EP1653996A2 - Utilisation de galactose oxydase pour la conjugaison chimique selective de molecules d'extraction a des proteines d'interet therapeutique - Google Patents

Utilisation de galactose oxydase pour la conjugaison chimique selective de molecules d'extraction a des proteines d'interet therapeutique

Info

Publication number
EP1653996A2
EP1653996A2 EP04739027A EP04739027A EP1653996A2 EP 1653996 A2 EP1653996 A2 EP 1653996A2 EP 04739027 A EP04739027 A EP 04739027A EP 04739027 A EP04739027 A EP 04739027A EP 1653996 A2 EP1653996 A2 EP 1653996A2
Authority
EP
European Patent Office
Prior art keywords
glycoprotein
fvii
group
ethoxy
galactose
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04739027A
Other languages
German (de)
English (en)
Inventor
Carsten Behrens
Patrick William Garibay
Magali Zundel
Niels Kristian Klausen
Sören E. BJÖRN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk Health Care AG
Original Assignee
Novo Nordisk Health Care AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk Health Care AG filed Critical Novo Nordisk Health Care AG
Publication of EP1653996A2 publication Critical patent/EP1653996A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)

Definitions

  • galactose oxidase for selective chemical conjugation of protractor molecules to proteins of therapeutic interest.
  • This invention relates to the use of galactose oxidase in combination with terminal galactose-containing glycoproteins (such as, e.g., sialidase treated glycoproteins (asialo glycoproteins)), for selective chemical conjugation of protractor molecules.
  • terminal galactose-containing glycoproteins such as, e.g., sialidase treated glycoproteins (asialo glycoproteins)
  • the sequential enzymatic treatment of natural glycoproteins with sialidases and galactose oxidase produces reactive aldehyde functionalities which chemically can be reacted with nucleophilic conjugation agents to produce novel modified glycoproteins with enhanced pharmacological properties, such as increased circulation half-life or increased distribu- tion volume.
  • Proteins of biological origin hold great promise as therapeutical agents as they often possess high efficacy and high selectivity towards their natural ligands. Being of biological origin makes them non-toxic and thus safer to use than conventional small molecular drugs, as the organism al ready posses well defined clearing mechanisms as well as metabolic pathways for their disposal. This in combination with the fact, that proteins now can be produced by recombinant DNA techniques in a variety of different expression systems, allowing for large scale production, makes proteins ideal drug candidates. How- ever, therapeutically interesting proteins such as hormones, soluble receptors, cytokines, enzymes, etc., often have short circulation half-life in the body which generally reducing their therapeutically utility. Therapeutic proteins may be removed from circulation by a number of routes.
  • a problem solved by the present invention is the prolongation of the circulating half-life of soluble glycoprotein derivatives, thus reducing the quantity of injected material and frequency of injection required for maintenance of therapeutically effective levels of circulating glycoprotein for treatment or prophylaxis.
  • the short in vivo plasma half-life of certain therapeutically glycoproteins is undesirable from the stand point of the frequency and the amount of soluble protein which would be required in treatment or prophylaxis.
  • the present invention provides means to prolong the circulating half-life of such glycoproteins with a conservative but still effective change to the glycoprotein structure and with the substantial maintenance of biological activity.
  • the present invention provide a general chemo enzymatic methodology for the modification of glycanes (in particular asparagines- or N-linked glycanes) of glycoproteins, in order to improve or enhance their pharmaceutically properties.
  • the method involves optional treatment of the glycoprotein with sialidases (neuraminidases) in order to remove any terminal sialic acid, and oxidization of the exposed galactose residues on the glycoprotein with galactose oxidase in the presence of a hydrogen peroxide scavenger such as, e.g., catalase or horseradish peroxidases.
  • the galactose oxidase treatment When preformed under appropriate conditions, the galactose oxidase treatment provides glycoproteins, with reactive aldehyde functionalities on the glycan termini, which subsequently can be chemically reacted with nucleophilic reagents to produce glycoconjugates. With glycoproteins which contain a high content of glycans with terminal galactose residues, the optional treatment with sialidases can be omitted.
  • the present invention provides a method for producing a conjugate of a glycoprotein having increased in vivo plasma half-life compared to the non-conjugated glycoprotein, the conjugate comprising a glycoprotein having at least one terminal galactose or derivative thereof, and a protractor group covalently bonded thereto, the method comprising the steps of: (a) contacting a glycoprotein having at least one terminal galactose or derivative thereof with galactose oxidase to create a glycoprotein comprising an oxidized terminal galactose or derivative thereof having a reactive aldehyde functionality; and (b) contacting the glycoprotein product produced in step (a) with a reactant X capable of reacting with an aldehyde group; wherein reactant X comprises a protractor group to create a conjugate represented by the formula (glycoprotein)- (protractor group).
  • the present invention provides a method for producing a conjugate of a glycoprotein having increased in vivo plasma half-life compared to the non-conjugated glycoprotein, the conjugate comprising a glycoprotein having at least one terminal galactose or derivative thereof, and a protractor group covalently bonded to the thereto through a linking moiety; the method comprising the steps of: (a) contacting a glycoprotein having at least one terminal galactose or derivative thereof with galactose oxidase to create a glycoprotein comprising an oxidized terminal galactose or derivative thereof having a reactive aldehyde functionality; (b) contacting the glycoprotein product produced in step (a) with a reactant X capable of reacting with an aldehyde group, wherein reactant X comprises a linking moiety further comprising a second reactive, optionally protected, group to create a conjugate of the glycoprotein and the linking moiety; and (c) contacting the product of step (b)
  • the method further comprising step (al) and step (a2) to be carried out before step (a) or step (c) of the method, respectively: (al) contacting a glycoprotein with one or more of sialidases, galacosidases, N- acetylhexosaminidases, fucosidases,mannosidases, endo H and endo F3 to create a glycoprotein where part of the glycan structure is removed, (a2) contacting the product of step (al) with a galactosyltransferase and a galactose substrate to create a glycoprotein with at least one terminal residue of galactose or a derivative thereof.
  • the method further comprises a first step
  • step (a3) to be carried out before step (a), in which: (a3) contacting a glycoprotein having at least one terminal sialic acid residue with sialidase or another reagent capable of removing sialic acid from the glycans to create an asialo glycoprotein comprising at least one terminal galactose or derivative thereof.
  • the present invention provides a glycoprotein conjugate having increased in vivo plasma half-life compared to the non-conjugated glycoprotein, said conjugate comprising a glycoprotein having at least one terminal galactose or derivative thereof, and a protractor group covalently bonded thereto, optionally through a linking moiety.
  • the present invention provides a glycoprotein conjugate obtainable by a method for producing a conjugate of a glycoprotein having increased in vivo plasma half-life compared to the non-conjugated glycoprotein, the conjugate comprising a glycoprotein having at least one terminal galactose or derivative thereof, and a protrac- tor group covalently bonded thereto, the method comprising the steps of: (a) contacting a glycoprotein having at least one terminal galactose or derivative thereof with galactose oxidase to create a glycoprotein comprising an oxidized terminal galactose or derivative thereof having a reactive aldehyde functionality; and (b) contacting the glycoprotein product produced in step (a) with a reactant X capable of reacting with an aldehyde group; wherein reactant X comprises a protractor group to create a conjugate represented by the formula (glycoprotein)- (protractor group).
  • the present invention provides a glycoprotein conjugate obtainable by a method for producing a conjugate of a glycoprotein having increased in vivo plasma half-life compared to the non-conjugated glycoprotein, the conjugate comprising a glycoprotein having at least one terminal galactose or derivative thereof, and a protractor group covalently bonded to the thereto through a linking moiety; the method com- prising the steps of: (a) contacting a glycoprotein having at least one terminal galactose or derivative thereof with galactose oxidase to create a glycoprotein comprising an oxidized terminal galactose or derivative thereof having a reactive aldehyde functionality; (b) contacting the glycoprotein product produced in step (a) with a reactant X capable of reacting with an aldehyde group, wherein reactant X comprises a linking moiety further comprising a second reactive, optionally protected, group to create a conjugate of the glycoprotein and the linking moiety;
  • the present invention provides a pharmaceutical composition comprising a glycoprotein conjugate according to the invention. DESCRIPTION OF FIGURES:
  • Figure 1 shows the amounth of exposed galactose residues on FVIIa after neuraminidase treatment (Vibro cholerae and Clostridium perfringens on agarose), as determined by galactose oxidase / amplex red assay from Molecular Probes (A-22179).
  • Figure 2 shows an IEF-Gel analysis of FVIIa and FVIIa treated with neuramidase to produce asialo-FVIIa.
  • Figure 3 shows MALDI-TOF spectra of FVIIa and asialo-FVIIa.
  • Figure 4 shows an IEF-gel analysis (Invitrogen procedure, pH 3-7) with the following setup: lane 1: pi standard, lane 2: CP FVIIa - in MES - buffer, lane 3: CP FVIIa + aga- rose bound neuraminidase Vibro cholerae, 16h, rt, lane 4: CP FVIIa + agarose bound neuraminidase Vibro Cholerae, 36h, rt, lane 5: Asialo CP FVIIa + Galactose oxidase + aminoxyacetic acid, lane 6: CP FVIIa - in MES - buffer, lane 7: CP FVIIa + agarose bound neuraminidase - Clostridium perfringens, 16h, rt, lane 8: CP FVIIa + agarose bound neuraminidase - Clostridium perfringens, 36h, r
  • Figure 5 shows a SDS-PAGE gel (Invitrogen procedure, non-reducing conditions) with asialo FVIIa, and two fractions of PEG-5000 derivatized FVIIa obtained after galactose oxidase - catalase and subsequent reaction with PEG-5000 derivatized hydroxylamine .
  • Figure 6 shows a IEF-gel analysis (Invitrogen procedure, pH 3-7) with the following setup: lane 1, pi standard; lane 2: FVIIa; lane 3: asialo FVIIa; lane 4-12 fractions of aminoxyacetic acid derivatized FVIIa after HiTrap ion-exchange purification.
  • Figure 7 shows a MALDI-TOF spectrum of derivatized N-glycanes released by PNGase F treatment of FVIIa, which has been derivatized according to the process with nitrobenzy- loxyamine.
  • Figure 8 shows a IEF gel analysis (Invitrogen procedure, pH 3-7) with the following setup: lane 1: pi standard, lane 2: FVIIa, lane 3: FVIIa afterbuffer exchange to MES buffer, lane 4, asialo FVIIa (Vibro cholerae treatment); lane 5: blank; lane 6: galactose oxidase / catalase / aminoxyacetic acid treatment performed with 30 mU galactose oxidase; lane 7: galactose oxidase / catalase / aminoxyacetic acid treatment performed with 300 mU galactose oxidase.
  • Figure 9 shows the amino acid sequence of wild-type human blood coagulation factor VII.
  • Figure 10 illustrates the convergent solution phase synthesis of a first generation den- drimer capped with 2-(2-[2-methoxyethoxy]ethoxy)acetic acid.
  • Figure 11 illustrates the solution phase synthesis of a second generation capped den- drimer with t-butyl protected carboxylic acid at the focal point.
  • Figure 12 Illustrates the solid phase synthesis of a second generation dendrimer.
  • Figure 13 illustrates the divergent solution phase synthesis of a second generation den- drimer, with free amino terminals and t-butyl protected carboxylic acid at the focal point.
  • Figure 14 illustrates the solution phase end capping of a second generation dendrimer f.ex made as illustrated in Figure 12 og 13.
  • Figure 15 illustrates the end capping of a second generation dendrimer using succinic acid mono tert-butyl ester and subsequent acid mediated deprotection to create a poly anionic glyco mimic polymer.
  • Figure 16 illustrates solution phase functionalization of a second generation dendrimer, in order to create a handle compatible with aldehyde functionalities as obtainable by the invention.
  • Figure 17 SDS-PAGE electrophoresis gel (non-denaturating).
  • Lane 1 Mark-12 Mw standard (Invitrogen);
  • lane 2 rFVIIa,
  • lane 3 asialo rFVIIa,
  • lane 4-10 fractions containing modified rFVIIa with bands at 50 KDa + several 1.7 KDa band additions.
  • Natural peptides obtained from eukaryote expression systems such as mammalian, insect or yeast cells, are frequently isolated in their glycosylated forms.
  • the glycosyl moiety also called the glycan moiety on such peptide, are themselves polyalcohols which either directly can be used for conjugation purposes, or by appropriate conditions can be converted into suitable attachment moieties for conjugation.
  • the glycans of interest are either O-linked glycanes, i.e. glycoproteins where the glycan is linked via the amino acids residues serine or threonine; or N-glycans where the glycan moiety is linked to aspar- agine residues of the peptide.
  • glycoprotein is intended to encompass peptides, oligopeptides and polypeptides containing one or more sugar residues (glycans) attached to one or more amino acid residues of the "back bone” amino acid sequence.
  • the glycans may be N- linked or O-linked.
  • w glycan or, interchangeable, “oligosaccharide chain” refers to the entire oligosaccharide structure that is covalently linked to a single amino acid residue.
  • Glycans are normally N-linked or O-linked, e.g., glycans are linked to an asparagine residue (N-linked glycosylation) or a serine or threonine residue (O-linked glycosylation).
  • N-linked oligosaccharide chains may be multi-antennary, such as, e.g., bi- , tri, or tetra-antennary and most often contain a core structure of GlcNAc-GlcNAc-Man 3 .
  • glycoproteins when produced in a human in situ, have a glycan structure with terminal, or "capping", sialic acid residues, i.e., the terminal sugar of each antenna is N-acetylneuraminic acid linked to galactose via an ⁇ 2->3 or ⁇ 2->6 linkage.
  • Other gly- coproteins have glycans end-capped with other sugar residues.
  • glycoproteins may contain oligosaccharide chains having different terminal structures on one or more of their antennae, such as, e.g., lacking sialic acid residues; containing N-glycolylneuraminic acid (Neu5Gc) residues; containing a terminal N-acetylgalactosamine (GalNAc) residue in place of galactose; and the like.
  • Patterns of N-linked and/or O-linked oligosaccharides may be determined using any method known in the art, including, without limitation: high-performance liquid chromatography (HPLC); capillary electrophoresis (CE); nuclear magnetic resonance (NMR); mass spectrometry (MS) using ionization techniques such as fast-atom bombardment, electrospray, or ma- trix-assisted laser desorption (MALDI); gas chromatography (GC); and treatment with exoglycosidases in conjunction with anion-exchange (AIE)-HPLC, size-exclusion chromatography (SEC), mass spectroscopy (MS), gel electrophoresis (SDS-PAGE, CE-PAGE), isoelectric focusing gels, or iso-electric focusing capillary electrophoresis (CE-IEF) See, e.g., Weber et al., Anal.
  • HPLC high-performance liquid chromatography
  • CE capillary electrophoresis
  • terminal sialic acid or, interchangeable, “terminal neuraminic acid” is thus intended to encompass sialic acid residues linked as the terminal sugar residue in a glycan, or oligosaccharide chain, i.e., the terminal sugar of each antenna is N- acetylneuraminic acid linked to galactose via an ⁇ 2->3 or ⁇ 2->6 linkage.
  • galactose or derivative thereof means a galactose residue, such as natural D-galactose or a derivative thereof, such as an N-acetylgalactosamine residue. In one embodiment, the galactose derivative is N-acetylgalactosamine.
  • terminal galactose or derivative thereof means the galactose or derivative thereof linked as the terminal sugar residue in a glycan, or oligosaccharide chain, e.g., the terminal sugar of each antenna is galactose or N-acetylgalactosamine.
  • asialo glycoprotein is intended to include glycoproteins wherein one or more terminal sialic acid residues have been removed, e.g., by treatment with a sialidase or by chemical treatment, exposing at least one galactose or N-acetylgalactosamine residue from the underlying "layer" of galactose or N-acetylgalactosamine ("exposed galactose residue").
  • oxidized asialo glycoprotein is intended to encompass asialo glycopro- teins that have been oxidized by galactose oxidase, optionally in combination with a hydrogen peroxide scavenger such as, e.g., catalase or horseradish peroxidase, thus creating a reactive aldehyde functionality or moiety located on the terminal galactose residue.
  • a hydrogen peroxide scavenger is intended to include compounds or substances able to react with, absorb, or neutralise hydrogen peroxide.
  • reactant X capable of reacting with an aldehyde group means a nucleophilic agents or reagents as well as other agents capable of reacting with an aldehyde group located in the oxidized terminal galactose residue of the glycoprotein, thus creating a covalent bond (or bonds) between the terminal galactose residue and the reactant (X).
  • the reactant may in some embodiments of the invention include a polymeric group.
  • Non- limiting examples of reactants X are hydroxylamines, O-alkylated hydroxylamines, amines, stabilized carbanions, stabilized enolates, hydrazides, alkyl hydrazides, hydrazi- nes, acyl hydrazines, as well as ring forming (e.g. thiazolidine forming) nucleophiles such as, thioethanamines, cystein or cystein derivatives.
  • the term "protractor group” as used herein means a group which upon conjugation to a protein or peptide increase the circulation half-life of said protein or peptide, when compared to the un-modified protein or peptide.
  • the specific principle behind the protractive effect may be caused by increased size, shielding of peptide sequences that can be recognized by peptidases or antibodies, or masking of glycanes in such way that they are not recognized by glycan specific receptores present in e.g. the liver or on macrophages, preventing or decreasing clearance.
  • the protractive effect of the protractor group can e.g. also be caused by binding to blood components sush as albumin, or un- specific adhesion to vascular tissue.
  • the conjugated glycoprotein should substantially preserve its biological activity.
  • the terms "electron withdrawing group” or “electron withdrawing groups” are used as defined in March, Advanced Organic Chemistry, 3rd edition, John Wiley & Sons, N.Y. 1985.
  • the protractor group is selected from the group consisting of :
  • a low molecular organic charged radical (15-1000 Da), which may contain one or more carboxylic acids, amines sulfonic acids, phosphonic acids, or combination thereoff.
  • a low molecular (15-1000 Da) hydrophobic molecule such as a fatty acid or cholic acid or derivatives theroff.
  • nucleophilic agent is interchangeable with “nucleophilic reagent” and
  • nucleophilic conjugation agent As used in the present context, the term “covalent attachment” is meant to encompass that the oligosaccharide moiety (glycan) and the reactant X is either directly covalently joined to one another, or else is indirectly covalently joined to one another through an intervening moiety or moieties, such as a bridge, spacer, or linkage moiety or moieties.
  • glycoconjugate or, interchangeably, “conjugate” or “conjugate protein” is intended to indicate a heterogeneous (in the sense of composite or chimeric) molecule formed by the covalent attachment of one or more terminal galactose resi- due(s) of a glycoprotein to one or more nucleophilic agents (reactants X).
  • protein is intended to include peptides, oligopeptides, and polypeptides having sequences of at least 4 amino acid residues, preferably having between 4 and about 1000 residues.
  • the protractor group is a polymeric mole- cule.
  • polymeric molecule means a molecules formed by covalent linkage of two or more monomers wherein none of the monomers is an amino acid residue.
  • the polymeric molecule is selected from the group consisting of dendrimers, polyalkylene oxide (PAO), including polyalkylene glycol (PAG), such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEGs, polyvinyl alcohol (PVA), polycarboxylate, poly-vinylpyrolidone, polyethylene-co- maleic acid anhydride, polystyrene-co-maleic acid anhydride, and dextran, including car- boxymethyl-dextran.
  • PAO polyalkylene oxide
  • PAG polyalkylene glycol
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • PVA polyvinyl alcohol
  • polycarboxylate poly-vinylpyrolidone
  • polyethylene-co- maleic acid anhydride polystyrene-co-
  • the polymeric molecule is a dend rimer with a molecular weight in the range of 700-10.000 Da
  • the polymer molecule is part of, or is attached to, a Reactant X capable of reacting with an aldehyde group in the oxidized galactose residue located in the glycopeptide.
  • a group present on the polymer may be activated to create a reactant X, as described above, before reaction with the oligosaccharide moiety or glycan.
  • the activated group, whether present on the oligosaccharide- or polymer moiety may be in the form of an activated leaving group.
  • Commonly used methods for activation of polymers include activation of functional groups with cyanogen bromide, periodate, glutaraldehyde, biepox- ides, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc. (see, for example, Taylor (1991).
  • electrophilic activated polymers can then be converted to reagents of type reagent X by reaction with e.g. binucleophilic linker moieties.
  • branched polymer or interchangebly "dendritic polymer”, "dendrimer” or “dendritic structure” means an organic polymer assembled from a selection of monomer building blocks of which, some contains branches.
  • the term "generation" means a single uniform layer, created by reacting one or more identical functional groups on an organic molecule with a particular monomer building block.
  • the number of reactive groups in a generation is given by the formula (2*(m - 1))2, where m is an integer of 1,2,3...8 representing the particular generation.
  • the number of reactive groups is given by the formula (3*(m-l))3
  • the number of reactive groups is given by (n*(m-l))n.
  • the number of reactive groups in a particular layer or generation can be calculated recursively knowing the layer po-sition and the number of branches of each individual monomers in the dendritic structure.
  • the protractor group is a selected from the goup consisting of serum protein binding-ligands, such as compounds which bind to albumin, like fatty acids, C5-C24 fatty acid, aliphatic diacid (e.g. C5-C24).
  • protractor groups includes small organic molecules containing moieties that under physiological conditions alters charge properties, such as carboxylic acids or amines, or neutral substituents that prevent glycan specific recognition such as smaller alkyl sub- stituents (e.g., C1-C5 alkyl).
  • the protractor group is albumin.
  • the protractor group is selected from the group consisting of: dendrimer, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polyethylene glycol (PEG), polypropylene glycol (PPG), branched PEGs, polyvinyl alcohol (PVA), poly- carboxylate, poly-vinylpyrolidone, polyethylene-co-maleic acid anhydride, polystyrene- co-maleic acid anhydride, dextran, carboxymethyl-dextran; serum protein binding- ligands, such as compounds which bind to albumin, such as fatty acids, C5-C24 fatty acid, aliphatic diacid (e.g.
  • C5-C24 a structure (e.g. sialic acid derivatives or mimetics) which inhibits the glycans from binding to receptors (e.g. asialoglycoprotein receptor and mannose receptor), a small organic molecule containing moieties that under physiological conditions alters charge properties, such as carboxylic acids or amines, or neutral substituents that prevent glycan specific recognition such as smaller alkyl substituents (e.g., C1-C5 alkyl), a low molecular organic charged radical (e.g. C1-C25), which may contain one or more carboxylic acids, amines sulfonic, phosphonic acids, or combination thereof; a low molecular neutral hydrophilic molecule (e.g.
  • C1-C25 such as cyclodextrin, or a polyethylene chain which may optionally branched; polyethyleneglycol with a avarage molecular weight of 2-40 KDa; a well defined precission polymer such as a dendrimer with an excact molecular mass ranging from 700 to 20.000 Da, or more preferably be- tween 700-10.000 Da; and a substantially non-imunogenic polypeptide such as albumin or an antibody or part of an antibody optionally containing a Fc-domain.
  • activated leaving group includes those moieties which are easily displaced in organic- or enzyme-regulated substitution reactions.
  • Reactive groups and classes of reactions useful in practising the present invention are generally those which proceed under relatively mild conditions. These include, but are not limited to nucleophilic substitutions (e.g., reaction of amines and alcohols with acyl halides, active esters).
  • the reactive functional groups can be selected such that they do not participate in, or interfere with, the reactions necessary to assemble the oligosaccharide and the polymer moiety.
  • a reactive functional group can be protected from partici- pating in the reaction by the presence of a protective group.
  • glycosylation site in FVII is intended to indicate the glycosylation sites at positions Asn-145 (N145), Asn-322 (N322), Ser-52 (S52), and Ser-60 (S60) of the amino acid sequence as shown in FIG.3.
  • the term “naturally occurring in vivo O-glycosylation site” includes the positions S52 and S60, whereas the term “naturally occurring in vivo -glycosylation site” includes the positions N145 and N322.
  • amino acid residues corresponding to amino acid residues S52, S60, N145, N322 of FIG: 3 is intended to indicate the Asn and Ser amino acid residues corresponding to the sequence of wild-type Factor VII (FIG: 3) when the sequences are aligned.
  • Amino acid sequence homology/identity is conveniently determined from aligned sequences, using a suitable computer program for sequence alignment, such as, e.g., the ClustalW program, version 1.8, 1999 (Thompson et al., 1994, Nucleic Acid Research, 22: 4673-4680).
  • the term "functional in vivo half-life” is used in its normal meaning, i.e., the time at which 50% of the biological activity of the polypeptide or conjugate is still present in the body/target organ, or the time at which the activity of the polypeptide or conjugate is 50% of its initial value.
  • "in vivo plasma half-life” may be determined, i.e., the time at which 50% of the polypeptide or conjugate molecules circulate in the plasma or bloodstream prior to being cleared. Determination of plasma half-life is often more simple than determining functional half-life and the magnitude of plasma half-life is usually a good indication of the magnitude of functional in vivo half-life.
  • plasma half-life alternatives include serum half-life, circulating half-life, circulatory half-life, serum clearance, plasma clearance, and clearance half-life.
  • the functionality to be retained is normally selected from procoagulant, proteolytic, co-factor binding, receptor binding activity, or other type of biological activity associated with the particular protein.
  • the term "increased" as used about the functional in vivo half-life or plasma half-life is used to indicate that the relevant half-life of the polypeptide or conjugate is statistically significantly increased relative to that of a reference molecule, such as non- conjugated glycoprotein as determined under comparable conditions.
  • the relevant half-life may be increased by at least about 25%, such as by at lest about 50%, e.g., by at least about 100%, 150%, 200%, 250%, or 500%.
  • the preparations of the present invention exhibit an increase in half-life of at least about 0.25 h, preferably at least about 0.5 h, more preferably at least about 1 h, and most preferably at least about 2 h, relative to the half-life of a reference preparation.
  • "Immunogenicity" of a preparation refers to the ability of the preparation, when administered to a human, to elicit a deleterious immune response, whether humoral, cellular, or both. In any human sub-population, there may exist individuals who exhibit sensitivity to particular administered proteins.
  • Immunogenicity may be measured by quanti- fying the presence of anti-glycoprotein antibodies and/or glycoprotein responsive T-cells in a sensitive individual, using conventional methods known in the art.
  • the preparations of the present invention exhibit a decrease in immunogenicity in a sensitive individual of at least about 10%, preferably at least about 25%, more preferably at least about 40% and most preferably at least about 50%, relative to the immu- nogenicity for that individual of a reference preparation.
  • linker moiety or “Ll” is meant any biocompatible molecule functioning as a means of linking a protractor group to "reactant X", which is capable of reacting with an aldehyde group. It is to be understood that in the final conjugate of a glycoprotein the “linker moiety” is linking via chemical bonds the glycoprotein to the protractor group. It is to be understood, that the linker moiety may contain both covalent and non-covalent chemical bonds or mixtures thereof.
  • Suitable linker moieties comprise group(s) such as, but are not limited to, peptides; polynucleotides; sacharides including monosaccharides, di- and oligosaccharides, cydodextrins and dextran; polymers including polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydrocarbons, polyacrylates and amino-, hydroxy-, thio- or carboxy- functionalised silicones, other biocompatible material units; and combinations thereof.
  • Such linker moiety materials described above are widely commercially available or obtainable via synthetic organic methods commonly known to those skilled in the art.
  • the linker moiety may, for example, be selected among the following structures: straight or branched C 1-50 -alkyl, straight or branched C 2-50 -alkenyl, straight or branched C 2-50 -alkynyl, a 1 to 50 -membered straight or branched chain comprising carbon and at least one N, O or S atom in the chain, C 3-8 cycloalkyl, a 3 to 8 -membered cyclic ring comprising carbon and at least one N, O or S atom in the ring, aryl, heteroaryl, amino acid, the structures optionally substituted with one or more of the following groups: H, hydroxy, phenyl, phenoxy, benzyl, thienyl, oxo, amino, C ⁇ -4 -alkyl, -CONH 2 , -CSNH 2 ⁇ C ⁇ - 4 monoalkylamino, C 1-4 dialkylamino, acylamino, sulfonyl
  • the linker moiety may be straight chained or branched and may contain one or more double or tri- pie bonds.
  • the linker moiety may contain one or more heteroatoms like N,0 or S. It is to be understood, that the linker moiety can comprise more than one class of the groups described above, as well as being able to comprise more than one member within a class. Where the linker moiety comprises more than one class of group, such linker moiety is preferably obtained by joining different units via their functional groups. Methods for forming such bonds involve standard organic synthesis and are well known to those of ordinary skill in the art.
  • alkyl or alkylene refer to a CI 6 alkyl or -alkylene, representing a satu-rated, branched or straight hydrocarbon group having from 1 to 6 carbon atoms.
  • Typical CI 6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopro- pyl, butyl, isobu-tyl, sec-butyl, tert-butyl, pentyl, hexyl and the corresponding divalent radicals.
  • alkenyl or “alkenylene” refer to a C2-6 alkenyl or -alkenylene, rep- resent-ing a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one double bond.
  • Typical C2 6 alkenyl groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1,3 butadienyl, 1-butenyl, 2-butenyl, 1- pentenyl, 2-pentenyl, 1-hexenyl, 2-hexenyl, l-ethylprop-2-enyl, l,l-(dimethyl)prop-2- enyl, l-ethylbut-3-enyl, l,l-(dimethyl)but-2-enyl, and the corresponding divalent radicals.
  • alkynyl or “alkynylene” refer to a C2 6 alkynyl or -alkynylene, rep- resent-ing a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one triple bond.
  • Typical C2 6 alkynyl groups include, but are not limited to, vinyl, 1-propynyl, 2-propynyl, isopropynyl, 1,3 butadynyl, 1-butynyl, 2-butynyl, 1- pentynyl, 2-pentynyl, 1-hexynyl, 2-hexynyl, l-ethylprop-2-ynyl, l,l-(dimethyl)prop-2- ynyl, l-ethylbut-3-ynyl, l,l-(dimethyl)but-2-ynyl, and the corresponding divalent radicals.
  • alkyleneoxy or "alkoxy” refer to "Cl-6-alkoxy” or -alkyleneoxy repre-senting the radical -O-Cl-6-alkyl or -O-Cl-6-alkylene, wherein CI 6 alkyl(ene) is as defined above.
  • Representative examples are methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like.
  • heteroaryl as used herein is intended to include heterocyclic aromatic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulfur such as furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothia- zolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5- triazinyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5- thiadiazolyl, 1,3,4-thiadiazolyl,
  • Heteroaryl is also intended to include the partially hydro- genated derivatives of the heterocyclic systems enumerated above.
  • Non-limiting examples of such partially hydrogenated derivatives are 2,3-dihydrobenzofuranyl, pyrrolinyl, pyrazolinyl, indolinyl, oxazolidinyl, oxazolinyl, oxazepinyl and the like.
  • increased half-life is achieved in such glycoproteins by treatments which block or inhibit removal of the protein by sugar-specific receptor, such as the galactose and mannose receptors in the liver.
  • sugar-specific receptor such as the galactose and mannose receptors in the liver.
  • the prolonging of soluble glycoprotein derivatives in circulation is described.
  • a "protractor” moiety such as, e.g., a serum protein binding ligand, such as albumin binding ligands, or a large polymer such as PEG.
  • a "protractor” moiety such as, e.g., a serum protein binding ligand, such as albumin binding ligands, or a large polymer such as PEG.
  • prolonged circulating half-lives are desirable in therapeutic proteins because frequency and/or size of dose can be reduced when half-life is longer.
  • Galactose oxidase is a copper enzyme that catalyzes the two-electron oxi- dation of a large number of primary alcohols to their corresponding aldehydes, coupled with the reduction of dioxygen to hydrogen peroxide: RCH 2 OH + 0 2 -> RCHO + H 2 0 2 .
  • the enzyme exhibits a broad specificity for reductant, and a wide variety of primary alcohols serve as effective substrates, including substituted benzyl alcohols.
  • Galactose oxidase together with catalase or horseradish peroxidase offers a mild and highly selective method for introducing aldehyde functionalities into glycoproteins, and has been extensively used for either radio or fluorescence labelling of purified glycoproteins, as well as glycoproteins embedded in lipid membranes of full cellular systems (see for example Baumann H. and Doyle D., J. Biol. Chem. 1979, 254 (7) 2542-2550 and Wilchek M.; Spiegel S. and Spiegel Y., Biochem. Biophys Res. Commun, 1980, 92(4) 1215-1222).
  • Ga- lactose oxidase selectively oxidizes galactose (Gal) and acetylated galactosamine (Gal- NAc).
  • Neuraminidase and galactose oxidase both have been immobilized on beads in order to produce cost efficient high storage and operational stable enzyme reactors, see for example Bilkova Z. et.al., J. Chromatography B. 2002, 770/1-2 (177-181).
  • the appli- cation of such immobilized enzyme optionally in connection with similar immobilized enzyme like catalase of horseradish peroxidase for the removal of hydrogen peroxide side product, are within the scope of the invention.
  • Beside increased stability immobilized en- zymes offers advantages in subsequent purification processes because they are easily removed by simple filtration.
  • one or more of the enzymes involved in the process are immobilized on support.
  • glycoproteins contain terminal sialic acid residues.
  • the glycoprotein which either can be obtained from natural sources, or from genetically modified mammalian cells such as COS-, BHK- or CHO cells, or from remodelled proteins obtained from yeast cells, is initially treated with sialidase, in order to expose at least one galactose residue from the inner layer of galactose residues.
  • Some glycoproteins are by nature not end-sialylated (does not contain terminal sialic acid residues), and sialidase treatment is in such cases obsolete.
  • Enzymatic de-sialylation can be performed either by use of soluble sialidases of commercial origin, by use of recombinant genetically modified sialidases or by use of sialidases bound to solid supports such as, e.g., agarose.
  • the re- action progress can conveniently be monitored by IEF-gel monitoration as described in WO 2003031464 (Neose), or by capillary electrophoresis techniques such as those described in N.K. Klausen and T. Kornfelt, J. Chromatography A, 718, 195-202 (1995).
  • a purification step can be necessary. Suitable purification techniques are known to the skilled person, and can involve, e.g., ion- exchange chromatography, or other similar technique.
  • Galactose oxidase The glycoprotein, or the asialo-glycoprotein obtained by treatment with sialidase, is subsequently treated with galactose oxidase using molecular oxygen as oxidant.
  • the by-product of the reaction is hydrogen peroxide, some measure to avoid its destructive power (e.g. oxidation of methionine residues, etc.) is generally taken, for example by adding a hydrogen peroxide scavenger. Both catalase and horseradish peroxidase are suitable, but other scavengers are known to the skilled person.
  • the reaction is more difficult to monitor.
  • Chemical conjugation with reporter molecules such as dansyl hydrazid is an option.
  • reporter molecules such as dansyl hydrazid
  • horseradish peroxidase or catalase is omitted, the reaction progress can be colorimetricly estimated using a commercially available kit, such as Amplex Red from Molecular Probes.
  • the glycoprotein for the chemical conjugation step is a glycoprotein which has been treated with sialidase to remove sufficient sialic acid to expose at least one galactose residue and which has been further treated, e.g., with galactose oxidase and horseradish peroxidase to produce a free reactive aldehyde functionality.
  • sialidase to remove sufficient sialic acid to expose at least one galactose residue
  • galactose oxidase and horseradish peroxidase to produce a free reactive aldehyde functionality.
  • Sia denotes a sialic acid linked to a galactose or galactose derivative (Gal) in either alpha-2,3-, or alpha-2,6-configuration.
  • Gal-OH represent galactose in which case,
  • Gal-OH represent the galactose derivative N-acetyl galactosamine and the galactose oxidase oxidizes the acetylated galactosamine residues in which case,
  • X is any type of molecule containing a chemical functionality that can react covalently with an aldehyde to form a C-6 modified galactose or N-acetyl galactosamine resi- due (such as, e.g., a nucleophile agent).
  • L is a divalent organic radical linker which may be any organic di-radical including those containing one or more carbohydrate moiety(-ies) consisting of natural monosaccharide ⁇ ), such as fucose, mannose, N-acetyl glycosamine, xylose, and arabinose, interlinked in any order and with any number of branches. L may also be a valence bond.
  • reactant X has the formula nuc-R, wherein nuc is a functional group which can react with an aldehyde to form a covalent bond, and R is a protractor group.
  • reactant X has the formula nuc-Ll-R, wherein nuc is a functional group which can react with an aldehyde to form a covalent bond, R is a protractor group, and Ll is a linking moiety.
  • reactant X is selected from the group consisting of : H 2 N-R, HR1N-R, H 2 N-0-R, HRIN-O-R, H 2 N-NH-CO-R, H 2 N-CHR1-CHR-SH, H 2 N-CHR-CHR1-SH, H 2 N-NH-S0 2 -R, and Z'-CH 2 -Z"-R; wherein R is a protractor group; RI is H or a second protractor group; Z' and Z" represent electron withdrawing groups, such as, e.g., COOEt, CN, N0 2 , and wherein one or both of the Z groups can be connected to the R group.
  • reactant X is selected from the group of : H 2 N-L1 -R, HR1N-L1-R, H 2 N-0-Ll-R, HRIN-O-Ll-R, H 2 N-NH-CO-Ll-R, H 2 N-CHR1-CH(L1- R)-SH, H 2 N-CH(L1-R)-CHR1-SH, H 2 N-NH-S0 2 -L1-R, Z'-CH 2 -Z"-L1-R; wherein Ll is a link- ing moiety, R is a protractor group, RI is H or a second protractor group; Z' and Z" represent electron withdrawing groups, such as, e.g., COOEt, CN, N0 2 , and wherein one or both of the Z groups can be connected to the R group.
  • nuc is a group that can react with an aldehyde group.
  • Non-limiting examples for illustration include hydroxylamines, O-alkylated hydroxylamines, amines, stabilized car- banions, stabilized enolates, hydrazides, alkyl hydrazides, hydrazines, acyl hydrazines etc.
  • Other embodiments includes ring forming (e.g. thiazolidine forming) nucleophiles such as, e.g., thioethanamines, cystein or cystein derivatives, ⁇ -mercaptoacylhydrazides ect.
  • nuc can be:
  • hydrazine derivatives -NH-NH 2 hydrazine carboxylate derivatives -0-C(0)-NH-NH 2
  • semicarbazide derivatives -NH-C(0)-NH-NH 2 thiosemicarbazide derivatives -NH-C(S)-NH-NH 2
  • carbonic acid dihydrazide derivatives -NHC(0)-NH-NH-C(0)-NH-NH 2 carbazide derivatives -NH-NH-C(0)-NH-NH 2
  • thiocarbazide derivatives -NH-NH-C(S)-NH-NH 2 aryl hydrazine derivatives -NH-C(0)-C 6 H 4 -NH-NH 2
  • hydrazide derivatives -C(0)-NH-NH 2 and oxylamine derivatives, such as -0-NH 2 , -C(0)-0-NH 2 , -NH-C(0)-0-NH 2 and -NH-C(S)-0-
  • R of general formula nuc-R may be an organic radical selected from one of the groups below: a) straight, branched and/or cyclic C ⁇ - 30 alkyl, C 2-30 alkenyl, C 2-30 alkynyl, Ci- 30 heteroalkyl, C 2-30 heteroalkenyl, C 2-30 heteroalkynyl, wherein one or more homo- cyclic aromatic compound biradical or heterocyclic compound biradical may be inserted, and wherein said C 1-30 or C 2-30 radicals may optionally be substituted with one or more substituents selected from -C0 2 H, -S0 3 H, -P0 2 OH, -S0 2 NH 2 , -NH 2 , - OH, -SH, halogen, or aryl, wherein said aryl is optionally substituted with -C0 2 H, - S0 3 H, -P0 2 OH, -S0 2 NH 2 , -NH 2 , -NH 2
  • dextrans ⁇ - ⁇ - or ⁇ -cyclodextrin, polyamide radicals e.g. polyamino acid radicals; PVP radicals; PVA radicals; poly(l-3-dioxalane); poly(l,3,6-trioxane); ethylene/maleic anhydride polymer; c) Cibacron dye stuffs, such as Cibacron Blue 3GA, and polyamide chains of specified length, as disclosed in WO 00/12587, which is incorporated herein by reference.
  • polyamide radicals e.g. polyamino acid radicals; PVP radicals; PVA radicals; poly(l-3-dioxalane); poly(l,3,6-trioxane); ethylene/maleic anhydride polymer; c) Cibacron dye stuffs, such as Cibacron Blue 3GA, and polyamide chains of specified length, as disclosed in WO 00/12587, which is incorporated herein by reference.
  • a substantially non-immunogenic protein residue such as a blood component like albuminyl derivative, or a antibody or a domain thereoff such as a Fc domain from human normal IgGl, as described in Kan, SK et al in The Journal of Immunology 2001, 166(2), 1320-1326 or in Stevenson, GT, The Journal of Immunology 1997, 158, 2242-2250 .
  • R may also represent -C(R5) 3 , wherein each RI independently represents hydrogen or a moiety selected from amongst -D-((CH 2 ) q 0) r -0R6, -D-CH 2 -0-((CH 2 ) q O) r - OR6, -D-0-((CH 2 ) q O) r -OR6; wherein q represents 1-6, r represent 10 to 500, and R6 represent hydrogen or C ⁇ -C 6 -alkyl; and wherein D represents a bond or Ci- 8 alkyl or C 1-8 heteroalkyl; g) moieties that are known to bind to plasma proteins, such as e.g.
  • albumin where the albumin binding property may be determined as described in J. Med. Chem, 43, 2000, 1986-1992, which is incorporated herein by reference, or an albumin binding moiety such as a peptide comprising less than 40 amino acid residues such as moieties disclosed in J. Biol Chem. 277, 38 (2002) 35035-35043, which is incorporated herein by reference, h) C ⁇ -C 20 -alkyl, such as C ⁇ -C ⁇ 8 -alkyl.
  • C_ 4 -, C_ 6 - and C ⁇ 8 -alkyl which optionally may be substituted with in particular charged groups, polar groups and/or halogens. Examples of such substituents include -C0 2 H and halogen.
  • all hydrogens in the C ⁇ -C 20 -alkyl are substituted with fluoro to form perfluoroalkyl.
  • R-nuc which is PEG - 2-40K derivatives is shown in the following by illustration and not limitation:
  • the reactant X has the formula nuc-R, wherein nuc is a functional group which can react with an aldehyde to form a covalent bond, and R is a PEG group.
  • the nuc-R is selected from the group consisting of :
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa, wherein mPEG has a molecular weight of 20 kDa,
  • mPEG has a molecular weight of 20 kDa
  • mPEG wherein mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa, wherein mPEG has a molecular weight of 20 kDa,
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa, wherein mPEG has a molecular weight of 20 kDa,
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa, wherein mPEG has a molecular weight of 20 kDa,
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa, wherein mPEG has a molecular weight of 20 kDa,
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa
  • mPEG has a molecular weight of 20 kDa, wherein mPEG has a molecular weight of 20 kDa.
  • mPEG has a molecular weight of 10 kDa.
  • mPEG has a molecular weight of 5 kDa.
  • mPEG has a molecular weight of 10 kDa, wherein mPEG has a molecular weight of 10 kDa,
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa, wherein mPEG has a molecular weight of 10 kDa,
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa, wherein mPEG has a molecular weight of 10 kDa,
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa, wherein mPEG has a molecular weight of 10 kDa,
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa, wherein mPEG has a molecular weight of 10 kDa,
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa
  • mPEG has a molecular weight of 10 kDa
  • the protractor group is a compound which bind to albumin.
  • the phrase "compound which bind to albumin” is used herein interchangeable with "albumin binder”.
  • the reactant X has the formula nuc-R, wherein nuc is a functional group which can react with an aldehyde to form a covalent bond, and R is a compound which bind to albumin.
  • the nuc-R is selected from the group consisting of :
  • k is an integer of between 0 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • reactant X is a nucleophile, which can form a covalent linkage upon dehydration.
  • Non-limiting examples for illustration include hydroxylamines, O- alkylated hydroxylamines, amines, stabilized carbanions, stabilized enolates, hydrazides, alkyl hydrazides, hydrazines, acyl hydrazines, ⁇ -mercaptoacylhydrazides etc.
  • Other embodiments includes ring forming (e.g. thiazolidine forming) nucleophiles such as, e.g., thioethanamines, cystein or cystein derivatives.
  • the product of the reaction may be further reacted with a reducing agent (a reductant) to form reduced products as indicated below:
  • reducing agents include sodium cyanoborohydride, pyridine borane, and sodium borohydride
  • x includes hydrazides, primary and secondary amines.
  • alkyl hydrazines Although more reactive, and in some cases directly destructive to the protein in question, alkyl hydrazines also react efficiently with aldehydes to produce hydrazones. Hydrazones are stable in aqueous solution and may therefore be considered as an alternative to hydroxylamines for derivatization:
  • Y OH or NHAc Hydrazides on the other hand, also react spontaneously with aldehydes, but the acyl hydrazone product is less stable in aqueous solution.
  • the resultant hydrazone is therefore frequently reduced to N-alkyl hydrazide using mild reduction reagents such as sodium cyanoborohydride or pyridine borane. See for example Butler T. et al. Chembiochem. 2001, 2(12) 884-894.
  • C6-oxidized galactose residues also react efficiently with amino thiols such as cystein or cystein derivatives or aminoethane thiol to produce thiazolidines as depicted below:
  • C6-oxidized galactose residues can also react with carbanionic organophosphorus reagents in a Horner-Wadsworth-Emmons reaction.
  • the reaction forms an alkene as depicted below.
  • the strength of the nucleophile can be varied by employing different organophosphorus reagents, like those employed in the Wittig reaction.
  • C6-oxidized galactose residues can also react with carbanion nucleophiles.
  • An example of this could be an aldol type reaction as illustrated below.
  • the Z' and Z" groups represent electron withdrawing groups, such as COOEt, CN, N0 2 (see March, Advanced Organic Chemistry, 3rd edition, John Wiley & Sons, N.Y. 1985), which increase the acidity of the methylene protons.
  • one or both of the Z groups would also be connected to an R group (protractor), which could improve the properties of the glycoprotein.
  • Linker molecules Modification of the oxidized (asialo) glycoprotein may also proceed in more than one step, before reaching to the final product.
  • the C6 oxidized galactose residue is initially reacted with a linker molecule possessing specificity for the aldehyde moiety.
  • the linker molecule itself containing an additional chemical handle (bifunctional), is then reacted further by attaching another molecule (e.g. the protractor group) to give the final product:
  • Suitable bifunctional linkers are well known to the skilled person, or can easilly be con- ceived. Examples include, but are not limited to bifunctional linkeres containing hydroxylamine-, amine-, or hydrazied in combination with malimides, , succimidyl ester, thiols hydroxylamines, amines, hydrazides or the like.
  • nucleophile directly into the reaction mixture when performing the oxidation using the galactose oxidase - catalase or the galactose oxidase horseradish peroxidase enzyme couple.
  • Such one-pot conditions can prevent any inter- molecular protein protein reactions of the aldehyde functionalities on one protein with the amino groups (e.g. epsilon amines in lysine residues) on the other.
  • Intra and intermo- lecular Schiff base (imine) formation between proteins can lead to incomplete reaction with the nucleophile, or precipitation of the protein in question.
  • concentration ratio of nucleophile to protein may depend on the protein in question and the type of nucleophile (e.g. hydroxylamine, hydrazide, amine, etc.) selected for conjugation.
  • Optimal conditions may be found by experiments, e.g. perform variation in the concentration ratio of nucleophile to protein, perform variation in the overall concentration of protein in solution, etc.
  • the invention can be used to covalently bind a protractor group to any terminal galactose moiety.
  • One example could be the addition of terminal galactose residues to a glycan by the use of galactosyl transferases, and such terminal galactose residues could be modified by the technology described by the invention.
  • Glomerular filtration and renal clearance Glomerular filtration is an important clearance pathway for proteins. Glomerular filtration is generally considered to be related to size and overall charge characteristics of the protein.
  • the basement membrane of the glomerular capillary wall is an ion filter, composed of negatively charged proteoglycans, which is able to differentiate between neutral blood components i.e. water and urea, and charged blood components such as salts, peptides and proteins. Charges on the protein surface can interact with the fixed negatively charged heparin sulphate proteoglycan of the glomerular basement membrane.
  • Proteins with surplus of negative surface charges tends to be repulsed by the fixed negatively charged heparin sulphate proteoglycan constituting the glomerular basement membrane and therefore are less likely to pass into the urine.
  • the fractional clearance of negatively charged horseradish peroxidase has been estimated to 0.007, compared to the fractional clearance for neutral horseradish peroxidase which has been estimated to 0.061 (H.G. Rennke, Y. Patel, and M.A. Venkatachalam, Kidney Int. 13 1978 278-288). Changes of surface charges therefore can have a marked effect on the proteins susceptibility towards glomerular filtration.
  • a protein is treated with neuraminidase and galactose oxidase according to the invention and then reacted with a molecular ligand containing one or more positively or negatively charged moieties, to produce a protein with altered surface charge properties.
  • the size of the protein also determines its ability to pass the glomerular capillary wall. Larger proteins (e.g. proteins with molecular mass above 50 KDa) have low tendency for renal clearance compared to smaller proteins. Increasing the size of a given protein thus may increase circulation half life, by minimizing renal clear- ance.
  • a protein is treated with neuraminidase and galactose oxidase according to the invention and then reacted with a reactant X, e.g, in the form of a nucleophile, containing a protractor group such as, e.g., PEG or one or more dendrimers to produce a protein with considerable increased size, in order to pre- vent renal clearance.
  • a reactant X e.g, in the form of a nucleophile, containing a protractor group such as, e.g., PEG or one or more dendrimers to produce a protein with considerable increased size, in order to pre- vent renal clearance.
  • the monomers of dendrimers are in general linear or branched bi-, tri- or tetrafurcated building blocks of the general structure A-L2-C-(L3-B) n (general formula I) where C serves as attachemt moiety for A-L2 as well as branching moiety for n number of L3-B, in which L2 and L3 both are linker moieties:
  • a and B both are functional groups selected in such way, that they together under appropriate condition can form a covalent bond.
  • the nature of the newly formed covalent bond depend upon the selection of A and B, and include but is not limitted to: amide bonds, carbamate bonds, ester bonds, phosphate ester bonds, thiophosphate ester bonds, phosphoramidates, ether, and thioether bonds.
  • A may be selected from (but is not limitted to): COOH, COOR, OCOOR, OP(NR2)OR, OP(OR)2, COCI, COBr, OCOCI, OCOBr, CHO, Br, CI, I, OTs, OMs, alkynes,
  • R is alkyl, aryl or substituted aryl
  • the moiety A of general formula I represent an activated moiety that can react with nucleophiles either on the peptide or of type B.
  • A is selected from the group of: Functional groups capable of reacting with amino and hydroxy groups such as a) carbonates such as the p-nitrophenyl, or succinimidyl; b) carbonyl imidazoles or carbonyl chlorides; c) carboxylic acids that are activated in situ; d) carbonyl halides, activated esters such as N-hydroxysuccinimide esters, N- hydroxybenzotriazole esters, esters of l,2,3-benzotriazin-4(3 )-one e) phosphoramidites and H-phosphonates or f) isocyanates or isothiocyanates.
  • B may be selected from NH 2 , OH, N 3 , NHR', OR', 0-NH 2 , O-NHR',
  • R' is a protection group including, but not limmited to:
  • the moiety B of general formula I represent a protected nucleophile moiety that can react with electrophiles preferably of type A.
  • B is selected from the group of: a) Fmoc protected amino groups b) free amino groups c) azides, that can be reduced to amino groups d) azides, that may participate together with alkynes to form triazoles e) O-substituted hydroxylamines f) hydroxyl groups g) DMT, MMT or trityl- protected hydroxyl groups
  • C is either a linear (divalent organic radical) or a branched (multivalent branched organic radical) linker, preferably of hydrophilic nature. It preferably includes a multiply- functionalized alkyl group containing up to 18, and more preferably between 1-10 carbon atoms. Several heteroatoms, such as nitrogen, oxygen or sulfur may be included within the alkyl chain. The alkyl chain may also be branched at a carbon or a nitrogen atom. In one aspect of the invention, C is a single nitrogen atom
  • Example of C include but is not limitted to divalent organic radicals such as ethylene, ary- lene, propylene, ethyleneoxy,
  • multivalent organic radicals such as propan-l,2,3-triyl, benzen-l,3,4,5-tetrayl, 1,1,1- nitrogentriyl, or a multivalent carbocyclic ring including, but is not limited to the following structures:
  • C is
  • linker L2 and L3 which preferably are of hydrophilic nature.
  • linkers include but is not limmited to
  • n is an integer between 0 and 10 and RI
  • R2, R3 and R4 independently can be H, Me, Et, Pr . ((CH 2 ) m O) n -, where m is ..2, 3, 4, 5, 6, and n is an integer between 0 and 10,
  • L2, L3 or both can also be a valence bond.
  • L2 and L3 are selected from water soluble organic divalent radicals.
  • either L2 or L3 or both are divalent organic radicals containing about 1 to 5 PEG (-CH 2 CH 2 0-) groups.
  • L2 is -oxy- or -oxymethyl-
  • L3 is (CH 2 CH 2 0-) 2 :
  • A is a carboxyl group and B is a protected amino group which after deprotection may be coupled to a new monomer of same type via its carboxy group to form an amide.
  • A is a phosphoramidite and B is a hydroxyl group suitable protected, which upon deprotection can be coupled to an other monomer of same type to form a phosphite triester which subsequently are oxidized to form a stable phosphate tri- ester or thio phosphate triester.
  • A is an reactive carbonate such as nitrophenyl carbamate
  • B is an amino group, preferably in its protected form.
  • A is an acyl halide such as COCI or COBr and B is an amino group, preferably in its protected form.
  • A-L2-C-(L3-B) n is
  • A-L2-C-(L3-B) n is
  • A-L2-C-(L3-B) n is
  • A-L2-C-(L3-B) n is
  • Branched polymers can in general be assembled from the monomers described above using one of two fundamentally different oligomerization strategies called the divergent approach and the convergent approach.
  • the branched polymers are assembled by an iterative process of synthesis cycles, where each cycle use suitable activated, reactive bi - tri or multi furcated monomers, them self containing functional end groups - allowing for further elongation (i.e. polymer growth).
  • the functional end groups usually needs to be pro- tected in order to prevent self polymerization and a deprotection step will in such cases be needed in order to generate a functional end group necessary for further elongation.
  • One such cycle of adding a activated (reactive) monomer and subsequent deprotection, in the iterative process completes a generation.
  • the divergent approach is illustrated in Fig. 13 using solution phase chemistry and in Fig. 12 using solid phase chemistry.
  • the conver- gent approach to building macromolecules involves building the final molecule by beginning at its periphery, rather than at its core as in the divergent approach. This avoid problems, such as incomplete formation of covalent bonds, typically associated with the reaction at progressivly larger numbers of sites.
  • the final branched polymer if desired may consist of different types of monomer building block in each of its layers or generations.
  • branched polymers with tailored properties can be made. That way the overall properties of the polymer, and the polymer-peptide conjugate can be controlled.
  • Fine tuning, or adjustment of the rigity can then be obtained by using the rigid monomer in one or more specific layers intermixed with monomers of more flexible nature. It also could be of interest to fine tune the overall hydrophilic nature of the polymer. This could be realized by choosing monomers with more hy- drophobic core structure (C) or more hydrophobic linker moieties (L2 & L3), in one or more of the dendritic layers.
  • C hy- drophobic core structure
  • L2 & L3 hydrophobic linker moieties
  • polyanionic biopolymers are well known in e.g. natural occuring glycoproteins, which commonly has multiple anionic charged sialic acids as termination groups on the antenna structure of their N-glucans.
  • glucans can be imi- tated with respect to their poly anionic nature.
  • Fig. 15 where the dendritic layer is capped with succinic acid mono tert butyl estes which upon deprotection with acids renders a polymer surface that are negatively charged under physiological conditions.
  • the assembly of monomers into polymers may be conducted either on solid support as described by N.J. Wells, A. Basso and M. Bradley in Biopolymers 47, 381-396 (1998) or in and appropriate organic solvent by classical solution phase chemistry as de- scribed by Frechet et al. in U.S. Patent 5,041,516.
  • the branched polymer is assembled on a solid support derivatized with a suitable linkage, in an iterative divergent process as described above and illustrated in Fig. 12.
  • solid phase protocols useful for conventional peptide synthesis can conveniently be adapted.
  • This type of solid support oxidation which is convineinetly achieved using iodine in an inert organic solvent, requires that the monomers with or without protection groups resist iodine oxidation.
  • the phosphor amidite methodology also allows for convenient synthesis of thiophosphates by simple replacement of the iodine with elementary sulfur in pyridine or organic thiolation reagents such as 3H-1,2- benzodithiole-3-one-l,l-dioxide (see for example M. Dubber and J.M.J. Frechet in Bioconjugate chem. 2003, 14, 239-246).
  • the resin attached branched polymer when complete, can then be cleaved from the resin under suitable conditions. It is important, that the cleavable linker between the growing polymer and the solid support is selected in such way, that it will stay intact during the oligomerization process of the individual monomers, including any deprotection steps, oxidation or reduction steps used in the individual synthesis cycle, but when de- sired under appropriate conditions can be cleaved leaving the final branched polymer intact.
  • the skilled person will be able to make suitable choices of linker and support, as well as reaction conditions for the oligomerisation process, the deprotection process and optionally oxidation process, depending of the monomers in question.
  • Resins derivatized with appropriate functional groups that allows for attachment of monomer units and later act as cleavable moieties are commercial available (see f.ex the cataloge of Bachem and NovoBiochem).
  • the branched polymer is synthesised on a resin with a suitable linker, which upon cleavage generates a branched polymer product furnished with a functional group that directly can act as an attatchment group in a sub- sequent solution phase conjugation process to a peptide as described below, or alternatively, by appropriate chemical means can be converted into such an attachment group.
  • the branched polymer is assembled in an appropriate solvent, by sequential addition of suitable activated monomers to the growing polymer. After each addition, a deprotection step may be needed before construction of the next layer or generation can be initiated. It may be desirable to use excess of monomer in order to reach complete reactions.
  • the removal of excess monomer takes advantages of the fact that hydrophilic polymers have low solubility in diethyl ether or similar types of solvents.
  • the growing polymer can thus be precipitated leaving the excess of monomers, coupling reagents, biproducts etc. in solution. Phase separation can then be performed by simple decantation, of more preferably by centrifugation followed by decantation.
  • Polymers can also be separated from biproducts by conventional chromatographic techniques on e.g. silica gel, or by the use of HPLC or MPLC systems under either normal or reverse phase conditions as described in P.R. Ashton et al. J. Org. Chem. 1998, 63, 3429-3437.
  • the considerbly larger polymer can be separated from low molecular components, such as excess monomers and biproducts using size exclusion chromatography optionally in combination with dialysis as described in E.R.Gillies and J.M.J. Frechet in J.Am.Chem.Soc. 2002, 124, 14137-14146.
  • solution phase also makes it possible to use the convergent approach for assembly of branched polymers as described above and further reviewed in S.M.Grayson and J.M.J. Frechet, Chem. Rev. 2001,101,3819-3867.
  • this approach it is desirable to initiate the synthesis with monomers, where the protected func- tional end groups (B) initially is converted into moieties that eventually will be present on the outer surface of the final branched polymer. Therefore the functional moiety (A) of general formula I in most cases will need suitable protection, that allows for stepwise chemical manipulation of the end groups (B). Protection groups for the functional moiety (A) depend on the actually functional group.
  • a tert-butyl ester derivate that can be removed by TFA would be an appropriate choice.
  • Suitable protection groups are known to the skilled person, and other examples can be found in Green & Wuts "Protection groups in organic synthesis", 3.ed. Wiley-interscience.
  • the convergent assembly of branched polymers is illustrated in Fig. 10 and Fig. 11.
  • step (i) of Fig. 10 a tertbutyl ester functionallity (A) is prepared by reaction of a suitable precurser with t-butyl ⁇ -bromoacetate.
  • step (ii) the terminal end groups (B) is manipulated in such way that they allows for the acylation of step (iii), with a carboxylic acid that is converted into a acyl halid in step (iv).
  • step (v) the t-butyl ester functionality (A) is removed creating a end (B) capped monomer.
  • This end capped monomer serves as starting material for preparing the second generation product in Fig. 11, where 2 equivalents is used in an acylation reaction with the product of step (ii) in Fig. 10.
  • the product of this reaction is a new t-butyl ester, which after deprotection can re-enter in the initial step of Fig. 11 in a itterative manner creating higher generation materials.
  • the polymer To effect covalent attachment of the polymer molecule(s) to the peptide in solution, the polymer must be provided with a reactive handle, i.e. furnished with a reactive functional group examples of which includes by illustration and not limitation, primary amino groups, hydrazides, hydrazides, ⁇ -and ⁇ aminothiols or a hydroxylamine.
  • a reactive handle i.e. furnished with a reactive functional group examples of which includes by illustration and not limitation, primary amino groups, hydrazides, hydrazides, ⁇ -and ⁇ aminothiols or a hydroxylamine.
  • Suitable attachment moieties on the branched polymer may be created after the polymer has been assembled using either conventional solution phase chemistry or solid phase chemistry.
  • Non-limitted examples on ways to create nucleophilic attachment moieties on a branched polymer containing a carboxylic acid group are listed in Fig. 16
  • One or more of the activated branched polymers can be attached to a biologically active polypeptides by standard chemical reactions according to the invention.
  • the conjugate is represented by the general formula II:
  • (branched polymer) branched polymer
  • (branched polymer) is a water-soluble substantially nonantigenic polymer consisting of monomers according to general formula I
  • L 4 is an linking moiety essentially defined as for L 2 and L 3 of general formula I
  • (z) is an integer > 1 representing the number of branched polymers conjugated to the biologically active polypeptide.
  • the upper limit for (z) will be determined by the number of available attachment sites on the polypeptide, and the degree of polymer attachment sought by the artisan.
  • the degree of conjugation can, as previously mentioned, be modified by varying the reaction stoiehiometry using well-known techniques.
  • More than one branched polymer conjugated to the polypeptide can be obtained by reacting a stoiehiometric excess of the activated polymer with the polypeptide.
  • the biologically active polypeptide can be reacted with the activated branched polymers in an aqueous reaction medium which can be buffered, depending upon the pH requirements of the polypeptide.
  • the optimum pH for the reaction is generally between about 6.5 and about 8 and preferably about 7.4 for most polypeptides.
  • the optimum reaction conditions for the polypeptides stability, reaction efficiency, etc. is within level of ordinary skill in the art.
  • the preferred temperature range is between 4°C and 37°C. The temperature of the reaction medium cannot exceed the temperature at which the polypeptide may denature or decompose.
  • the polypeptide be reacted with an excess of the activated branched polymer.
  • the conjugate is recovered and purified such as by diafiltration, column chromatography including size exclussion chromatotrapy, ion-exchange chromatograph, affinity chromatography, electrophoreses, or combinations thereof, or the like.
  • Proteolytical shielding A major problem when using proteins and peptides as therapeutics, is their susceptibility to proteolytical degradation by proteases present in plasma. Degradation plays an obvious role in the elimination of damaged or abnormal polypeptides, but also affects half-lives of normal proteins and peptides, and individual turnover rates can be strongly dependent on the peptide sequence, the structure and in the case of proteins, the surface properties. Shielding effects against proteolysis not only rely on structural or sequential factors but also global biophysical factors, such as overall hydrophobicity or overall charge characteristics. Consequently even small changes in overall charge properties of the protein can have effect on protease mediated proteolysis in serum. Such changes in glycan moieties of glycoproteins, by e.g.
  • moieties that are charged under physiological conditions are included in the invention.
  • moieties that are negatively charged under physiological conditions includes carboxylic acids, sulfonic acids, phosphonic acids, phosphates, phosphoramidates ect.
  • moieties that are positively charges under physiological conditions includes primary-, secondary-, tertiary- and quaternary amino groups, guanidines, and heterocycles such as pyridine, imidazole, quinoline, etc.
  • a glycoprotein treated with sialidase (optionally) and galactose oxidase according to the invention is reacted with a nucleophile containing a moiety which is either positively or negatively charged under physiological conditions, in order to create a modified glycoprotein with enhanced stability towards serum proteases.
  • a nucleophile containing a moiety which is either positively or negatively charged under physiological conditions in order to create a modified glycoprotein with enhanced stability towards serum proteases.
  • Other alternatives that can increase stability towards proteolytical degradation includes, the attachment of hydrophobic side chains such as long chain alkanes and pol- yaromates, or attaching bulky hydrophilic polymers such as, e.g., polyethyleneglycol (PEG).
  • ligands that bind to serum proteins such as albumins may be conjugated to the glycan part of a glycoprotein therapeutic using the procedures described in this invention.
  • the glycoprotein when modified in such way will thereby be able to form a specific non covalent complex to e.g. albumin, which due to the overall size can escape glomerular filtration.
  • non covalent complexes may reduce the biological activity of the glycoprotein counterpart; it may be desirable to adjust the affinity of the ligand to the particular serum protein chosen, in order to retain biological activity.
  • a glycoprotein treated with galactose oxidase according to the invention is reacted with a reactant X, e.g., in the form of a nucleophile, containing a long chain fatty acid residue (e.g. C12, C14, C16 or C18) which can bind to human serum albumin, or diacids or lithocholic acids.
  • a reactant X e.g., in the form of a nucleophile, containing a long chain fatty acid residue (e.g. C12, C14, C16 or C18) which can bind to human serum albumin, or diacids or lithocholic acids.
  • a glycoprotein treated with galactose oxidase is treated, according to the invention, with a reactant X, e.g., in the form of a nucleophile, containing a portion of a protein having a long circulating half-life, such as an im- munoglobulin.
  • the glycoprotein is treated with a reactant X, e.g., in the form of a nucleophile, containing a F c domain of IgG
  • Mammalian glycoproteins often have N-acetyl-neuraminic acid (sialic acid) as the external (terminal) residue of the oligosaccharide chains which may be N-linked or O- linked (See, e.g., Osawa and Tsuji (1987) Ann. Rev. Biochem. 56:21). Where the nature of the oligosaccharide is the primary determinant for clearance from circulation, generally glycoproteins with terminal sialic acid residues removed (asialoglycoproteins) are cleared more quickly than their intact counterparts.
  • sialic acid N-acetyl-neuraminic acid
  • Circulating glycoproteins are exposed to sialidase(s) (or neuraminidase) which can remove terminal sialic acid residues.
  • sialidase(s) or neuraminidase
  • the removal of the sialic acid exposes galactose residues, and these residues are recognized and bound by galactose-specific receptors in hepatocytes (reviewed in Ashwell and Harford (1982) Ann. Rev. Biochem. 51 :531).
  • the liver also contains other sugar-specific receptors which mediate removal of glycoproteins from circulation. Specificities of such receptors also include N-acetylglucosamine, mannose, fucose and phosphomannose.
  • Modifications that result in increased half-life include, but are not limited to, exposure of galactose residues followed by oxidation or derivatization of the galactose such that binding of the modified glycoprotein to galactose receptor is inhibited or blocked.
  • a glycoprotein treated with galactose oxidase according to the invention is reacted with a nucleophile containing a steric moiety that prevents glycan specific receptor-recognition.
  • One embodiment is one in which the terminal sialic acid residues of the oligosaccharide side chains of the soluble protein or soluble protein derivative have been removed with neuraminidase treatment, and then the exposed galactose residues are oxidized by galactose oxidase (optionally also with horseradish peroxidase treatment).
  • the oxidation of the exposed galactose residues has the effect of preventing rapid clearance of the modified glycoprotein from circulation by specific galaetose receptors in the liver.
  • terminal galactose residues including but not limited to addition of a functional group or small molecule or mild oxidation treatment, which have the effect of blocking, inhibiting or preventing recognition of terminal galactose residues without destroying the biological activity are functionally equivalent.
  • Modified glycoproteins made in accordance with the present invention include those with structural alterations (modifications) of the oligosaccharide portions of the glycoprotein which result in prolonged circulating half-life by blocking or inhibiting clear- ance via sugar-specific receptors of the liver, by reducing renal clearance, or by minimizing proteolytically degradation. Modifications which substantially decrease the biological activity of the glycoprotein are to be avoided.
  • the glycoproteins are synthesized in a recombinant mammalian host.
  • the chemical and enzymatic treatments to produce the structural modifications of the oligosaccharide should not substantially alter the binding reaction of the glycoprotein to its biological target protein. It is preferred that the circulating half-life of modified glycoprotein of the present invention be at least about.
  • the structural modification of the oligosac- charide so as to prolong circulating half-life is the most conservative structural change which will achieve the end.
  • a variety of chemical derivatization procedures, or chemical and/or enzymatic procedures, as understood in the art, may be employed to produce the modified glycoprotein of the present invention.
  • any modified glycoprotein made in accordance with the present invention it is most desirable that an immunological response will not be elicited in a human patient exposed to the modified glycoprotein.
  • the biological activity is not significantly decreased to detrimentally affect the therapeutic function of the glycoprotein by the structural modification employed to confer prolonged circulation. It is also most desirable, that any structural modification of a glycoprotein does not result in toxicity in a patient to which that modified glycoprotein is administered.
  • the modified glycoprotein should have minimal toxic, irritant or other side effects upon administration to humans. Strategies for prolonging the circulation of a particular glycoprotein must therefore be evaluated on a case-by-case basis.
  • Chemical modification or derivatization of the C6 position of galactose is pre- ferred to maximize half-life and minimize clearance without significantly affecting biological function and without eliciting negative physiological reactions; minimal and mild treatment is preferred.
  • Functional groups or other structure-modifying molecules that may be added to the oligosaccharide portion of a glycoprotein in accordance with the present invention include any chemical group from the size of a single methyl group to larger polymeric groups such as, e.g., polyethylene glycol. Modifications which substantially decrease the biological activity of the glycoprotein are to be avoided. In general, the mechanism for clearance most be evaluated and the strategies for slowing or avoiding clearance must take into account maintenance of desired biological activity or function, potential toxicity, potential immunogenicity and cost.
  • a uniform modified glycoprotein may be incorporated in a therapeutic composition or a mixture of modified glycoprotein may be formulated in such a composition, so long as the desired therapeutic action is achieved by those molecules and so long as clearance by sugar-specific receptors mediating clearance, by proteolysis or by renal clearance, is inhibited or prevented by the modification or modifications made to said glycoprotein.
  • the methodology is generally applicable to therapeutic glycoproteins which are cleared from circulation by renal clearance, sugar specific receptors, or by proteases present in serum. It will be readily apparent that those of ordinary skill in the art that assays, reagents, procedures and technique other than those specifically described herein, can be employed to obtain the same or equivalent results and achieve the goals described herein. For example, chemical means of oxidation by removal of sialic acid can be readily substituted for enzymatic means specifically described. All such alternatives are encompassed by the spirit and scope of this invention.
  • a modified soluble glycoprotein derivative with increased plasma half-life as compared with the unmodified derivative is produced .
  • Increased circulating half-life of glycoproteins is achieved in general by means which block or inhibit removal of glycoprotein by galactose, mannose or other sugar-specific receptors, or by means that inhibits renal clearance, proteolytical degradation or immunological neutralization.
  • a protein containing an biantennary N-glucan is reacted with sialidase, galactose oxidase and a hydrogen peroxide scavenger (e.g., catalase) according to the invention and the resultant protein product treated with O- pegylated hydroxylamine to produce a glycol-glycoconjugate with reduced renal clearance.
  • a galactose oxidized glycoprotein prepared according to the invention is reacted with O-carboxymethyl hydroxylamine to produce a glycoprotein with isoelectric properties similar to wild type glycoprotein, but without any sialidase labile neuraminic acids.
  • a galactose oxidized glycoprotein prepared accord- ing to the invention is reacted with O-diethylaminoethyl hydroxylamine to produce a glycoprotein with altered binding properties to the hepatic lectine receptors.
  • a glycoprotein is optionally reacted with a sialidase followed by galactose oxidase and a nucleophile to produce a glycoconjugate with reduced renal clearance compared to the unmodified glycoprotein.
  • the produced glycoconjugate has a reduction in renal clearance of at least 50% compared to the unmodified glycoprotein.
  • the produced glycoconjugate has a reduction in renal clearance of at least 100% compared to the unmodified glycoprotein.
  • a glycoprotein is optionally reacted with a sialidase followed by galactose oxidase and a nucleophile to produce a glycoconjugate with reduced binding to the asialoglycoprotein receptor compared to the unmodified glycoprotein.
  • a glycoprotein is optionally reacted with a sialidase followed by galactose oxidase and a nucleophile to produce a glycoconjugate with reduced binding to the mannose receptor compared to the unmodified glycoprotein.
  • a glycoprotein is optionally reacted with a sialidase followed by galactose oxidase and a nucleophile to produce a glycoconjugate with reduced clearence by the liver compared to the unmodified glycoprotein.
  • the produced glycoconjugate has a reduction in liver clearance of at least 50% compared to the unmodified glycoprotein.
  • the produced glycoconjugate has a reduction in liver clearance of at least 100% compared to the un- modified glycoprotein.
  • a glycoprotein is optionally reacted with a sialidase followed by galactose oxidase and a nucleophile to produce a glycoconjugate with increased serum stability compared to the unmodified glycoprotein.
  • a glycoprotein is optionally reacted with a sialidase fol- lowed by galactose oxidase and a nucleophile to produce a glycoconjugate with increased circulation half-life in an animal model compared to the unmodified glycoprotein.
  • the produced glycoconjugate has 100% increased circulation half- life in an animal model compared to the unmodified glycoprotein.
  • the galactose oxidized glycoprotein prepared according to the invention is reacted with a nucleophile which is connected to a fatty acid (e.g. C 5 -C 24 ). The fatty acid can be connected to the nucleophile through a linker moiety.
  • the linker moiety can either be a simple structure designed to connect the fatty acid to the nucleophile, or it may contain functional groups (e.g. carboxylic acids, amines, alco- hoi, etc.) which enhance the in vivo properties of the embodiment.
  • the galactose oxidized glycoprotein prepared according to the invention is reacted with a nucleophile which is connected to an aliphatic diacid (e.g. C 5 -C 2 ).
  • the aliphatic diacid can be connected to the nucleophile through a linker moiety.
  • the linker moiety can either be a simple structure designed to connect the aliphatic diacid to the nucleophile, or it may contain functional groups (e.g.
  • the galactose oxidized glycoprotein prepared according to the invention is reacted with a reactant X, e.g., in the form of a nucleophile, which is connected to a structure that binds to serum proteins, like albumin.
  • a reactant X e.g., in the form of a nucleophile
  • the struc- ture that binds to serum proteins can be connected to the nucleophile through a linker moiety.
  • the linker moiety can either be a simple structure designed to connect the aliphatic diacid to the nucleophile, or it may contain functional groups (e.g.
  • the galactose oxidized glycoprotein prepared according to the invention is reacted with a reactant X, e.g., in the form of a nucleophile, which is connected to a structure (e.g. sialic acid derivatives) which inhibits the glycans from binding to receptors (e.g. asialoglycoprotein receptor and mannose receptor) that may remove the glycoprotein from circulation.
  • a reactant X e.g., in the form of a nucleophile, which is connected to a structure (e.g. sialic acid derivatives) which inhibits the glycans from binding to receptors (e.g. asialoglycoprotein receptor and mannose receptor) that may remove the glycoprotein from circulation.
  • the glycoprotein is first treated with a series of enzymes e.g. neuramidases, galactosidases, mannosidases, endo H and endo F3 sequentially, or directly by endo H or endo F3 to remove part of the glycan structure (see for example K. Witte et al. J. Am. Chem. Soc, 119, 2114 (1997)).
  • a series of enzymes e.g. neuramidases, galactosidases, mannosidases, endo H and endo F3 sequentially, or directly by endo H or endo F3 to remove part of the glycan structure (see for example K. Witte et al. J. Am. Chem. Soc, 119, 2114 (1997)).
  • a galactose moiety can then be added to the new terminus by employing a galactosyltransferase and the appropriate galactose substrate, or a series of transferases together with various carbohy- drate substrates may be employed before employing the galactosyltransferase.
  • the new glycan structure can then be oxidised and reacted with a nucleophile as described by the invention, thus yielding a glycoprotein with both an alternative glycan structure and a galactose modification which improves its therapeutic properties.
  • Glycoproteins suitable for conjugation in accordance with the present invention are described as "biologically active".
  • the term is not limited to physiological or pharmacological activities.
  • some inventive polymer conjugates containing proteins such as immu- noglobulin, enzymes with proteolytical activities and the like are also useful as laboratory diagnostics, i.e., for in vivo studies etc.
  • a key feature of all of the conjugates is that at least same portion of the activity associated with the unmodified bio-active peptide is maintained.
  • the conjugates thus are biologically active and have numerous therapeutic applications.
  • Humans in need of treatment which includes a biologically active peptide can be treated by administering an effective amount of a branched polymer conjugate containing the desired bioactive peptide.
  • Biologically active peptides of interest of the present invention include, but are not limited to proteins, peptides, peptides and enzymes. Enzymes of interest include carbohydrate-specific enzymes, proteolytic enzymes, oxidoreductases, transferases, hy- drolases, lyases. isomerases and ligasese.
  • examples of enzymes of interest include asparaginase, arginase, arginine deaminase, adenosine deaminase, superoxide dismutase. endotoxinases. cataiases. chymotrypsin, lipases, uricases, adenosine diphosphatase. tyrasinases, and bilirubin oxidase.
  • Carbohydrate-specific enzymes of interest include glucose oxidases, glycosidases, glucocerebrosi- dases. glucouronidases. etc.
  • Peptides and proteins, that do not contain glycan moieties can be glycosylated either enzymatically as described in Li Shao et all. Glycobiology 12(11) 762-770 (2002) using glycosyltransferases, or chemically synthesised , for example by using standard peptide chemistry and glycosylated amino acid components such as N-galactosylated as- paragine. Alternatively glycosylation sites may be engineered into proteins or peptides which in vivo normally are produced in their non-glycosylated form.
  • insertion of the consensus sequence Cys-XXX-Ser-XXX-Pro-Cys in an EGF repeat allows for selective O-glycosylation of serine using UDP-Glucose and glucosyltransferase Li Shao et all. Glycobiology 12(11) 762-770 (2002), whereas insertion of the consensus sequence Asn-XXX-Ser/Thr allows for N-glycosylation R.A. Dwek, Chem. Rev. 1996, 96, 683-720.
  • Peptide sequences containing threonine or serine also undergoes glycosylation in the presence of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase and UDP-GalNAc in a sequence dependent manner (see for example B.C. O'Connell, F.K.Hagen and L.A. Tabak in J. Biol. Chem. 267(35), 25010-25018 (1992)).
  • site directed mutagenesis introducing cystein mutations can be used for introsuction of galactose or galactose containing sugar structures via mixed disulphide fromation as described by D.P. Gamblin et al. in Angew. Chem. Int.
  • Galactose or N- acetylgalactosamine containing peptide and proteins can also be made by conjugation to proteins or peptides containing non-biogenical handles such as methods described by P.G. Schultz in J.Am.Chem.Soc, 125, 1702 (2003), or unspecifically by direct glycosylation of peptides using glycosyl donor substrates such as trichloroacetamidyl galactosides ect.
  • glycosidase inhibitores to fermentation cultures, thereby producing glycoproteins with truncated glycan structures as described in US 4925796A / US 5272066Alis also a possibility for obtaining galactose or N-acetylgalactosamine containing proteins, as well as enzymatic modification of glutamine residues using TGase (see for example M. Sato et al. Angew. Chem. Int. Ed. 43, 1516-1520, (2004). Production og N-glycosylated proteins are not limited to the use of mammalian host cells such as CHO or BHK cells, but also can be performed using bacterial cells as described by M. Wacker et al.
  • Proteins and peptides of interest include, but are not limited to, hemoglobin, serum proteins such as blood factors including Factors VII, , FX, FII, FV, protein C, protein S, tPA, PAI-1, tissue factor, FXI, FXII, and FXIII, as well as sequence FVIII, FIX variants thereof; immunoglobulins, cytokines such as interleukins, alpha-, beta-, and gamma- interferons, colony stimulating factors including granulocyte colony stimulating factors, platelet derived growth factors and phospholipase-activating protein (PUP).
  • hemoglobin serum proteins
  • serum proteins such as blood factors including Factors VII, , FX, FII, FV, protein C, protein S, tPA, PAI-1, tissue factor, FXI, FXII, and FXIII, as well as sequence FVIII, FIX variants thereof
  • immunoglobulins such as interleukins, al
  • proteins and peptides of general biological and therapeutic interest include insulin, plant pro- teins such as lectins and ricins, tumor necrosis factors and related alleles, soluble forms of tumor necrosis factor receptors, interleukin receptors and soluble forms of interleukin receptors, growth factors such as tissue growth factors, such as TGFa's or TGFps and epidermal growth factors, hormones, somatomedins, erythropoietin, pigmentary hor- mones, hypothalamic releasing factors, antidiuretic hormones, prolactin, chorionic go- nadotropin, follicle-stimulating hormone, thyroid-stimulating hormone, tissue plasmino- gen activator, and the like.
  • tissue growth factors such as TGFa's or TGFps and epidermal growth factors
  • hormones, somatomedins, erythropoietin, pigmentary hor- mones include hypothala
  • Immunoglobulins of interest include IgG, IgE. IgM. IgA, IgD and fragments thereof.
  • the glycoprotein is selected from the group consisting of: aprotinin, tissue factor pathway inhibitor or other protease inhibitors, insulin or insulin precursors, human or bovine growth hormone, interleukin, glucagon, oxyn- tomodulin,GLP-l, GLP-2, IGF-I, IGF-II, tissue plasminogen activator, transforming growth factor y or ⁇ , platelet-derived growth factor, GRF (growth hormone releasing factor), human growth factor, immunoglobulines, EPO, TPA, protein C, blood coagulation factors such as FVII, FVIII, FIX, FX, FII, FV, protein C, protein S, PAI-1, tissue factor, FXI, FXII, and FXIII, exendin-3, exentidin-4, and enzymes or functional analogues thereof.
  • the term "functional analogue” is meant to indicate a protein with a similar function as the native protein.
  • the protein may be structurally similar to the native protein and may be derived from the native protein by addition of one or more amino acids to either or both the C and N-terminal end of the native protein, substitution of one or more amino acids at one or a number of different sites in the native amino acid sequence, deletion of one or more amino acids at either or both ends of the native protein or at one or several sites in the amino acid sequence, or insertion of one or more amino acids at one or more sites in the native amino acid sequence.
  • the protein may be acylated in one or more positions, see, e.g., WO 98/08871, which discloses acylation of GLP-1 and analogues thereof, and WO 98/08872, which discloses acylation of GLP-2 and analogues thereof.
  • An example of an acylated GLP-1 derivative is Lys26(N epsilon -tetradecanoyl)-GLP-l (7-37) which is GLP-1 (7-37) wherein the epsilon-amino group of the Lys residue in position 26 has been tetradecanoylated.
  • the glycoprotein is selected from the group consisting of: FVII, FVIII, FIX, FX, FII, FV, protein C, protein S, tPA, PAI-1, tissue factor, FXI, FXII, FXIII, as well as sequence variants thereof; immunoglobulins, cytokines such as interleukins, alpha-, beta-, and gamma-interferons, colony stimulating factors including granulocyte colony stimulating factors, platelet derived growth factors and phospholi- pase-activating protein (PUP).
  • proteins and peptides of general biological and therapeutic interest include insulin, plant proteins such as lectins and ricins, tumor necrosis factors and related alleles, soluble forms of tumor necrosis factor receptors, inter- leukin receptors and soluble forms of interleukin receptors, growth factors such as tissue growth factors, such as TGFa's or TGFps and epidermal growth factors, hormones, soma- tomedins, erythropoietin, pigmentary hormones, hypothalamic releasing factors, antidiu- retic hormones, prolactin, chorionic gonadotropin, follicle-stimulating hormone, thyroid- stimulating hormone, tissue plasminogen activator, and the like.
  • tissue growth factors such as TGFa's or TGFps and epidermal growth factors
  • hormones soma- tomedins
  • erythropoietin pigmentary hormones
  • hypothalamic releasing factors antidiu- retic hormones
  • Immunoglobulins of interest include IgG, IgE. IgM. IgA, IgD and fragments thereof.
  • the glycoprotein is FVII.
  • the glycoprotein is FVIII.
  • the glycoprotein is FIX.
  • the glycoprotein is FXIII.
  • the proteins or portions thereof can be prepared or isolated by using techniques known to those of ordinary skill in the art such as tissue culture, extraction from animal sources, or by recombinant DNA methodologies. Transgenic sources of the proteins, peptides, amino acid sequences and the like are also contemplated. Such materials are obtained form transgenic animals, i.
  • mice e., mice, pigs, cows, etc.
  • proteins ex-pressed in milk, blood or tissues.
  • Transgenic insects and baculovirus expression systems are also contemplated as sources.
  • mutant versions, of proteins, such as mutant TNF's and/or mutant interferons are also within the scope of the invention.
  • Other proteins of interest are allergen proteins such as ragweed, Antigen E, honeybee venom, mite allergen, and the like.
  • allergen proteins such as ragweed, Antigen E, honeybee venom, mite allergen, and the like.
  • the protein is not CD4 protein; in another embodiment the protein is not soluble CD4 protein.
  • the glycoprotein is FVII having the amino acid sequence of wild-type Factor VII (figure 9).
  • the polypeptides are wild-type Factor Vila.
  • the glycoprotein is a Factor VII polypeptide.
  • the Factor VII polypeptide is a Factor VII variant having substantially the same biological activity as wild-type Factor VII including S52A-FVIIa, S60A-FVIIa ( Lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998); FVIIa variants exhibiting increased proteolytic stability as disclosed in U.S. Patent No.
  • FVII variants as disclosed in PCT/DK02/00189; and FVII variants exhibiting increased prote- olytic stability as disclosed in WO 02/38162 (Scripps Research Institute); FVII variants having a modified Gla-domain and exhibiting an enhanced membrane binding as disclosed in WO 99/20767 (University of Minnesota); and FVII variants as disclosed in WO 01/58935 (Maxygen ApS) and WO 04/029091 (Maxygen ApS).
  • the Factor VII polypeptide is a FVII variant having increased biological activity compared to wild-type FVIIa
  • FVII variants as disclosed in WO 01/83725, WO 02/22776, WO 02/077218, PCT/DK02/00635, Danish patent application PA 2002 01423, Danish patent application PA 2001 01627; WO 02/38162 (Scripps Research Institute); and FVIIa variants with enhanced activity as disclosed in JP 2001061479 (Chemo-Sero-Therapeutic Res Inst.).
  • the Factor VII polypeptides are selected from the group consisting of: L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P- FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, K157A- FVII, E296V
  • the Factor VII polypeptides are selected from the list consisting of: Factor VII variants having increased biological activity compared to wild- type FVIIa as disclosed in WO 01/83725, WO 02/22776, WO 02/77218, WO 03/27147, and WO 03/37932; L305V/K337A-FVII, L305V/V158D-FVII, L305V/E296V-FVII, L305V/M298Q-FVII, L305V/V158T-FVII, L305V/K337A/V158T-FVII, L305V/K337A/M298Q-FVII, L305V/K337A/E296V-FVII, L305V/K337A/V158D-FVII, L305V/V158D/M298Q-FVII, L305V/V158D/E296V-FVII, L305V/V158T/M298Q
  • the Factor Vll-related polypeptides are selected from the group consisting of: R152E-Factor VII, S344A- Factor VII, FFR- Factor VII, and Factor Vila lacking the Gla domain. In one embodiment, the Factor Vll-related polypeptide exhibit at least about
  • the Factor Vll-related polypeptide exhibit less than about
  • the conjugated polypeptide exhibits a bioavailability that is at least about 110% of the bioavailability of a reference preparation, such as at least about 120%, or at least about 130%, or at least about 140% of the bioavailability of the reference preparation.
  • the conjugated polypeptide exhibits a serum half-life that is at least about 125% of the half-life of a reference preparation, such as at least about 150%, or at least about 200%, or at least about 250% of the half-life of the reference preparation.
  • the glycoconjugates prepared according to the present invention exhibit improved functional properties relative to reference preparations.
  • the improved functional properties may include, without limitation, a) physical properties such as, e.g., storage stability; b) pharmacokinetic properties such as, e.g., bioavailability and half-life; and c) immunogenicity in humans.
  • a reference preparation refers to a preparation comprising a polypeptide that has an amino acid sequence identical to that contained in the preparation of the invention to which it is being compared (such as, e.g., non-conjugated forms of wild-type Factor VII or a particular variant or chemically modified form) but which is not conjugated to any polymer molecule(s) found in the preparation of the invention.
  • reference preparations typically comprise non-conjugated glycoprotein.
  • Storage stability of a glycoprotein may be assessed by measuring (a) the time required for 20% of the bioactivity of a preparation to decay when stored as a dry powder at 25°C and/or (b) the time required for a doubling in the proportion of predetermined degradation products, such as, e.g., aggregates, in the preparation.
  • the preparations of the invention exhibit an increase of at least about 30%, preferably at least about 60% and more preferably at least about 100%, in the time required for 20% of the bioactivity to decay relative to the time required for the same phenomenon in a reference preparation, when both preparations are stored as dry powders at 25°C.
  • Bioactivity measurements may be performed in accordance with the kind of bio- activity associated with the particular protein; in case of, e.g., FVII, bioactivity may be measured using any of a clotting assay, proteolysis assay, TF-binding assay, or TF- independent thrombin generation assay.
  • the preparations of the invention exhibit an increase of at least about 30%, preferably at least about 60%, and more preferably at least about 100%, in the time required for doubling of predetermined degradation products, such as, e.g., aggregates, relative to a reference preparation, when both preparations are stored as dry powders at 25°C.
  • the content of aggregates may, for example, be determined by gel permeation HPLC, or another type of well-known chromatography methods.
  • aggregates may be determined by gel permeation HPLC on a Protein Pak 300 SW column (7.5 x 300 mm) (Waters, 80013) as follows. The column is equilibrated with Eluent A (0.2 M ammonium sulfate, 5 % isopropanol, pH adjusted to 2.5 with phosphoric acid, and thereafter pH is adjusted to 7.0 with triethylamine), after which 25 ⁇ g of sample is applied to the column.
  • Eluent A 0.2 M ammonium sulfate, 5 % isopropanol, pH adjusted to 2.5 with phosphoric acid, and thereafter pH is adjusted to 7.0 with triethylamine
  • Bioavailability refers to the proportion of an administered dose of a glycoconjugate that can be detected in plasma at predetermined times after administration. Typically, bioavailability is measured in test animals by administering a dose of between about 25-250 ⁇ g/kg of the preparation; obtaining plasma samples at predetermined times after administration; and determining the content of glycoprotein in the samples using a suitable bioassay, or immunoassay, or an equivalent.
  • the data are typically displayed graphically as [glycoprotein] v. time and the bioavailability is expressed as the area under the curve (AUC).
  • AUC area under the curve
  • Relative bioavailability of a test preparation refers to the ratio between the AUC of the test preparation and that of the reference preparation.
  • the preparations of the present invention exhibit a relative bioavailability of at least about 110%, preferably at least about 120%, more preferably at least about 130% and most preferably at least about 140% of the bioavailability of a reference preparation.
  • the bioavailability may be measured in any mammalian species, preferably dogs, and the predetermined times used for calculating AUC may en- compass different increments from 10 min- 8 h.
  • o2-3,6,8,9-Neuraminidase (E.C. 3.2.1.18) from Anthrobacter urefaciens was obtained from Calbiochem, CA, USA, soluble neuraminidase from Vibreo cholerae, agarose supported neuraminidase from Clostridium perfringens and bovin catalase (E.C. 1.11.1.6) were obtained from Sigma-Aldrich.
  • Galactose oxidase (E.C. 1.1.3.9) was obtained Wor- thington Biochemical Corporation, USA.
  • PNGase F was from New England Biolabs Inc. MA, USA. All other chemicals were of standard grade and obtained from Sigma-Aldrich, Bachem or Fluka.
  • Galactose oxidase kit A22179 was obtained from Molecular Probes, OR, USA. Proton and carbon nuclear magnetic resonance ( H and 13 C NMR) were recorded on a Bruker 300 MHz or 400 MHz NMR apparatus, with chemical shift ( ⁇ ) reported down field from tetramethylsilane.
  • MALDI-TOF spectra were obtained using an Autoflex MALDI-TOF mass spectrophotometer from Bruker Daltonics Inc. Spectra were recorded in the linear mode using o-cyano-4-hydroxycinnamic acid as matrix.
  • the following method can be used to assay Factor Vila bioactivity.
  • the assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark).
  • the absorbance at 405 nm is measured continuously in a SpectraMaxTM 340 plate reader (Molecular Devices, USA).
  • the absorbance developed during a 20-minute incubation, after subtraction of the ab- sorbance in a blank well containing no enzyme, is used to calculate the ratio between the activities of a test and a reference Factor Vila.
  • the following method can be used to assay Factor Vila bioactivity.
  • the assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark).
  • Factor Vila (10 nM) and Factor X (0.8 uM) in 100 ⁇ l 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 5 mM CaCI 2 and 1 mg/ml bovine serum albumin, are incubated for 15 min.
  • Factor X cleavage is then stopped by the addition of 50 ⁇ l 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 20 mM EDTA and 1 mg/ml bovine serum albumin.
  • the amount of Factor Xa generated is meas- ured by addition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden), final concentration 0.5 mM.
  • the absorbance at 405 nm is measured continuously in a SpectraMaxTM 340 plate reader (Molecular Devices, USA).
  • the absorbance developed during 10 minutes, after subtraction of the absorbance in a blank well containing no FVIIa, is used to calculate the ratio between the proteolytic activities of a test and a reference Factor Vila.
  • Measurement of in vivo biological half-life can be carried out in a number of ways as described in the literature.
  • An example of an assay for the measurement of in vivo half-life of rFVIIa and variants thereof is described in FDA reference number 96-0597. Briefly, FVIIa clotting activity is measured in plasma drawn prior to and during a 24-hour period after administration of the conjugate, polypeptide or composition. The median apparent volume of distribution at steady state is measured and the median clearance determined.
  • Bioavailability may, for example, be measured in a dog model as follows: The experimen- tis performed as a four leg cross-over study in 12 Beagle dogs divided in four groups. All animals receive a test preparation A and a reference preparation B at a dose of about 90 ⁇ g/kg in a glycylglycine buffer (pH 5.5) containing sodium chloride (2.92 mg/ml), calcium chloride dihydrate (1.47 mg/ml), mannitol (30 mg/ml) and polysorbate 80. Blood samples are withdrawn at 10, 30, and 60 minutes and 2, 3, 4, 6 and 8 hours following the initial administration. Plasma is obtained from the samples and Factor VII is quanti- fied by ELISA.
  • CE-analysis Capillary electrophoresis was performed on a Hewlett-Packard HP 3DCE sys- tem equipped with a UV-VIS DAD with a range from 200 to 600 nm and complete pneumatic system, enabling automated flushing of capillaries and replenishment of buffer vials.
  • the instrument was operated in the normal polarity mode at +7.0-7.5 kV/ + 80-100 uA and detection was performed on column, at 214 nm with 280 nm as reference wavelength.
  • CE analysis was carried out using fused-silica capillaries of 64,5 cm (55,5 cm ef- fective length) x 75 um I.D. The capillary was thermostated at 30 °C.
  • Electrophoretic data were collected at a rate of 10 Hz. Electrolytes were prepared by dissolving known amount of putrescein dihydrochloride in 100 mM phosphate buffer (pH 8.0) and subsequently adjusting to pH 8.0 with IN sodium hydroxide solutions (see N.K. Klausen and T. Kornfelt, J. Chromatography A, 718, 195-202 (1995)).
  • LC-MS mass spectra were obtained using apparatus consisting of a Hewlett Packard series 1100 G1312A Bin Pump, a Hewlett Packard series 1100 Column compartment, a Hewlett Packard series 1100 G13 15A DAD diode array detector and a Hewlett Packard series 1100 MSD.
  • the instrument was controlled by HP Chemstation software.
  • the HPLC pump was connected to two eluent reservoirs containing 0.01% TFA in water (A) and
  • N-glycan analysis procedure was modified from D.I. Papac et al. Glycobiology 8(5), 445-454 (1998) and illustrated for FVIIa as followes: The pH of the FVIIa solution (lmg/ml in lOmM glycylglycin, lOmM CaCI 2 , 50mM NaCl, pH 6.5) (50 ⁇ l) is adjusted to 8.3 by addition of NaOH 50mM.
  • PNGase F 500U/ml in lOmM Tris acetate buffer pH8.3 is added (20 ⁇ l, 10U), followed by Tris acetate pH 8.3 buffer (total volume of the reaction mixture: 80 ⁇ l).
  • the reaction mixture is incubated overnight at 37°C.
  • PVDF membrane Immobilon-P from Millipore, Millipore Corporation, Bedford, Ma, USA
  • Acetic acid 10% is added (10 ⁇ l) and the reaction mixture shaken for 2h at RT.
  • the reaction mixture is then added to the resin (Dowex 50W-X8, H form) (0.3ml). Water (280 ⁇ l) is added, and the mixture is vortexed. The supernatant is taken out, evaporated, re- dissolved in water (20 ⁇ l), and analyzed by MALDI-TOF as described by Papac et al.
  • Example 1-4 illustrates the synthesis of representative O-substituted hydroxylamine nucleophiles suitable for conjugation to galactose oxidized glycans according to the invention.
  • ⁇ /-hydroxyphthalimide (10.0 g, 62.5 mmol) was dissolved in THF (100 ml) and potassium carbonate (18.63 g; 0.134 mol) was added. Diethylaminoethylchloride (10.54 g; 61.5 mmol) was added and the mixture was heated to reflux for 4h. The mixture was cooled and added water (100 ml) and stirred until all solid had dissolved. The mixture was then extracted twice with DCM. The organic phase was dried with anhydrous sodium sulphate and evaporated to give 8.63 g (53%) of ⁇ /-( ⁇ / / ⁇ /-diethylaminoethoxy)phthalimide as a clear yellow oil.
  • 3-Aminopropionic acid te/t-butyl ester hydrochloride (7.27 g. 40 mmol) was dissolved in DMF (40 ml). Triethylamine was added (4.05 g, 40 mmol). A solution of tert-butyl acry- late (5.13 g, 40 mmol) in DMF (40 ml) was added to the solution under inert atmosphere. After stirring at rt for 16 h, the precipitate was filtered off, and the filtrate is concentrated, dissolved in ethyl acetate (100 ml), washed with sat. NaHC0 3 (2 x 50ml), dried over MgS0 4 and concentrated.
  • Example 3 mPEG 500 o-(CH 2 ) 2 -CONH-(CH 2 ) 4 -0-NH 2
  • Step 1 0-(4-Aminobutyl)-W-(tert-butoxycarbonyl)hydroxylamine (120 mg, 0.584 mmol) was dissolved in DCM (25 ml) under nitrogen. mPEG (50 oo ) -succinimidyl propionate (1 g, 0.195 mmol) was added, and the solution was stirred for 16 h. Diethyl ether (150 ml) was added, and the resulting precipitate was filtered off and washed with diethyl ether (2 x 60 ml). The precipitate was dried under vacuum for 16 h to yield a white solid (911 mg).
  • Step 2 mPEG 50 oo-(CH 2 ) 2 -CONH-(CH 2 ) 4 -0-NH-Boc (911 mg, 0.174 mmol) was dissolved in TFA (8 ml). The solution was stirred under nitrogen for 30 min at rt. Diethyl ether (150 ml) was added, and after stirring for 30 min the resulting precipitate was filtered off and washed with diethyl ether (2 x 50 ml). The solid was dried in a vacuum oven at 35 °C for 16 h. to yield a white solid (800 mg, 90% yield).
  • Step 1 16-Bromo-hexadecanoic acid methyl ester (1.05 g, 3 mmol) and N-tert- butoxycarbonyl hydroxylamine (1 g, 7.5 mmol) were placed in a flask, and dissolved in DBU (2.25 ml). The solution was stirred under nitrogen for 3 h. DCM (100 ml) was added, and the solution was washed with 1 N HCl (3 x 25 ml) using some brine and methanol to assist in phase separation. The solution was dried over MgS0 4 , and concentrated to yield an oily crystalline residue (1.09 g, 90% yield).
  • Step 2 16-(/V-te/t-butoxycarbonylaminooxy)hexadecanoic acid methyl ester (1.05 g, 2.62 mmol) was placed in THF (100 ml) and 1 N NaOH (2.75 ml) was added. The sample was stirred at rt for 16 h then refluxed for 2 h. After cooling to rt, 4N NaOH (20 ml) and methanol (50 ml) were added and the solution was stirred at rt for 45 min. The solution was neutralized with cone HCl and cooled in an ice bath.
  • Example 5-15 illustrates enzymatic protocols and chemical conjugation methods for obtaining glycoproteins with modified glycan structures according to the invention.
  • the elution buffer was then changed to 10 mM Tris, 50 mM NaCl, 25 mM CaCI 2 , pH 8.0 (10 vol, flow: 1 ml/min). Pure FVIIa samples were pooled. The buffer was then ex-changed to 10 mM glycylglycin, 10 mM CaCI 2 , 50 mM NaCl, pH 6 using NAP-10 columns (Amersham), and samples stored at - 80 °C until later use. Amidolytic activity toward the peptide substrate S2288 (see in vitro hydrolysis assay section) was measuered to be identical to non-modified rFVIIa.
  • the galactose assay kit from Molecular Probes (A22179), used to quantify the amount of exposed galactose residues on proteins (see the analytical procedure section), gave a ration of 3.70 umol galactose / umol FVIIa (figure 1) equivalent to complete removal of all sialic acids from FVIIa when assuming the presence of two N-linked (N145/322) bi-antenna structures.
  • Example 7 Method using agarose supported neuraminidase from Clostridium perfringens.
  • Neuraminidase-agarose resin (Clostridium perfringens, Type VI-A, Sigma N 5254) as ammonium sulfate suspension (2 ml), was washed extensively with MilliQ water, then drained and added to a 5 ml solution of FVIIa (1.4 mg/ml in 10 mM glycylglycin, 10 mM CaCI 2 , 50 mM NaCl, pH 6). The mixture was shaken gently at room temperature for 48h, then neuraminidase-agarose resin was filtered off.
  • the galactose assay kit from Molecular Probes (A22179), used to quantify the amount of exposed galactose residues on proteins (see the analytical procedure section), gave a ratio of 3.90 umol galactose / umol FVIIa (figure 1) equivalent to complete removal of all sialic acids from FVIIa when assuming the presence of two N-linked (N145/322) bi-antenna structures. Removal of sialic acids can alternatively by analyzed by the CE-protocol as described in the analytical section.
  • Step A buffer eschange: FVIIa (1 ml, 28 nmol, 1.4 mg/ml) in 10 mM glycylglycin buffer (pH 6.0) containing 10 M CaCI2 and 50 mM NaCl is added to a 1 ml NAP-10 column (Amersham Bioscience) previously callibrated with 15 ml of 10 mM MES buffer (pH 6.0) containing 10 mM CaCI2 and 50 mM NaCl. The solution is allowed to pass into the bed of the column.
  • Step B neuroaminidase treatment: The eluate is added 75 mU neuraminidase (from Vibro cholerae, EC 3.2.1.18), and the mixture is incubated at room temperature for 24h.
  • Step C galactose oxidase + reaction with nucleophile: A freshly prepared solution of 12 U galactose oxidase (EC. 1.1.3.9) and 240 U of catalase (EC.
  • N-glycanes from a small aliquot are subsequently cleaved using the PNGase F protocol and analysed using procedures described in the analytical section, and the FVIIa modified end product is then purified as described in example 6 and stored at - 80 °C until later use.
  • the progress of the reaction is monitored either by capillary electrophoresis using the method specified in the material section, or by performing an IEF gel (or SDS-PAGE gel depending on the type of hydroxylamine) on small aliquots according to Invitrogen's procedure.
  • the glycan derivatized FVIIa analogue is purified by ion-exchange chromatography according to L. Thim et al. Biochemistry, 1988, 27, 7785-7793, or as described in example 6.
  • the product is finally characterized by capillary electrophoreses as described in the analytical section, MALDI-TOF or other appropriate methods for protein analysis. Modified N-glycans are cleaved using the PNGase F protocol and analyzed by MALDI-TOF method described in the analytical section.
  • the progress of the reaction is monitored either by capillary electrophoresis using the method specified in the material section, or by per- forming an IEF gel (or SDS-PAGE gel depending on the type of hydroxylamine) on small aliquots according to Invitrogen's procedure.
  • the glycan derivatized glycoprotein is purified by appropriate chromatographically techniques.
  • the product is finally characterized by capillary electrophoreses, MALDI-TOF or other appropriate methods for protein analysis. Modified N-glycans is analysed by the PNGase F - MALDI-TOF method described in the analytical section.
  • a solution of 250 ul desialylated glycoprotein (24 uM) in a 10 mM MES buffer (pH 6.0) is reacted with agarose immobilised galactose oxidase (100 mg, with a typically activity of 70 U/g support) and agarose immobilised catalase (100 mg, with a typically activity of 3000 U/g support) in MES buffer (pH 6.0) containing 1.20 mM (50x) of nucleophilic agent (e.g. R-CO-NHNH2, R-NHNH2 or R-0-NH 2 ).
  • nucleophilic agent e.g. R-CO-NHNH2, R-NHNH2 or R-0-NH 2
  • the progress of the reaction is monitored either by capillary electrophoresis using the method specified in the material section, or by performing an IEF gel (or SDS-PAGE gel depending on the type of hydroxylamine) on small aliquots according to Invitrogen's procedure.
  • IEF gel or SDS-PAGE gel depending on the type of hydroxylamine
  • bead supported enzymes are removed by filtration and the glycan derivatized glycoprotein subsequently purified by appropriate chromatographically techniques.
  • the product is finally characterized by capillary electrophoreses, MALDI-TOF or other appropriate methods for protein analysis. Modified N- glycans is analysed by the PNGase F - MALDI-TOF method described in the analytical section.
  • Example 12 mPEG 50 oo-(CH 2 ) 2 -CONH-(CH 2 ) 4 -0-NH 2 ligation to FVIIa.
  • Step 1 preparation of asialo FVIIa: Neuraminidase (Clostridium Perfringens on agarose, Sigma N5254) was washed with milli-Q water (3 x 15 ml), and was added to a solution of FVIIa (11 ml, 1.4 mg/ml, in Gly-gly buffer). The sample was shaken gently for 16 h.
  • Neuraminidase Clostridium Perfringens on agarose, Sigma N5254
  • the neuraminidase was filtered off, and the buffer was exchanged to a MES buffer (10 mM MES, 10 mM CaCI 2 , 50 mM NaCl, pH 6) using NAP-25 columns and a NAP-10 column (Amersham biosciences), yielding the asialo FVIIa in a MES buffer. Analysis using an IEF- gel indicates a change in the proteins pi.
  • Step 2 One-pot Galactose oxidation and oxime formation with mPEG (500 o)-(CH 2 ) 2 -CONH- (CH 2 ) -0-NH 2 ):
  • mPEG 500 o
  • 5 ml Some of the asialo FVIIa from above (5 ml) was added Galactose oxidase (1.28 mg, of 51 U/mg), Catalase (9.24 mg of 1183 U/mg) and mPEG (50 oor(CH 2 ) 2 -CONH- (CH 2 ) -0-NH 2 (9.53 mg). The sample was allowed to stand at rt for 20 h.
  • the fractions containing FVIIa were collected and analysed by SDS-PAGE. The increased mass of the products was visible by SDS-PAGE (figure 5)
  • Asialo FVIIa (10.5 mg, in Gly-Gly buffer pH 6.0 (7.5 ml)), as prepared in example 2 or 3 was submitted to buffer exchanged to a 10 mM MES, 10 mM CaCI2, 50 mM NaCl, pH 6.0 buffer, using three NAP-25 (Amersham) column previously equilibrated with 10 mM MES, 10 mM CaCl 2 , 50 mM NaCl, pH 6.0.
  • the elution buffer was then changed to 10 mM Tris, 50 mM NaCl, 25 mM CaCI 2 , pH 8.0 (10 vol, flow: 1 ml/min). Fractions were analyzed by IEF gels (figure 6) and pure aminoxyacetic acid modified FVIIa samples (having pi of approxi- matly 5.8 on the gel) were pooled. The buffer was then ex-changed to 10 mM Gly-Gly, 10 mM CaCI 2 , 50 mM NaCl, pH 7 using NAP-10 columns (Amersham), and samples stored at - 80 °C until later use. The peptidolytical activity using the S2288 peptide substrate (see the in vitro assay section) was measured to 54% of starting material.
  • Example 14 One pot method for derivatization with p-nitrobenzyloxyamine.
  • Step A buffer exchange: FVIIa (1 ml, 28 nmol, 1.4 mg/ml) in 10 mM GlyGly buffer (pH 6.0) containing 10 mM CaCl2 and 50 mM NaCl was added to a 1 ml NAP-10 column (Amersham Bioscience) pre- viously calibrated with 15 ml of 10 mM MES buffer (pH 6.0) containing 10 mM CaC12 and
  • Step B neuroaminidase treatment: The eluate was added 75 mU neuraminidase (from
  • Step C galactose oxidase + reaction with nucleophile: A freshly prepared solution of 12
  • Step D analysis of derivatized N-glycanes: All the sample was used for analysis. The N- glycanes were cleaved using PNGase F, and the released N-glycanes identified by MALDI- TOF spectroscopy using the methods described in the analytical section. Two derivatized glycan structures was identified (figure 7) corresponding to modified biantenna with Gal and GalNAc modifications respectively.
  • Example 15 One-pot derivatization with aminoxyacetic acid.
  • Step A buffer exchange: FVIIa (1 ml, 28 nmol, 1.4 mg/ml) in 10 mM GlyGly buffer (pH 6.0) containing 10 mM CaCI2 and 50 mM NaCl was added to a 1 ml NAP-10 column (Amersham Bioscience) previously callibrated with 15 ml of 10 mM MES buffer (pH 6.0) containing 10 mM CaCI2 and 50 mM NaCl. The solution was allowed to pass into the bed of the column. Then 1.5 ml of 10 mM MES buffer (pH 6.0) containing 10 mM CaCI 2 and 50 mM NaCl was added, while collecting the 1.5 ml of eluate.
  • Step B neuroaminidase treatment: 1 ml of the eluate was added 150 mU neuraminidase (from Vibro cholerae, EC 3.2.1.18), and the mixture was incubated at room temperature for 24h.
  • Step C galactose oxidase + reaction with nucleophile: A freshly prepared solution of 300 mU galactose oxidase (EC. 1.1.3.9) and 1316 U of catalase (EC.
  • Desialylation of sialylated glycoproteins using solid supported neuraminidases (preparation of asialo glycoproteins) : 250 ul of a solution of a sialylated glycoprotein (1.24 mg/ml, 24 uM) in a 10 mM MES buffer (pH 6.0) is added o2-3,6,8,9-neuraminidase immobilised on agarose support and the reaction mixture is incubated for 32h at 4°C. To monitor the reaction, a small aliquot of the reaction is diluted with the appropriate buffer and an IEF gel performed according to Invitrogen's procedure. Samples is also analysed by capillary electrophoresis according to the methods described in the material section.
  • the instrument was controlled by HP Chemstation software.
  • the HPLC pump was connected to two eluent reservoirs containing :
  • the analysis was performed at 40 °C by injecting an appropriate volume of the sample (preferably 1 ⁇ L) onto the column, which was eluted with a gradient of acetonitrile.
  • an appropriate volume of the sample preferably 1 ⁇ L
  • the HPLC conditions, detector settings and mass spectrometer settings used are given in the following table.
  • Boc tert-butoxy carbonyl
  • CDI carbonyldiimidazole
  • DCM dichloromethane, methylenechloride
  • DIPEA diisopropylcarbodiimide
  • DhbtOH 3-hydroxy-l,2,3-benzotriazin-4(3r/)-one
  • DMAP 4-dimethylaminopyridine
  • DMF /V / ⁇ /-dimethylformamide
  • DMSO dimethyl sulphoxide
  • DTT Dithiothreitol EtOH : ethanol
  • Fmoc 9-fluorenylmethyloxycarbonyl
  • HOBt 1-hydroxybenzotriazole MeOH: methanol
  • 2-(2-Chloroethoxy)ethanol (100.00 g; 0.802 mol) was dissolved in dichloromethane (100 ml) and a catalytical amount of boron trifluride etherate (2.28 g; 16 mmol).
  • the clear solution was cooled to 0 °C, and epibromhydrin (104.46 g; 0.762 mol) was added drop- wise maintaining the temperature at 0 °C.
  • the clear solution was stirred for an additional 3h at 0 °C, then solvent was removed by rotary evaporation.
  • Trichloroacetylchloride (1,42 g, 7.85 mmol) was dissolved in THF (10 ml), and the solu- tion was cooled to 0 °C.
  • a solution of l,3-Bis[2-(2-azidoethoxy)ethoxy]propan-2-ol (1.00 g; 3.3 mmol) and triethylamine (0,32 g, 3.3 mmol) in THF (5 ml) was slowly added drop wise over 10 min. Cooling was removed, and the resulting suspension was stirred for 6h at ambient temperature. The mixture was filtered, and the filtrate was evaporated to give a light brown oil. The oil was treated twice with acetonitril following evaporation, and the product was used without further purification.
  • Tritylchloride (lOg, 35.8 mmol) was dissolved in dry pyridine, diethyleneglycol (3.43 mL, 35.8 mmol) was added and the mixture was stirred under nitrogen overnight. The solvent was removed in vacuo. The residue was dissolved in dichloromethane (100 mL) and washed with water. The organic phase was dried over Na 2 S0 4 and solvent was removed in vacuo. The crude product was purified by recrystallization from heptane/toluene (3:2) to yield the title compound.
  • 2-(2-Trityloxyethoxy)ethanol (6.65 g, 19 mmol) was dissolved in dry THF (100 mL). 60 % NaH - oil suspension (0.764 mg, 19 mmol) was added slowly. The suspension was stirred for 15 min. Epibromohydrin (1.58 mL, 19 mmol) was added and the mixture was stirred under nitrogen at room temperature overnight. The reaction was quenched with ice, separated between diethyl ether (300 mL) and water (300 mL). The water phase was extracted with dichloromethane.
  • 2-(2-Trityloxyethoxy)ethanol (1.14 g, 3.28 mmol) was dissolved in dry DMF (5 mL). 60 % NaH - oil suspension (144 mg, 3.61 mmol) was added slowly and the mixture was stirred under nitrogen at room temperature for 30 min. The mixture was heated to 40°C. 2-[2-(2-Trityloxyethoxy)ethoxymethyl]oxirane (1.4 g, 3.28 mmol) was dissolved in dry DMF (5 mL) and added drop wise to the solution under nitrogen while stirring was maintained. After ended addition the mixture was stirred under nitrogen at 40°C overnight.
  • ⁇ /, ⁇ /-Bis(2-(2-phthalimidoethoxy)ethyl)-0-tert-butylcarbamate is dissolved in a polar solvent such as ethanol. Hydrazine (or another agent known to remove the phthaloyl protecting group) is added. The mixture is stirred at room temperature (or if necessary ele- vated temperature) until the reaction is complete. The mixture is concentrated under vacuum as much as possible. The crude compound is purified by standard column chromatography or if possible by vacuum destination.
  • ⁇ /, ⁇ /-Bis(2-(2-aminoethoxy)ethyl)-0-te t-butylcarbamate is dissolved in a mixture of aqueous sodium hydroxide and THF or in a mixture of aqueous sodium hydroxide and acetonitrile.
  • Benzyloxychloroformate is added.
  • the mixture is stirred at room tempera- ture until the reaction is complete. If necessary, the volume is reduced in vacuo.
  • Ethyl acetate is added.
  • the organic phase is washed with brine.
  • the organic phase is dried, filtered, and subsequently concentrated in vacuo as much as possible.
  • the crude compound is purified by standard column chromatography.
  • EXAMPLE 45 ll-Oxo-17-phthalimido-12-(2-(2-phthalimidoethoxy)ethyl)-3,6,9,15-tetraoxa-12- azaheptadecanoic acid
  • 3,6,9-Trioxaundecanoic acid is dissolved in dichloromethane.
  • a carbodiimide e.g., ⁇ /. /V- dicyclohexylcarbodiimide or /V, ⁇ -diisopropylcarbodiimide
  • the solution is stirred over night at room temperature.
  • the mixture is filtered.
  • the filtrate can be concentrated in vacuo if necessary.
  • the acylation of amines with the formed intramolecular anhydride is known from literature (e.g., Cook, R. M.; Adams, J. H.; Hudson, D.
  • EXAMPLE 46 5-Oxo-l l-phthalimido-6-(2-(2-phthalimidoethoxy)ethyl)-3,9-dioxa-6-azaundecanoic acid
  • the reaction is known (Schneider, S.E. et al. Tetrahedron, 1998, 54(50) 15063-15086) and can be performed by treating the support bound azide with excess of triphenyl phosphine in a mixture of THF and water for 12-24 hours at room temperature.
  • trimethylphosphine in aqueous THF as described by Chan, T.Y. et al Tetrahedron Lett. 1997, 38(16), 2821-2824 can be used.
  • Reduction of azides can also be performed on solid phase using sulfides such as dithiothreitol (Meldal, M. et al. Tetrahedron Lett.
  • Solid phase carbamate formation The reaction is known and is usually performed by reacting an activated carbonate, or a halo formiate derivative with an amine, preferable in the presence of a base.
  • This example uses the 2-(l,3-Bis[azidoethoxyethyl]propan-2-yloxy)acetic acid monomer building block prepared in example 6 in the synthesis of a second generation amide based dendrimer capped with 2-[2-(2-methoxyethoxy)ethoxy]acetic acid.
  • the coupling chemistry is based on standard solid phase peptide chemistry, and the protection methodology is based on a solid phase azide reduction step as described above.
  • Step 1 Fmoc- ⁇ ala-Wang resin (100 mg; loading 0.31 mmol/g BACHEM) was suspended in dichloromethane for 30 min, and then washed twice with DMF. A solution of 20% piperidine in DMF was added, and the mixture was shaken for 15 min at ambient temperature. This step was repeated, and the resin was washed with DMF (3x) and DCM (3x).
  • Step 2 Coupling of monomer building blocks: A solution of 2-(l,3- bis[azidoethoxyethyl]propan-2-yloxy)acetic acid (527 mg; 1,4 mmol, 4x) and DhbtOH (225 mg; 1,4 mmol, 4x) were dissolved in DMF (5 ml) and DIG (216 ul, 1,4 mmol, 4x) was added, The mixture was left for 10 min (pre-activation) then added to the resin to- gether with DIPEA (240 ul; 1,4 mmol, 4x). The resin was shaken for 90 min, then drained and washed with DMF (3x) and DCM (3x).
  • Step 3 Capping with acetic anhydride: The resin was then treated with a solution of ace- tic anhydride, DIPEA, DMF (12:4:48) for 10 min. at ambient temperature. Solvent was removed and the resin was washed with DMF (3x) and DCM (3x).
  • Step 4 Deprotection (reduction of azido groups): The resin was treated with a solution of DTT (2M) and DIPEA (IM) in DMF at 50 °C for 1 hour. The resin was then washed with DMF (3x) and DCM (3x). A small amount of resin was redrawn and treated with a solution of benzoylchloride (0.5 M) and DIPEA (1 M) in DMF for lh. The resin was cleaved with 50% TFA/DCM and the dibenzoylated product analyzed with NMR and LC-MS.
  • DTT DTT
  • DIPEA DIPEA
  • Step 5-7 was performed as step 2-4 using a double molar amount of reagents but same amount of solvent.
  • Step 8 capping with 2-[2-(2-methoxyethoxy)ethoxy]acetic acid: A solution of 2-[2-(2- methoxyethoxy)ethoxy] acetic acid (997 mg; 5.6 mmol, 16x with respect to resin loading) and DhbtOH (900 mg; 5.6 mmol, 16x) are dissolved in DMF (5 ml) and DIC (864 ul, 5.6 mmol, 16x) is added. The mixture is left for 10 min (pre-activation) then added to the resin together with DIPEA (960 ul; 5.6 mmol, 16x). The resin is shaken for 90 min, then drained and washed with DMF (3x) and DCM (3x).
  • Step 9 Cleavage from resin: The resin is treated with a 50% TFA - DCM solution at ambient temperature for 30 min. The solvent is collected and the resin is washed an additional time with 50% TFA - DCM. The combined filtrates are evaporated to dryness, and the residue purified by chromatography.
  • This example uses the l,3-Bis[2-(2-azidoethoxy)ethoxy]porpan-2-yl-p- nitrophenylcarbonate monomer building block prepared in example 4 in the synthesis of a second generation carbamate based dendrimer capped with 2-[2-(2- methoxyethoxy)ethoxy]acetic acid.
  • the coupling chemistry is based on standard solid phase carbamate chemistry, and the protection methodology is based on a solid phase azide reduction step as described above.
  • Step 1 Fmoc- ⁇ ala-Wang resin (100 mg; loading 0.31 mmol/g BACHEM) was suspended in dichloromethane for 30 min, and then washed twice with DMF. A solution of 20% piperidine in DMF was added, and the mixture was shaken for 15 min at ambient temperature. This step was repeated, and the resin was washed with DMF (3x) and DCM (3x).
  • Step 2 Coupling of monomer building blocks: A solution of 1,3-
  • Step 3 Capping with acetic anhydride: The resin was then treated with a solution of acetic anhydride, DIPEA, DMF (12:4:48) for 10 min. at ambient temperature. Solvent was removed and the resin was washed with DMF (3x) and DCM (3x).
  • Step 4 Deprotection (reduction of azido groups): The resin was treated with a solution of DTT (2M) and DIPEA (IM) in DMF at 50 °C for 1 hour. The resin was then washed with DMF (3x) and DCM (3x). A small amount of resin was redrawn and treated with a solution of benzoylchloride (0.5 M) and DIPEA (1 M) in DMF for lh. The resin was cleaved with 50% TFA/DCM and the dibenzoylated product analyzed with NMR and LC-MS.
  • DTT DTT
  • DIPEA DIPEA
  • Step 5-7 was performed as step 2-4 using a double molar amount of reagents but same amount of solvent.
  • Step 8 capping with 2-[2-(2-methoxyethoxy)ethoxy]acetic acid: A solution of 2-[2-(2- methoxyethoxy)ethoxy]acetic acid (997 mg; 5.6 mmol, 16x with respect to resin loading) and DhbtOH (900 mg; 5.6 mmol, 16x) are dissolved in DMF (5 ml) and DIC (864 ul, 5.6 mmol, 16x) is added. The mixture is left for 10 min (pre-activation) then added to the resin together with DIPEA (960 ul; 5.6 mmol, 16x). The resin is shaken for 90 min, then drained and washed with DMF (3x) and DCM (3x).
  • Step 9 Cleavage from resin: The resin is treated with a 50% TFA - DCM solution at am- bient temperature for 30 min. The solvent is collected and the resin is washed an additional time with 50% TFA - DCM. The combined filtrates are evaporated to dryness, and the residue purified by chromatography.
  • Step 1 Fmoc- ⁇ -alanine linked Wang resin (A22608, Nova Biochem, 3.00 g; with loading 0.83 mmol/g) was svelled in DCM for 20 min. then washed with DCM (2x20 ml) and NMP (2x20 ml). The resin was then treated twice with 20% piperidine in NMP (2x15 min). The resin was washed with NMP (3x20 ml) and DCM (3x20 ml).
  • Step 2 2-(l,3-Bis[2-(2-azidoethoxy)ethoxy]propan-2-yloxy)acetic acid (3.70 g; 10 mmol) was dissolved in NMP (30 ml) and DhbtOH (1.60 g; 10 mmol) and DIC (1.55 ml; 10 mmol) was added. The mixture was stirred at ambient temperature for 30 min, then added to the resin obtained in step 1 together with DIPEA (1.71 ml; 10 mmol). The reac- tion mixture was shaken for 1.5 h, then drained and washed with NMP (5x20 ml) and DCM (3x20 ml).
  • Step 3 A solution of SnCl 2 .2H 2 0 (11.2 g; 49.8 mmol) in NMP (15 ml) and DCM (15 ml) was then added. The reaction mixture was shaken for lh. The resin was drained and washed with NMP: MeOH (5x20 ml; 1: 1). The resin was then dried in vacuo.
  • Step 4 A solution of 2-[2-(2-methoxyethyl)ethoxy]acetic acid (1.20 g; 6.64 mmol), DhbtOH (1.06 g; 6.60 mmol) and DIC (1.05 ml; 6.60 mmol) in NMP (10 ml) was mixed for 10 min, at room temperature, and then added to the 3-[2-(l,3-bis[2-(2- aminoethoxy)ethoxy]propan-2-yloxy)acetylamino]propanoic acid tethered wang resin (1.0 g; 0.83 mmol/g) obtained in step 3. DIPEA (1.15 ml, 6.60 mmol) was added, and the reaction mixture was shaken for 2.5 h. Solvent was removed, and the resin was washed with NMP (5x20 ml) and DCM (10x20 ml).
  • Step 5 The resin product of step 4 was treated with TFA: DCM (10 ml, 1: 1) for 1 hour. The resin was filtered and washed once with TFA: DCM (10 ml, 1 : 1). The combined filtrate and washing was then taken dryness, to give a yellow oil (711 mg). The oil was dissolved in 10% acetonitril-water (20 ml), and purified over two runs on a preparative HPLC appa- ratus using a C18 column, and a gradient of 15-40% acetonitril-water. Fractions were subsequently analysed by LC-MS. Fractions containing product were pooled and taken to dryness. Yield: 222 mg (37%).
  • This material was prepared from 3-[2-(l,3-bis[2-(2-aminoethoxy)ethoxy]propan-2- yloxy)acetylamino]propanoic acid tethered wang resin (1.0 g; 0.83 mmol/g), obtained in step 3 of example 50 by repeating step 2-5, doubling the amount of reagents used. Yield: 460 mg (33%).
  • MALDI-MS ⁇ -cyanohydroxycinnapinic acid matrix
  • m/z 1670 (M+Na + ).
  • This material was prepared from 3-[2-(l,3-bis[2-(2-aminoethoxy)ethoxy]propan-2- yloxy)acetylamino]propanoic acid tethered wang resin (1.0 g; 0.83 mmol/g), obtained in step 3 of example 50 by repeating step 2-3 with 2x the amount of reagents used, then repeating step 2-5 with 4x the amount of reagent used. Yield: 84 mg (4%).
  • ⁇ - R (CDCI 3 ): ⁇ 1.42 ppm (s, 9H); 2.40 (t, 2H) ; 3.21 (dd, 2H) ; 3.33 (s, 12H); 3.38-3.72 (m, 99H); 3.80 (m, 2H); 3.95 (s, 8H); 4.08 (s, 6H); 6.99 (m, IH); 7.23 (m, 4H); 7.69 (m, 2H); 7.85 (m, IH); 8.00 (m, IH).
  • the material is prepared from two equivalents of N-hydroxysuccimidyl 2-(l,3-bis[2-(2- ⁇ 2-[2-(2-methoxyethoxy)ethoxy]acetamino ⁇ ethoxy)ethoxy]propan-2-yloxy)acetate and one equivalent of t-Butyl 2-(l,3-bis[2-(2-aminoethoxy)ethoxy]propan-2-yloxy)acetate, using the protocol and purification method described in example 58. Subsequent removal of t-butyl group is done as described in example 59 and N-hydroxysuccimidyl ester formation is done as described in example 60.
  • the (S)-2,6-Bis-(2-[2-(2-[2-((S)-2,6-bis-[2-(2-[2-(2-(2-(2-(2-(2-(2-(2-(2-methoxyethoxy)ethoxy) acetylamino)ethoxy]ethoxy)acetylamino]hexanoylamino)ethoxy]ethoxy) ethoxy]acetylamino)hexanoic acid methyl ester can be saponified to the free acid and attached to an amino group of a peptide or protein using via an activated ester.
  • the activated ester may be produced and coupled to the amino group of the peptide or protein by standard coupling methods known in the art such as diisopropylethylamine and N- hydroxybenzotriazole or other activating conditions.
  • Asialo rFVIIa (10.2 mg, 0.2 umol) in 13.5 ml TRIS buffer (10 mM Cacl2, 10 mM TRIS, 50 mM NaCl, 0.5% Tween 80, pH 7.4) was cooled on an icebath.
  • the elution buffer was then changed to 10 mM Tris, 50 mM NaCl, 25 mM CaCI 2 , pH 7.4 (10 vol, flow: 1 ml/min).
  • the eluates were monitored by UV, and each fraction containing protein was analyzed by SDS-PAGE gel electrophoresis. Pure samples of N-glycan modified rFVII were pooled and stored at - 80 °C.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Cette invention concerne de nouveaux composés, des procédés de conjugaison chimique sélective de molécules d'extraction et leur utilisation à des fins diagnostiques et/ou thérapeutiques.
EP04739027A 2003-08-08 2004-08-09 Utilisation de galactose oxydase pour la conjugaison chimique selective de molecules d'extraction a des proteines d'interet therapeutique Withdrawn EP1653996A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DKPA200301145 2003-08-08
US49444703P 2003-08-12 2003-08-12
DKPA200301646 2003-11-05
US51921203P 2003-11-12 2003-11-12
PCT/DK2004/000530 WO2005014035A2 (fr) 2003-08-08 2004-08-09 Utilisation de galactose oxydase pour la conjugaison chimique selective de molecules d'extraction a des proteines d'interet therapeutique

Publications (1)

Publication Number Publication Date
EP1653996A2 true EP1653996A2 (fr) 2006-05-10

Family

ID=34139681

Family Applications (2)

Application Number Title Priority Date Filing Date
EP04739027A Withdrawn EP1653996A2 (fr) 2003-08-08 2004-08-09 Utilisation de galactose oxydase pour la conjugaison chimique selective de molecules d'extraction a des proteines d'interet therapeutique
EP04739028A Withdrawn EP1654004A2 (fr) 2003-08-08 2004-08-09 Synthese et utilisation de nouveaux polymeres ramifies structuraux bien definis comme agents de conjugaison de peptides

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP04739028A Withdrawn EP1654004A2 (fr) 2003-08-08 2004-08-09 Synthese et utilisation de nouveaux polymeres ramifies structuraux bien definis comme agents de conjugaison de peptides

Country Status (3)

Country Link
EP (2) EP1653996A2 (fr)
JP (3) JP2007501811A (fr)
WO (2) WO2005014049A2 (fr)

Families Citing this family (70)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7723296B2 (en) 2001-01-18 2010-05-25 Genzyme Corporation Methods for introducing mannose-6-phosphate and other oligosaccharides onto glycoproteins and its application thereof
DE10209821A1 (de) 2002-03-06 2003-09-25 Biotechnologie Ges Mittelhesse Kopplung von Proteinen an ein modifiziertes Polysaccharid
PL1615945T3 (pl) 2003-04-09 2012-03-30 Ratiopharm Gmbh Sposoby glikopegylacji i białka/peptydy wytwarzane tymi sposobami
US9005625B2 (en) 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
WO2005014655A2 (fr) 2003-08-08 2005-02-17 Fresenius Kabi Deutschland Gmbh Conjugues d'amidon d'hydroxyalkyle et de proteine
ATE481420T1 (de) * 2003-10-10 2010-10-15 Novo Nordisk As Il-21-derivate
US20060228331A1 (en) 2003-10-10 2006-10-12 Novo Nordisk A/S IL-21 Derivatives and variants
US20080305992A1 (en) 2003-11-24 2008-12-11 Neose Technologies, Inc. Glycopegylated erythropoietin
ATE455861T1 (de) 2004-05-04 2010-02-15 Novo Nordisk Healthcare Ag O-verknüpfte glykoformen von faktor vii und verfahren zu deren herstellung
EP1771066A2 (fr) 2004-07-13 2007-04-11 Neose Technologies, Inc. Remodelage de peg ramifie et glycosylation de peptide-1 semblable a glucagon glp-1
WO2006050247A2 (fr) 2004-10-29 2006-05-11 Neose Technologies, Inc. Remodelage et glycopegylation du facteur de croissance des fibroblastes (fgf)
NZ556436A (en) 2005-01-10 2010-11-26 Biogenerix Ag Glycopegylated granulocyte colony stimulating factor
EP1843790A2 (fr) * 2005-01-27 2007-10-17 Novo Nordisk A/S Derives d'insuline conjugues avec des polymeres ramifies structurellement bien definis
CN101123992A (zh) * 2005-02-16 2008-02-13 诺和诺德公司 缀合有结构完全确定的支化聚合物的促胰岛素剂的衍生物
WO2006094826A2 (fr) * 2005-03-11 2006-09-14 Fresenius Kabi Deutschland Gmbh Procede permettant de coupler des glycoconjugues actives sur le plan enzymatique a un compose de modification
AU2006222187A1 (en) * 2005-03-11 2006-09-14 Fresenius Kabi Deutschland Gmbh Production of bioactive glycoproteins from inactive starting material by conjugation with hydroxyalkylstarch
TWI372629B (en) * 2005-03-18 2012-09-21 Novo Nordisk As Acylated glp-1 compounds
WO2006097538A1 (fr) 2005-03-18 2006-09-21 Novo Nordisk A/S Composes glp-1 avances
EP2386571B1 (fr) 2005-04-08 2016-06-01 ratiopharm GmbH Compositions et méthodes utilisées pour la préparation de mutants par glycosylation de l'hormone de croissance humaine résistant à la protéase
PT2279758E (pt) 2005-06-16 2015-05-27 Nektar Therapeutics Conjugados possuindo uma ligação degradável e reagentes poliméricos úteis na preparação de tais conjugados
US20070105755A1 (en) 2005-10-26 2007-05-10 Neose Technologies, Inc. One pot desialylation and glycopegylation of therapeutic peptides
DK1767545T3 (da) 2005-09-22 2010-03-15 Biocompatibles Uk Ltd GLP-1 (Glucagon-lignende peptid-1)-fusionspolypeptider med forøget peptidaseresistens
US20090048440A1 (en) 2005-11-03 2009-02-19 Neose Technologies, Inc. Nucleotide Sugar Purification Using Membranes
CU23556A1 (es) * 2005-11-30 2010-07-20 Ct Ingenieria Genetica Biotech Estructura polimérica semejante a dendrímero para la obtención de conjugados de interés farmacéutico
JP5096363B2 (ja) * 2005-12-16 2012-12-12 ネクター セラピューティックス Ｇｌｐ−１のポリマ複合体
ATE444741T1 (de) 2006-05-10 2009-10-15 Biocompatibles Uk Ltd Glp-1 peptide enthaltende kugelförmige mikrokapseln, deren produktion und deren verwendung
EP2044150B1 (fr) 2006-07-21 2014-01-15 Nektar Therapeutics Réactifs polymères comprenant un groupe vinylique terminal et conjugués formés à partir de ceux-ci
ES2516694T3 (es) 2006-07-21 2014-10-31 Ratiopharm Gmbh Glicosilación de péptidos a través de secuencias de glicosilación con unión en O
ES2531934T3 (es) 2006-09-01 2015-03-20 Novo Nordisk Health Care Ag Glicoproteínas modificadas
WO2008057683A2 (fr) 2006-10-03 2008-05-15 Novo Nordisk A/S Méthodes de purification de conjugués de polypeptides
CA2670618C (fr) 2006-12-15 2016-10-04 Baxter International Inc. Conjugue du facteur viia et de l'acide (poly)sialique presentant une demi-vie in vivo prolongee
US8133865B2 (en) 2006-12-27 2012-03-13 Nektar Therapeutics von Willebrand factor- and factor VIII-polymer conjugates having a releasable linkage
NZ577397A (en) 2006-12-27 2012-01-12 Nektar Therapeutics Factor ix moiety-polymer conjugates having a releasable linkage
DK2121713T3 (da) 2007-01-18 2013-06-24 Genzyme Corp Oligosaccharider, omfattende en aminooxygruppe, og konjugater deraf
BRPI0809670A8 (pt) 2007-04-03 2018-12-18 Biogenerix Ag métodos para aumentar a produção de célula tronco, para aumentar o número de granulócitos em um indivíduo, para prevenir, tratar e aliviar a mielossupressão que resulta de uma terapia contra o câncer, para tratar uma condição em um indivíduo, para tratar neutropenia e trombocitopenia em um mamífero, para expandir células tronco hematopoiéticas em cultura, para aumentar a hematopoiese em um indivíduo, para aumentar o número de célulars progenitoras hematopoiéticas em um indivíduo, e para fornecer enxerto estável da medula óssea, e, forma de dosagem oral.
EP2157967B1 (fr) * 2007-04-23 2013-01-16 Intarcia Therapeutics, Inc Formulations en suspension de peptides insulinotropes et leurs utilisations
CN101778859B (zh) 2007-06-12 2014-03-26 诺和诺德公司 改良的用于生产核苷酸糖的方法
CN103497246B (zh) 2008-02-27 2016-08-10 诺沃—诺迪斯克有限公司 缀合的因子viii分子
CA2733200A1 (fr) 2008-08-06 2010-02-11 Novo Nordisk Health Care Ag Proteines conjuguees a efficacite in vivo prolongee
EP2889043B1 (fr) 2008-12-16 2019-04-10 Genzyme Corporation Intermédiaires synthétiques utiles dans la préparation des conjugués oliogosaccharide-protéine
CN102292349B (zh) 2009-01-22 2016-04-13 诺沃—诺迪斯克保健股份有限公司 稳定的生长激素化合物
JP5418360B2 (ja) 2009-03-27 2014-02-19 日油株式会社 ポリオキシアルキレン誘導体の製造方法
US8642737B2 (en) 2010-07-26 2014-02-04 Baxter International Inc. Nucleophilic catalysts for oxime linkage
KR101912335B1 (ko) 2009-07-27 2018-10-26 리폭센 테크놀로지즈 리미티드 비혈액 응고 단백질의 글리코폴리시알화
ES2597954T3 (es) 2009-07-27 2017-01-24 Baxalta GmbH Conjugados de proteína de la coagulación sanguínea
US8809501B2 (en) 2009-07-27 2014-08-19 Baxter International Inc. Nucleophilic catalysts for oxime linkage
NZ623810A (en) 2009-07-27 2015-10-30 Lipoxen Technologies Ltd Glycopolysialylation of non-blood coagulation proteins
CN102612376A (zh) 2009-08-06 2012-07-25 诺沃-诺迪斯克保健股份有限公司 具有延长的体内功效的生长激素
CA2782320A1 (fr) 2009-12-02 2011-06-09 Acceleron Pharma Inc. Compositions et procedes pour augmenter la demi-vie serique de proteines de fusion fc
WO2011080102A2 (fr) * 2009-12-16 2011-07-07 Novo Nordisk A/S Analogues et dérivés de glp-1
EP2525833A2 (fr) 2010-01-22 2012-11-28 Novo Nordisk Health Care AG Composés stables d'hormone de croissance
CN106139158A (zh) 2010-01-22 2016-11-23 诺沃—诺迪斯克保健股份有限公司 体内功效延长的生长激素
KR20230156435A (ko) 2010-07-09 2023-11-14 바이오버라티브 테라퓨틱스 인크. 인자 iｘ 폴리펩티드 및 이들의 사용 방법
RU2600440C3 (ru) 2010-12-16 2021-12-10 Ново Нордиск А/С Твердые композиции, содержащие агонист glp-1 и соль n-(8-(2-гидроксибензоил)амино)каприловой кислоты
BR112013015898A2 (pt) 2010-12-22 2018-06-26 Baxter International Inc. derivado de ácido graxo solúvel em água, e, métodos para preparar um derivado de ácido graxo e uma proteína terapêutica conjugada.
US8901341B2 (en) * 2011-02-11 2014-12-02 Bezwada Biomedical, Llc Amino acid derivatives and absorbable polymers therefrom
CN106117344B (zh) 2011-04-12 2020-11-03 诺沃—诺迪斯克有限公司 双酰化glp-1衍生物
US8883982B2 (en) 2011-06-08 2014-11-11 Acceleron Pharma, Inc. Compositions and methods for increasing serum half-life
US9790305B2 (en) * 2011-09-09 2017-10-17 Franklin And Marshall College Site specifically incorporated initiator for growth of polymers from proteins
ES2715308T3 (es) 2012-03-22 2019-06-03 Novo Nordisk As Composiciones que comprenden un agente de suministro y su preparación
US10933120B2 (en) 2012-03-22 2021-03-02 Novo Nordisk A/S Compositions of GLP-1 peptides and preparation thereof
EP2863895B1 (fr) 2012-06-20 2021-04-14 Novo Nordisk A/S Composition de comprimé comprenant un peptide et un agent délivrant
JP2015533152A (ja) 2012-10-15 2015-11-19 ノヴォ・ノルディスク・ヘルス・ケア・アーゲー 第ｖｉｉ因子コンジュゲート
US11045523B2 (en) 2013-04-05 2021-06-29 Novo Nordisk Healthcare Ag Formulation of growth hormone albumin-binder conjugate
BR112015026325A2 (pt) 2013-05-02 2017-07-25 Novo Nordisk As dosagem oral de compostos glp-1
WO2015055692A1 (fr) 2013-10-15 2015-04-23 Novo Nordisk Health Care Ag Polypeptides du facteur vii de la coagulation
KR20230033737A (ko) * 2014-05-27 2023-03-08 아카데미아 시니카 증진된 항체 효능을 위한 범용 당형태에 관한 조성물 및 방법
EP3265827A4 (fr) * 2015-03-06 2018-07-25 Tymora Analyticaly Operations LLC Matrice fonctionnalisée chimiquement pour analyser des modifications protéiniques
CN106478806B (zh) * 2016-10-24 2019-08-30 合肥国肽生物科技有限公司 一种索玛鲁肽的固相合成方法
EP3746111B1 (fr) 2018-02-02 2023-07-19 Novo Nordisk A/S Compositions solides comportant un agoniste glp-1, du sel d'acide n-(8-(2-hydroxybenzoyl)amino)caprylate et un lubrifiant

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0175617A2 (fr) * 1984-09-13 1986-03-26 Cytogen Corporation Conjugués d'anticorps et d'agent thérapeutique
AU720860B2 (en) * 1996-10-08 2000-06-15 Macquarie Research Limited Glycan derivatives

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2101918A1 (fr) * 1991-03-18 1992-09-19 Samuel Zalipsky Conjugues de polypeptides et de glycopolypeptides avec des polymeres contenant de l'hydrazine
US6180134B1 (en) * 1993-03-23 2001-01-30 Sequus Pharmaceuticals, Inc. Enhanced ciruclation effector composition and method
US6326353B1 (en) * 1993-03-23 2001-12-04 Sequus Pharmaceuticals, Inc. Enhanced circulation effector composition and method
WO1994028024A1 (fr) * 1993-06-01 1994-12-08 Enzon, Inc. Polymere modifie aux glucides presentant une activite erythropoïetique
MX9504664A (es) * 1994-03-07 1997-05-31 Dow Chemical Co Conjugados de dendrimeros bioactivos y/o dirigidos.
US5932462A (en) * 1995-01-10 1999-08-03 Shearwater Polymers, Inc. Multiarmed, monofunctional, polymer for coupling to molecules and surfaces
AU5920096A (en) * 1995-05-15 1996-11-29 Constantin A. Bona Carbohydrate-mediated coupling of peptides to immunoglobulins
JP2927291B2 (ja) * 1997-09-01 1999-07-28 東洋インキ製造株式会社 ビニル基含有デンドリマー
US6485718B1 (en) * 1999-04-13 2002-11-26 Pharmacia Corporation Site specific ligation of proteins to synthetic particles
WO2001002861A1 (fr) * 1999-06-29 2001-01-11 Dako A/S Detection a l'aide de sondes et de marqueurs a dendrimeres
RU2278123C2 (ru) * 2000-02-11 2006-06-20 Максиджен Холдингз Лтд. Молекулы, подобные фактору vii или viia
BR0112428A (pt) * 2000-07-12 2003-11-25 Gryphon Therapeutics Inc Quimiocinas sintéticas boativas modificadas por polìmero e métodos para sua produção e uso
US20030091640A1 (en) * 2001-02-08 2003-05-15 Srinivasan Ramanathan Enhanced oral and transcompartmental delivery of therapeutic or diagnostic agents
KR100948532B1 (ko) * 2001-11-07 2010-03-23 넥타르 테라퓨틱스 분지형 중합체 및 그의 공액체

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0175617A2 (fr) * 1984-09-13 1986-03-26 Cytogen Corporation Conjugués d'anticorps et d'agent thérapeutique
AU720860B2 (en) * 1996-10-08 2000-06-15 Macquarie Research Limited Glycan derivatives

Also Published As

Publication number Publication date
WO2005014035A3 (fr) 2005-07-28
JP2011157365A (ja) 2011-08-18
WO2005014035A2 (fr) 2005-02-17
JP2007501812A (ja) 2007-02-01
JP2007501811A (ja) 2007-02-01
EP1654004A2 (fr) 2006-05-10
WO2005014049A2 (fr) 2005-02-17
WO2005014049A3 (fr) 2005-09-01

Similar Documents

Publication Publication Date Title
US20140274903A1 (en) Use of galactose oxidase for selective chemical conjugation of protractor molecules to proteins of therapeutic interest
EP1653996A2 (fr) Utilisation de galactose oxydase pour la conjugaison chimique selective de molecules d'extraction a des proteines d'interet therapeutique
ES2363205T3 (es) Glicoformas del factor vii pegilado.
US8053410B2 (en) Pegylated factor VII glycoforms
US20080108557A1 (en) Modified Proteins
EP2059527B1 (fr) Glycoprotéines modifiées
EP2532369B1 (fr) Conjugué facteur VIIa-acide (poly)sialique présentant une demi-vie in vivo prolongée
CN102719508A (zh) 糖基聚乙二醇化的因子vii和因子viia
US20150225711A1 (en) Factor VII Conjugates
CN101374861A (zh) 糖基聚乙二醇化的因子ⅶ和因子ⅶa

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20060308

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20110203

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: NOVO NORDISK HEALTH CARE AG

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20140718