is to treat a tumor, for example, inducing apoptosis, the administration can be directly to the tumor and / or tissues surrounding the tumor. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. The term "decrease" or "reduce" refers to any indication of success in the treatment of a condition or pathology, including any objective or subjective parameter such as abatement, remission or reduction of symptoms or an improvement in a physical or mental of the patient. The reduction of symptoms can be based on objective or subjective parameters; including the results of a physical examination and / or a psychiatric evaluation. The term "therapy" refers to "treating" or "treatment" of a disease or condition that includes preventing the occurrence of the disease or condition in an animal that could be predisposed to disease but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), inhibit the disease (slow down or stop its development), provide relief of the symptoms or side effects of the disease (including palliative treatment) and alleviate the disease (cause regression of the disease). The term "effective amount" or "an effective amount of" or a "therapeutically effective amount" or any grammatically equivalent term means the amount that, when administered to an animal to treat a disease, is sufficient to carry out the treatment of an animal. that disease.

The term "isolated" refers to a material that is substantially or essentially free of components, which are used to produce the material. For peptide conjugates of the invention, the term "isolated" refers to a material that is substantially or essentially free of components which normally accompany the material in the mixture used to prepare the peptide conjugate. "Isolated" and "pure" are used interchangeably. Typically, the isolated peptide conjugates of the invention have a level of purity which is preferably expressed as a scale. The lower end of the purity scale for the peptide conjugates is about 60%, about 70% or about 80% and the upper end of the purity scale is about 70%, about 80%, about 90 % and more than around 90%. When the peptide conjugates are more than about 90% pure, their purities are also preferably expressed as a scale. The lower end of the purity scale is about 90%, about 92%, about 94%, about 96% or about 98%. The upper end of the purity scale is about 92%, about 94%, about 96%, about 98% or about 100% purity. The purity is determined by a method of analysis recognized in the art (e.g., intensity of bands on a silver stained gel, polyacrylamide gel electrophoresis, HPLC or a similar medium). "Essentially each member of the population", as used herein, describes a characteristic of a population of peptide conjugates of the invention in which a selected percentage of the modified sugars added to a peptide are added to several identical receptor sites in the peptide. "Essentially any member of the population" refers to the "homogeneity" of the sites in the peptide conjugated to a modified sugar and refers to conjugates of the invention, which are at least about 80%, preferably at least about 20%, 90% and most preferably at least about 95% homogeneous. "Homogeneity" refers to the structural consistency through a population of receptor portions to which the modified sugars are conjugated. Thus, in a peptide conjugate of the invention in which each modified sugar portion is conjugated to a receptor site having the same structure as the receptor site to which each other modified sugar is conjugated, the peptide conjugate is said to be approximately 100% homogeneous. Homogeneity is typically expressed as a scale. The lower end of the homogeneity scale for the peptide conjugates is about 60%, about 70% or about 80% and the upper end of the purity scale is about 70%, about 80%, around 90% or more of approximately 90%. When the peptide conjugates are more than or equal to about 90% homogeneous, their homogeneity is also preferably expressed as a scale. The lower end of the homogeneity scale is around 90%, about 92%, about 94%, about 96% or about 98%. The upper end of the purity scale is about 92%, about 94%, about 96%, about 98% or about 100% homogeneity. The homogeneity of the peptide conjugates is typically determined by one or more methods known to those skilled in the art, for example, liquid chromatography-mass spectrometry (LC-MS), the matrix-assisted laser desorption of the mass of flight spectrometry (MALDITOF), capillary electrophoresis and the like. The above description is equally relevant for other sites of O-glycosylation and N-glycosylation. "Substantially uniform glycoform" or a "substantially uniform glycosylation standard", when referring to a glycopeptide species, refers to the percentage of receptor portions that are glycosylated by the glycosyltransferase of interest (eg, fucosyltransferase). For example, in the case of an al, 2-fucosyltransferase, a substantially uniform fucosylation pattern exists if substantially all (as defined below) Galßl, 4-GlcNAc-R and sialylated analogs thereof are fucosylated in a peptide conjugate. of the invention. It will be understood by one skilled in the art that the starting material may contain glycosylated (eg, fucosylated) and glycosylated receptor portions • Galßl, fucosylated 4-GlcNAc-R). Thus, the calculated percentage glycosylation will include receptor portions that are glycosylated by the methods of the invention, as well as receptor portions that are already glycosylated in the starting material. The term "substantially" in the above definitions of "substantially uniform" generally refers to about 40%, at least about 70%, at least about 80% or most preferably at least about 90%, and even more preferably at least about 95% of the receptor portions for a particular glycosyltransferase are glycosylated. For example, if a factor IX peptide conjugate includes a glycosyl residue bound by Ser, at least about 70%, 80%, 90%, 95%, 97%, 99%, 99.2%, 99.4%, 99.6 % or most preferably 99.8% of the peptides in the population will have the same glycosyl residue covalently linked to the same residue Ser. When the substituent groups are specified by their conventional chemical formulas, written from left to right, they also include the chemically identical substituents, the which would result from writing the structure from right to left, for example, -CH20- is intended to also describe -OCH2-. The term "alkyl" in itself or as part of another substituent member, unless otherwise indicated, means a straight or branched chain, a cyclic hydrocarbon radical, or combination thereof, which may be completely saturated, mono - or polyunsaturated and may include di- and multivalent radicals, having the designated carbon atom number (ie, C? -C? 0 means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, cyclohexylmethyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), ethynyl, - and 3-propynyl, 3-butynyl and the higher homologs and isomers. The term "alkyl", unless otherwise indicated, also attempts to include those alkyl derivatives defined in more detail below, such as "heteroalkyl". Alkyl groups that are limited to hydrocarbon groups are called "homoalkyl". The term "alkylene" in itself or as part of another substituent means means a divalent radical derived from an alkane, as exemplified, but not limited by -CH 2 CH 2 CH 2 CH 2 -, and further includes those groups described below as "heteroalkylene". Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, which generally has eight or fewer carbon atoms. The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the rest of the molecule by means of an oxygen atom, an amino group or a sulfur atom, respectively. The term "heteroalkyl", in itself or in combination with another term, means, unless otherwise indicated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the indicated number of carbon atoms and at least one heteroatom selected from the group consisting of 0, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom optionally quaternized. The heteroatoms 0, N, S and Si can be placed in any interior position of the heteroalkyl group or in the position in which the alkyl group is attached to the rest of the molecule. Examples include, but are not limited to, -CH2-CH2-0-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N (CH3) -CH3, -CH2-S-CH2-CH3, -CH2 -CH2-S (O) -CH3, -CH2-CH2-S (0) 2-CH3, -CH = CH-0-CH3, -Si (CH3) 3, -CH2CH = N-0CH3 and -CH = CH -N (CH3) -CH3. Up to two heteroatoms can be consecutive, such as, for example, -CH2-NH-OCH3 and ~ CH2-0-Si (CH3) 3. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a heteroalkyl-derived divalent radical, as exemplified, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, the heteroatoms may also occupy either or both of the chain terms (eg, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Moreover, for the alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C (0) 2R'- represents both -C (0) 2R'- and -R'C (0) 2-. The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise indicated, cyclic versions of "alkyl" and "heteroalkyl", respectively. In addition, for heterocycloalkyl, a heteroatom can occupy the position in which the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1, 2, 5, 5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl , tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl and the like. The terms "halo" or "halogen", by themselves or as part of another substituent, mean, unless otherwise indicated, a fluorine, bromine or iodine atom. In addition, terms such as "haloalkyl", are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "C1-C4 haloalkyl" is intended to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term "aryl" means, unless otherwise indicated, a polyunsaturated and aromatic substituent which may be single ring or several rings (preferably 1 to 3 rings), which are fused together or covalently linked. The term "heteroaryl" refers to aryl groups (or rings) containing one to four heteroatoms selected from N, 0 and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atoms are optionally quaternized. A heteroaryl group can be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, -oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl , 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl , 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, tetrazolyl, benzo [b] furanyl, benzo [b] thienyl, 2,3-dihydrobenzo [1,4] dioxin-6-yl, benzo [1, 3] dioxol -5-yl and 6-quinolyl. The substituents for each of the aryl and heteroaryl ring systems indicated above are selected from the group of acceptable substituents described below. For brevity, the term "aryl", when used in combination with other terms "eg, aryloxy, arylthioxy, arylalkyl", includes both aryl and heteroaryl rings as defined above, Thus, the term "arylalkyl" is intended to include those radicals in which an aryl group is attached to an alkyl group (eg, benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (eg, a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl, and the like) Each of the above terms (e.g., "alkyl", "heteroalkyl", "aryl" and "heteroaryl") ") is intended to include both substituted and unsubstituted forms of the indicated radical The preferred substituents for each type of radical are given below: Substituents for the alkyl and heteroalkyl radicals (including those groups commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl) are generally referred to as "alkyl group substituents", and may be one or more of a variety of groups selected from, but not limited to: -OR ', = 0, = NR', = N-0R ', -NR'R ", -SR', -halogen, -SiR'R" R "', -0C (0) R' , -C (0) R ', -C02R', -CONR'R ", -OC (O) R'R", -NR "C (0) R ', -NR' -C (O) NR" R "', -NR" C (0) 2R', -NR-C (NR'R "R" ') = NR "", -NR-C (NR'R ") = NR"', -S (0 ) R ', -S (0) 2' _ S (0) 2NR'R ", -NRS02R ', -CN and -N02 in a number that varies from zero to (2m' + l), where m 'is the total number of carbon atoms in this radical. R ', R ", R"' and R "" independently each preferably refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkyl, for example, aryl substituted with 1-3 halogens, alkyl, alkoxy or substituted or unsubstituted thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as each group R ', R ", R"' and R "" when more than one of these groups is present. . When R 'and R "are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6- 6-membered ring, for example, -NR'R" is intended to include, but is not is limited to, 1-pyrrolidinyl and 4-morpholinyl. From the previous description of the substituents, one of skill in the art will understand that the term "alkyl" is intended to include groups that include carbon atoms attached to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2-CF3) and acyl (eg, example, -C (0) CH3, -C (0) CF3, -C (0) CH2OCH3 and the like). Similar to the substituents described by the alkyl radical, substituents for the aryl and heteroaryl groups are generically referred to as "aryl group substituents". The substituents are selected from, for example: halogen, -OR ', = 0, = NR', = N-0R ', -NR'R ", -SR', -halogen, -SiR'R" R "', -0C (0) R ', -C (0) R', -C02R ', -CONR'R ", -0C (0) NR'R", -NR "C (0) R', .BR '- C (0) NR "R" ', -NR "C (0) 2R', -NR-C (NR'R" R "') = NR" ", -NR-C (NR'R") = NR "', -S (0) R', -S (0) 2R ', S (0) 2NR'R", -NRS02R', -CN and -N02, -R ', -N3, -CH (Ph) 2, C? -C4 fluoroalkoxy, and C? -C4 fluoroalkyl, in a number ranging from zero to the total number of open valencies in the aromatic ring system and where R ', R ", R"' and R "" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When a compound of the invention includes more than one group R, for example, each of the groups R is independently selected as each group R ', R ", R"' and R "" when one or more of these groups is present. . In the following schemes, the symbol X represents "R" as described above. Two of the substituents on adjacent atoms of the aryl or heteroaryl ring can be optionally replaced with a substituent of the formula -TC (O) - (CRR ') qU-, wherein T and U are independently -NR-, -0-, -CRR'- or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring. they can optionally be replaced with a substituent of the formula -A- (CH2) rB-, where A and B are independently -CRR'-, -O-, -NR-, -S-, -S (0) -, -S (0) 2-, -S (0) 2NR'- or a single bond, and r is an integer from 1 to 4. One of the individual bonds of the new ring formed in this way can be optionally replaced by a double bond . Alternatively, two of the substituents on adjacent atoms of the aryl o-heteroaryl ring can be optionally replaced with a substituent of the formula - (CRR ') sX- (CR "R"') a-, where syd are independently integers from 0 to 3, and X is -O-, -NR'-, -S-, -S (0) -, -S (0) 2- or -S (0) 2NR'-. The substituents R, R ', R "and r"' are preferably independently selected from hydrogen or substituted or unsubstituted C 1 -C 6 alkyl. As used herein, the term "heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

Introduction As described above, factor IX is vital in the coagulation cascade in the blood. The structure and sequence of factor IX are given in figure 1. A deficiency of factor IX in the body characterizes a type of hemophilia (type B). The treatment of this disease is usually limited to the intravenous transfusion of factor IX concentrates into human plasma protein. However, in addition to the practical disadvantages of time and cost, the transfusion of blood concentrates includes the risk of transmission of viral hepatitis, acquired immunodeficiency syndrome or thromboembolic diseases to the recipient. Although factor IX has shown itself to be an important and useful compound for therapeutic applications, the present methods for the production of factor IX from recombinant cells (U.S. Patent No. 4,770,999) result in a product with a life shorter biological average and a non-precise glycosylation pattern that could potentially lead to immunogenicity, loss of function, an increased need for both larger and more frequent doses to achieve the same effect, and the like. To improve the effectiveness of recombinant factor IX used for therapeutic purposes, the present invention provides conjugates of glycosylated and non-glycosylated factor IX peptides with polymers, for example, PEG (m-PEG), PPG (m-PPG), etc. The conjugates may be additionally or alternatively modified by further conjugation with various species such as therapeutic portions, diagnostic portions, steering portions and the like. The conjugates of the invention are formed by the enzymatic attachment of a sugar modified to the glycosylated or non-glycosylated peptide. Glycosylation sites and glycosylated residues provide sites for conjugating modifying groups to the peptide, for example, by glycoconjugation. An exemplary modifying group is a water soluble polymer, such as poly (ethylene glycol), for example, methoxy-poly (ethylene glycol). Modification of factor IX peptides can improve the stability and retention time of recombinant factor IX in the circulation of a patient, and / or reduce the antigenicity of recombinant factor IX. The methods of the invention make it possible to assemble peptides and glycopeptides having a substantially homogeneous derivatization pattern. The enzymes used in the invention are generally selective for a particular amino acid residue, the combination of amino acid residues or particular glycosyl residues of the peptide. The methods are also practical for the large-scale production of modified peptides and glycopeptides. Thus, the methods of the invention provide a practical means for the large-scale preparation of glycopeptides having preselected uniform derivation patterns. The present invention also provides conjugates of glycosylated and non-glycosylated peptides with increased therapeutic average life due to, for example, reduced elimination rate, or reduced rate of absorption by the immune or reticuloendothelial system (RES). In addition, the methods of the invention provide a means to hide antigenic determinants in peptides, thereby reducing or eliminating the immune response of a host against the peptide. The selective binding of targeting agents can also be used to direct a peptide to a particular tissue or cell of a particular cell or tissue surface receptor, which is specific to the particular targeting agent.

The Conjugates In a first aspect, the present invention provides a conjugate between a selected modifier group and a factor IX peptide. The linkage between the peptide and the modifier group includes a glycosyl linking group interposed between the peptide and the selected portion. As described in this, the selected portion is essentially any species that can be linked to a saccharide unit, resulting in a "modified sugar" that is recognized by a suitable transfer enzyme, which binds the sugar modified on the peptide. The saccharide component of the modified sugar, when interposed between the peptide and a selected portion, becomes a "glycosyl linking group", for example, an "intact glycosyl linking group". The glycosyl linkage group is formed from any mono- or oligosaccharide which, after modification with the modifier group, is a substrate for an enzyme to be added the modified sugar to an amino acid or glycosyl residue of a peptide. The glycosyl linkage group may be, or may include, a portion of saccharide that is modified in a degrading manner before or during the addition of the modifier group. For example, the glycosyl linkage group can be derived from a saccharide residue that is produced by oxidative degradation of an intact saccharide to the corresponding aldehyde, for example, by the action of metaperiodate, and subsequently converted to a Schiff base with an appropriate amine, which is then reduced to the corresponding amine. Exemplary conjugates of the invention correspond to the following structure: in which the symbols a, b, c, d and s represent a positive integer and that is not zero; and t is either 0 or a positive integer. The "agent" is a therapeutic agent, a bioactive agent, a detectable marker, water-soluble portion (e.g., PEG, m-PEG, PPG and m-PPG) or the like. The "agent" can be a peptide, for example, enzyme, antibody, antigen, etc. The linker can be any of a wide range of link groups, cited below. Alternatively, the linker can be a single link or a "zero order linker". In an exemplary embodiment, the selected modifier group is a water soluble polymer, for example, m-PEG. The water soluble polymer is covalently linked to the peptide by means of a glycosyl linking group. The glycosyl linkage group is covalently linked to an amino acid residue or a glycosyl residue of the peptide. The invention also provides conjugates in which the amino acid residue and a glycosyl residue are modified with a glycosyl linking group. An exemplary water soluble polymer is poly (ethylene glycol), for example, methoxypoly (ethylene glycol). The poly (ethylene glycol) used in the present invention is not restricted in any particular form or scale of molecular weight. For unbranched poly (ethylene glycol) molecules the molecular weight is preferably between 500 and 100,000. A molecular weight of 2,000-60,000 daltons is used, preferably and most preferably around 5,000 to about 30,000 daltons. In another embodiment the poly (ethylene glycol) is a branched PEG having more than one bound PEG portion. Examples of branched PEGs are described in US Pat. Do not. 5,932,462; patent of E.U.A. No. 5,342,940; patent of E.U.A.

No. 5,643,575; patent of E.U.A. No. 5,919,455; patent of E.U.A. No. 6,113,906; patent of E.U.A. No. 5,183,660; WO 02/09766; Kodera Y., Bioconjugate Chemistry 5: 283-288 (1994); and Yamasaki et al., Agrie. Biol. . Chem., 52: 2125-2127, 1998.

Useful and additional branched polymer species are described herein. In a preferred embodiment the molecular weight of each poly (ethylene glycol) of the branched PEG is equal to or greater than about 2,000, 5,000, 10,000, 15,000, 20,000, 40,000, 50,000 and 60,000 daltons. In addition to providing conjugates that are formed through an enzymatically added glycosyl linking group, the present invention provides conjugates that are highly homogeneous in their substitution patterns.

Using the methods of the invention, it is possible to form peptide conjugates in which essentially all the sugar portions modified through a population of conjugates of the invention bind to several copies of structurally identical amino acid or glycosyl residues. Thus, in a second aspect, the invention provides a peptide conjugate having a population of water soluble polymer portions, which are covalently linked to the peptide through an intact glycosyl linking group. In a conjugate of the invention that is preferred, essentially each member of the population is linked via the glycosyl linking group to a glycosyl residue of the peptide, and each glycosyl residue of the peptide to which the glycosyl linking group is attached have the same Structure A peptide conjugate having a population of water soluble polymer portions covalently linked thereto through a glycosyl linking group is also provided. In a preferred embodiment, essentially each member of the population of water soluble polymer portions is linked to an amino acid residue of the peptide by means of a glycosyl linking group, and each amino acid residue having a glycosyl linking group attached to it. same has the same structure. The present invention also provides conjugates analogous to those described above in which the peptide is conjugated to a therapeutic moiety, diagnostic moiety, targeting moiety, toxin moiety or the like via an intact glycosyl linking group.

Each of the portions described above may be a small molecule, natural polymer (e.g., polypeptide) or synthetic polymer. The peptides of the invention include at least one N- or O-linked glycosylation site, which is glycosylated with a glycosyl residue that includes a PEG portion. The PEG is covalently linked to the factor IX peptide by means of an intact glycosyl linkage group. The glycosyl linkage group is covalently linked to either "an amino acid residue or a glycosyl residue of the factor IX peptide." Alternatively, the glycosyl linking group is linked to one or more glycosyl units of a glycopeptide. conjugates in which the glycosyl linkage group is linked to both an amino acid residue and a glycosyl residue In an exemplary embodiment, the factor IX peptide comprises a portion having the formula: In the above formula, D is a member selected from -OH and RX-L-HN-; G is a member selected from R1-L- and -C (O) (C? -C6 alkyl; R1 is a portion comprising a member selected from a portion comprising a straight or branched chain poly (ethylene glycol) residue and L is a linker which is a member selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, such that when D is OH, G is R1-L-, and when G is C (0) ( Cx-C6 alkyl), D is R1-L-NH- In one embodiment, an Rx-L has the formula: where a is an integer from 0 to 20. In an exemplary embodiment, R1 has a structure that is a member selected from: CH3 wherein e and f are selected integers independently from 1 to 2,500; and q is an integer from 1 to 20. In other embodiments, R1 has a structure that is a member selected from: wherein e, f and f are selected integers independently from 1 to 2,500; and q and q 'are selected integers independently from 1 to 20. In yet another embodiment, the invention provides a factor IX peptide conjugate wherein R1 has a structure that is a member selected from: wherein e, f and f are selected integers independently from 1 to 2,500; and q, q 'and q "are integers selected independently from 1 to 20. In other embodiments, R1 has a structure that is a member selected from: k-C (0) CH2CH2 (OCH2CH2) eOCH3 and -C (0) OCH2CH2 (OCH2CH2) .OCH3 wherein e and f are independently selected integers from 1 to 2.500. In another exemplary embodiment, the invention provides a peptide comprising a portion having the formula: Gal can be attached to an amino acid residue or to a glycosyl residue that is directly or indirectly linked (for example, via a glycosyl residue) ) to an amino acid. In other modalities, the portion has the formula: The Gal can be linked to an amino acid residue or to a glycosyl residue that is directly or indirectly (for example, through a glycosyl residue) attached to an amino acid. In an exemplary embodiment, this structure is associated with the glycopegilation of an O-glycosylation site in factor IX (Figure 2B). In a further exemplary embodiment, the peptide comprises a portion according to the formula: wherein AA is an amino acid residue of the peptide and, in each of the above structures, D and G are as described herein. Exemplary amino acid residues of the peptide in which one or more of the above species can be conjugated include serine and threonine, for example, serine 53 or 61 or threonine 159, 162 or 172 of SEQ ID NO: 1. In another exemplary embodiment, The invention provides a factor IX conjugate that includes a glycosyl residue having the formula: where a, b, c, d, i, r, s, tyu are integers selected independently of 0 and 1. The index q is 1. The efgyh indices are selected independently of the integers 0 to 6. The indices j, k , 1 and m are independently selected from the integers from 0 to 100. The indices of v, w, x and y are independently selected from 0 and 1, and at least one of v, w, xyy is 1. The symbol AA represents a remainder of amino acid of the factor IX peptide. The symbol Sia- (R) represents a group that has the formula: wherein D is selected from -OH and R1-L-HN-. The symbol G represents Rx-L- or -C (0) (C? -C6 alkyl). R1 represents a portion that includes a straight or branched chain poly (ethylene glycol) residue. L is a linker that is a member selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl. In general, when D is OH, G is Rx-L-, and when G is -C (0) (C _-C alquilo alkyl), D is RX-L-NH-. In another exemplary embodiment, the sialic acid portion modified with PEG in the conjugate of the invention has the formula: in which the index "s" represents an integer from 0 to 20, and n is an integer from 1 to 2,500. In a selected mode, s is 1, and the PEG is approximately 20 kD. In an exemplary additional embodiment, the PEG-modified sialic acid has the formula: wherein L is a substituted or unsubstituted alkyl linker or substituted or unsubstituted heteroalkyl portion linking the sialic acid portion and the PEG portion. In an alternative and exemplary embodiment, in which the glycosyl residue has the structure shown above, it is conjugated to one or both Asn 157 and Asn 167. Factor IX has been cloned and sequenced. Essentially any factor IX peptide having any sequence is useful as the factor IX peptide component of the conjugates of the present invention. In one exemplary embodiment, the peptide has the sequence presented herein as SEQ ID NO: 1. YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGS CKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKWCSCTEGYRLAENQK SCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRWG GEDAKPGQFPWQWLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITWAGEHNIEETEHT EQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSG YVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSG GPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT. The present invention is in no way limited to the sequence shown herein. Factor IX variants are well known in the art, such as those described, for example, in the U.S. Patents. Nos. 4,770,999, 5,521,070 in which a tyrosine is replaced by an alanine in the first position, patent of E.U.A. No. 6,037,452, in which factor XI is linked to a group of alkylene oxide, and U.S. No. 6,046,380, in which the DNA encoding factor IX is modified in at least one splice site. As demonstrated herein, factor IX variants are well known in the art, and the present disclosure encompasses those variants known or to be developed or discovered in the future. Methods for determining the activity of a mutant or modified factor IX can be carried out using the methods described in the art, such as a phase activated partial thromboplastin time trial as described in, for example, Biggs (1972). , Human Blood Coagulation Haemostasis and Thrombosis (Ed. 1), Oxford, Blackwell, Scientific, page 614). Briefly, to test the biological activity of a factor IX molecule developed in accordance with the methods of the present invention, the assay can be carried out with equal volumes of activated partial thromboplastin reagent, factor IX deficient plasma isolated from a patient with hemophilia B using sterile phlebotomy techniques well known in the art, and plasma pooled as normal, or sample. In this assay, one unit of activity is defined as the amount present in one milliliter of normal pooled blood. In addition, an assay for biological activity based on the ability of factor IX to reduce the plasma coagulation time from factor IX deficient patients can be carried out as described in, for example, Proctor and Rapaport (AMER. J. Clin.Path 36: 212 (1961) The peptides of the invention include at least one N-linked or O-linked glycosylation site, at least one of which is conjugated to a glycosyl residue that includes a PEG moiety The PEG is covalently bound to the peptide by means of an intact glycosyl linking group.The glycosyl linking group is covalently linked either to an amino acid residue or to a glycosyl residue of the peptide., the glycosyl linkage group is linked to one or more glycosyl units of a glycopeptide. The invention also provides conjugates in which the glycosyl linking group is linked to both an amino acid residue and a glycosyl residue.

The PEG portion is linked to an intact glycosyl linker directly, or via a non-glycosyl linker, for example, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl.

Modified Sugars The present invention uses modified sugars and modified sugar nucleotides to form conjugates of the modified sugars. In the modified sugar compounds of the invention, the sugar portion is preferably a saccharide, a deoxyssaccharide, an aminosaccharide or an N-acylsaccharide. The term "saccharide" and its equivalents, "saccharyl", "sugar" and "glycosyl" refer to monomers, dimers, oligomers and polymers. The sugar portion is also functionalized with a modifying group. The modifying group is conjugated to the sugar portion, typically, through conjugation as an amine, sulfhydryl or hydroxyl, for example, primary hydroxyl, portion on the sugar. In an exemplary embodiment, the modifying group is linked through an amine portion on the sugar, for example, through an amide, a urethane or a urea that is formed through the reaction of the amine with a reactive derivative of the modifier group. Any sugar can be used as the sugar core of the conjugates of the invention. Exemplary sugar cores that are useful for forming the compositions of the invention include, but are not limited to, glucose, galactose, mannose, fucose and sialic acid. Other useful sugars include amino sugars such as glucosamine, galactosamine, mannosamine, the 5-amine analogue of sialic acid and the like. The sugar core can be a structure found in nature or can be modified to provide a site to conjugate the modifier group. For example, in one embodiment, the invention provides a sialic acid derivative in which the 9-hydroxy portion is replaced with an amine. The amine is easily derived with an activated analog of a selected modifier group. In an exemplary embodiment, the invention utilizes a modified sugar amine having the formula: wherein G is a glycosyl moiety, L is a bond or a linker and R1 is a modifying group. Exemplary linkages are those that are formed between an NH2 in the glycosyl moiety and a complementary reactivity moiety in the modifying group. Thus, exemplary links include, but are not limited to NHR1, OR1, SR1 and the like. For example, when R1 includes a carboxylic acid moiety, this moiety can be activated and coupled with an NH2 moiety on the glycosyl residue to give a bond having the structure NHC (0) R1. Similarly, the OH and SH groups can be converted to the corresponding ether or thioether derivatives, respectively. Exemplary linkers include alkyl and heteroalkyl portions. The linkers include linking groups, for example acyl-based linking groups, for example, -C (0) NH-, -0C (0) NH- and the like. The linking groups are bonds formed between components of the species of the invention, for example, between the glycosyl portion and the linker (L), or between the linker and the modifying group (R1). Other linking groups are ethers, thioethers and amines. For example, in one embodiment, the linker is an amino acid residue, such as a glycine residue. The carboxylic acid portion of the glycine is converted into the corresponding amide by reaction with an amine in the glycosyl residue, and the glycine amine is converted to the corresponding amide or urethane by reaction with an activated carboxylic acid or carbonate of the group modifier Another exemplary linker is a PEG linker or a portion of PEG that is functionalized with an amino acid residue. PEG is linked to the glycosyl group through the amino acid residue in a PEG term and linked to R1 through the other PEG term. Alternatively, the amino acid residue binds to R1 and the PEG term not attached to amino acid is linked to the glycosyl group. An exemplary species for NH-L-R1 has the formula: -NH. { C (0) (CH2) aNH} s. { C (0) (CH2) b (OCH2CH2) c0 (CH2) in which the indices s and t are independently 0 or 1. The indices a, b and d are independently integers from 0 to 20, and c is an integer from 1 to 2,500. Other similar linkers are based on species in which the -NH portion is replaced by another group, for example, -S-, -O or -CH2. More particularly, the invention uses compounds in which NH-L-R1 is: NHC (O) (CH2) aNHC (0) (CH2) b (OCH2CH2) c0 (CH2) dNHRa, NHC (O) (CH2) b ( OHC2CH2) cO (CH2) dNHRx, NHC (O) O (CH2) b (OCH2CH2) cO (CH2) dNHRx, NH (CH2) aNHC (0) (CH2) b (OCH2CH2) cO (CH2) dNHR1, NHC (O ) (CH ^ aNHR1, NHÍCH ^ aNHR1 and NHR1.) In these formulas, indices a, b and d are independently selected from the integers from 0 to 20, preferably from 1 to 5. The index c is an integer from 1 to 2,500. In the following description, the invention is illustrated by a reference to the use of the selected sialic acid derivatives, Those skilled in the art will recognize that the focus of the discussion is for clarity of illustration and that the structures and compositions described are generally applicable through of the genus of saccharide groups, modified saccharide groups, modified and activated saccharide groups and conjugates of modified saccharide groups In an illustrative embodiment, G is an ac sialic acid and the compounds selected for use in the invention have the formulas: As will be appreciated by those skilled in the art, the sialic acid moiety in the above exemplary compounds can be replaced with any other aminosaccharide including, but not limited to, glucosamine, galactosamine, mannosamine, its N-acetyl derivatives and the like.

In another illustrative embodiment, a primary hydroxyl portion of the sugar is functionalized with the modifying group. For example, the 9-hydroxyl of sialic acid can be converted to the corresponding amine and functionalized to provide a compound according to the invention. The formulas according to this modality include: In a further exemplary embodiment, the invention utilizes modified sugars in which the 6-hydroxy position is converted to the corresponding amine moiety, which carries a linker modifying group such as those described above. Exemplary saccharyl groups that can be used as the nucleus of these modified sugars include Gal, GalNAc, Glc, GlcNAc, Fue, Xyl, Man and the like. A representative sugar modifier according to this modality has the formula: wherein R3-R5 and R7 are members independently selected from H, OH, C (0) CH3, NH and NH C (0) CH3. R6 is OR1, NHR1 or NH-L-R1, which is as described above. The conjugates selected for use in the invention are based on glucose, galactose or glucose, or on species that have the stereochemistry of glucose, galactose or glucose. The general formulas of these conjugates are: In another exemplary embodiment, the invention uses compounds such as those described above that are activated as the corresponding nucleotide sugars. Exemplary sugar nucleotides which are used in the present invention in their modified form include mono- or di-triphosphates of nucleotides or analogs thereof. In a preferred embodiment, the modified sugar nucleotide is selected from a UDP-glycoside, CMP-glycoside or a GDP-glycoside. Even more preferably, the sugar nucleotide portion of the modified sugar nucleotide is selected from UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid or CMP -NeuAc In an exemplary embodiment, the nucleotide phosphate is linked to C-1. Thus, in an illustrative embodiment in which the glycosyl portion is sialic acid, the invention uses compounds having the formulas: wherein L-R1 is as described above, and L1-R1 represents a linker attached to the modifying group. As with L, exemplary linker species according to L1 include a bond, alkyl or heteroalkyl moieties.

The sugar nucleotide compounds exemplarily modified in accordance with these embodiments are described in Figure 7 and Figure 8. In another exemplary embodiment, the invention provides a conjugate formed between a modified sugar of the invention and a factor IX substrate peptide. . In this embodiment, the sugar portion of the modified sugar becomes a glycosyl linking group interposed between the substrate and the modifying group. An exemplary glycosyl linker group is an intact glycosyl linker group, in which the glycosyl portion or portions that form the linker group are not degraded by chemical (eg, sodium metaperiodate) or enzymatic (eg, oxidase) processes. Selected conjugates of the invention include a modifying group that is linked to the amine portion of an amino-saccharide, for example, mannosamine, glucosamine, galactosamine, sialic acid, etc. Cassettes of intact glycosyl linkage groups by exemplary modifying groups according to this motif are based on a structure of sialic acid, such as those having the formulas: In the above formulas, R1 and L1 are as described above. In a further exemplary embodiment, the conjugate is formed between a substrate factor IX and a saccharyl portion in which the modifying group is linked through a linker at the carbon 6 position of the saccharyl position. Thus, the illustrative conjugates according to this modality have the formulas: in which the radicals are as described above. Those skilled in the art will appreciate that the modified saccharyl portions described above can also be conjugated to a substrate through an oxygen or nitrogen atom at 2, 3, 4 or 5 carbon atoms. Illustrative compounds for use in this embodiment include compounds having the formulas: in which the R groups and indices are as described above. The invention also provides the use of sugar nucleotides modified with L-R1 at the carbon position 6. Exemplary species according to this embodiment include: in which the groups R, and L, represent portions as described above. The "y" index is 0, 1 or 2. Another exemplary and additional nucleotide sugar for use in the invention is based on a species having the tricky stereochemistry of GDP. The exemplary species according to this modality have the structure: In a further exemplary embodiment, the invention provides a conjugate wherein the modified sugar is based on the stereochemistry of UDP galactose. An exemplary nucleotide sugar for use in this invention has the structure: In another exemplary embodiment, the nucleotide sugar is based on the glucose stereochemistry. The exemplary species according to this modality have the following formulas: The modifying group, R1, is any of a number of species including, but not limited to, water soluble polymers, water insoluble polymers, therapeutic agents, diagnostic agents and the like. The nature of exemplary modifying groups is described in more detail below herein.

Modifying Groups Water-Soluble Polymers Many water-soluble polymers are known to those skilled in the art and are useful for practicing the present invention. The term water-soluble polymer encompasses species such as saccharides (eg, dextran, amylose, hyaluronic acid, polysialic acid, heparans, heparins, etc.); polyamino acids, for example, polyaspartic acid, and polyglutamic acid; nucleic acids, synthetic polymers (for example, polyacrylic acid, polyethers, for example poly (ethylene glycol), peptides, proteins and the like) The present invention can be carried out with any water-soluble polymer with the only limitation being that the polymer it should include a point at which the remainder of the conjugate can be bound in. Methods for activating polymers can also be found in WO 94/17039, U.S. Patent No. 5,324,844, WO 94/18247, WO 94 / 0.4193, Patent No. 5,219,564, U.S. Patent No. 5,122,614, WO 90/13540, U.S. Patent No. 5,281,698 and in addition WO 93/15189, and for conjugation between activated polymers and peptides, e.g., Coagulation Factor VIII (WO. 94/15625), hemoglobin (WO 94/09027), oxygen carrier molecule (US Patent No. 4,412,989), ribonuclease and superoxide dismutase (Veronese et al., App. Biochem. Biotech 11: 141-45 (1985) The soluble polymers it is in water that are preferred are those in which a substantial proportion of the polymer molecules in a sample of the polymer have approximately the same molecular weight; these polymers are "homodispersos". ! The present invention is further illustrated by reference to a conjugate of poly (ethylene glycol). Several reviews and monographs on the functionalization and conjugation of PEG are available. See, for example, Harris, Macronol. Chem. Phys. C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987); Wong et. Al. , Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et. al , Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky, Bioconjugate Chem. 6: 150-165 (1995); and Bhadra, et. Al. , Pharmazie, 57: 5-29 (2002). The routes for preparing the reactive PEG molecules and forming conjugates using the reactive molecules are known in the art. For example, the patent of E.U.A. No. 5,672,662 discloses a water soluble and isolable conjugate of an active ester of a polymeric acid selected from linear or branched polyalkylene oxides, polyoxyethylated polyols, polyolefin alcohols and polyacrylomorpholine. The patent of E.U.A. No. 6,376,604 illustrates a method for preparing a hydrosoluble 1-benzotriazolylcarbonate ester of a water-soluble and non-peptidic polymer by reacting a terminal hydroxyl of the polymer with di- (1-benzotriazolyl) carbonate in an organic solvent. The active ester is used to form conjugates with a biologically active agent such as a protein or peptide. WO 99/45964 describes a conjugate comprising a biologically active agent and an activated water soluble polymer comprising a polymer base structure having at least one term linked to the base structure of the polymer through a stable bond, in wherein at least one term comprises a branching portion having nearby reactive groups linked to the branching portion, in which the biologically active agent is linked to at least one of the nearby reactive groups. Other poly (ethylene glycol) is branched are described in WO 96/21469, U.S. Pat. No. 5,932,462 discloses a conjugate formed with a branched PEG molecule that includes a branched term that includes reactive functional groups. Free reactive groups are available to react with a biologically active species, such as a protein or peptide, forming conjugates between the poly (ethylene glycol) and the biologically active species. The patent of E.U.A. No. 5,446,090 discloses a bifunctional PEG linker and its use to form conjugates having a peptide in each of the PEG linker terms. Conjugates that include degradable PEG bonds are described in WO 99/34833; and WO 99/14259, as well as in the patent of E.U.A. No. 6,348,558. These degradable linkages are applicable in the present invention. The methods recognized in the polymer activation art described above are useful in the context of the present invention in the formation of the branched polymers shown herein and also for the conjugation of these branched polymers to other species, for example, sugars, sugar nucleotides and the like. Exemplary poly (ethylene glycol) molecules for use in the invention include, but are not limited to, those having the formula: wherein R8 is H, OH, NH2, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroalkyl, eg, acetal, OHC-, H2N - (CH2) q-, HS- (CH2) g- or - (CH2) qC (Y) Z1. The index "e" represents an integer from 1 to 2,500. The indices b, dyq independently represent integers from 0 to 20. The symbols Z and Z1 independently represent OH, NH2, leaving groups, for example, imidazole, p-nitrofenyl, HOBT, tetrazole, halide, S-R9, the portion of alcohol of activated esters; - (CH2) PC (Y1) V, or - (CH2) PU (CH2) SC (Y1) v. The symbol Y represents H (2), = 0, = S, = N-R10. The symbols X, Y, Y1, A1 and U independently represent the portions O, S, N-R11. The symbol V represents OH, NH2, halogen, S-R12, the alcohol component of activated esters, the amine component of activated amides, sugar nucleotides and proteins. The indices p, q, syv are members selected independently of the integers from 0 to 20. The symbols R9, R10, R11 and R12 independently represent H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl and substituted or unsubstituted heteroaryl. In other exemplary embodiments, the poly (ethylene glycol) molecule is selected from the following: The poly (ethylene glycol) useful for forming the conjugate of the invention is either linear or branched. Branched poly (ethylene glycol) and branched poly (ethylene glycol) molecules suitable for use in the invention include, but are not limited to, those described by the following formula: wherein R8 and R8 'are members selected by the groups defined for R8, above. A1 and A2 are members selected independently of the groups defined for A1, above. The indices e, f, o and q are as described above. Z and Y are as described above. X1 and X1 'are members selected independently from S, SC (0) NH, HNC (0) S, SC (0) 0, O, NH, NHC (O), (0) CNH and NHC (0) O, OC (0) NH. In other exemplary embodiments, the branched PEG is based on a core of cysteine, serine or di-lysine. Thus, exemplary and additional branched PEGs include: In yet another embodiment, the branched P? G portion is based on a tri-lysine peptide. The tri-lysine can be mono-, di-, tri- or tetra-PEGylated. Exemplary species according to this modality have the formulas: in which e, f and f are selected integers independently from 1 to 2,500; and q, q 'and q "are integers selected independently from 1 to 20. In exemplary embodiments of the invention, the PEG is m-PEG (5 kD, 10 kD, 15 kD, 20 kD or 30 kD) A species of branched PEG exemplary is a serine- or cysteine- (m-PEG) 2 in which m-PEG is a 20 kD m-PEG As will be apparent to those skilled in the art, the branched polymers for use in the invention include variations In the subjects shown above, for example, the di-lysine-PEG conjugate shown above may include three polymeric subunits, the third linked to the α-amine shown as unmodified in the above structure. tri-lysine functionalized with three to four polymeric subunits is within the scope of the invention Additional exemplary species useful in the invention include: and active carbonates and asters of these species, such as: Other activating groups, or projections, suitable for activating linear PEGs for use in preparing the compounds shown herein include, but are not limited to, the species: PEG molecules that are activated with these and other species and methods for making the PEGs activated are shown in WO 04/083259. Those skilled in the art will appreciate that one or more of the m-PEG arms of the branched polymer can be replaced by a PEG portion with a different term, for example, OH, COOH, NH2, C2-C_ alkyl, 0, etc. Moreover, the above structures are easily modified by inserting alkyl linkers (or removing carbon atoms) between the α-carbon atom and the functional group of the "amino acid" side chain. In this manner, "homo" derivatives and higher homologs, as well as lower homologs are useful as "nuclei of" amino acids "for branched PEGs useful in the present invention. The branched PEG species shown herein are readily prepared by methods such as those shown in the following reaction scheme: in which Xa is O or S and r is an integer from 1 to 5. The indices e and f are integers selected independently from 1 to 2,500. Thus, according to this reaction scheme, a natural or unnatural amino acid is contacted with an activated m-PEG derivative, in this case the tosylate, by forming 1 by alkylating the side chain heteroatom Xa. The monofunctionalized m-PEG amino acid is subjected to N-acylation conditions with a reactive m-PEG derivative, thereby assembling the branched m-PEG 2. As those skilled in the art will appreciate, the tosylate leaving group can be replaced with any group Suitable projection, for example, halogen, mesylate, triflate, etc. Similarly, the reactive carbonate used to acylate the amine can be replaced with an active ester, for example, N-hydroxysuccinimide, etc., or the acid can be activated in situ using a dehydrating agent such as dicyclohexylcarbodiimide, carbonyldiimidazole, etc. In an exemplary embodiment, the modifying group is a PEG portion, however, any modifying group, eg, water soluble polymer, water insoluble polymer, therapeutic portion, etc., may be incorporated in a glycosyl portion through an appropriate link. The modified sugar is formed by enzymatic means, chemical means or a combination thereof, thus producing a modified sugar. In an exemplary embodiment, the sugars are substituted with an active amine in any position that allows fixation of the modifier portion, but still allows the sugar to function as a substrate for an enzyme capable of coupling the modified sugar to the peptide. In an exemplary embodiment, when the galactosamine is the modified sugar, the amine moiety is attached to the carbon atom in the 6-position.

Species Modified with Water-Soluble Polymers The nucleotide sugar species modified with water-soluble polymers in which the sugar portion is modified with a water-soluble polymer are useful in the present invention. An exemplary modified sugar nucleotide carries a sugar group that is modified through a portion of amine or in sugar. Modified sugar nucleotides, for example, sacchalamine derivatives of sugar nucleotides, are also useful in the methods of the invention. For example, a saccharilamine (without the modifying group) can be enzymatically conjugated to a peptide (or other species) and the free amine portion of saccharyl subsequently conjugated to a desired modifying group. Alternatively, the modified sugar nucleotide can function as a substrate for an enzyme that transfers the modified sugar to a saccharyl receptor on a substrate, for example, a peptide, glycopeptide, lipid, aglycone, glycolipid, etc. In one embodiment, in which the saccharide core is galactose or glucose, R5 is NHC (O) Y. In an exemplary embodiment, the modified sugar is based on a 6-amino-N-acetyl-glycosyl moiety. As shown below for N-acetylgalactosamine, the 6-a-sugar portion is easily prepared by standard methods.

In the above reaction scheme, the index n represents an integer from 1 to 2,500, preferably from 10 to 1,500, most preferably from 10 to 1,200. The symbol "A" represents an activating group, for example, a halo, a component of an activated ester (for example, an N-hydroxysuccinimide ester), a component of a carbonate (for example, p-nitrophenyl carbonate) and Similar. Those skilled in the art will appreciate that other sugars of P? G-amide nucleotides are readily prepared by this and analogous methods. In other exemplary embodiments, the amide portion is replaced by a group such as a urethane or a urea. In other additional embodiments, R1 is a branched PEG, for example, one of those species shown above. Illustrative compounds according to this embodiment include: wherein X4 is a bond or O. Furthermore, as described above, the present invention provides nucleotide sugars that are modified with a water soluble polymer, which is either straight or branched chain. For example, compounds having the formula shown below are within the scope of the present invention: in which X is O or a link. Similarly, the invention provides nucleotide sugars of those modified sugar species in which the cn in the 6-position is modified: wherein X4 is a bond or 0. Conjugates of peptides and glycopeptides, lipids and glycolipids including the compositions of the invention are also provided. For example, the invention provides conjugates having the following formulas: Water-Insoluble Polymers In another embodiment, analogous to those described above, the modified sugars include a water-insoluble polymer, instead of a water-soluble polymer. The conjugates of the invention may also include one or more water-insoluble polymers. This embodiment of the invention is illustrated by the use of the conjugate as a vehicle with which a therapeutic peptide is delivered in a controlled manner. Polymeric drug delivery systems are known in the art. See, for example, Dunn et al. , Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series vol. 469, American Chemical Society, Washington, D.C. 1991. Those skilled in the art will appreciate that substantially any delivery and any known drug delivery system is applicable to the conjugates of the present invention. Representative water-insoluble polymers include, but are not limited to, polyphosphazines, poly (vinyl) alcohols, polyamides, polycnates, polyalkylenes, polycarilamides, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinyl pyrrolidone, polyglycolides, polysiloxanes, polyurethanes, poly (methyl) methacrylate, poly (ethyl) methacrylate, poly (butyl) methacrylate, poly (isobutyl) methacrylate, poly (hexyl) methacrylate, poly (isododecyl) methacrylate, methacrylate poly (lauryl), poly (phenyl) methacrylate, poly (methyl) acrylate, poly (isopropyl) acrylate, poly (isobutyl) acrylate, poly (octadecyl) acrylate, polyethylene, polypropylene, poly (ethylene glycol), oxide of poly (ethylene), poly (ethylene) terephthalate, poly (vinyl acetate), polyvinyl chloride, polystyrene, polyvinylpyrrolidone, pluronic and poly vinylphenol, and copolymers thereof. Synthetically modified natural polymers for use in the conjugates of the invention include, but are not limited to, alkylcelluloses, hydroxyalkylcelluloses, cellulose ethers, cellulose esters and nitrocelluloses. Particularly preferred members of the broad classes of synthetic polymers include, but are not limited to, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxybutylmethylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, cxymethylcellulose , cellulose triacetate, sodium salt of cellulose sulfate, and polymers of acrylic and methacrylic esters and alginic acid. These and other polymers described herein can be readily obtained from commercial sources such as Sigma Chemical Co. (St. Louis, MO), Polysciences (Warrenton, PA), Aldrich (Milwaukee, Wl), Fluka (Ronkonkoma, NY) and BioRad (Richmond, CA), or otherwise synthesized from monomers obtained from these suppliers using standard techniques. Representative biodegradable polymers for use in the conjugates of the invention include, but are not limited to, polylactides, polyglycolides and copolymers thereof, poly (ethylene) terephthalate, poly (butyric acid), poly (valeric) acid, poly (lactide-co-caprolactone), poly (lactide-co-glycolide), polyanhydrides, polyorthoesters, blends and copolymers thereof. Of particular utility are compositions that form gels, such as those that include collagens, pluronic and the like. Polymers for use in the invention include "hybrid" polymers that include water-insoluble materials that have within a portion of their structure a bioresorbable molecule. An example of this polymer is one that includes a water-insoluble copolymer, which has a bioresorbable region, a hydrophilic region and a plurality of crosslinkable functional groups per polymer chain. For the purposes of the present invention, "water-insoluble materials" include materials that are substantially insoluble in water or environments containing water. Thus, although certain regions or segments of the copolymer can be hydrophilic or even soluble in water, the polymer molecule, completely, does not dissolve to a substantial extent in water. For the purposes of the present invention, the term "bioresorbable molecule" includes a region that is capable of being metabolized or degraded and resorbed and / or eliminated through normal excretory routes of the body. These metabolites or degradation products are preferably substantially non-toxic to the body. The bioresorbable region can be either hydrophobic or hydrophilic, so long as the composition of the copolymer as a whole does not become soluble in water. In this way, the bioresorbable region is selected based on the preference that the complete polymer remain insoluble in water. Accordingly, the relative properties, ie, the types of functional groups contained by, and the relative properties of the bioresorbable region, and the hydrophilic region are selected to ensure that the useful bioresorbable compositions remain insoluble in water. Exemplary resorbable polymers include, for example, resorbable and synthetically produced block copolymers of poly (α-hydroxy-carboxylic acid) / poly (oxyalkylene) (see, Cohn et al., U.S. Patent No. 4,826,945). These copolymers are not entangled and are soluble in water so that the body can excrete degraded block copolymer compositions. See, Younes et al. , J. Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al. , J. Biomed. Mater. Res. 22: 993-1009 (1988). Currently preferred bioresorbable polymers include one or more components selected from poly (esters), poly (hydroxy acids), poly (lactones), poly (amides), poly (steramides), poly (amino acids), poly (anhydrides), poly ( orthoesters), poly (carbonates), poly (phosphacins), poly (phosphoesters), poly (thioesters), polysaccharides and mixtures thereof. More preferably still, the bioresorbable polymer includes a polyhydroxy acid component. Of the polyhydroxy acids, polylactic acid, polyglycolic acid, polycaproic acid, polybutyric acid, polyvaleric acid and copolymers and mixtures thereof are preferred. In addition to forming fragments that are absorbed in vivo ("bioresorbed"), the polymeric coatings that are preferred to be used in the methods of the invention can also form a removable and / or metabolizable fragment. The higher order copolymers can also be used in the present invention. For example, Casey et al., Patent of E.U.A. No. 4,438,253, which was issued on March 20, 1984, describes copolymers of three blocks produced from the transesterification of poly (glycolic acid) and a poly (alkylene glycol) of hydroxyl ends. These compositions are described for use as resorbable monofilament structures. The flexibility of these compositions is controlled by the incorporation of an aromatic orthocarbonate, such as tetra-p-tolyl orthocarbonate into the structure of the copolymer. Other polymers based on lactic acid and / or glycolic acids can also be used. For example, Spinu, patent of E.U.A. No. 5,202,413, which was issued on April 13, 1993, describes copolymers of various biodegradable blocks having sequentially ordered polylactide and / or polyglycolide blocks produced by opening polymerization of lactide rings and / or glycolide on either a diol oligomeric or a diamine residue followed by chain extension with a difunctional compound, such as, for example, a disocyanate, diacyl chloride or dichlorosilane. The bioresorbable regions of the coatings useful in the present invention can be designed to be hydrolytically and / or enzymatically cuttable. For the purposes of the present invention, "hydrolytically cuttable" refers to the susceptibility of the copolymer, especially the bioresorbable region, to hydrolysis in water or in a water-containing environment. Similarly, "enzymatically cuttable" as used herein, refers to the susceptibility of copolymer, especially the bioresorbable region, to cleavage by endogenous or exogenous enzymes. When placed inside the body, the hydrophilic region can be processed into excretable and / or metabolizable fragments. Thus, the hydrophilic region can include, for example, polyethers, polyalkylene oxides, polyols, poly (vinylpyrrolidone), poly (vinyl) alcohol, poly (alkyloxazolines), polysaccharides, carbohydrates, peptides, proteins and copolymers and mixtures thereof. Furthermore, the hydrophilic region can also be, for example, a poly (alkylene) oxide. These poly (alkylene) oxides may include, for example, poly (ethylene) oxide, poly (propylene) oxide and mixtures and copolymers thereof. Polymers that are components of hydrogels are also useful in the present invention. Hydrogels are polymeric materials that are capable of absorbing relatively large amounts of water. Examples of hydrogel-forming compounds include, but are not limited to, polyacrylic acids, sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, gelatin, carrageenan and other polysaccharides, hydroxyethylene methacrylic acid (HEMA), as well as derivatives thereof, and the like. Hydrogels can be produced that are stable, biodegradable and bioresorbable. In addition, hydrogel compositions can include subunits that exhibit one or more of these properties. Biocompatible hydrogel compositions are known whose integrity can be controlled through entanglement, and are currently preferred for use in the methods of the invention. For example, Hubbell et al. US patents Nos. 5,410,016, which was issued on April 25, 1995 and 5,529,914, which was issued on June 25, 1996, describe water-soluble systems, which are interlaced block copolymers having a central block segment soluble in water. water sandwiched between two hydrolytically labile extensions. These copolymers also have blocked ends with photopolymerizable acrylate functionalities. When they are intertwined, these systems become hydrogels. The water-soluble core block of these copolymers can include poly (ethylene glycol); while the hydrolytically labile extensions may be a poly (α-hydroxy) acid, such as polyglycolic acid or polylactic acid. See, Sawhney et al., Macromolecules 26: 581-587 (1993). In another preferred embodiment, the gel is a thermoreversible gel. Thermoreversible gels include components, such as pluronics, collagen, gelatin, hyaluronic acid, polysaccharides, polyurethane hydrogel, polyurethane-urea hydrogel and combinations thereof are currently preferred. In yet another exemplary embodiment, the conjugate of the invention includes a component of a liposome. Liposomes can be prepared according to methods known to those skilled in the art, for example, as described in Eppstein et al., U.S. Pat. No. 4,522,811, which was issued June 11, 1985. For example, liposome formulations can be prepared by dissolving suitable lipids (such as stearoylphosphatidylethanolamine, stearoylphosphatidylcholine, aracadoylphosphatidylcholine and cholesterol) in an inorganic solvent which is then evaporated, leaving behind a thin film of dried lipids on the surface of the container. An aqueous solution of the active compound or its pharmaceutically acceptable salt is then introduced into the container. The container is then rotated by hand to release lipid material from the sides of the container and to disperse lipid aggregates, thus forming the liposome suspension. The microparticles and methods described above for preparing the microparticles are offered by way of example and are not intended to define the scope of microparticles for use in the present invention. It will be apparent to those skilled in the art that an arrangement of microparticles, manufactured by different methods, are useful in the present invention. The structural formats described above in the context of water soluble polymers, both straight chain and branched, are generally applicable with respect to the water insoluble polymers as well. Thus, for example, the nuclei of cysteine, serine, dilisin and trilisin can be functionalized with two portions of water-insoluble polymer. The methods used to produce these species are closely analogous generally to those used to produce the water soluble polymers. The degree of PEG substitution of the conjugates can be controlled by the choice of stoichiometry, number of glycosylation sites available, selection of an enzyme that is selective for a particular site, and the like (Figure 2F). The glycoPEGylated Factor IX species displays an increased average circulatory life relative to non-labeled factor IX (Figure 3, Figure 6).

The Methods In addition to the conjugates described above, the present invention provides methods for preparing these and other conjugates. Moreover, the invention provides methods for preventing, curing or decreasing a disease state by administering a conjugate of the invention to a subject at risk of developing the disease or to a subject having the disease. Thus, the invention provides a method for forming a covalent conjugate between a selected portion and a factor IX peptide.

In exemplary embodiments, the conjugate is formed between a water-soluble polymer, a therapeutic moiety, a targeting moiety or a biomolecule, and a glycosylated or non-glycosylated factor IX peptide. The polymer, therapeutic moiety or biomolecule is conjugated to the peptide by means of a glycosyl linking group, which is interposed between, and covalently linked to both the peptide and the modifier group (e.g., water soluble polymer). The method incs contacting the peptide with a mixture containing a modified sugar and an enzyme, e.g., a glycosyltransferase, which conjugates the modified sugar to the substrate (e.g., peptide, aglycone, glycolipid). The reaction is carried out under suitable conditions to form a covalent bond between the modified sugar and the factor IX peptide. The factor IX receptor peptide is typically synthesized again, or expressed recombinantly in a prokaryotic cell (eg, bacterial cell, such as E. coli) or in a eukaryotic cell, such as mammalian cell, yeast, insect , fungal or vegetable. The peptide can be either a full-length protein or a fragment. Moreover, the peptide can be a wild-type or mutated peptide. In an exemplary embodiment, the peptide incs a mutation that adds or removes one or more N- or O-linked glycosylation sites to the peptide sequence.

In an exemplary embodiment, factor IX is 0-glycosylated and functionalized with a water-soluble polymer in the following manner. The peptide is either produced with an available amino acid glycosylation site or, if glycosylated, the glycosyl moiety is cut to expose the amino acid. For example, a serine or threonine is α-1 N-acetylamino galactosylated (GalNAc) and the NAc-galactosylated peptide is sialylated with a cassette of sialic acid modifying groups using ST6GalNAcTl. Alternatively, the NAc-galactosylated peptide is galactosylated using Core-1-GalT-1 and the product is sialylated with a cassette of sialic acid modifying groups using ST3GalTl. An exemplary conjugate according to this method has the following bonds: Thr-a-l-GalNAc-β-1, 3-Gal-a2, 3-Sia *, where Sia * is the cassette of sialic acid modifying groups. In the methods of the invention, such as those described above, the use of various enzymes and saccharyl donors, the individual glycosylation steps can be carried out separately, or combined in a single-vessel reaction. For example, in the three enzyme reaction shown above GalNAc transferase, GalT and SiaT and their donors can be combined in a single vessel. Alternatively, the GalNAc reaction can be carried out alone and both the GalT and the SiaT and the appropriate saccharyl donors added, as a single step. Another way of running the reactions incs adding each enzyme and a suitable donor sequentially and carrying out the reaction in a "single-container" motif. Combinations of each of the methods shown above are useful for preparing the compounds of the invention In the conjugates of the invention, particularly the glycoPEGylated N-linked glycans, the cassette of Sia modifying groups can be linked to the Gal in a -2.6 or a-2.3 The method of the invention also provides for the modification of incompletely glycosylated factor IX peptides that are produced recombinantly Using a modified sugar in a method of the invention, the peptide can be simultaneously glycosylated and further derivatized with, for example, a water soluble polymer, therapeutic agent or the like. The sugar portion of the modified sugar may be the residue that could properly be conjugated to the receptor in a fully glycosylated peptide, or other portion of sugar with desirable properties. - Exemplary methods for modifying the peptides useful in the present invention are shown in WO04 / 099231, WO 03/031464, and the references mentioned therein.

In an exemplary embodiment, the invention provides a method for making a pegylated factor IX comprising the portion of: wherein D is -OH or R1-L-NH-. The symbol G represents R1-L-o-C (0) (C? -C6 alkyl). R1 is a portion comprising a straight or branched chain poly (ethylene glycol) residue.

The symbol L represents a linker selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl. In general, when D is OH, G is R1-L-, and when G is -C (0) (C_-C3 alkyl), D is R1-L-NH-.

The method of the invention includes, (a) contacting a factor IX substrate peptide with an acid donor.

PEG-sialic and an enzyme that is capable of transferring the PEG-sialic acid portion of the donor to the factor IX polypeptide of the substrate. An exemplary PEG-sialic acid donor is a nucleotide sugar such as that having the formula: and an enzyme that transfers the PEG-sialic acid onto an amino acid or glycosyl residue of the factor IX peptide, under conditions suitable for transfer. In one embodiment the substrate factor IX peptide is expressed in a host cell prior to the formation of the conjugate of the invention. An exemplary host cell is a mammalian cell. In other embodiments, the host cell is an insect cell, plant cell, bacterium or fungus. The method presented herein can be applied to each of the factor IX conjugates shown in the previous sections. The factor IX peptides modified by the methods of the invention can be synthetic or wild-type peptides, or they can be mutated peptides, produced by methods known in the art such as site-directed mutagenesis. The glycosylation of the peptides is typically either N-linked or O-linked. An exemplary N-bond is the fixation of the modified sugar to the side chain of an asparagine residue. The sequences of three asparagine-X-serine and asparagine-X-threonine peptides, wherein X is any amino acid except proline, are the recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these three peptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of a sugar (eg, N-acetylgalactosamine, galactose, mannose, GlcNAc, fucose or xylose) to the hydroxy side chain of a hydroxy amino acid, preferably serine or threonine, although they can also use unusual or unnatural amino acids, for example, 5-hydroxyproline or 5-hydroxylysine. The addition of glycosylation sites to a peptide or other structure is conveniently achieved by altering the amino acid sequence such that it contains one or more glycosylation sites. Addition can also be made by incorporating one or more species having an -OH group, preferably serine or threonine residues, within the peptide sequence (for O-linked glycosylation sites). The addition can be done by mutation or by complete chemical synthesis of the peptide. The amino acid sequence of the peptide is preferably altered through changes at the DNA level, particularly by changing the DNA and by mutating the DNA encoding the peptide at preselected bases such that the codons are generated that are translated into the amino acids. desired. DNA mutations are preferably made using methods known in the art. In an exemplary embodiment, the glycosylation site is added by intermixing polynucleotides. Polynucleotides encoding a candidate peptide can be modulated with DNA intermixing protocols. The intermingling of DNA is a process of recursive recombination and mutation, carried out by random fragmentation of a group of related genes, followed by the reassembly of the fragments by a polymerase chain reaction type process. See, for example, Stemmer, Proc. Nati Acad. Sci. USA 91: 10747-10751 (1994); Stemmer, Nature 370: 389-391 (1994); and patents of E.U.A. Nos. 5,605,793, 5,837,458, 5,830,721 and 5,811,238. Exemplary methods for adding or removing glycosylation sites, and adding or removing structures or glycosyl substructures are described in detail in WO04 / 099231, WO03 / 031464 and in U.S.A. and related PCT. The present invention also utilizes means for adding (or removing), one or more selected glycosyl residues to a factor IX peptide, after which a modified sugar is conjugated to at least one of the glycosyl residues selected from the peptide. These techniques are useful, for example, when it is desired to conjugate the modified sugar to a selected glycosyl residue that is neither present in a factor IX peptide or that is not present in a desired amount. Thus, before coupling a modified sugar to a peptide, the selected glycosyl residue is conjugated to the peptide by enzymatic or chemical coupling. In another embodiment, the glycosylation pattern of a glycopeptide is altered prior to conjugation of the modified sugar by removal of a glycopeptide carbohydrate residue. See, for example WO 98/31826. For example, the sialic acid groups can be removed from factor IX, forming asialo-factor IX, before glycopeglycerin using a PEG-modified sialic acid (Figure 2E). Exemplary attachment sites for the selected glycosyl residue include, but are not limited to: (a) consensus sites for N-linked glycosylation, and sites for O-linked glycosylation; (b) terminal glycosyl moieties that are receptors for a glycosyltransferase; (c) arginine, asparagine and histidine; (d) free carboxyl groups; (e) free sulfhydryl groups such as those of cysteine; (f) free hydroxyl groups such as those of serine, threonine or hydroxyproline; (g) aromatic residues such as those of phenylalanine, tyrosine or tryptophan or (h) the amide group of glutamine. Exemplary methods for use in the present invention are described in WO 87/05330, published September 11, 1987, and in Aplin and Wriston, CRC CRIT, REV. BIOCHEM., Pgs. 259-306 (1981). The sugars modified with PEG are conjugated to a glycosylated or unglycosylated peptide using a suitable enzyme to mediate conjugation. Preferably, the concentrations of the sugars, donor enzymes and receptor peptides are selected in such a way that the glycosylation proceeds until the receptor is consumed. The considerations described below, which detail the context of a sialyltransferase, are generally applicable to other glycosyltransferase reactions. A number of methods of using glycosyltransferases to synthesize desired oligosaccharide structures are known and are generally applicable to the present invention. Exemplary methods are described, for example, in WO 96/32491, Ito et al. , Puré Appl. Chem. 65: 753 (1993), patents of E.U.A. Do not . 5,352,670, 5,374,541, 5,545,553 and in the U.S. Patents. in co-ownership Nos. 6,399,336 and 6,440,703 which are incorporated herein by reference. The present invention is carried out using a single glycosyltransferase or a combination of glucosyltransferases. For example, a combination of a sialyltransferase and a galactosyltransferase can be used. In those embodiments that use more than one enzyme, the enzymes and substrates are preferably combined in an initial reaction mixture, or the enzymes and reagents for a second enzymatic reaction are added to the reaction medium once the first enzymatic reaction is complete. or almost complete. By carrying out two enzymatic reactions in sequence in a single vessel, total yields are improved over the procedures in which an intermediate species is isolated. In addition, the cleaning and disposal of additional solvents and byproducts is reduced. In a preferred embodiment, each of the first and second enzymes is a glycosyltransferase. In another preferred embodiment, an enzyme is an endoglycosidase. In a further preferred embodiment, more than two enzymes are used to assemble the modified glycoprotein of the invention. Enzymes are used to alter a saccharide structure on the peptide at any point either before or after the addition of the modified sugar to the peptide. In another embodiment, the method makes use of one or more exo- or endoglucosidase. Glucosidase is typically a mutant, which is manipulated to form glycosyl bonds instead of breaking them. The mutant glucanase typically includes a substitution of an amino acid residue for an acidic amino acid residue is active on site. For example, when the endoglucanase in endo-H, substituted active site residues will typically be Asp in position 130, Glu in position 132 or a combination thereof. The amino acids are usually replaced with serine, alanine, asparagine or glutamine. The mutant enzyme catalyzes the reaction, usually by a synthesis step that is analogous to the reverse reaction of the endoglucanase hydrolysis step. In these embodiments, the glycosyl donor molecule (eg, a desired oligo- or mono-saccharide structure) containing a leaving group and the reaction proceeds with the addition of the donor molecule to a GlcNAc residue in the protein. For example, the outgoing group may be a halogen, such as fluoride. In other embodiments, the leaving group is an Asn, or an Asn-peptide portion. In further preferred embodiments, the GlcNAc residue in the glycosyl donor molecule is modified. For example, the GlcNAc residue may comprise a 1,2-oxazoline moiety. In a preferred embodiment, each of the enzymes used to produce a conjugate of the invention are present in a catalytic amount. The catalytic amount of a particular enzyme varies according to the substrate concentration of that enzyme as well as to the reaction conditions such as temperature, time and pH value. The means for determining the catalytic amount for a given enzyme under preselected substrate concentrations and reaction conditions are well known to those skilled in the art. The temperature at which a previous process is carried out can vary from just above the freezing temperature to the temperature at which the most sensitive enzyme denatures. Preferred temperature scales are from about 0 ° C to about 55 ° C, and most preferably at about 20 ° C to about 37 ° C. In another exemplary embodiment, one or more components of the present method are carried out at an elevated temperature using a thermophilic enzyme. The reaction mixture is maintained for a period of time sufficient for the receptor to be glycosylated, thus forming the desired conjugate. Some of the conjugates can commonly be detected after a few hours, with recoverable amounts normally controlled at 24 hours or less. Those skilled in the art understand that the reaction rate depends on a number of variable factors (eg, enzyme concentration, donor concentration, receptor concentration, temperature and volume of solvent), which are used for a selected system. The present invention also provides the production on an industrial scale of modified peptides. As used herein, an industrial scale generally produces at least 250 mg, preferably at least 500 mg and most preferably at least 1 gram of finished and purified conjugate. In the following description, the invention is exemplified by the conjugation of modified sialic acid portions to a glycosylated peptide. The exemplary modified sialic acid is labeled with PEG. The approach of the following discussion on the use of modified sialic acid with PEG and glycosylated peptides is for clarity of illustration and is not intended to imply that the invention is limited to the conjugation of these two partners. Someone skilled in the art understands that the description is generally applicable to the additions of modified glucosyl portions that are not sialic acid. In addition, the description is equally applicable to the modification of a glycosyl unit with non-PEG agents including other PEG portions, therapeutic portions and biomolecules. An enzymatic approach can be used for the selective introduction of PEGylated or PPGylated carbohydrates onto a peptide or glycopeptide. The method uses modified sugars containing PEG, PPG or a hidden reactive functional group, and combined with the appropriate glycosyltransferase or glycosylate. By selecting the glycosyltransferase that will make the desired carbohydrate link and using the modified sugar as the donor substrate, the PEG or PPG can be introduced directly into the base structure of the peptide, onto existing sugar residues of a glycopeptide or on sugar residues that have been added to a peptide. A receptor for the sialyltransferase is present in the peptide which will be modified by the methods of the present invention either as a structure that occurs naturally or a set therein recombinantly, enzymatically or chemically. Suitable receptors include, for example, galactosyl receptors such as Galßl, 4GlcNAc, Galßl, 4GalNAc, Galßl, 3GalNAc, lacto-N-tetraose, Galßl, 3GlcNAc, Galßl, 3Ara, Galßl, 6GlcNAc, Galßl, 4Glc (lactose) and other receptors such as those described in the art (see, for example , Paulson et al., J. Biol. Chem. 253: 5617-5624 (1978)). In one embodiment, an acceptor for the sialyltransferase is present in the glycopeptide that will be modified after the in vivo synthesis of the glycopeptide. These glycopeptides can be sialylated using the methods claimed without a prior modification of the glycosylation pattern of the glycopeptide. Alternatively, the methods of the invention can be used to sialylate a peptide that has not included a suitable receptor; one first modifies the peptide to include a receptor by means known to those skilled in the art. The technique. In an exemplary embodiment, a GalNAc residue is added by the action of a GalNAc transferase. In an exemplary embodiment, the galactosyl receptor is assembled by attaching a galactose residue to a suitable receptor linked to the peptide. For example, a GlcNAc. The method includes incubating the peptide to be modified in a reaction mixture containing a suitable amount of galactosyltransferase (eg, galßl, 3 or galßl, 4) and a suitable galactosyl donor (eg, UDP-galactose). The reaction is allowed to proceed substantially to completion or, alternatively, the reaction is concluded when a preselected amount of the galactose residue is added. Other methods for assembling a selected saccharide receptor will become apparent to those skilled in the art. In yet another embodiment, oligosaccharides linked to glycopeptides are first "clipped", either complete or in part, to expose either a receptor for the sialyltransferase or a portion for the. which one or more suitable residues can be added to obtain a suitable receptor. Enzymes such as glycosyltransferases and endoglycosidases (see, for example, U.S. Patent No. 5,716,812) are useful for binding and trimming reactions. In the following description, the method of the invention is exemplified by the use of modified sugars having a PEG moiety attached thereto. The focus of the description is for clarity and illustration. Those skilled in the art will appreciate that the description is equally relevant to those embodiments in which the modified sugar carries a therapeutic portion, biomolecule and the like. In an exemplary embodiment of the invention in which a carbohydrate residue is "trimmed" before the addition of the modified sugar cane high, it is trimmed back to the first generation biantennary structure. A modified sugar that carries a PEG portion is conjugated or otherwise sugar residues exposed by "back-trimming". In one example, a portion of PEG is added via a GlcNAc portion conjugated to the PEG portion. The modified GlcNAc is bound to one or both of the terminal mannose residues of the biantennary structure. Alternatively, an unmodified GlcNAc can be added to one or both of the terms of the branched species. In another exemplary embodiment, a portion of PEWG is added to one or both of the terminal mannose residues of the biantennary structure with a modified sugar such that a galactose residue can be conjugated to a residue of GlcNAc either on the residues of crafty terminals. Alternatively, an unmodified Gal can be added to one or both terminal GlcNAc residues. In a further example, a PEG portion is added over a Gal residue using a modified sialic acid. In another exemplary embodiment, a high-masonry structure is "retro-cut" to the mafia from which the biantenary structure branches. In some example, a portion of PEG is added by means of a GlcNAc modified with the polymer. Alternatively, an unmodified GlcNAc is added to mannose, followed by a Gal with a portion of bound PEG. In yet another embodiment, unmodified GlcNAc and Gal residues are loaded sequentially into the mannose, followed by a modified sialic acid portion with a PEG portion. In a further exemplary embodiment, high crafty is "retro-trimmed" to the GlcNAc to which the first mat is attached. The GlcNAc is conjugated to a Gal residue carrying a PEG portion. Alternatively, an unmodified Gal is added to the Gal followed by the addition of a sialic acid modified with water-soluble sugar. In yet another example, the terminal GlcNAc is conjugated with Gal and the GlcNAc is subsequently fucosylated with a modified fucose carrying a PEG portion. The tall mannose can also be retro-cut to the first GlcNAc bound to the Asn of the peptide. In an example, the GlcNAc of the GlcNAc residue (Fue) a, is conjugated with a GlcNAc carrying a water soluble polymer. In another example, the GlcNAc of the GlcNAc- (Fue) a residue is modified with Gal, which carries a water-soluble polymer. In a further embodiment, the GlcNAc is modified with Gal, followed by conjugation to the Gal of a sialic acid modified with a PEG portion. Other exemplary embodiments are shown in the patent application publications of E.U.A. in co-ownership: 2004132640; 20040063911; ' 20040137557; patent applications of E.U.A. Nos: 10 / 369,979; 10 / 410,913; 10 / 360,770; 10 / 410,945 and PCT / US02 / 32263 each of which is incorporated herein by reference. The examples shown above provide an illustration of the potency of the methods shown herein. Using the methods described herein, it is possible to "retro-clip" and accumulate a carbohydrate residue of substantially any desired structure. The modified sugar can be added to the terms of the carbohydrate moiety as described above, or it can be intermediate between the peptide core and the carbohydrate terminus. In an exemplary embodiment, an existing sialic acid is removed from a factor IX glycopeptide using a sialidase, thus unclogging all or part of the underlying galactosyl residues. Alternatively, a peptide or glycopeptide is labeled with galactose residues, or an oligosaccharide residue that ends in a galactose unit. After exposure or addition of the galactose residues, a suitable sialyltransferase is used to add a modified sialic acid. The approach is summarized in the reaction scheme 1.

Reaction scheme 1 In a further approach, summarized in reaction scheme 2, a hidden or masked reactive functionality is present in the sialic acid. The hidden or masked reactive group is preferably unaffected by the conditions used to bind the modified sialic acid to factor IX. After the covalent attachment of the modified sialic acid to the peptide, the mask is removed and the peptide is conjugated with such an agent. as PEG. The agent is conjugated to the peptide in a specific manner by its reaction with the reactive group not hidden in the modified sugar residue.

Reaction scheme 2 of PPG Any modified sugar shown herein can be used with its suitable glycosyltransferase, depending on the terminal sugars of the oligosaccharide light chains of the glycopeptide (Table 1). As described above, the terminal or glycopeptide sugar required for the introduction of the PEGylated structure can be introduced naturally during expression or can be introduced after expression using the glucosidase, glucosyltransferase or mixtures of glucosidase and glucosyltransferase.

Table 1 Derivatives of UDP-galactosamine (when A = NH, R can be acetyl) Derivatives of UDP-glucosamine (when A = NH, R4 can be acetyl) X = O, NH, S, CH-., N-CRi ^. Y = X; Z = X; A = X; B = X. Q '= H2,?, S, NH, N-R. R, Rl-4 = H. Linker-M, M M = PEG, p. e. , m-PEG In a further exemplary embodiment, UDP-galactose-PEG is reacted with bovine milk and with β1, 4-galactosyltransferase, thereby transferring the modified galactose to the appropriate terminal N-acetylglucosamine structure. The terminal GlcNAc residues in the glycopeptide can be produced during expression, as can occur in expression systems such as mammals, insects, plants or fungi, but can also be enhanced by reacting and treating the glycopeptide with a sialidase and / or glucosidase and / or glucosyltransferase, as required. In another exemplary embodiment, a GlcNAc transferase, such as GNT-5, is used to transfer PEGylated GlcN to a terminal maize residue on a glycopeptide. In a further exemplary embodiment, an N- and / or 0-linked glycan structure is enzymatically removed from a glycopeptide to expose an amino acid or a terminal glycosyl residue that is subsequently conjugated to the modified sugar. For example, an endoglucanase is used to remove the N-linked structures of a glycopeptide pair to expose a terminal GlcNAc, such as a GlcNAc-linked-Asn in the glycopeptide. UDP-Gal-PEG and the appropriate galactosyltransferase is used to introduce the functionality of PEG-galactose onto the exposed GlcNAc. In an alternative embodiment, the modified sugar is added directly to the polymer base structure using a known glycosyltransferase to transfer sugar residues to the base structure of the peptide. This exemplary embodiment is shown in Figure 3. Exemplary glycosyltransferases useful for practicing the present invention include, but are not limited to, GalNAc transferase (GalNAc Tl-14), GlcNAc transferases, fucosyltransferases, glucosyltransferases, xylosyltransferases, mannosyltransferases and similar. The use of this approach allows the direct addition of modified sugars on peptides lacking any carbohydrate or, alternatively, in existing glycopeptides. In both cases, the addition of the modified sugar occurs at specific positions in the base structure of the polymer and the polypeptide as defined in substrate specificity of the glycosyltransferase and not randomly such as occurs during a modification of a patient's protein and the base structure of a peptide using chemical means. An agent assay can be introduced into proteins or glycoproteins that may lack the proper glycosyltransferase and the peptide sequence of the glycosyltransferase sequence by manipulating the appropriate amino acid sequence in the polypeptide chain.

Reaction scheme 3 Othene, GalNH-CO (CH2) 4NH-PEG '< ? GaiNH-CO (CH2) 4NH-PEG In each of the exemplary embodiments shown above, one or more additional chemical or enzymatic modification steps can be used after conjugation of the modified sugar to the peptide. In an exemplary embodiment, an enzyme (e.g., fucosyltransferase) is used to attach a glycosyl unit (e.g., fucose) to the terminal modified sugar attached to the peptide. In another example, an enzymatic reaction is used to "plug" sites to which the modified sugar could not be conjugated. Alternatively, a chemical reaction is used to alter the structure of the conjugated modified sugar. For example, the conjugated modified sugar is reacted with agents that stabilize or destabilize its binding to the above peptide component to which the modified acid and the modified sugar bind. In another example, a component of the modified sugar is deprotected according to its conjugation to the peptide. Someone of skill in the art and in the field will appreciate that it is an arrangement of enzymatic chemical procedures that are useful in the methods of the invention in a step subsequent to the modified sugar when the peptide is conjugated. A further elaboration of the modified sugar-peptide conjugate is within the scope of the invention.

Enzymes In addition to the enzymes described above in the context of forming the acetyl-linked conjugate, the glycosylation pattern of the conjugate and the starting substrates (eg, peptides, lipids) can be elaborated, trimmed or otherwise modified by methods using other enzymes. Methods of remodeling peptides and lipids using enzymes that transfer a sugar donor to a receptor are described in great detail in DeFrees, WO 03/031464 A2, published on April 17, 2003. A brief summary of enzymes selected for use in the This method is detailed below.

Glucosyltransferase Glucosyltransferases catalyze the addition of activated sugars (NDP- or NMP-donating sugars) in a stepwise manner, to a protein, glycopeptide, lipid or glycolipid or to the non-reducing end of a growing oligosaccharide. The N-linked glycopeptides. they are synthesized by means of a transferase and an oligosaccharide donor linked to lipids Dol-PP-NAG2Glc3Man9 in a block transfer followed by nucleus trimming. In this case, the nature of the "core" saccharide is a little different from that of the subsequent fixations. A very large number of glucosyltransferases are known in the art.

The glycosyltransferases to be used in the present invention can be any as long as they can use the modified sugar as a sugar donor. Examples of these enzymes include the glycosyltransferase of the Leloir pathway, such as galactosyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, fucosyltransferase, sialyltransferase, mannosyltransferase, xylosyltransferase, glucurononyltransferase and the like. For the synthesis of enzymatic saccharides that include the glucosyltransferase reactions, the glucosyltransferase can be cloned, or isolated from any source. Many cloned glycosyltransferases are known, and their polynucleotide sequences. See, for example, "The WWW Guide To Cloned Glycosiltransferases" (http: //www.vei.co.uk/ TGN / gt guide.htm). The amino acid sequences and glycosyltransferase nucleotide sequences encoding glycosyltransferases from which the amino acid sequences can be deduced are also found in several publicly available databases, including GenBank, Swiss-Prot, EMBL, and others. The glycosyltransferases that can be employed in the methods of the invention include, but are not limited to, galactosyltransferases, fucosyltransferases, glucosyltransferases, N-acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases, glucuronyltransferases, sialyltransferases, mannosyltransfrass, glucuronic acid transferases, galacturonic acid transferases and oligosaccharyltransferases. Suitable galactosyltransferases include those obtained from eukaryotes as well as from prokaryotes. The DNA encoding glycosyltransferases can be obtained by chemical synthesis, by screening inverse transcripts of mRNA from suitable cells or cultures or cell lines, by screening genomic libraries from suitable cells, or by combinations of these methods. Screening of mRNA or genomic DNA can be carried out with oligonucleotide probes generated from the sequence of the glycosyltransferase gene. The probes can be labeled with a detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group according to known procedures used in conventional hybridization assays. Alternatively, the glycosyltransferase gene sequences can be obtained by using the polymerase chain reaction method (PCR), with the PCR oligonucleotide primers being produced from the glycosyltransferase gene sequence. See, patent of E.U.A. No. 4,683,195 to Mullis et al. , and patent of E.U.A. No. 4,683,202 to Mullís. The glycosyltransferase can be synthesized in host cells transformed with vectors containing DNA encoding the enzyme glucosyltransferase. The vectors are used either to amplify DNA encoding the glycosyltransferase enzyme and / or to express DNA encoding the glycosyltransferase enzyme. An expression vector is a replicable DNA construct in which a DNA sequence encoding the enzyme glycosyltransferase is operably linked to suitable control sequences capable of carrying out the expression of the enzyme glucosyltransferase in a suitable host. The need for these control sequences will vary depending on the selected host and the selected transformation method. Generally, the control sequences include a transcription promoter, an optional operator sequence for controlling transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control transcription and translation termination. Amplification vectors do not require expression control domains. All that is required is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate the recognition of transformants.

In an exemplary embodiment, the invention utilizes a prokaryotic enzyme. These glycosyltransferases include enzymes involved in the synthesis of lipooligosaccharides (LOS), which are produced by many gram-negative bacteria (Preston et al., Critique Reviews in Microbiology 23 (3).-139-180 (1996)). These enzymes include, but are not limited to, rfa operone proteins from species such as E. coli and Salmonella typhimurium, which include a β1, 6-galactosyltransferase and a β1, 3-galactosyltransferase (see, for example, EMBL). Registration Nos. M80599 and M86935 (E. coli); EMBL Registration No.

S56361 (S. typhimurium)), a glycosyltransferase (Swiss-Prot Registration No. P25740 (E. coli) an ßl, 2-glucosyltransferase (rfaJ) (Swiss-Prot Registration No. P27129 (E. coli) and Swiss-Prot Registration No. P19817 (S. typhimurium)), and a βl, 2-N-acetylglucosaminyltransferase (rfaK) (EMBL No. U00039 record (E. coli) Other glycosyltransferases for which amino acid sequences are known include those which are encoded by operons such as rfaB, which have been characterized in organisms such as Klebsiella pneumoniae, E. coli, Salmonella typhimurium, Salmonella enterica , Yersinia enterocolitica, Mycobacterium leprosum and the Rhl operon of Pseudomonas aeruginosa Also suitable for use in the present invention are glycosyltransferases which are involved in producing structures containing lacto-N-neotetraose, D-galactosyl-β-1, 4- N-acetyl-D-glucosaminyl-β-1,3-D-galactosyl-β-1,4-D-glucose and the trisaccharide sequence of the blood group Pk, D ~ galactosyl-al, 4-D-galactosyl-β -l, 4-D-glucose, which have been identified in the LOS of the patóg mucosal tissues Neisseria gonnorhoeae and N. meningi tidis (Scholten et al. , J. Med. Microbiol. 41: 236-243 (1994)). The genes of N. meningi tidis and N. gonorrhoeae coding for the glycosyltransferases involved in the biosynthesis of these structures have been identified from immunotypes L3 and Ll (Jennings et al., Mol.Microbiol.18: 729-740 (1995 )) and the F62 mutant of N. gonorrinoae (Gotschlich, J. Exp. Med. 180: 2181-2190 (1994)). In N. meningitidis, a locus consisting of three genes, lgtA, lgtB and Ig E, codes for the glycosyltransferase enzymes required for the addition of the last three of the sugars in the lacto-N-neotetraose chain (Wakarchuk et al., J. Biol. . Chem. 271: 19166-73 (1996)). Recently, the enzymatic activity of the lgtB and IgtA gene product was demonstrated, providing the first direct evidence of its proposed glycosyltransferase function (Wakarchuk et al., J. Biol. Chem. 271 (45): 28271276 (1996)). In N. Gonorrhoeae there are two additional genes, IgtD which adds β-D-Gal? Ac to position 3 of the terminal galactose of the lacto-N-neotetraose structure and IgtC which adds a terminal aD-Gal to the element of lactose from a truncated LOS, thus creating the structure of the blood group antigen Pk (Gotschlich (1994), cited above). In N. meningi tidis, a separate immunotype Ll also expresses the blood group antigen P and has shown to carry an IgtC gene (Jennings et al., (1995), cited above). Neisseria glycosyltransferases and associated genes are also described in USP? 5,545,553 (Gotschlich). The genes for al, 2-fucosyltransferase and al, 3-fucosyltransferase from Helicobacter pylori have also been characterized (Martin et al., J. Biol. Chem. 272: 31349-21356 (1997)). Likewise, the use of the Campylobacter j ejuni glucosyltransferases is contemplated in the present invention (see, for example, http: // afmb.C? RS-mrs. Fr / ~ pedro / CAZY / gtf_42.html).

Fucosyl transfrases In some embodiments, a glycosyltransferase used in the method of the invention is a fucosyltransferase. Fucosyltransferases are known to those skilled in the art. Exemplary fucosyltransferases include enzymes, which transfer GDP-fucose L-fucose to a hydroxy position of a receptor sugar. Fucosyltransferases that transfer non-nucleotide sugars to a receptor are also useful in the present invention.

In some embodiments, the receptor sugar is, for example, the GlcNAc in a Galβ (1-- 3,4) GlcNAcβ group in an oligosaccharide glycoside. Formulations of fucosyltransferases suitable for this reaction include the Galß (- 3, 4) GlcNAcßl-a (l -3,4) fucosyltransferase (FTIII EC No. 2.4.1.65), which was first characterized from human milk ( see, Palcic, et al., Carbohydrate Res. 190: 1-11 (1989), Prieels, et al., J. Biol. Chem. 256: 10456-10463 (1981), and Nunez, et al., Can J. Chem. 59: 2086-2095 (1981) and the Galß (l- »4) GlcNAcß-afucosyltransferases (FTIV, FIV, FTVI) which are found in human serum FTVII (EC No. 2.4.1.65) , a sialyl a (2-3) Galß ((1-3) GlcNAcß fucosyltransferase, has also been characterized.) A recombinant form of Galß (l-3, 4) GlcNAcß-a (l? 3,4) fucosyltransferase also has been characterized (See, Dumas, et al., Bioorg, Med. Letters 1: 425-428 (1991) and Kukowska-Latallo, et al., Genes and Development 4: 1288-1303. (1990)). Other fucosyltransfers include, for example, an al, 2-fucosyltransferase (E.C. No. 2.4.1.69). Enzymatic fucosylation can be carried out by means of the methods described in Mollicone, et al. , Eur. J. Biochem. 191: 169-176 (1990) or patent of E.U.A. No. 5,374,655. The cells that are used to produce fucosyltransferase will also include an enzymatic system to synthesize GDP-fucose.

Galactosil transf erases In another group of modalities, the glucosyltransferase is a galactosyltransferase. Exemplary galactosyltransferases include (1,3) galactosyltransferases (EC No. 2.4.1.151, see, for example, Dabkowski et al., Transplant Proc. 25: 2921 (1993) and Yamamoto et al., Nature 345: 229-233. (1990), bovine (GenBank J04989, Joziasse et al., J. Biol. Chem. 264: 14290-14297 (1989)), murine (GenBank m26925; Larsen et al., Proc. Nat'l. Acad. Sci. USA 86: 8227-8231 (1989)), swine (GenBank L36152, Strahan et al., Immunogenetics 41: 101-105 (1995)). Another suitable 3-galactosyltransferase is that which is involved in the synthesis of the antigen of the blood group B (EC 2.4.1.37, Yamamoto et al., J. Biol. Chem. 265: 1146-1151 (1990) (human)). A traditional exemplary galactosyltransferase is Gal-core Ti.They are also suitable for use in the methods of the invention the β (1,4) galactosyltransferases, which include, for example, EC 2.4.1.90 (LacNAc synthetase) and EC 2.4.1.22 (lactose synthetase) (bovine (D'Agostaro et al., Eur.

J. Biochem. 183: 2.11-217 (1989)), human (Masri et al., Biochem. Biophys. Res. Commun. 157: 657-663 (1988)), murine (Nakazawa et al., J. Biochem 104: 165-168 (1988)), as well as E.C.2.4.1.38 and the ceramide galactosyltransferase (EC 2.4.1.45, Stahl et al., J. Neurosci. Res. 38: 234-242 (1994)).

Other suitable galactosyltransferases include, for example, al, 2-galactosyltransferases (e.g., Schizosaccharomyces pombe, Chapell et al., Mol. Biol. Cell 5: 519-528 (1994)).

Sialiltransfrases Sialyltransferases are another type of glycosyltransferase that are useful in the recombinant cells and reaction mixtures of the invention. Cells that produce recombinant sialyltransferases will also produce CMP-sialic acid, which is a sialic acid donor for sialyltransferases. Examples of sialyltransferases that are suitable for use in the present invention include ST3Gal III (for example, rat or human ST3Gal III), ST3Gal IV ST3Gal I, ST3GalII, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, STdGalNAc II and ST6GalNAc III (the sialyltransferase nomenclature used herein is as described in Tsuji et al., Glycobiology 6: v-xiv (1996)). An exemplary a (2,3) sialyltransferase referred to as an a (2, 3) sialyltransferase (EC2.4.99.6) transfers sialic acid to the non-reducing terminal Gal of a disaccharide or Galßl-3Glc glycoside. See, Van den Eijnden et al. , J. Biol. Chem. 256: 3159 (1981), Weinstein et al. , J. Biol. Chem. 257: 13845 (1982) and Wen et al. , J. Biol. Chem. 267: 21011 (1992). Another exemplary a2, 3-sialyltransferase (EC 2.4.99.4) transfers sialic acid to the nonreducing terminal Gal of the disaccharide or glycoside. See, Rearick et al. , J. Biol. Chem. 254: 444 (1979) and Gillespie et al. , J. Biol. Chem. 267: 21004 (1992). Additional exemplary enzymes include Gal-β-1,4-GlcNAc α2,6 siallyltrasferase (See, Kurosawa et al., Eur. J. Biochem 219: 375-381 (1994)). Preferably, for the glycosylation of glycopeptide carbohydrates the sialyltransferase will be able to transfer sialic acid to the sequence Galßl, 4GlcNAc-, the penultimate most common sequence underlying terminal sialic acid on fully sialylated carbohydrate structures (see Table 2). Table 2 Sialyltransferases using the Galßl, 4GlcNAc sequence as a receptor substrate 1) Goochee et al. , Bio / Technology 9: 1347-1355 (1991) 2) Yamamoto et al. , J. Biochem. 120: 104-110 (1996) 3) Gilbert et al. , Biol. Chem. 271: 28271-28276 (1996) Other sialyltransferases for use in the present invention include those shown in the table of Figure 4. Sialyltransferases can be used to transfer a portion of PEGylated sialic acid from a sialic acid donor species. pegylated on an N-linked glycosyl residue of a peptide (Figure 2C) or a 0-linked glycosyl residue on Factor IX (Figure 2D). An example of a sialyltransferase that is useful in the claimed methods is ST3Gal III, which is also referred to as a (2, 3) sialyltransferase (EC 2.4.99.6). This enzyme catalyzes the transfer of sialic acid to Gal from a Galßl, 3GlcNAc or Galßl, 4GlcNAc glycoside (see, for example, Wen et al., J. Biol., Chem.267: 21011 (1992); Van den Eijnden et al. ., J. Biol. Chem. 256: 3159 (1991)) and is responsible for the sialylation of oligosaccharides linked to sparagin in glycopeptides. The sialic acid is bound to a Gal with the formation of a bond between the two saccharides. The binding (linkage) between the saccharides is between position 2 of NeuAc and position 3 of Gal. This particular enzyme can be isolated from rat liver (Weinstein et al., J. Biol. Chem. 257: 13845 (1982)), the human cDNA sequences are known (Sasaki et al. (1993) J. Biol. Chem. 268: 22782-22787; Kitagawa &Paulson (1994) J. Biol. Chem. 269: 1394-1401) and genomics (Kitagawa et al. (1996) J. Biol. Chem. 271: 931-938), facilitating the production of this enzyme by recombinant expression. In a preferred embodiment, the claimed sialylation methods use a rat ST3Gal III. Other exemplary sialyltransferases for use in the present invention include those isolated from Campylobacter jejuni, including a (2,3). See, for example, WO99 / 49051. Sialyltransferases other than those listed in Table 2 are also useful in a large-scale economical and efficient process for the sialylation of important commercial glycopeptides. As a simple test to find the utility of these other enzymes, several amounts of each enzyme (1-100 mU / mg protein) are reacted with asialo-ai AGP (at 1-10 mg / ml) to compare the capacity of the sialiltra? sferasa of interest for sialylating glycopeptides in relation with either ST6Gal I, ST3Gal III of bovine or both sialyltransferases. Alternatively, other glycopeptides or glycopeptides, or N-linked oligosaccharides enzymatically released from the peptide base structure can be used in place of asialo-ai AGP for this evaluation. Sialyltransferases with the ability to sialylate N-linked oligosaccharides of glycopeptides more efficiently than ST6Gal I are useful in a practical large-scale process for the sialylation of peptides.

GalNAc transf erases The N-acetylgalactosaminyltransferases are useful for practicing the present invention, particularly for attaching a GalNAc portion to an amino acid of the O-linked glycosylation site of the peptide. Suitable N-acetylgalactosaminyltransferases include, but are not limited to, (1, 3) N-acetylgalactosaminyltransferase, β (1,4) N-acetylgalactosaminyltransferases (Nagata et al., J. Biol. Chem. 267: 12082-12089 (1992 ) and Smith et al., J. Biol. Chem. 269: 15162 (1994)) and N-acetylgalactosaminyltransferase polypeptide (Homa et al., J. Biol. Chem. 268: 12609 (1993)). The production of proteins such as the enzyme GalNAc T _.- ?? from genes cloned by genetic engineering is well known. See, for example, US patent. No. 4,761,371. One method includes the collection of sufficient samples, then the amino acid sequence of the enzyme is determined by N-terminal sequencing. This information is then used to isolate a cDNA clone that codes for a full-length transferase (membrane bound) which after expression in the Sf9 insect cell line results in the synthesis of a fully active enzyme. The receptor specificity of the enzyme is then determined using a semiquantitative analysis of the amino acids surrounding the known glycosylation sites in sixteen different proteins followed by glycosylation studies in synthetic peptides. This work has shown that certain amino acid residues are overrepresented in segments of glycosylated peptides and that residues in specific positions surrounding serine and glycosylated serine and threonine residues may have a more marked influence on the efficiency of the receptor than in other portions of amino acids .

Cell-bound glucosyltransferases In another embodiment, the enzymes used in the method of the invention are cell-bound glycosyltransferases. Although many soluble glucosyltransferases are known (see, for example, U.S. Patent No. 5,032,519), glycosyltransferases are generally in membrane-bound form when associated with cells. Many of the membrane-bound enzymes studied to date are considered to be intrinsic proteins; that is, they are not released from the membranes by sonification and require detergents for their solubilization. Surface glycosyltransferases have been identified on the surfaces of vertebrate and invertebrate cells, and it has also been recognized that these surface transferases maintain catalytic activity under physiological conditions. However, the most recognized function of cell surface glycosyltransferases is for intercellular recognition (Roth, MOLECULAR APPROACHES to SUPRACELLULAR PHENOMENA, 1990). Methods have been developed to alter the glycosyltransferases expressed by cells. For example, Larsen et al., Proc. Nati Acad. Sci. USA 86: 8227-8231 (1989), report a genetic approach to isolate cloned cDNA sequences that determine the expression of oligosaccharide structures on cell surfaces and their cognate glucosyltransferases. A cDNA library generated from mRNA isolated from a murine cell line that is known to express UDP-galactose: β-D-galactosyl-1,4-N-acetyl-D-glucosaminide a-1,3-galactosyltransferase was transfected in COS-1 cells. The transfected cells were then cultured and assayed for activity at 1-3 galactosyltransferase. Francisco et al., Proc. Nati Acad. Sci. USA 8: 2713-2717 (1992), describe a method for anchoring β-lactamase to the outer surface of Escherichia coli. A tripartite fusion occurs which consists of (i) a signal sequence from an outer membrane protein, (ii) a membrane spanning section of an outer membrane protein, and (iii) a complete mature ß-lactamase sequence. , resulting in a β-lactamase molecule bound to active surface. However, Francisco's method is limited only to prokaryotic cell systems and, as recognized by the authors, requires a complete tripartite fusion for proper functioning.

Sulfotransfrases The invention also provides methods for producing peptides that include sulfated molecules, including, for example, sulfated polysaccharides such as heparin, heparan sulfate, carrageenan and related compounds. Suitable sulfotransferases include, for example, chondroitin-6-sulfotransferase (chicken cDNA described by Fukuta et al., J. Biol. Chem. 270: 18575-18580 (nineteen ninety five); GenBank Registration No. D49915), glycosaminoglucan N-acetylglucosamine N-deacetylase / N-sulfotransferase I (Dixon et al., Genomics 26: 239-241 (1995); UL18918) and glycosaminoglycan N-acetylglucosamine N-deacetylase / N-sulfotransferase 2 (murine cDNA described in Orellana et al. al., J. Biol. Chem. 269: 2270-2276 (1994) and Eriksson et al., J. Biol. Chem. 269: 10438-10443 (1994); human cDNA described in GenBank Registration No. U2304).

Glucosidase This invention also encompasses the use of mutant wild-type glycosidates. The mutant β-galactosidase enzymes have been shown to catalyze the formation of disaccharides through the coupling of an α-glycosyl fluoride to a galactosyl receptor molecule. (Withers, U.S. Patent No. 6,284,494, issued September 4, 2001). Other glycosidases useful in this invention include, for example, β-glucosidases, β-galactosidases, β-mannosidases, β-acetylglucosaminidases, β-N-acetylgalactosaminidases, β-xylosidases, β-fucosidases, cellulases, xylanases, galactanases, manases, hemicellulases, amylases, glucoamylases, a-glucosidases, a-galactosidases, a-mannosidases, α-N-acetylglucosaminates, α-N-acetyl-galactose-amidases, α-xylosidases, α-fucosidases and neuraminidases / sialidases. In an exemplary embodiment, a sialidase is used to remove sialic acid from a factor IX N-glycan (Figure 2A) prior to glycoPEGylation. The invention also provides a method that does not require the prior removal of sialic acid. Thus, a method incorporating a sialic acid exchange reaction using a modified sialic acid portion and ST3Gal3 is useful in the present invention. Immobilized Enzymes The present invention also provides the use of enzymes that are immobilized on a solid and / or soluble support. In an exemplary embodiment, a glycosyltransferase is provided which is conjugated to a PEG by means of an intact glycosyl linker according to the methods of the invention. The PEG-linker enzyme conjugate is optionally attached to a solid support. The use of enzymes supported on solids in the methods of the invention simplifies the treatment of the reaction mixture and purification of the reaction product, and also makes possible the easy recovery of the enzyme. The glucosyltransferase conjugate is used in the methods of the invention. Other combinations of enzymes and supports will be apparent to those skilled in the art.

Fusion proteins In other exemplary embodiments, the methods of the invention utilize fusion proteins that have more than one enzymatic activity that is involved in the synthesis of a desired glycopeptide conjugate. The fusion polypeptides may be composed of, for example, a catalytically active domain of a glycosyltransferase that is linked to a catalytically active domain of an accessory enzyme. The catalytic domain of the accessory enzyme can, for example, catalyze a step in the formation of a nucleotide sugar that is a donor for the glycosyltransferase, or catalyze a reaction involved in a glycosyltransferase cycle. For example, a polynucleotide encoding a glycosyltransferase can be bound, in frame, or polynucleotide that codes for an enzyme involved in the synthesis of nucleotide sugars. The resulting fusion protein can then catalyze not only the synthesis of the nucleotide sugar, but also the transfer of the sugar portion to the receptor molecule. The fusion protein can be two or more cyclic enzymes linked in an expressible nucleotide sequence. In other embodiments, the fusion protein includes the catalytically active domains of two or more glycosyltransferases. See, for example, 5,641,668. The modified glycopeptides of the present invention can be designed and manufactured easily using various suitable fusion proteins (see, for example, PCT patent application PCT / CA98 / 01180, which was published as WO 99/31224 on June 24, 1999 ).

Preparation of Modified Sugars In general, the portion of sugar or linker cassette of sugar portion and the groups of cassettes of PEG or PEG-linker are linked together through the use of reactive groups, which are typically transformed by the linking process in a new organic functional group or non-reactive species. Reactive sugar functional groups are located at any position in the sugar portion. The reactive groups and classes of reactions useful for practicing the present invention are generally those that are well known in the bioconjugate chemistry art. The classes of presently preferred reactions available with reactive sugar moieties are those that proceed under relatively mild conditions. These include, but are not limited to, nucleophilic substitutions (eg, reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (eg, enamine reactions) and additions to carbon-carbon and carbon-carbon multiple bonds. heteroatom (for example, Michael's reaction, addition of Diels-Alder). These and other useful reactions are described, for example, in March, ADVANCED ORGANIC CHEMISTRY, 3rd ed. , John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al. , MODIFICATION OF PROTEINS; Advances in Chemistry Series, vol. 198, American Chemical Society, Washington, DC, 1982. Useful reactive functional groups that hang from a sugar core or modifier group include, but are not limited to: (a) carboxyl groups and various derivatives thereof including, but not limited to, limited to N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b) hydroxyl groups, which can be converted, for example, into esters, ethers, aldehydes, etc. (c) haloalkyl groups, in which the halide can be subsequently displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, a thiol anion, a carbanion or an alkoxide ion, thus resulting in the binding covalent of a new group in the functional group of the halogen atom; (d) dienophile groups, which are capable of participating in Diels-Alder reactions such as, for example, maleimido groups; (e) aldehyde or ketone groups, such that subsequent derivatization is possible by means of the formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or by mechanisms such as the addition of Grignard or addition of alkyl lithium; (f) sulfonyl halide groups for the subsequent reaction with amines, for example, to form sulfonamides; (g) thiol groups, which can, for example, be converted to disulfides or reacted with acyl halides; (h) amine or sulfhydryl groups, which may, for example, be acylated, alkylated or oxidized; (i) alkenes, which may suffer, for example, cycloadditions, acylation, addition of Michael, etc .; and (j) epoxides, which may react, for example, with amines and hydroxyl compounds. The reactive functional groups can be selected such that they do not participate in, or interfere with, the reactions necessary to assemble the reactive sugar core or modification group. Alternatively, a reactive functional group can be protected from participating in the reaction by the presence of a protecting group. Those skilled in the art understand, to protect a particular functional group in such a way that it does not interfere with a selected set of reaction conditions. For examples of useful protecting groups, see, for example, Greene et al. , PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991. In an exemplary embodiment, the peptide that is modified by a method of the invention is a glycopeptide that is produced in mammalian cells (e.g., CHO cells) or in a transgenic animal and thus, contains N- and / or 0-linked oligosaccharide chains, which are incompletely sialylated. The oligosaccharide chains of the glycopeptide lacking a sialic acid and containing a terminal galactose residue can be PEGylated, PPGylated or otherwise modified with a modified sialic acid. In reaction scheme 4, aminoglycoside 1 is treated with the active ester of a protected amino acid (eg, glycine), converting the sugar amine residue to the corresponding protected amino acid amide adduct. The adduct is treated with an aldolase to form an α-hydroxycarboxylate 2. The compound 2 is converted to the corresponding CMP derivative by the action of CMP-SA synthetase, followed by the catalytic hydrogenation of the CMP derivative to produce compound 3. The amine introduced via the formation of the glycine adduct is used as a PEG binding locus by reacting compound 3 with an activated PEG or PPG derivative (eg, PEG-C (0) NHS, PEG-OC (O) Op-nitrophenyl), producing species such as 4 or 5, respectively.

Reaction scheme 4 CMP-SA-5-NHCOCH2JSIH-C (0) 0-PEG 5 Table 3 illustrates representative examples of sugar monophosphates which are derivatives with a PEG moiety. Certain of the compounds of Table 3 are prepared by the method of reaction scheme 4. Other derivatives are prepared by methods known in the art. See, for example, Keppler et al. , Glycobiology ll.llR (2001) and Charter et al. , Glycobiology 10: 1049 (2000)). Other PEG and PPG analogs reactive with .amine are commercially available, or can be prepared by methods readily available to those skilled in the art.

Table 3 CMP-NeuAc-4-NH-R CMP-NeuAc-4-O-R The modified sugar phosphates for use in the practice of the present invention can be substituted in other positions as well as those described above. The sialic acid substitutions that are especially preferred are shown in the following formula: wherein X is a linking group, which is preferably selected from -O-, -N (H) -, -S, CH2- and -N (R) 2, in which each R is a member independently selected of R1-R5. The symbols Y, Z, A and B each represent a group that is selected from the group shown above for the identity of X. X, Y, Z, A and B are each independently selected and, therefore, may be the same. same or different. The symbols R1, R2, R3, R4 and R5 represent H, a PEG portion, a therapeutic portion, biomolecule or other portion. • Alternatively, these symbols represent a linker that is linked to a PEG portion, therapeutic portion, biomolecule, or other portion. Exemplary moieties attached to the conjugates described herein include, but are not limited to, PEG derivatives (eg, acyl-PEG, acyl-alkyl-PEG, alkyl-acyl-PEG carbamoyl-PEG, aryl-PEG), derivatives of PPG (for example, acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-PPG carbamoyl-PPG, aryl-PPG), therapeutic portions, diagnostic moieties, mannose-6-phosphate, heparin, heparan, Slex, mannose , Mannose-6-phosphate, Sialyl Lewis X, FGF, VFGF, proteins, chondroitin, keratan, dermatan, albumin, integrins, antennal oligosaccharides, peptides and the like. Methods for conjugating the different modifying groups to a saccharide moiety are readily available to those skilled in the art (POLY (ETHYLENE GLYCOL CHEMISTRY: BIOTECHNICAL AND BIOMEDICAL APPLICATIONS, J. Milton Harris, Ed., Plenum Pub. Corp., 1992; POLY (ETHYLENE GLYCOL) CHEMICAL AND BIOLOGICAL APPLICATIONS, J. Milton Harris, Ed., ACS Symposium Series No. 680, American Chemical Society, 1997, Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996 and Dunn et al., Eds POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series vol.491, American Chemical Society, Washington, DC 1991).

Linker Groups (Linker Groups) The preparation of the modified sugar for use in the methods of the present invention includes fixing a PEG portion to a sugar residue and preferably forming a stable adduct, which is a substrate for a glycosyltransferase. Thus, it is commonly preferred to use a linker, for example, one formed by the reaction of the PEG portion and sugar with an interlacing agent to conjugate the PEG and the sugar. Exemplary bifunctional compounds that can be used to link modifying groups to carbohydrate moieties include, but are not limited to, poly (ethylene glycol) is bifunctional, polyamides, polyethers, polyesters, and the like. Similar approaches for linking carbohydrates to other molecules are known in the literature. See, for example, Lee et al. , Biochemistry 28: 1856 (1989); Bhatia et al. , Anal. Biochem. 178: 408 (1989); Janda et al. , J. Am. Chem. Soc. 112: 8886 (1990) and Bednarski et al. , WO 92/18135. In the following description, the reactive groups are treated as benign in the sugar portion of the nascent modified sugar. The focus of the description is for clarity of illustration. Those skilled in the art will appreciate that the description is relevant to reactive groups in the modifier group as well. A variety of reagents are used to modify the modified sugar components with intramolecular chemical entanglements (for revisions of interlacing reagents and entanglement procedures see: Wold, F., eth. Enzymol. 25: 623-651, 1972; Weetall, H. H., and Cooney, D. A., In: ENZYMES AS DRUGS. (Holcenberg and Roberts, ed.) Pages. 395-442, Wiley, New York, 1981; Ji, T. H., Meth. Enzymol. 91: 580-609, 1983; Mattson et al. Mol. Biol. Rep. 17: 167-183, 1993, all of which are incorporated herein by reference). Preferred crosslinking reagents are derived from various zero-length, homo-bifunctional and hetero-bifunctional crosslinking reagents. The zero-length entanglement reagents include the direct conjugation of two intrinsic chemical groups without introduction of extrinsic material. The agents that catalyze the formation of a disulfide bond belong to this category. Another example are reagents that induce the condensation of a carboxyl and a primary amino group to form an amide bond such as carbodiimides, ethyl chloroformate, Woodward's Reagent K (3'-sulfonate, 2-ethyl-5-phenylisoxazolium sulfonate). ) and carbonyldiimidazole. In addition to these chemical reagents, the enzyme transglutaminase (glutamyl-peptide? -glutamyltransferase; EC 2.3.2.13) can be used as a zero-length entanglement reagent. This enzyme catalyzes the acyl transfer reactions in carboxamide groups of glutaminyl residues bound to proteins, usually with a primary amino group as a substrate. The homo- and hetero-bifunctional reagents that are preferred contain two identical sites or two dissimilar sites, respectively, which may be reactive for amino, sulfhydryl, guanidino, indole or non-specific groups.

Purification of Factor IX Conjugates The products produced by the above processes can be used without purification. However, it is usually preferred to recover the product. Standard and well known techniques for the recovery of glycosylated saccharides such as thin or thick layer chromatography, column chromatography, ion exchange chromatography or membrane filtration can be used. It is preferred to use membrane filtration, most preferably using a reverse osmotic membrane, or one or more column chromatographic techniques for recovery as described hereinafter and in the literature cited herein. For example, membrane filtration in which the membranes have a molecular weight limit of about 3,000 to about 10,000 can be used to remove proteins such as glycosyltransferases. The nanofiltration or reverse osmosis can then be used to remove salts and / or purify saccharides from the product (see, for example, WO 98/15581). Nanofiltration membranes are a class of reverse osmosis membranes that pass monovalent salts but retain polyvalent salts and uncharged solutes of more than about 100 to about 2,000 daltons, depending on the membrane used. Thus, in a typical application, the saccharides prepared by the methods of the present invention will be retained in the membrane and the contaminating salts will pass through it.

If the modified glycoprotein is produced intracellularly, as a first step, the remains of particles, either host cells or fragments used, are removed, for example, by centrifugation or ultrafiltration; optionally, the protein can be concentrated with a commercially available protein concentration filter, followed by the separation of the polypeptide variant from other impurities by one or more steps selected from immunoaffinity chromatography, fractionated on ion exchange column (e.g. diethylaminoethyl (DEAE) or matrices containing carboxymethyl or sulfopropyl groups), chromatography on Blue-Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S, lentil lectin-Sepharose, WGA-Sepharose, With A-Sepharose, Toyopearl Ether, Butyl Toyope rl, Toyopearl Phenyl or Protein A Sepharose, SDS-PAGE chromatography, silica chromatography, chromatofocusing, reverse phase HPLC (for example, silica gel with attached aliphatic groups), gel filtration using, for example, a molecular sieve Sephadex or size exclusion chromatography, chromatography on columns that selectively bind to the polypeptide, and precipitation with ammonium sulfate or or ethanol. Modified glycopeptides produced in culture are usually isolated by initial extraction from cells, enzymes, etc., followed by one or more of concentration, desalification, aqueous ion exchange or size exclusion chromatography. In addition, the modified glycoprotein can be purified by affinity chromatography. Finally, HPLC can be used for final purification steps. A protease inhibitor, for example, methylsulfonyl fluoride (PMSF) may be included in any of the above steps to inhibit proteolysis and antibiotics may be included to prevent the growth of upstart contaminants. In another embodiment, supernatants of systems that produce the modified glycopeptide of the invention are first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be applied to a suitable purification matrix. For example, a suitable affinity matrix may comprise a ligand for the peptide, a lectin or antibody molecule bound to a suitable support. Alternatively, an anion exchange resin may be used, for example, a matrix or substrate that has hanging DEAE groups. Suitable matrices include acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include several insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are particularly preferred. Finally, one or more RP-HPLC steps employing hydrophobic RP-HPLC media, for example, silica gel having pendant methyl groups or other aliphatic groups, can be used to further purify a variant composition of polypeptides. Some or all of the above purification steps, in various combinations, may also be employed to provide a homogenous modified glycoprotein. The modified glycopeptide of the invention resulting from large-scale fermentation can be purified by methods analogous to those described by Urdal et al. , J. Chromatog. 296: 171 (1984). This reference describes two sequential RP-HPLC steps for the purification of recombinant human IL-2 on a preparative HPLC column. Alternatively, techniques such as affinity chromatography can be used to purify the modified glycoprotein.

Pharmaceutical Compositions In another aspect, the invention provides a pharmaceutical composition. The pharmaceutical composition includes a pharmaceutically acceptable diluent and a covalent conjugate between a non-naturally occurring portion of PEG, therapeutic portion or biomolecule and a glycosylated or non-glycosylated factor IX peptide. The polymer, therapeutic moiety or biomolecule is conjugated to the peptide by means of an intact glycosyl linker group interposed between and covalently linked to both the peptide and the polymer, therapeutic moiety or biomolecule. The pharmaceutical compositions of the invention are suitable for use in a variety of drug delivery systems. Formulations suitable for use in the present invention are found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985). For a brief review of drug delivery methods, see, Langer, Science 249: 1527-1533 (1990). The pharmaceutical compositions can be formulated for any suitable manner of administration, including, for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the vehicle preferably comprises water, saline, alcohol, a fat, a wax or a pH regulator. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate may be employed. Biodegradable microspheres (eg, polylactate polyglycolate) can also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are described, for example, in the U.S. Patents. Nos. 4,897,268 and 5,075,109. Commonly, the pharmaceutical compositions are administered parenterally, for example, intravenously. Thus, the invention provides compositions for parenteral administration comprising the dissolved compound suspended in an acceptable vehicle, preferably an aqueous vehicle, for example, water, pH-regulated water, saline, PBS and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required for approximately physiological conditions, such as pH adjusting agents and pH regulators, tonicity adjusting agents, wetting agents, detergents and the like. These compositions can be sterilized by conventional sterilization techniques, or they can be sterile filtered. The resulting aqueous solutions can be packaged for use as such, or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicle prior to administration. The pH of the preparations will typically be between 3 and 11, most preferably 5 to 9 and more preferably 7 and 8. In some embodiments, the glycopeptides of the invention can be incorporated into liposomes formed from standard vesicle-forming lipids. A variety of methods available to prepare liposomes, as described, for example, in Szoka et al. , Ann. Rev. Biophys. Bioeng. 9: 467 (1980), patent of E.U.A. Nos. 4,235,871, 4,501,728 and 4,837,028. Addressing liposomes using a variety of targeting agents (eg, the sialyl galactosides of the invention) is well known in the art (see, for example, U.S. Patent Nos. 4,957,773 and 4,603,044). Standard methods for coupling targeting agents to liposomes can be used. These methods generally include the incorporation into liposomes of lipid components, such as phosphatidylethanolamine, which can be activated for binding of targeting agents, or derived lipophilic compounds, such as glycopeptides derived from lipids of the invention. The steering mechanisms generally require that the steering agents be placed on the surface of the liposome in such a way that the steering portions are available for their interaction with the target., for example, a receptor on the cell surface. The carbohydrates of the invention can be attached to a lipid molecule before the liposome is formed using methods well known to those skilled in the art (eg, alkylation or acylation of a hydroxyl group present on the carbohydrate with an alkyl halide of long chain or with a fatty acid, respectively). Alternatively, the liposome can be designed in such a way that a connecting portion is first incorporated into the membrane at the time of forming the membrane. The connecting portion must have a lipophilic portion, which is firmly embedded and anchored in the membrane. It must also have a reactive portion, which is chemically available on the aqueous surface of the liposome. The reactive portion is selected such that it is chemically adequate to form a stable chemical bond with the targeting agent or carbohydrate, which is then added. In some cases it is possible to attach the target agent directly to the linker molecule, but in most cases it is more appropriate to use a third molecule to act as a chemical bridge, thereby binding the linker molecule that is in the membrane with the target agent or carbohydrate that is extended, three-dimensionally, outside the surface of the vesicle. The compounds prepared by the methods of the invention can also find utility as diagnostic reagents. For example, labeled compounds can be used to locate areas of inflammation or tumor metastasis in a patient suspected of having inflammation. For this use, the compounds can be labeled with 125 I, 14 C or tritium. The active ingredient used in the pharmaceutical compositions of the present invention is glycopegylated factor IX and its derivatives which have the biological properties of participating in the blood coagulation cascade. The liposomal dispersion of the present invention is useful as a parenteral formulation for treating coagulation disorders characterized by low or defective coagulation such as various forms of hemophilia. Preferably, the factor IX composition of the present invention is administered parenterally (eg, IV, IM, SC or IP). Effective doses are expected to vary considerably depending on the condition being treated and the route of administration but are expected to be on the scale of about 0.1 to 000 μg / kg body weight of the active material. Preferred doses for the treatment of coagulation disorders are about 50 to about 3,000 μg / kg three times a week. Most preferably, about 500 to about 2,000 μg / kg three times a week. More preferably, about 750 to about 1,500 μg / kg three times a week and most preferably about 1,000 μg / kg three times a week. Because the present invention provides an XI factor with an increased in vivo residence time, the doses indicated are optionally reduced when a composition of the invention is administered. The following examples are provided to illustrate the conjugates, and methods of the present invention, but not to limit the reclining invention.

Eg emplos Example 1 Preparation of UOP-GalNAc-6-CHO UDP-GalNAc (200 mg, 0.30 mmol) was dissolved in a solution of 1 mM CuS04 (20 mL) and a 25 mM NaH2P0 solution. (pH 6.0, 20 mL). Then galactose oxidase was added (240 OR; 240 μL) and catalase (13,000 U; 130 μL), the reaction system was equipped with a balloon filled with oxygen and stirred at room temperature for seven days. The reaction mixture was then filtered (centrifugal cartridge, MWCO 5K) and the filtrate (-40 mL) was stored at 4 ° C until required. TLC (silica; EtOH / water (7/2); Rf = 0.77, visualized with anisaldehyde stain).

Example 2 Preparation of UDP-GalNAc-6 '-NH2 Ammonium acetate (15 mg, 0.194 mmol) and NaBH3CN (solution in 1M THF, 0.17 mL, 0.17 mmol) were added to the solution of UDP-GalNAc-6 '-CHO above (2 mL or -20 mg) at 0 ° C and allowed to warm to room temperature during the night. The reaction was filtered through a G-10 column with water and the product was collected. The appropriate fractions were freeze-dried and stored frozen. TLC (silica: ethanol / water (7/2); Rf = 0.72, visualized with ninhydrin reagent).

Example 3 Preparation of UDP-GalNAc-6-NHCO (CH2) 2-0-PEG-OMe (1 KDa) Galactosaminyl-l-phosphate-2-NHCO (CH2) 2-0-PEG-OMe (1 KDa) (58 mg, 0.045 mmol) was dissolved in DMF (6 mL) and pyridine (1.2 mL). Then UMP-morfolidate (60 mg, 0. 15 mmol) and the resulting reaction mixture was stirred at 70 ° C for 48 hours. The solvent was removed by bubbling nitrogen through the reaction mixture and the residue was purified by reverse phase chromatography (C-18 silica, step gradient between 10 to 80%, methanol / water). The desired fractions were collected and dried under reduced pressure to give 50 mg (70%) of a white solid. TLC (silica, propanol / H20 / NH40H, (30/20/2), Rf = 0.54). MS (MALDI): Observed, 1485, 1529, 1618, 1706.

Example 4 Preparation of cysteine-PEG2 (2) 4. 1 Synthesis of (1) Potassium hydroxide (84.2 mg, 1.5 mmol, as a powder) was added to a solution of L-cysteine (93.7 mg, 0.75 mmol) in anhydrous methanol (20 mL) under argon. The mixture was stirred at room temperature for 30 min. , and then mPEG-O-tosylate with a molecular weight of 20 kilodaltons (Ts, 1.0 g, 0.05 mmol) was added in several portions over 2 hours. The mixture was stirred at room temperature for 5 days and concentrated by rotary evaporation. The residue was diluted with water (30 mL) and stirred at room temperature for two hours to destroy any excess of 20 kilodalton mPEG-O-tosylate. The solution was then neutralized with acetic acid, adjusted to pH 5.0 and loaded onto reverse phase chromatography (silica C-18 column). The column was eluted with a methanol / water gradient (the product elutes at about 70% methanol), elution of the product was monitored by evaporative light scattering, and the appropriate fractions were collected and diluted with water (500 mL). This solution was subjected to chromatography (ion exchange, XK 50 Q, BIG beads, 300 mL, hydroxide form, water to water / acetic acid gradient-0.75N) and the pH of the appropriate fractions was lowered to 6.0 with acetic acid. This solution was then captured on a reverse phase column (C-18 silica) and eluted with a methanol / water gradient as described above. The product fractions were pooled, concentrated, redissolved in water and freeze-dried to give 453 mg (44%) of a white solid solvent (1). The structural data for the compound were as follows: ^ • H-NMR (500 MHz; D20) d 2.83 (t, 2H, 0-C-CH2-S), 3.05 (q, 1H, S-CHH-CHN), 3.18 (q, ÍH, (q, 1H, S-CHH-CHN), 3.38 (s, 3H, CH30), 3.7 (t, OCH2CH20), 3.95 (q, 1H, CHN) The purity of the product was confirmed by SDS PAGE. 4. 2 Synthesis of (2) Triethylamine (-0.5 mL) was added dropwise to a solution of 1 (440 mg, 22 μmol) dissolved in anhydrous CHC12. (30 mL) until the solution became basic. A solution of 20 kilodaltons of mPEG-O-p-nitrophenyl carbonate (660 mg, 33 μmol) and N-hydroxysuccinimide (3.6 mg, 30.8 μmol) in CH2C12 (20 mL) was added in several portions for one hour at room temperature. The reaction mixture was stirred at room temperature for 24 hours. The solvent was then stirred by rotary evaporation, the residue was dissolved in water (100 mL); and the pH was adjusted to 9.5 with 1.0 N NaOH. The basic solution was stirred at room temperature for two hours and then neutralized with acetic acid to a pH of 7.0. The solution was then loaded onto a reverse phase chromatography column (silica C-18). The column was eluted with a gradient of methanol / water (the product elutes at about 70% methanol), the product elution was monitored by evaporative light scattering, and the appropriate fractions were collected and diluted with water (500 mL). This solution was subjected to chromatography (ion exchange, XK 50 Q, BIG Beads, 300 mL, hydroxide form, water to water / acetic acid-0.75N gradient) and the pH of the appropriate fractions was lowered to 6.0 with acetic acid. This solution was then captured on a reverse phase column (silica C-18) and eluted with a methanol / water gradient as described above. The product fractions were pooled, concentrated, redissolved in water and freeze dried to give 575 mg (70%) of a white solid (2). The structural data for the compound were the following: ^ -RMN (500 MHz; D20) d 2.83 (t, 2H, 0-C-CH2-S), 2.95 (t, 2H, 0-C-CH2-C), 3.12 (q, 1H, S-CHH-CHN), 3.39 (s, 3H CH30), 3.71 (t, 0CH2CH20). The purity of the product was confirmed by SDS PAGE.

Example 5 Preparation of UDP -GalNAc- 6 -NHCO (CH2) 2-0- PEG-OMe (1 KDa) I-Phosphosate of galactosaminyl-2-NHCO (CH2) 2-0-PEG-OMe (1 kilodalton) ( 58 mg, 0.045 mmol) was dissolved in DMF (6 mL) and pyridine (1.2 mL). Then UMP-morfolidate (60 mg, 0.15 mmol) was added and the resulting mixture was stirred at 70 ° C for 48 hours. The solvent was removed by bubbling nitrogen through the reaction mixture and the residue was purified by reverse phase chromatography (silica C-18, step gradient between 10 to 80%, methanol / water). The desired fractions were collected and dried under reduced pressure to give 50 mg "(70%) of a white solid. TLC (silica, propanol / H20 / NH40H, (30/20/2), Rf = 0.54). MS (MALDI): Observed, 1485, 1529, 1618, 1706.

Example 6 GlycoPEGylation of factor IX produced in CHO cells This example shows the preparation of asialof actor IX and its sialylation with CMP-sialic acid-PEG. 6. 1 Desialylation of rFactor IX A recombinant form of the Coagulation Factor-IX (rFactor XI) was made in CHO cells. 6,000 International Units of rFactor IX were dissolved in a total of 12 mL of H20 USP. This solution was transferred to a centrifugal Centricon Plus 20 filter, PL-10 with another 6 ml of H20 USP. The solution was concentrated to 2 mL and then diluted with 15 mL of 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 5 mM CaCl 2, 0.05% NaN 3 and then re-concentrated. The dilution / concentration was repeated four times to effectively change the pH regulator to a final volume of 3.0 mL. From this solution, 2.9 mL (approximately 29 mg of rFactor IX) were transferred to a small plastic tube and to this was added 530 mU of the a2-3,6,8-neuraminidase-agarose conjugate (Vibrio cholerae, Calbiochem, 450 μL). The reaction mixture was gently rotated for 26.5 hours at 32 ° C. The mixture was centrifuged two minutes at 10,000 rpm and the supernatant was collected. The agarose beads (containing neuraminidase) were washed six times with 0.5 mL of 50 mM Tris-HCl, pH 7.12, 1 M NaCl, 0.05% NaN3. The pooled washes and supernatants were centrifuged again for two minutes at 10,000 rpm to remove any residual residual agarose. The desililated and pooled protein solution was diluted to 19 mL with the same pH regulator and concentrated to - 2 mL in a Centricon Plus 20 PL-10 centrifugal filter. The solution was diluted twice with 15 mL of 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05% NaN3 and re-concentrated to 2 mL. The final desialylated rFactor IX solution was diluted to 3 mL final volume (-10 mg / mL) with the Tris pH regulator. Native and desialylated rFactor IX samples were analyzed by IEF-electrophoresis. Isoelectric focusing gels (pH 3-7) were run using 1.5 μL (15 μg) samples diluted first with 10 μL of Tris pH buffer and mixed with 12 μL of sample charge pH regulator. The gels were loaded, run and fixed using standard procedures. The gels were stained with Colloidal Blue stain (figure 154), demonstrating a band for desialylated factor IX.

Example 7 Preparation of PEG (1 kDa and 10 kDa) -SA-Factor IX desialylated rFactor-IX (29 mg, 3 mL) was divided into two 1.5 mL samples (14.5 mg) in two 15 mL centrifuge tubes. Each solution was diluted with 12.67 mL of 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05% NaN3 and either CMP-SA-PEG-lk or lOk (7.25 μmol) was added. The tubes were gently inverted to mix and 2.9 U of ST3Gal3 (326 μL) (total volume 14.5 mL) were added. The tubes were inverted again and rotated gently for 65 hours at 32 ° C. The reactions were stopped by freezing at -20 ° C. Samples of 10 μg of the reactions were analyzed by SDS-PAGE. The PEGylated proteins were purified on a Tosa Haas Biosep G3000SW HPLC column (21.5 x 30 cm, 13 um) with Dulbeco phosphate pH regulated saline, pH 7.1 (Gibco), 6 mL / min. The reaction and purification were monitored using SDS Page and IEF gels. The 1 mm gels of Novex Tris-Glycine 4-20% were loaded with 10 μL (10 μg) of samples after dilution with 2 μL of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% regulator NaN3 pH and mixing with a pH regulator sample charge of 12 μL and 1 μL of 0.5 M DTT and heated for 6 minutes at 85 ° C. The gels were stained with Colloidal Blue stain (figure 155) showing a band for PEG (1 kDa and 10 kDa) -SA-Factor ix.

Example 8 Direct Sialyl-GlycopePEGylation of Factor IX This example describes the preparation of sialyl-PEGylation of Factor IX without previous treatment with sialidase. 8. 1 Sialyl-PEGylation of Factor IX with CMP-SA-PEG- (10 KDa) Factor IX (1100 IU), which was expressed in CHO cells and was completely sialylated, dissolved in 5 mL of 20 mM histidine, 520 mM glycine , 2% sucrose, 0.05% NaN3 and 0.01% polysorbate 80, pH 5.0. The CMP-SA-PEG- (10 kDa) (27 mg, 2.5 μmol) was then dissolved in the solution and 1 U of ST3Gal3 was added. The reaction was completed after mixing gently for 28 hours at 32 ° C. The reaction was analyzed by SDS-PAGE as described by Invitrogen. The product protein was purified on an Amersham Superdex 200 HPLC column (10 x 300 mm, 13 μm) pH phosphate buffered saline, pH 7.0 (PBS), 1 mL / min. R5 = 9.5 min.

Example 9 Sialyl-PEGylation of Factor IX with CMP-SA-PEG- (20 kDa) Factor IX (1100 IU), which was expressed in CHO cells and completely sialylated, was dissolved in 5 mL of 20 mM histidine, glycine 520 mM, 2% sucrose, 0.05% NaN3 and 0.01% polysorbate 80, pH 5.0. The CMP-SA-PEG- (20 kDa) (50 mg, 2.3 μmol) was then dissolved in the solution and CST-II was added. The reaction mixture was complete after mixing gently for 42 hours at 32 ° C. The reaction was analyzed by SDS-PAGE as described by Invitrogen. The product protein was purified on an Amersham Superdex 200 HPLC column (10 x 300 mm, 13 μm) with phosphate buffered saline, pH 7.0 (Fisher), 1 mL / min. R = 8.6 min.

EXAMPLE 10 Blockade of glycosylated factor IX sialic acid ends These examples show the procedure for sialic acid blocking of the sialyl glycopegylated peptide ends. Here, factor IX is the exemplary peptide. 10. 1 Blocking of sialic acid ends of N-linked and O-linked factor-IX-SA-PEG (10 kDa) glycans r-Factor-IX-PEG (10 kDa) (2.4 mg) was concentrated in a centrifugal filter Centricon "Plus 20 PL-10 (Millipore Corp., Bedford, MA) and the pH regulator was changed to 50 mM Tris-HCl pH 7.2, 0.15 M NaCl, 0.05% NaN3 for a final volume of 1.85 mL. The protein solution was eluted with 372 μL of the same pH regulator Tris and 7.4 mg of CMP-SA (12 μmoles) were added as a solid. The solution was inverted gently to mix and 0.1 U ST3Gall and 0.1 U ST3Gal3 were added. The reaction mixture was gently rotated for 42 hours at 32 ° C. A 10 μg sample of the reaction was analyzed by SDS-PAGE. 1 mm Novex Tris-Glycine 4-12% gels were carried out and stained using Colloidal Blue as described by Invitrogen. Briefly, samples, 10 μL (10 μg), were mixed with 12 μL of sample charge pH regulator and 1 μL of 0.5 M DTT and heated for 6 minutes at 85 ° C (Figure 156, line 4).

EXAMPLE 11 Pharmacokinetic Study of Glicopiglylated Factor IX Four glycoPEGylated FIX variants (variants PEG-9) were tested in a PK study in normal mice. The activity of the four compounds had previously been established in vi tro by the coagulation assays, endogenous thrombin potential (ETP) and thromboelastography (TEG). The activity results are summarized in table 1.

To evaluate the prolongation of the activity of the four PEG-9 compounds in the circulation, a PK study was designed and carried out. Non-hemophilic mice were used, two animals per time point, three samples per animal. The sampling time points were 0, 0.08, 0.17, 0.33, 1, 3, 5, 8, 16, 24, 30, 48, 64, 72 and 96 hours after the administration of the compound. The blood samples were centrifuged and stored in two aliquots; one for clot analysis and one for ELISA. Due to material restrictions, the PEG-9 compounds were dosed in different quantities: BeneFIX 250 U / kg; 2K (low substitution: "LS" (1-2 substitutions with PEG per peptide molecule) 200 U / kg; 2K (high substitution: "HS" (3-4 PEG substitutions per peptide molecule) 200 U / kg; 10K 100 U / kg; 30K 100 U / kg All doses were based on measured coagulation units, the results are delineated in figure 6 and table II.

Table II The results demonstrate a trend towards prolongation for all PEG-9 compounds. The values of AUC and Cmax are not directly compared. However, the elimination (CL) was compared and the CL is lower for the PEG-9 compounds compared to BeneFIX, indicating a longer residence time in the mice. The time for less detectable clot activity is increased for the PEG-9 compounds compared to BeneFIX, even though BeneFIX was administered at the highest dose.

Example 12 Preparation of glycoPEGylated Factor IX of LS and HS glycoPEGylated Factor IX with a low degree of substitution with PEG was prepared from native factor IX of an exchange reaction catalyzed by ST3Gal-III. The reactions were carried out in a pH regulator of 10 mM histidine, 260 mM glycine, 1% sucrose and 0.02% Tween 80, pH 7.2. For PEGylation with CMPSA-PEG (2 kD and 10 kD), Factor XI (0.5 mg / mL) was incubated with ST3GalIII (50 mU / mL) and CMP-SA-PEG (0.5 mM) for 16 hours at 32 ° C . For PEGylation with CMP-SA-PEG 30 kD, the concentration of factor IX was increased to 1.0 mg / mL, and the concentration of CMP-SA-PEG was reduced to 0.17 mM. Under these conditions, more than 90% of the factor IX molecules were substituted with at least one portion of PEG.

GlycoPEGylated Factor IX with a high degree of substitution with PEG was prepared by enzymatic desialylation of native factor IX. The factor IX peptide was exchanged in pH buffer at 50 mM mES, pH 6.0, using a PD10 column, adjusted to a concentration of 0.66 mg / mL. and treated with AUS sialidase (5 mU / mL) for 16 hours at 32 ° C. The desialylation was verified by glucan analysis SDS-PAGE, HPLC and MALDI. Asialofactor IX was purified on Q Sepharose FF to remove the sialidase. The CaCl2 fraction was concentrated using an Ultral5 concentrator and the pH regulator was exchanged for MES, pH 6.0 using a PD10 column. PEGylation of 2kD and 10 kD of asialo-factor IX (0.5 mg / mL) was carried out by incubation with ST3Gal-III (50 mU / mL) and CMP-SA-PEG (0.5 mM) at 32 ° C for 16 days. hours. For PEGylation with CMPSA-PEG-30kD, the concentration of factor IX was increased to 1.0 mg / mL and the concentration of CMP-SA-PEG was reduced to 0.17 mM. After 16 hours of PEGylation, the glucans with terminal galactose were capped with sialic acid by adding 1 mM of CMP-SA and continuing the incubation for an additional 8 hours at 32 ° C. Under these conditions, more than 90% of the factor IX molecules were substituted with at least one portion of PEG. Factor IX produced by this method has a higher apparent molecular weight on SDS-PAGE.

EXAMPLE 13 Preparation of O-GlycoPEGylated Factor IX Chains of O-glycan were reintroduced into native factor IX (1 mg / mL) by incubation of the peptide with GalNAcT-II (25mU / mL) and 1 mM of UDP-GalNAc at 32 ° C. After four hours of incubation, the PEGylation reaction was initiated by adding CMPSA-PEG (2Kd or 10 Kd at 0.5 mM or 30 kDd at 0.17 mM) and ST6GalNAc-I (25 mU / mL) and incubating for an additional 20 hours. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in view thereof will be suggested to those skilled in the art and should be included within the spirit and scope of this application and scope of the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (33)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A Factor IX peptide characterized in that it comprises at least one portion having the formula: wherein D is a member selected from -OH and R1-L-HN-; G is a member selected from Rx-L- and C (O) (C? -C6 alkyl); R1 is a portion comprising a member selected from straight or branched chain poly (ethylene glycol) residue and L is a linker which is a member selected from a bond, substituted or unsubstituted alkyl or unsubstituted heteroalkyl, such that when D is OH, G is Rx-L-, and when G is -C (O) (C __-C3 alkyl), D is RX-L-NH-. 2. The Factor IX peptide according to claim 1, characterized in that L-R1 has the formula: wherein a is an integer from 0 to 20. 3. The Factor IX peptide according to claim 1, characterized in that R1 has a structure that is a member selected from: CH3 wherein e and f are independently selected integers from 1 to 2500 and q is an integer from 0 to 20. 4. The Factor IX peptide according to claim 1, characterized in that R1 has a structure that is a member selected from: wherein e, f and f are selected integers independently from 1 to 2500 and q and q 'are integers selected independently from 1 to 20. 5. The Factor IX peptide according to claim 1, characterized in that R1 has a structure that is a member selected from : wherein e, f and f are selected integers independently from 1 to 2500 and q, q 'and q "are integers selected independently from 1 to 20. The Factor IX peptide according to claim 1, characterized in that R1 has a structure that is a member selected from: | -C (0) CH2CH2 (OCH2CH2) eOCH3 and | -C (0) OCH2CH2 (OCH2CH2) fOCH3 where e and f are selected integers independently from 1 to 2500. 7. The Factor IX peptide according to claim 1, characterized in that the portion has the formula: 8. The Factor IX peptide according to claim 1, characterized in that the portion has the formula: 9. The Factor IX peptide according to claim 1, characterized in that the portion has the formula: wherein AA is an amino acid residue of the peptide. 10. The Factor IX peptide according to claim 9, characterized in that the amino acid residue is a member selected from serine or threonine. 11. The Factor IX peptide according to claim 1, characterized in that the peptide has the amino acid sequence of SEQ ID N0: 1. 12. The Factor IX peptide according to claim 11, characterized in that the amino acid residue is serine at position 61 of SEQ ID NO: 1. 13. The Factor IX peptide according to claim 1, characterized in that the portion has the formula: wherein a, b, c, d, i, r, s, t and u are integers selected independently of 0 and 1; q is 1; e, f, g and h are members selected independently from the integers from 0 to 6; j, k, 1 and m are members selected independently from the integers from 0 to 100; v, w, x and y are independently selected from 0 and 1, and at least one of v, w, x and y is 1; AA is an amino acid residue of the factor IX peptide; Sia- (R) has the formula wherein D is a member selected from -OH and RX-L-HN-G is a member selected from Rx-L- and -C (O) (CL-C6 alkyl); R1 is a portion comprising a member selected from a straight or branched chain poly (ethylene glycol) residue and L is a linker that is a member selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, such so that when D is OH, G is Rx-L-, and when G is -C (O) (C_-C6 alkyl), D is R1-L-NH-. 14. The Factor IX peptide according to claim 7, characterized in that the glycosyl residue is bound to a member selected from Asn 157, Asn 167 and combinations thereof. 15. A pharmaceutical composition characterized in that it comprises Factor IX according to claim 1 and a pharmaceutically acceptable carrier. 16. A method for stimulating blood coagulation in a mammal, characterized in that it comprises administering to the mammal the Factor IX peptide according to claim 1. 17. A method for treating hemophilia in a subject, characterized in that it comprises administering to the subject the peptide of Factor IX according to claim 1. 18. A method for making a Factor IX peptide conjugate comprising the portion: wherein D is a member selected from -OH and RX-L-HN-; G is a member selected from R1-L- and C (O) (C_-C6 alkyl); R1 is a portion comprising a member selected from straight or branched chain poly (ethylene glycol) residue and L is a linker which is a member selected from a bond, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl, in such a manner that when D is OH, G is R1-L-, and when G is -C (0) (C-C6 alkyl), D is R1-L-NH-, the method is characterized in that it comprises: (a) putting in contact with a substrate Factor IX peptide with a donor portion of PEG-sialic acid having the formula: and an enzyme that transfers the PEG-sialic acid onto an amino acid or glycosyl residue of the Factor IX peptide, under conditions suitable for transfer. 19. The method according to claim 18, characterized in that L-R1 has the formula: wherein a is an integer from 0 to 20. The method according to claim 18, characterized in that R1 has a structure that is a member selected from: CH3 wherein e and f are selected integers independently from 1 to 2500 and q is an integer from 0 to 20. 21. The method according to claim 18, characterized in that R1 has a structure that is a member selected from: wherein e, f and f are selected integers independently from 1 to 2500 and q and q 'are selected integers independently from 1 to 20. 22. The method according to claim 18, characterized in that R1 has a structure that is a member selected from: where e, f and f are selected integers independently from 1 to 2500 and q, q 'and q "are integers selected independently from 1 to 20. 23. The method according to the claim 18, characterized in that R1 has a structure which is a member selected from: - C (0) CH2CH2 (OCH2CH2) eOCH3 and - C (0) 0CH2CH2 (0CH2CH2) CH3 where e and f are integers independently selected from 1 to 2500. 24. The method according to claim 18, characterized in that the Factor IX peptide conjugate comprises a portion having the formula: 25. The method according to claim 18, characterized in that the Factor IX peptide conjugate comprises a portion having the formula: 26. The method according to claim 18, characterized in that the Factor IX peptide conjugate comprises a portion having the formula: wherein AA is an amino acid residue of the Factor IX peptide. 27. The method according to claim 26, characterized in that the amino acid residue is a member selected from serine or threonine. 28. The method according to claim 18, characterized in that the Factor IX substrate peptide has the amino acid sequence of SEQ ID NO: 1. 29. The Factor IX peptide according to claim 28, characterized in that the amino acid residue is serine in position 61 of SEQ ID NO: l. 30. The method according to claim 18, characterized in that the Factor IX conjugate comprises a glycosyl residue having the formula: wherein a, b, c, d, i, r, s, t and u are integers selected independently of 0 and 1; q is 1; e, f, g and h are members selected independently from the integers from 0 to 6; j, k, 1 and m are members selected independently from the integers from 0 to 100; v, w, x and y are independently selected from 0 and 1, and at least one of v, w, x and y is 1; AA is an amino acid residue of the factor IX peptide; Sia- (R) has the formula: wherein D is a member selected from -OH and R1-L-HN-G is a member selected from R1-L- and -C (0) (C6-C6 alkyl); R1 is a portion comprising a member selected from a straight or branched chain poly (ethylene glycol) residue and L is a linker that is a member selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, such so that when D is OH, G is R1-L-, and when G is -C (0) (C_-Ce alkyl), D is R1-L-NH-. 31. The method according to claim 30, characterized in that the glycosyl residue is bound to a member selected from Asn 157, Asn 167 and combinations thereof. 32. The method according to claim 18, characterized in that it further comprises, prior to step (a): (b) expressing the substrate Factor IX peptide in a sble host cell. 33. The method according to claim 32, characterized in that the host cell is selected from an insect cell and a mammalian cell.