WO2004024776A1 - Method of producing hydroxyalkyl starch derivatives - Google Patents

Method of producing hydroxyalkyl starch derivatives Download PDF

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Publication number
WO2004024776A1
WO2004024776A1 PCT/EP2003/008829 EP0308829W WO2004024776A1 WO 2004024776 A1 WO2004024776 A1 WO 2004024776A1 EP 0308829 W EP0308829 W EP 0308829W WO 2004024776 A1 WO2004024776 A1 WO 2004024776A1
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Prior art keywords
group
compound
functional group
hydroxyalkyl starch
reaction
Prior art date
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PCT/EP2003/008829
Other languages
French (fr)
Inventor
Norbert Zander
Harald Conradt
Wolfram Eichner
Original Assignee
Fresenius Kabi Deutschland Gmbh
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Priority claimed from EP02020425A external-priority patent/EP1400533A1/en
Application filed by Fresenius Kabi Deutschland Gmbh filed Critical Fresenius Kabi Deutschland Gmbh
Priority to JP2004535070A priority Critical patent/JP4688494B2/en
Priority to BR0314107-1A priority patent/BR0314107A/en
Priority to CA2496317A priority patent/CA2496317C/en
Priority to MXPA05002593A priority patent/MXPA05002593A/en
Priority to AU2003260393A priority patent/AU2003260393B2/en
Priority to EP10011836.3A priority patent/EP2272865A3/en
Priority to ES03020424T priority patent/ES2213506T3/en
Priority to DK03020424T priority patent/DK1398327T3/en
Priority to DE60323192T priority patent/DE60323192D1/en
Priority to DE0001398327T priority patent/DE03020424T1/en
Priority to PT03020424T priority patent/PT1398327E/en
Priority to DE20321836U priority patent/DE20321836U1/en
Priority to EP08012908A priority patent/EP2017287A3/en
Priority to AT03020424T priority patent/ATE406387T1/en
Priority to SI200331431T priority patent/SI1398327T1/en
Priority to EP03020424A priority patent/EP1398327B1/en
Publication of WO2004024776A1 publication Critical patent/WO2004024776A1/en
Priority to JP2006522324A priority patent/JP2007501870A/en
Priority to EP04763853A priority patent/EP1660134B1/en
Priority to SG200806055-0A priority patent/SG145746A1/en
Priority to BRPI0413450-8A priority patent/BRPI0413450A/en
Priority to MXPA06001359A priority patent/MXPA06001359A/en
Priority to DE602004030805T priority patent/DE602004030805D1/en
Priority to KR1020067002747A priority patent/KR101154343B1/en
Priority to CNA2004800295229A priority patent/CN1863549A/en
Priority to EP04763855A priority patent/EP1653991A2/en
Priority to AT04763853T priority patent/ATE493150T1/en
Priority to CA002534412A priority patent/CA2534412A1/en
Priority to DK04763853.1T priority patent/DK1660134T3/en
Priority to AU2004262921A priority patent/AU2004262921B2/en
Priority to SI200431617T priority patent/SI1660134T1/en
Priority to RU2006106926/15A priority patent/RU2370281C2/en
Priority to TW093123553A priority patent/TWI357337B/en
Priority to BRPI0412671-8A priority patent/BRPI0412671A/en
Priority to PCT/EP2004/008818 priority patent/WO2005014050A2/en
Priority to CN2004800226500A priority patent/CN1832762B/en
Priority to PL04763853T priority patent/PL1660134T3/en
Priority to PCT/EP2004/008821 priority patent/WO2005014655A2/en
Priority to ZA200600651A priority patent/ZA200600651B/en
Priority to PCT/EP2004/008820 priority patent/WO2005014024A2/en
Priority to US10/567,266 priority patent/US20080274948A1/en
Priority to US10/567,265 priority patent/US20080206182A1/en
Priority to MXPA06001358A priority patent/MXPA06001358A/en
Priority to CA002534418A priority patent/CA2534418A1/en
Priority to TW093123647A priority patent/TW200519128A/en
Priority to TW093123642A priority patent/TWI356065B/en
Priority to ARP040102853A priority patent/AR045450A1/en
Priority to ARP040102852A priority patent/AR045236A1/en
Priority to ARP040102851A priority patent/AR045235A1/en
Priority to HK04106173A priority patent/HK1063477A1/en
Priority to IL166930A priority patent/IL166930A/en
Priority to US11/078,098 priority patent/US20050238723A1/en
Priority to NO20051427A priority patent/NO20051427L/en
Priority to IL173187A priority patent/IL173187A/en
Priority to NO20061121A priority patent/NO20061121L/en
Priority to HK06110272.2A priority patent/HK1089683A1/en
Priority to US12/824,618 priority patent/US20120046240A9/en
Priority to HR20110056T priority patent/HRP20110056T1/en
Priority to JP2012130094A priority patent/JP2012211329A/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B31/00—Preparation of derivatives of starch
    • C08B31/08—Ethers
    • C08B31/12—Ethers having alkyl or cycloalkyl radicals substituted by heteroatoms, e.g. hydroxyalkyl or carboxyalkyl starch
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B31/00—Preparation of derivatives of starch
    • C08B31/08—Ethers
    • C08B31/10—Alkyl or cycloalkyl ethers
    • 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
    • A61K47/61—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 the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/10—Peptides being immobilised on, or in, an organic carrier the carrier being a carbohydrate
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B31/00—Preparation of derivatives of starch
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B31/00—Preparation of derivatives of starch
    • C08B31/003—Crosslinking of starch
    • C08B31/006—Crosslinking of derivatives of starch
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B31/00—Preparation of derivatives of starch
    • C08B31/08—Ethers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B31/00—Preparation of derivatives of starch
    • C08B31/18—Oxidised starch
    • C08B31/185—Derivatives of oxidised starch, e.g. crosslinked oxidised starch
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H1/00—Macromolecular products derived from proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to hydroxyalkyl starch derivates, particularly hydroxyalkyl starch derivatives obtainable by a process in which hydroxyalkyl starch is reacted with a primary or secondary amino group of a crosslinking compound or with two crosslinking compounds wherein the resulting hydroxaylkyl starch derivative has at least one functional group X which is capable of being reacted with a functional group Y of a further compound and wherein this group Y is an aldehyd group, a keto group, a hemiacetal group, an acetal group, or a thio group.
  • the present invention relates to hydroxyalkyl starch derivatives obtainable by a process according to which hydroxyalkyl starch is reacted with a primary or secondary amino group of a crosslinking compound, the resulting reaction product optionally being further reacted with a second crosslinking compound, wherein the resulting hydroxaylkyl starch derivative has at least one func- tional group X which is capable of being reacted with a functional group Y of a further compound and wherein this group Y is an aldehyd group, a keto group, a hemiacetal group, an acetal group, or a thio group, and the resulting reaction product is reacted with a polypeptide, preferably with a polypeptide such as AT III, IFN-beta or erythropoietin and especially preferably with erythropoietin, which comprises at least one of these functional groups Y.
  • a polypeptide such as AT III, IFN-bet
  • a hydroxyalkyl starch which is especially preferred is hydroxyethyl starch.
  • the hydroxyalkyl starch and preferably the hydroxyl ethyl starch is reacted with the linker compound at its reducing end which is optionally oxidized prior to said reaction.
  • HES Hydroxyethyl starch
  • Amylopectin consists of glucose moieties, wherein in the main chain alpha- 1,4- glycosidic bonds are present and at the branching sites alpha-l,6-glycosidic bonds are found.
  • the physical-chemical properties of this molecule are mainly determined by the type of glycosidic bonds. Due to the nicked alpha- 1,4-glycosidic bond, helical structures with about six glucose-monomers per turn are produced.
  • the physico- chemical as well as the biochemical properties of the polymer can be modified via substitution.
  • the introduction of a hydroxyethyl group can be achieved via alkaline hydroxyethylation.
  • DE 26 16 086 discloses the conjugation of hemoglobin to hydroxyethyl starch wherein, in a first step, a cross-linking agent, e.g. bromocyane, is bound to hydroxyethyl starch and subsequently hemoglobin is linked to the intermediate product.
  • a cross-linking agent e.g. bromocyane
  • HES HES-derived neuropeptides
  • polypeptides which are applied, e.g., to the circulatory system in order to obtain a particular physiologi- cal effect.
  • polypeptides erythropoietin, an acid gly- coprotein of approximately 34,000 kD which is essential in regulating the level of red blood cells in the circulation.
  • WO 94/28024 discloses that physiologically active polypeptides modified with polyethyleneglycol (PEG) exhibit reduced immunogenic- ity and antigenicity and circulate in the bloodstream considerably longer than uncon- jugated proteins, i.e. have a longer clearance rate.
  • PEG-drug conjugates exhibit several disadvantages, e.g. they do not exhibit a natural structure which can be recognized by elements of in vivo degradation pathways. Therefore, apart from PEG-conjugates, other conjugates and protein polymerates have been produced.
  • a plurality of methods for the cross-linking of different proteins and macromolecules such as polymerase have been described in the literature (see e.g. Wong, Chemistry of protein conjugation and cross-linking, 1993, CR.CS, Inc.).
  • WO 02/08079 A2 discloses compounds comprising a conjugate of an active agent and a hydroxyalkyl starch wherein active agent and hydroxyalykl starch are either linked directly or via a linker compound.
  • the reaction of active agent and hydroxyalkyl starch is carried out in an aqueous medium which comprises at least 10 wt.-% of water.
  • No examples are given which are directed to a hydroxyalkyl starch derivative which is linked to a carbonyl group comprised in the active reagent, neither an aldehyd or keto group nor a an acetal or a hemiacetal group.
  • hydroxyalkyl starch derivatives which are capable of forming a chemical linkage to a further compound, e.g. a polypeptide, which comprises, as functional group, a thio group or an aldehyd group, a keto group, a hemiacetal group, or an acetal group.
  • a further compound e.g. a polypeptide, which comprises, as functional group, a thio group or an aldehyd group, a keto group, a hemiacetal group, or an acetal group.
  • the aldehyd group, the keto group, the hemiacetal group, or the acetal group are comprised in a carbohydrate moiety of the further compound.
  • the present invention relates to a method of producing a hydroxyalkyl starch derivative, said hydroxyalkyl starch having a structure according to formula (I)
  • hydroxyalkyl starch refers to a starch derivative which has been substituted by at least one hydroxyalkyl group. Therefore, the term hydroxyalkyl starch as used in the present invention is not limited to compounds where the terminal carbohydrate moiety comprises hydroxyalkyl groups Ri, R 2 , and or R 3 as depicted, for the sake of brevity, in formula (I), but also refers to compounds in which at least one hydroxy group present anywhere, either in the terminal carbohydrate moiety and/or in the remaining part of the starch molecule, HAS', is substituted by a hydroxyalkyl group i, R , or R 3 .
  • the alkyl group may be a linear or branched alkyl group which may be suitably substituted.
  • the hydroxyalkyl group contains 1 to 10 carbon atoms, more preferably from 1 to 6 carbon atoms, more preferably from 1 to 4 carbon atoms, and even more preferably 2-4 carbon atoms.
  • "Hydroxyalkyl starch” therefore preferably comprises hydroxyethyl starch, hydroxypropyl starch and hydroxybutyl starch, wherein hydroxyethyl starch and hydroxypropyl starch are particularly preferred.
  • Hydroxyalkyl starch comprising two or more different hydroxyalkyl groups is also comprised in the present invention..
  • the at least one hydroxyalkyl group comprised in HAS may contain two or more hydroxy groups. According to a preferred embodiment, the at least one hydroxyalkyl group comprised HAS contains one hydroxy group.
  • hydroxyalkyl starch also includes derivatives wherein the alkyl group is mono- or polysubstituted.
  • the alkyl group is substituted with a halogen, especially fluorine, or with an aryl group, provided that the HAS remains soluble in water.
  • the terminal hydroxy group a of hydroxyalkyl group may be esterified or etherified.
  • alkyl instead of alkyl, also linear or branched substituted or unsubstituted alkene groups may be used.
  • Hydroxyalkyl starch is an ether derivative of starch.
  • ether derivatives also other starch derivatives can be used in the context of the present invention.
  • derivatives are useful which comprise esterified hydroxy groups. These derivatives may be, e.g., derivatives of unsubstituted mono- or dicarboxylic acids with 2-12 carbon atoms or of substituted derivatives thereof.
  • derivatives of unsubstituted monocarboxylic acids with 2-6 carbon atoms especially derivatives of acetic acid.
  • acetyl starch, butyl starch and propyl starch are preferred.
  • derivatives of dicarboxylic acids it is useful that the second carboxy group of the dicarboxylic acid is also esterified. Furthermore, derivatives of monoal- kyl esters of dicarboxylic acids are also suitable in the context of the present invention.
  • the substitute groups may be pref- erably the same as mentioned above for substituted alkyl residues.
  • Hydroxyethyl starch is most preferred for all embodiments of the present invention. Therefore, the present invention also relates to a method as described above wherein the hydroxyalkyl starch is hydroxyethyl starch.
  • HES is mainly characterized by the molecular weight distribution and the degree of substitution. There are two possibilities of describing the substitution degree:
  • substitution degree can be described relatively to the portion of substituted glucose monomers with respect to all glucose moieties (DS).
  • substitution degree can be described as the "molar substitution” (MS), wherein the number of hydroxyethyl groups per glucose moiety are described.
  • HES solutions are present as polydisperse compositions, wherein each molecule dif- fers from the other with respect to the polymerisation degree, the number and pattern of branching sites, and the substitution pattern. HES is therefore a mixture of compounds with different molecular weight. Consequently, a particular HES solution is determined by average molecular weight with the help of statistical means.
  • M n is calculated as the arithmetic mean depending on the number of mole- cules.
  • M w the weight mean, represents a unit which depends on the mass of the HES.
  • hydroxyethyl starch may have a mean molecular weight (weight mean) of from 1 to 300 kDa, wherein a mean molecular weight of from 5 to 100 kDa is more preferred. Hydroxyethyl starch can further exhibit a molar degree of substitution of from 0.1 to 0.8 and a ratio between C 2 : C 6 substitution in the range of from 2 to 20 with respect to the hydroxyethyl groups.
  • R 2 and R 3 are independently hydrogen or a hydroxyalkyl group, a hydroxyaryl group, a hydroxyaralkly group or a hydroxyalkarly group having of from 1 to 10 carbon atoms. Hydrogen and hydroxyalkyl groups having of from 1 to 6 carbon atoms are preferred.
  • the alkyl, aryl, aralkyl and/or alkaryl group may be linear or branched and suitably substituted.
  • the present invention also related to a method as described above wherein Ri, R and R 3 are independently hydrogen or a linear or branched hydroxyalkyl group with from 1 to 6 carbon atoms.
  • Ri, R 2 and R 3 may be hydroxyhexyl, hydroxypentyl, hydroxybutyl, hydroxypropyl such as 1 -hydroxypropyl, 2-hydroxypropyl, 3 -hydroxypropyl, 1- hydroxyisopropyl, 2-hydroxyisopropyl, hydroxyethyl such as 1 -hydroxyethyl, 2- hydroxyethyl, or hydroxymethyl. Hydrogen and hydroxyethyl groups are preferred, hydrogen and the 2-hydroxyethyl group being especially preferred.
  • the present invention also relates to a method as described above wherein Ri, R 2 and R 3 are independently hydrogen or a 2-hydroxyethyl group.
  • compound (D) or compound (L) is reacted with the reducing end of the hydroxyalkyl starch via the reaction of the functional group Zi with the reducing end where group Zi is comprised in compound (D) or compound (L).
  • compound (D) or compound (L) is reacted with the reducing end of the hydroxyalkyl starch and where the reducing end is oxidized prior to the reaction.
  • hydroxyalkyl starch in which the terminal carbohydrate group comprises a lactone group, or in which the terminal carbohydrate group, depending of the chemical reaction conditions and/or the oxidizing agents, has a non-cyclic structure comprising a carboxy group.
  • the hydroxyalkyl starch which is oxidized at its reducing end is present as a mixture of a compound comprising the lactone group and a compound comprising the carboxy group. In the mixture, the respective compounds may be present at any conceivable ratio.
  • the present invention also relates to a method as described above wherein he reducing end of the hydroxyalkyl starch is oxidized prior to the reaction with compound (D) or compound (L), said hydroxyalkyl starch thus having a structure according to formula (Ila)
  • the oxidation of the reducing end of the hydroxyalkyl starch may be carried out according to each method or combination of methods which result compounds having the above-mentioned structures (Ila) and/or (lib).
  • the oxidation may be carried out according to all suitable method or methods resulting in the oxidized reducing end of hydroxyalkyl starch, it is preferably carried out using an alkaline iodine solution as described, e.g., in 196 28 705 Al. Therefore, the present invention also relates to a method as mentioned above wherein the reducing end is oxidized by an alkaline iodine solution.
  • compound (D) or compound (L) is reacted with the reducing end of the hydroxyalkyl starch and where the reducing end is not oxidized prior to the reaction.
  • the present invention also relates to a method as mentioned above wherein the reducing end of the hydroxyalkyl starch is not oxidized prior to the reaction with compound (D) or compound (L), said hydroxyalkyl starch thus having a structure according to formula (I)
  • each functional group may be used which is capable of forming a chemical linkage with the optionally oxidized reducing end of the hydroxyalkyl starch.
  • the functional group Zi comprises the chemical structure -NH-. Therefore, the present invention also relates to a method as described above wherein the functional group Zi comprises the structure -NH-.
  • the functional group Zi is a group having the structure R'-NH- where R' is hydrogen or a alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue where the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directly to the NH group or, according to another embodiment, may be linked by an oxygen bridge to the NH group.
  • alkyl, cycloalkyl, aryl, aralkyl, arylcy- cloalkyl, alkaryl, or cycloalkylaryl residues may be suitably substituted.
  • halogenes such as F, CI or Br may be mentioned.
  • Especially preferred residues R' are hydrogen, alkyl and alkoxy groups, and even more preferred are hydrogen and unsubstituted alkyl and alkoxy groups.
  • alkyl and alkoxy groups groups with 1, 2, 3, 4, 5, or 6 C atoms are preferred. More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups. Especially preferred are methyl, ethyl, methoxy, ethoxy, and particular preference is given to methyl or methoxy.
  • the present invention also relates to a method as described above wherein R' is hydrogen or a methyl or a methoxy group.
  • R" is selected from the group consisting of
  • G where, if G is present twice, it is independently O or S.
  • the present invention also relates to a method as mentioned above wherein the fuctional group Zi is selected from the group consisting of
  • G is O or S and, if present twice, independently O or S, and R' is methyl.
  • the functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group
  • the functional group X preferably comprises the chemical structure -NH-. Therefore, the present invention also relates to a method as described above wherein the functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group, and the functional group X comprises the structure -NH-.
  • the functional group X is a group having the structure R'-NH- where R' is hydrogen or a alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue where the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directly to the NH group or, according to another embodiment, may be linked by an oxygen bridge to the NH group.
  • alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl, or cycloalkylaryl residues may be suitably substituted.
  • halogenes such as F, CI or Br may be mentioned.
  • Especially preferred residues R' are hydrogen, alkyl and alkoxy groups, and even more preferred are hy- drogen and unsubstituted alkyl and alkoxy groups.
  • alkyl and alkoxy groups groups with 1, 2, 3, 4, 5, or 6 C atoms are preferred. More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups. Especially preferred are methyl, ethyl, methoxy, eth- oxy, and particular preference is given to methyl or methoxy.
  • the present invention also relates to a method as described above wherein R' is hydrogen or a methyl or a methoxy group.
  • R" is selected from the group consisting of
  • G is present twice, it is independently O or S.
  • the present invention also relates to a method as mentioned above wherein the fuctional group X is selected from the group consisting of
  • G is O or S and, if present twice, independently O or S, and R is methyl.
  • the functional group X is preferably selected from the groups consisting of
  • Hal is CI, Br or I, preferably Br or I.
  • the present invention also relates to a method as described above where- inwherein the functional group Y is -SH and the functional group X is selected from the group consisting of wherein Hal is CI, Br or I.
  • hydroxyalkyl starch is reacted with a compound (D) and the resulting reaction product is further reacted with compound (L) where the chemical linkage between compound (L) and the reaction product is formed by reaction of functional group Z 2 comprised in compound (L) and functional group W comprised in compound (D) being part of the reaction product.
  • amino group -NH 2 or derivatives of the amino groups comprising the struc- ture unit -NH- such as aminoalkyl groups, aminoaryl group, aminoaralkyl groups, or alkarlyaminogroups; the hydroxylamino group -O-NH 2 , or derivatives of the hydroxylamino group comprising the structure unit -O-NH-, such as hydroxylalkylamino groups, hy- droxylarylamino groups, hydroxylaralkylamino groups, or hydroxalal- karylamino groups; alkoxyamino groups, aryloxyamino groups, aralkyloxyamino groups, or alkary- loxyamino groups, each comprising the structure unit -NH-O-; residues having a carbonyl group, -Q-C( G)-M, wherein G is O or S, and M is, for example,
  • the group the group where Z 2 and W, respectively, is a group capable of forming a chemical linkage with one of the above-mentioned groups.
  • both W and Z 2 are groups from the list of groups given above.
  • Z 2 or W is a thio group.
  • the functional group W is preferably selected from the group consisting of
  • Hal is CI, Br, or I, preferably Br or I.
  • the present invention also relates to a method as described above wherein the functional group W or the functional group Z 2 is -SH and the functional group Z 2 or the functional group W is selected from the group consisting of
  • Hal is CI, Br, or I.
  • Z 2 or W is selected from the group consisting of an activated ester, as described above, or a carboxy group which is optionally transformed into an activated ester.
  • the functional group W or Z 2 respectively, comprises the chemical structure -NH-.
  • the present invention also relates to a method as described above wherein Z 2 or W is selected from the group consisting of an activated ester, as described above, or a carboxy group which is optionally transformed into an activated ester, and the functional group W or Z 2 ., respectively, comprises the chemical structure - NH-.
  • the functional group W or Z 2 comprising the structure -NH- is a group having the structure R'-NH- where R' is hydrogen or a alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue where the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directly to the NH group or, according to an- other embodiment, may be linked by an oxygen bridge to the NH group.
  • alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl, or cycloalkylaryl residues may be suitably substituted.
  • halogenes such as F, CI or Br may be mentioned.
  • Especially preferred residues R' are hydrogen, alkyl and alkoxy groups, and even more preferred are hydrogen and unsubstituted alkyl and alkoxy groups.
  • alkyl and alkoxy groups groups with 1, 2, 3, 4, 5, or 6 C atoms are preferred. More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups. Especially preferred are methyl, ethyl, methoxy, ethoxy, and particular preference is given to methyl or methoxy.
  • the present invention also relates to a method as described above wherein W or Z 2 is selected from the group consisting of an activated ester, as described above, or a carboxy group which is optionally transformed into an activated ester, and the functional group W or Z 2 , respectively, is R'-NH- wherein R' is hydrogen or a methyl or a methoxy group.
  • the functional group R" is selected from the group consisting of
  • G where, if G is present twice, it is independently O or S.
  • the present invention also relates to a method as mentioned above wherein the functional group W or Z 2 is selected from the group consisting of
  • G is O or S and, if present twice, independently O or S, and R' is methyl.
  • the at least one functional group X, Z 2 and or W may be a group which is not capable of reacting directly with a given -further compound, but which may be chemically modified in order to be capa- ble of reacting in the desired way.
  • a functional group to be modified prior to the reaction with a further compound a 1,2-amino alcohol or a 1,2-diol may be mentioned which is modified, e.g., by oxidation to form an aldehyd or a keto group.
  • a functional group to be modified prior to the reaction with a further compound is a -NH 2 group which is modified by the reaction with, e.g., a compound according to the following formula
  • a functional group to be modified prior to the reaction with a further compound is a -NH 2 group which is modified by the reaction with, e.g., a compound according to the following formula
  • a functional group to be modified prior to the reaction with a further compound is an amino group which is reacted with bromoacetic anhydride or N-succinimidyl iodo acetate.
  • a compound (L) has the structure Zi-L'-X or Z 2 -L'-X, L' being an organic residue separating the functional groups and being optionally absent, the structure depending on whether a compound (D) is reacted with the hydroxyalkyl starch or not.
  • no compound (D) is involved and Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group.
  • L' may be a linear or branched alkyl or cycloalkyl or aryl or or aralkyl or arylcycloalkyl or alkaryl or cycloalkylaryl group, wherein L' may comprise at least one heteroatom such as N, O, S, and wherein L' may be suitably substituted.
  • the size of the group L' may be adapted to the specific needs.
  • the separating group L' generally has from 1 to 60, preferably from 1 to 40, more preferably from 1 to 20, more preferably from 1 to 10, more preferably from 1 to 6 and especially preferably from 1 to 4 carbon atoms.
  • the separating group comprises generally from 1 to 20, preferably from 1 to 8 and especially preferably from 1 to 4 heteroatoms.
  • the separating group L' comprises 1 to 4 oxygen atoms.
  • the separating group L' may comprise an optionally branched alkyl chain or an aryl group or a cycloalkyl group having, e.g., from 5 to 7 carbon atoms, or be a aralkyl group, an alkaryl group where the alkyl part may be a linear and/or cyclic alkyl group.
  • the separating group is an alkyl chain of from 1 to 20, preferably from 1 to 8, more preferably from 1 to 6, more preferably from 1 to 4 and especially preferably from 2 to 4 carbon atoms.
  • a chain comprising 1 to 4 oxygen atoms is particularly preferred.
  • a compound (D) is involved.
  • a compound (D) has the structure Zi-D'-W, D' being an organic residue separating the functional groups and being optionally absent.
  • a specific example of a compound D where D' is absent is NH 3 .
  • D' may be a linear or branched alkyl or cycloalkyl or aryl or or aralkyl or arylcycloalkyl or alkaryl or cycloalkylaryl group, wherein D' may comprise at least one heteroatom such as N, O, S, and wherein D' may be suitably substituted.
  • the size of the group D' may be adapted to the specific needs.
  • the separating group D' generally has from 1 to 60, preferably from 1 to 40, more preferably from 1 to 20, more preferably from 1 to 10, more preferably from 1 to 6 and especially preferably from 1 to 4 carbon atoms.
  • the separating group comprises generally from 1 to 20, preferably from 1 to 8 and especially preferably from 1 to 4 heteroatoms.
  • the separating group D' comprises 1 to 4 oxygen atoms.
  • the separating group D ! may comprise an optionally branched alkyl chain or an aryl group or a cycloalkyl group having, e.g., from 5 to 7 carbon atoms, or be an aralkyl group, an alkaryl group where the alkyl part may be a linear and/or cyclic alkyl group.
  • the separating group is an alkyl chain of from 1 to 20, preferably from 1 to 8, more preferably from 1 to 6, more preferably from 1 to 4 and especially preferably from 2 to 4 carbon atoms.
  • a chain comprising 1 to 4 oxygen atoms is particularly preferred.
  • preferred compounds (D) having the structure Zi-D'-W where D' is not absent are:
  • the fimctional group Y is a thio group and the functional group W comprises the structure -NH-, as described above in detail, the following types of compounds (L) are, among others, preferred:
  • the functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group
  • the functional group W is a thio group
  • the following types of compounds (L) are, among others, preferred:
  • the separating groups L' and/or D' may be suitably substituted.
  • Prefeixed substituents are, e.g, halides such as F, CI, Br or I.
  • the separating groups L' and/or D' may comprise one or more cleavage sites such as
  • a compound (D) or a compound (L) is reacted with the reducing end of the hydroxyalkyl starch which is not oxidised.
  • reaction product of a compound (D) or a compound (L) is reacted with the reducing end of the hydroxyalkyl starch which is not oxidised may have different constitutions. According to a preferred embodiment of the present invention, this reaction is carried out in an aqueous system.
  • aqueous system refers to a solvent or a a mixture of solvents comprising water in the range of from at least 10 % per weight, preferably at least 50 % per weight, more preferably at least 80 % per weight, even more preferably at least 90 % per weight or up to 100 % per weight, based on the weight of the solvents involved.
  • solvents such as DMSO, DMF, ethanol or methanol may be mentioned.
  • the hydroxyalkyl starch derivative may have a constitution according to formula (Ilia)
  • the hydroxyalkyl starch derivative may have a constitution according to formula (Ilia) or formula (Illb) or be a mixture of compounds according to formulae (Ilia) and (Illb)
  • the compounds according to formula (Ilia) may be present with the N atom in equatorial or axial position where also a mixture of both forms may be present having a certain equilibrium distribution.
  • the compounds according to formula (Illb) may be present with the C-N double bond in E or Z conformation where also a mixture of both forms may be present having a certain equilibrium distribution.
  • the present invention also relates to a hydroxyalkyl starch derivative as described above having a constitution according to formula (Illb) or according to fonnula (Illb) or according to formulae (Ilia) and (Illb).
  • acylation of the compound according to formula (Ilia) is particularly preferred, especially in the case where R' is hydrogen.
  • acylation reagent all suitable reagents may be used which result in the desired hydroxyalkyl starch derivative according to formula (INa)
  • the residue Ra being part of the acylation reagent is methyl.
  • acylation reagents carboxylic acid anhydrides, carboxylic acid halides, and carboxylic acid active esters are preferably used.
  • the acylation is carried at a temperature in the range of from 0 to 30 °C, preferably in the range of from 2 to 20 °C and especially preferably in the range of from 4 to 10 °C.
  • the present invention also relates to a hydroxyalkyl starch derivate obtainable by a method as described above wherein said derivative has a constitution according to formula (IV a).
  • boro hydrides such as ⁇ aC ⁇ BH 3 or NaBH are used.
  • the reduction is carried at a temperature in the range of from 4 to 100 °C, preferably in the range of from 10 to 90 °C and especially preferably in the range of from 25 to 80 °C.
  • the present invention also relates to a hydroxyalkyl starch derivate obtainable by a method as described above wherein said derivative has a constitution ac- cording to formula (IVb).
  • the present invention further relates to mixtures of compounds having constitutions according to formulae (ma) and (Illb), (IVa) and (INb), (Ilia) and (IVa), (Ilia) and (IVb), (Illb) and (IVa), (Illb) and (IVb), (Ilia) and (Illb) and (IVa), (Illb) and (IVb), (Ilia) and (Illb) and (IVa), (Ilia) and (Illb) and (IVb), (IVa) and (IVb) and (Ilia), and (IVa) and (IVb) and (Illb) wherein (Ilia) and/or (IVa) may be independently present in a conformation where the ⁇ atom .in equatorial or axial position and/or wherein (Illb) may be present with the C- ⁇ double bond in E ox Z conformation.
  • a compound (D) or a compound (L) is reacted with the reducing end of the hydroxyalkyl starch which is oxidised.
  • polar aprotic solvents are used which may also contain a cer- tain amount of water, such as up to 10 wt.-%.
  • Preferred aprotic solvents are, among others, DMSO or DMF.
  • An example of a preferred reaction temperature range is from room 20 to 65 °C, and the reaction times are generally in the range of 1 minute to several hours and up to several days, depending on the chemical nature of the functional group which is reacted with the oxidized reducing end og the hydroxyalkyl starch and the other reaction conditions.
  • the hydroxyalkyl starch derivative may have a constitution according to formula (Va)
  • the present invention also relates to a hydroxyalkyl starch derivate obtainable by a method as described above wherein said derivative has a constitution according to formula (Va).
  • a preferred embodiment of the present invention relates to a method as described above wherein hydroxyalkyl starch is reacted with a compoimd (L) via the reaction of functional group Zi with the optionally oxidized reducing end of the hydroxyalkyl starch and the resulting reaction product is reacted with a further compound (M) via the reaction of the functional group X comprised in compound (L) with the fimctional group Y comprised in compound (M).
  • Another embodiment of the present invention relates to a method as described above wherein hydroxyalkyl starch is reacted with a compoimd (L) via the reaction of func- tional group Zi with the optionally oxidized reducing end of the hydroxyalkyl starch, where compound (L), prior to the reaction with hydroxyalkyl starch, is reacted with a further compound (M) via the reaction of fimctional group X comprised in compound (L) with the functional group Y comprised in compound (M).
  • Still another embodiment of the present invention relates to a method as described above wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of the functional group Zi comprised in compound (D), with the optionally oxidized reducing end of the hydroxyalkyl starch to give a first hydroxyalkyl starch derivative, and where the first hydroxyalkyl starch derivative is reacted with a compound (L) via the reaction of functional group Z 2 comprised in compound (L) with the functional group W comprised in compound (D) to give a second hydroxyalkyl starch derivative.
  • Yet another embodiment of the present invention relates to the latter method wherein the second hydroxyalkyl starch derivative is reacted with a further compound (M) via the reaction of functional group X comprised in compound (L) with the fimctional group Y comprised in compound (M).
  • Still yet another embodiment of the present invention relates to a method as described above wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of functional group Zi comprised in compound (D) with the optionally oxidized reducing end of the hydroxyalkyl starch to give a first hydroxyalkyl starch derivative, and where the first hydroxyalkyl starch derivative is reacted, via the reaction of the functional group W, comprised in compound (D), and the functional group Z 2 , comprised in compound (L), with compoimd (L), where compound (L), prior to the reaction with the first hydroxyalkyl starch derivative, is reacted with a further compound (M) via the reaction of functional group X comprised in compound (L) with the functional group Y comprised in compound (M).
  • water is used as solvent, either alone or in combination with at least one other solvent.
  • DMSO, DMF, methanol and ethanol may be mentioned.
  • Preferred solvents other than water are DMSO, DMF, methanol and ethanol.
  • hydroxylalkyl starch is preferably reacted via the non-oxidized reducing end.
  • the temperature of the reaction is preferably in the range of from 5 to 45 °C, more preferably in the range of from 10 to 30 °C and especially preferably in the range of from 15 to 25 °C.
  • the temperature is preferably in the range of up to 100 °C, more preferably in the range of from 70 to 90 °C and especially preferably in the range of from 75 to 85 °C.
  • the temperature may be varied, preferably in the above-given ranges, or held essentially constant.
  • reaction time for the reaction of hydroxyalkyl starch with compound (D) or compound (L) may be adapted to the specific needs and is generally in the range of from 1 h to 7 d.
  • the reac- tion time is preferably in the range of from 1 h to 3 d and more preferably of from 2 h to 48 h.
  • the reaction time is preferably in the range of from 2 h to 7 d.
  • the pH value for the reaction of hydroxyalkyl starch with compound (D) or com- pound (L) may be adapted to the specific needs such as the chemical nature of the reactants.
  • the pH value is preferably in the range of from 4.5 to 6.5.
  • the pH value is preferably in the range of from 8 to 12.
  • the suitable pH value of the reaction mixture may be adjusted, for each reaction step, by adding at least one suitable buffer.
  • suitable buffers sodium acetate buffer, phosphate or borate buffers may be mentioned.
  • the at least one functional group X may be protected with at least one suitable protecting group prior to the reaction of hydroxyalkyl starch with compound (L) or prior to the reaction of compound (D) with compoimd (L) or prior to the reaction of compound (L) with the reaction product of the reaction of hydroxyalkyl starch with compound (D).
  • protecting groups are possible which prevent the protected compound (L) from reacting via the at least one functional group X.
  • the protecting group may be chosen depending from the chemical nature of the functional group X to be protected, from, e.g., the solvent the reaction is carried out in or the pH of the reaction mixture.
  • Preferred protecting groups are, among others, the benzyloxycarbonyl group, the tert-butoxycarbonyl group, the methoxyphenyl group, the 2,4-dimethoxyphenyl group, triarly methyl groups, trityl, the monomethoxytrityl group, the dimethoxytrityl group, the mono- methyltrityl group, the dimethyltrityl group, the trifluoracetyl group, phthalimin compounds, 2-(trialkylsilyl)ethoxy carbonyl compounds, Fmoc, the tert-butyl group, or trialkyl silyl groups. If two or more different functional groups X are present in compound (L), at least one group may be protected whereas at least one other group may be left unprotected.
  • the at least one protecting group may be left in the reaction product or removed by suitable methods such as conventional methods known to the person skilled in the art. If two different fimctional groups X are protected by suitable protecting groups, it is possible to remove at least one protecting group so as to make at least one fimctional group X available for fiirther reaction with at least one further compound (M), and leave at least one other functional group protected until the reaction product comprising compound (L) is reacted with the further compound (M). Afterwards, the protecting group of the functional group still protected may be removed to make the remaining fimctional group X available for reaction with yet a fiirther compound (M).
  • the use of at least one protecting group may be important for preventing the reaction from resulting in a hydroxyalkyl starch derivative comprising a compound (L) or compound (D) which has been reacted with two or more hydroxyalkyl starch mole- cules, i.e. a multiple HAS substituted compoimd (L) or (D).
  • the same result may be achieved by reacting hydroxyalkyl starch with an excess of compound (L) or (D). If an excess amount of compound (L) or (D) is used in the process of the present invention, the molar ratio of compound (L) or (D) to hydroxyalkyl starch is preferably in the range of from 2 to 100.
  • reaction product of the respective reaction step as described above, it may be isolated from the reaction mixture by at least one suitable method. If necessary, the reaction product may be precipitated prior to the isolation by at least one suitable method.
  • reaction product is precipitated first, it is possible, e.g., to contact the reaction mixture with at least one solvent or solvent mixture other than the solvent or solvent mixture present in the reaction mixture at suitable temperatures.
  • the reaction mixture is contacted with a mixture of ethanol and acetone, preferably a 1:1 mixture, indicating equal volumes of said compounds, at a temperature, preferably in the range of from -20 to +50 °C and especially preferably in the range of from 0 to 25 °C.
  • Isolation of the reaction product may be carried out by a suitable process which may comprise one or more steps.
  • the reaction product is first separated off the reaction mixture or the mixture of the reaction mixture with, e.g., the ethanol-acetone mixture, by a suitable method such as centrifugation or filtration.
  • the separated reaction product may be subjected to a further treatment such as an after-treatment like dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography, HPLC, MPLC, gel filtration and/or lyophilisation.
  • the separated reaction product is first dialysed, preferably against water, and then lyophilized until the solvent content of the reaction product is sufficiently low according to the desired specifications of the product. Lyophilisation may be carried out at temperature of from 20 to 35 °C, preferably of from 25 to 30 °C.
  • the hydroxyalkyl starch derivative comprising hydroxyalkyl starch and compound (L) or comprising hydroxyalkyl starch, compound (D) and compound (L) is fiirther reacted with the further compound (M) which comprises at least one functional group Y.
  • compound (M) there are no limitations regarding compound (M).
  • a polypeptide is used as compoimd (M) in the context of the present invention.
  • other compounds (M) are also possible, either polymers or oligomers or monomolecular compounds or mixtures of two or more thereof.
  • the polypeptide may be a naturally occuring compound or a polypeptide which does not occur naturally, the latter comprising naturally occuring amino acids and/or at least one amino acid which does not naturally occur.
  • the backbone of the polypeptide, the polypeptide chain may be fur- ther substituted with at least one suitable substituent thus having at least one side- chain.
  • the at least one functional group Y may be part of the polypeptide backbone or of at least one substituent of the backbone wherein embodiments are possible comprising at least one functional group being part of the polypeptide backbone and at least one functional group being part of at least one substituent of the polypeptide backbone.
  • the polypeptide comprises at least one functional group Y.
  • Said fimctional group Y may be linked directly to the polypeptide backbone or be part of a side-chain of the back- bone. Either side-chain or functional group Y or both may be part of a naturally occuring polypeptide or may be introduced into a naturally occuring polypeptide or into a polypeptide which, at least partially, does not occur naturally, prior to the reaction with the fimctional group X.
  • polypeptide can be, at least partly, of any human or animal source.
  • polypeptide is of human source.
  • the polypeptide may be a cytokine, especially erythropoietin, an antithrombin (AT) such as AT III, an interleukin, especially interleukin-2, IFN-beta, IFN-alpha, G-CSF, CSF, inte ⁇ eukin-6 and therapeutic antibodies.
  • AT antithrombin
  • interleukin especially interleukin-2, IFN-beta, IFN-alpha, G-CSF, CSF, inte ⁇ eukin-6 and therapeutic antibodies.
  • the polypeptide is an antithrombin (AT), preferably AT III (Levy JH, Weisinger A, Ziomek CA, Echelard Y, Recombinant Antithrombin: Production and Role in Cardiovascular Disorder, Seminars in Thrombosis and Hemostasis 27, 4 (2001) 405-416; Edmunds T, Van Patten SM, Pollock J, Hanson E, Bernasconi R, Higgins E, Manavalan P, Ziomek C, Meade H, McPherson J, Cole ES, Transgenically Produced Human Antithrombin: Structural and Functional Comparison to Human Plasma-Derived Antithrombin, Blood 91, 12 (1998) 4661- 4671; Minnema MC, Chang ACK, Jansen PM, Lubbers YTP, Pratt BM, Whittaker BG, Taylor FB, Bush CE, Friedman B, Recombinant human antithrombin III improves survival and attenuates inflammatory
  • AT antithrom
  • the polypeptide is human IFN-beta, in particular IFN-beta la (cf. Avonex®, REBIF®) and IFN-beta lb (cf. BETASERON®).
  • a further preferred polypeptide is human G-CSF (granulocyte colony stimulating factor).
  • G-CSF granulocyte colony stimulating factor
  • the at least two polypep- tides may differ, e.g., in the molecular mass, the number and/or sequence of amino acids, the number and/or chemical nature of the substituents or the number of polypeptide chains linked by suitable chemical bonds such as disulfide bridges.
  • the reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) is isolated, preferably according to at least one of the above-mentioned processes, and then re- acted with a polypeptide having at least one functional group Y.
  • the functional group Y is comprised in a carbohydrate moiety of the polypeptide.
  • carbohydrate moiety refers to hy- droxyaldehydes or hydroxyketones as well as to chemical modifications thereof (see Rompp Chemielexikon, Thieme Verlag Stuttgart, Germany, 9 th edition 1990, Volume 9, pages 2281-2285 and the literature cited therein). Furthermore, it also refers to derivatives pf naturally occuring carbohydrate moieties like glucose, galactose, mannose, sialic acid and the like. The term also includes chemically oxidized, naturally occuring carbohydrate moieties. The structure of the oxidized carbohydrate moiety may be cyclic or linear.
  • the carbohydrate moiety may be linked directly to the polypeptide backbone.
  • the carbohydrate moiety is part of a carbohydrate side chain. More preferably, the carbohydrate moiety is the terminal moiety of the carbohydrate side chain.
  • the carbohydrate moiety is a galactose residue of the carbohydrate side chain, preferably the terminal galactose residue of the carbohydrate side chain.
  • This galactose residue can be made available for reaction with the functional group X comprised in the reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L), by removal of terminal sialic acids, followed by oxidation, as described hereinunder.
  • reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is fiirther reacted with compound (L) is linked to a sialic acid residue of the carbohydrate side chains, preferably the terminal sialic acid residue of the carbohy- drate side chain. Oxidation of terminal carbohydrate moieties can be performed either chemically or enzymatically.
  • the carbohydrate moiety may be oxidized enzymatically.
  • Enzymes for the oxidation of the individual carbohydrate moieties are known in the art, e.g. in the case of galactose the enzyme is galactose oxidase. If it is intended to oxidize terminal galactose moieties, it will be eventually necessary to remove terminal sialic acids (partially or completely) if the polypeptide has been produced in cells capable of attaching sialic acids to carbohydrate chains, e.g. in mammalian cells or in cells which have been genetically modified to be capable of attaching sialic acids to carbohydrate chains.
  • the fimctional group of the polypeptide is the thio group. Therefore, the reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) may be linked to the polypeptide via a thioether group wherein the S atom can be derived from any thio group comprised in the polypeptide. In the context of this embodiment, it is particularly preferred to react the polypeptide with a reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L).
  • the present invention also relates to a method as described above wherein the reaction product of hydroxyalkyl starch and compound (D) is further reacted with compound (L) is reacted with the polypeptide via a thio group comprised in the polypeptide.
  • the present invention also relates to a method as described above wherein the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) is reacted with the polypeptide via an oxidized carbohydrate moiety and a thio group comprised in the polypeptide.
  • the thio group may be present in the polypeptide as such. Moreover, it is possible to introduce a thio group into the polyeptide according to a suitbale method. Among others, chemical methods may be mentioned. If a disulfide bridge is present in the polypeptide, it is possible to reduce the -S-S- structure to get a thio group. It is also possible to transform an amino group present in the polypeptide into a SH group by reaction the polypeptide via the amino group with a compound which has at least two different functional groups, one of which is capable of being reacted with the amino group and the other is an SH group or a precursor of an SH group.
  • This modification of an amino group may be regarded as an example where the protein is first reacted with a compoimd (L) which has at least two different functional groups, one of which is capable of being reacted with the amino group and the other is an SH group, and the resulting reaction product is then reacted with, e.g., a HAS derivative comprising HAS and a compound (D), said derivative comprising a functional group being capable of reacting with the SH group.
  • a compoimd (L) which has at least two different functional groups, one of which is capable of being reacted with the amino group and the other is an SH group
  • the resulting reaction product is then reacted with, e.g., a HAS derivative comprising HAS and a compound (D), said derivative comprising a functional group being capable of reacting with the SH group.
  • an SH group by mutation of the polypeptide such as by introducing a cystein or a suitable SH func- tional amino acid into the polypeptide or such as removing a cystein from the polypeptide so as to disable another cystein in the polypeptide to form a disulfide bridge.
  • erythropoietin EPO is used as an especially preferred polypeptide.
  • the present invention also relates to a method as described above wherein the polypeptide is erythropoietin.
  • the EPO can be of any human (see e.g. Inoue, Wada, Takeuchi, 1994, An improved method for the purification of human erythiOpoietin with high in vivo activity from the urine of anemic patients, Biol. Pharm. Bull. 17(2), 180-4; Miyake, Kung, Gold- wasser, 1977, Purification of human erythropoietin., J. Biol. Chem., 252(15), 5558- 64) or another mammalian source and can be obtained by purification from naturally occurring sources like human kidney, embryonic human liver or animal, preferably monkey kidney.
  • erythropoietin or "EPO” encompasses also an EPO variant wherein one or more amino acids (e.g. 1 to 25, preferably 1 to 10, more preferred 1 to 5, most preferred 1 or 2) have been exchanged by an- other amino acid and which exhibits erythropoietic activity (see e.g. EP 640 619 Bl).
  • the measurement of erythropoietic activity is described in the art (for measurement of activity in vitro see e.g. Fibi et al.,1991, Blood, 77, 1203 ff; Kitamura et al, 1989, J. Cell Phys., 140, 323-334; for measurement of EPO activity in vivo see Ph. Eur.
  • the EPO is recombinantly produced.
  • the EPO may be expressed in transgenic animals (e.g. in body fluids like milk, blood, etc.), in eggs of transgenic birds, especially poultry, preferred chicken, or in transgenic plants.
  • the recombinant production of a polypeptide is known in the art. In general, this includes the transfection of host cells with an appropriate expression vector, the cultivation of the host cells under conditions which enable the production of the polypeptide and the purification of the polypeptide from the host cells. For detailled information see e.g.
  • the EPO has the amino acid sequence of human EPO (see EP 148 605 B2).
  • the EPO may comprise one or more carbohydrate side chains, preferably 1 to 12, more preferably 1 to 9, even more preferably 1 to 6 and particularly 1 to 4, especially preferably 4 carbohydrate side chains, attached to the EPO via N- and/ or O-linked glycosylation, i.e. the EPO is glycosylated.
  • carbohydrate side chains may have been attached to the EPO during biosynthesis in mammalian, especially human, insect or yeast cells.
  • glycosylated EPO The structure and properties of glycosylated EPO have been extensively studied in the art (see EP 428 267 Bl; EP 640 619 Bl; Rush, Derby, Smith, Merry, Rogers, Rohde, Katta, 1995, Microhetero- geneity of erythropoietin carbohydrate structure, Anal Chem., 67(8), 1442-52; Ta- keuclii, Kobata, 1991, Structures and fimctional roles of the sugar chains of human erythropoietins, Glycobiology, 1(4), 337-46 (Review).
  • the hydroxyalkyl starch derivative according to the present invention may comprise at least one, preferably 1 to 12, more preferably 1 to 9, even more prefera- bly 1 to 6 and particularly preferably 1 to 4 HAS molecules per EPO molecule.
  • the number of HAS-molecules per EPO molecule can be determined by quantitative carbohydrate compositional analysis using GC-MS after hydrolysis of the product and derivatisation of the resulting monosaccharides (see Chaplin and Kennedy (eds.), 1986, Carbohydrate Analysis: a practical approach, IRL Press Practical approach series (ISBN 0-947946-44-3), especially Chapter 1, Monosaccharides, page 1-36; Chapter 2, Oligosaccharides, page 37-53, Chapter 3, Neutral Polysaccharides, page 55-96).
  • the car- bohydrate moiety linked to EPO is part of a carbohydrate side chain. More preferably, the carbohydrate moiety is the terminal moiety of the carbohydrate side chain. In an even more preferred embodiment, the carbohydrate moiety is a galactose residue of the carbohydrate side chain, preferably the terminal galactose residue of the carbohydrate side chain. This galactose residue can be made available for reaction with the reaction product of compound (I) and compound (II) by removal of terminal sialic acids, followed by oxidation, as described hereinunder.
  • reaction product of compound (I) and (II) is linked to a sialic acid residue of the carbohydrate side chains, preferably the terminal sialic acid residue of the carbohydrate side chain.
  • the sialic acid is oxidized as described hereinunder.
  • this galactose residue is made available for reaction with the reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) via the functional group X by removal of terminal sialic acid followed by oxidation.
  • this galactose residue is made available for reaction with the reac- tion product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) via the functional group X by oxidation wherein terminal sialic acid is not removed.
  • reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) be reacted with a thio group comprised in EPO.
  • reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is fur- ther reacted with compound (L) with a thio group as well as with a carbohydrate moiety each of them comprised in the at least one further compound, preferably a polypeptide, more preferably erythropoietin.
  • this SH group may be linked to a preferably oxidized carbohydrate moiety, e.g. by using a hydroxylamine derivative, e.g. 2-
  • the thio group is preferably introduced in an oxidized carbohydrate moiety of EPO, more preferably an oxidized carbohydrate moiety which is part of a carbohydrate side chain of EPO.
  • the thio group is derived from a naturally occurring cysteine or from an added cysteine. More preferably, the EPO has the amino acid sequence of human EPO and the naturally occurring cysteines are cysteine 29 and/or 33. In a more preferred embodiment, t the reaction product of hydroxyalkyl starch and compoimd (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) is reacted with cysteine 29 whereas cysteine 33 is replaced by another amino acid.
  • reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compoimd (D) which is further reacted with compound (L) is reacted with cysteine 33 whereas cysteine 29 is replaced by another amino acid.
  • the term "added cysteines” indicates that the polypeptides, preferably EPO, comprise a cysteine residue which is not present in the wild-type polypeptide.
  • cysteine may be an additional amino acid added at the N- or C-terminal end of EPO.
  • the added cysteine may have been added by replacing a naturally occuring amino acid by cysteine or a suitably substituted cysteine.
  • the EPO is human EPO and the replaced amino acid residue is serine 126.
  • water is used as solvent, either alone or in combination with at least one other solvent.
  • DMSO, DMF, methanol or ethanol may be mentioned.
  • Preferred solvents other than water are methanol and ethanol.
  • DMSO or DMF or methanol or ethanol or a mixture of two or more thereof is used as solvent.
  • reaction temperatur is preferably preferably in the range of from 4 to 37 °C, more preferably of from 10 to 30 °C and especially preferably of from 15 to 25 °C.
  • Isolation of the reaction product comprising the further compound (M), preferably the polypeptide and especially pererably erythropoietin can be performed by using known procedures for the purification of natural and recombinant EPO (e.g.size ex- elusion chromatography, ion-exchange chromatography, RP-HPLC, hydroxyapatite chromatography, hydrophobic interaction chromatography or combinations thereof). Isolation of the reaction product may be carried out by a suitable process which may comprise one or more steps.
  • the reaction product is first separated off the reaction mixture or the mix- ture of the reaction mixture with, e.g., the ethanol-acetone mixture, by a suitable method such as centrifugation or filtration.
  • a suitable method such as centrifugation or filtration.
  • the separated reaction product may be subjected to a further treatment such as an after-treatment like dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography such as, e.g., by a column containing Q-sepharose, HPLC, MPLC, gel filtration and/or ly- ophilisation.
  • the separated reaction product is first dialysed, preferably against water, and then lyophilized until the solvent content of the reaction product is sufficiently low according to the desired specifications of the product. Lyophilisation may be carried out at temperature of from 20 to 35 °C, preferably of from 25 to 30 °C.
  • the reaction mixture comprising the reaction product is applied to a column containing Q- Sepharose to give an eluate which is concentrated, e.g. by centrifugal filtration.
  • the present invention relates to a hydroxyalkyl starch derivative obtain- able by a method of producing a hydroxyalkyl starch derivative, said hydroxyalkyl starch having a structure according to fonnula (I)
  • hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end or a hydroxyalkyl starch derivative, obtainable by reacting hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end with a compound (D), said compound (D) comprising at least one functional group Zi capable of being reacted with the optionally oxidized reducing end of the hydroxyalkyl starch, and at least one functional group W, with a compound (L) comprising at least one functional group Zi capable of being reacted with said hydroxyalkyl starch, or at least one functional group Z 2 capable of being reacted with functional group W comprised in said hydroxyalkyl starch derivative, and at least one functional group X capable of being reacted with a functional group Y of a further compound (M), wherein said functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, an acetal group, or
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein Ri, R 2 and R 3 are independently hydrogen or a linear or branched hydroxyalkyl group.
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein Ri, R 2 and R 3 are inde- pendently hydrogen or a 2-hydroxyethyl group.
  • the present invention relates to a hydroxyalkyl starch derivative as described wherein the hydroxyalkyl starch is hydroxyethyl starch.
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein the functional group Zi comprises the structure -NH-.
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein Zi is selected from the group consisting of
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein the functional group Y is selected from the group consisting of an aldehyd group, a eto group, a hemiacetal group, and an acetal group, and the functional group X comprises the structure -NH-.
  • the present invention relates to a hydroxyalkyl starch derivative as described above X is selected from the group consisting of
  • G is O or S and, if present twice,' independently O or S, and R is methyl.
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein the functional group Y is - SH and the -functional group X is selected from the group consisting of
  • Hal is CI, Br or I.
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein the functional group W or the functional group Z 2 is -SFI and the functional group Z 2 or the functional group W is selected from the group consisting of wherein Hal is CI, Br, or I.
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein the functional group W or the functional group Z 2 is selected from the group consisting of an activated ester, as described above, or a carboxy group which is optionally transformed into an activated ester and the functional group Z 2 or the functional group W is selected from the group consisting of
  • G is O or S and, if present twice, independently O or S, and R is methyl.
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein the reducing end of the hydroxyalkyl starch is not oxidized prior to the reaction with compound (D) or compound (L), said hydroxyalkyl starch thus having a structure according to formula (I)
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein the reducing end of the hydroxyalkyl starch is oxidized prior to the reaction with compound (D) or compound (L), said hydroxyalkyl starch thus having a structure according to formula (Ila)
  • the present invention relates to a hy- droxyalkyl starch derivative as described above wherein the reducing end is oxidized by an alkaline iodine solution.
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein hydroxyalkyl starch is reacted with a compound (L) via the reaction of functional group Zi with the optionally oxidized reducing end of the hydroxyalkyl starch and the resulting reaction product is reacted with a further compound (M) via the reaction of the functional group X comprised in compoimd (L) with the functional group Y comprised in compound (M).
  • the present invention relates to a hydroxyalkyl starch derivative as described above hydroxyalkyl starch is reacted with a compound (L) via the reaction of functional group Zi with the optionally oxidized reducing end of the hydroxyalkyl starch, where compound (L), prior to the reaction with hydroxyalkyl starch, is reacted with a further compound (M) via the reaction of functional group X comprised in compound (L) with the functional group Y comprised in compound (M).
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of the functional group Zi comprised in compound (D), with the optionally oxidized reducing end of the hydroxyalkyl starch to give a first hydroxyalkyl starch derivative, and where the first hydroxyalkyl starch derivative is reacted with a compound (L) via the reaction of fimctional group Z 2 comprised in compound (L) with the functional group W comprised in compound (D) to give a second hydroxyalkyl starch derivative.
  • the present invention relates to the aforesaid hydroxyalkyl starch derivative wherein the second hydroxyalkyl starch derivative is reacted with a further compound (M) via the reaction of functional group X comprised in compound (L) with the functional group Y comprised in compound (M).
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of functional group Zi comprised in compound (D) with the optionally oxidized reducing end of the hydroxyalkyl starch to give a first hydroxyalkyl starch derivative, and where the first hydroxyalkyl starch derivative is reacted, via the reaction of the functional group W, comprised in compound (D), and the functional group Z 2 , comprised in compound (L), with compound (L), where compound (L), prior to the reaction with the first hydroxyalkyl starch derivative, is reacted with a further compound (M) via the reaction of fimctional group X comprised in compound (L) with the functional group Y comprised in compound CM).
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein the at least one further compound (M) is a polypeptide.
  • the present invention relates to a hydroxyalkyl starch derivative as described above wherein the polypeptide is erythiOpoietin.
  • the hydroxyalkyl starch derivative which in the following is referred to as HAS- EPO conjugate and which is formed by reaction of hydroxyalkyl starch with compound (L) and optionally compoimd (D) and erythrpoietin, has the advantage that it exhibits an improved biological stability when compared to the erythropoietin before conjugation. Furthermore, it exhibits a higher biological activity than standard BRP EPO. This is mainly due to the fact that this hydroxyalkyl starch derivative is less or even not recognized by the removal systems of the liver and kidney and therefore persists in the circulatory system for a longer period of time.
  • the HAS-EPO conjugate of the invention may exhibit essentially the same in- vitro . biological activity as recombinant native EPO, since the in-vitro biological activity only measures binding affinity to the EPO receptor. Methods for determining the in- vitro biological activity are known in the art.
  • the HAS-EPO exhibits a greater in-vivo activity than the EPO used as a starting material for conjugation (unconjugated EPO).
  • Methods for determining the in vivo biological activity are known in the art.
  • the HAS-EPO conjugate may exhibit an in vivo activity of from 110 % to 500 %, preferably of from 300 to 400 %, or preferably of from 110 to 300 %, more preferably from 110 % to 200 %, more preferably from 110 % to 180 % or from 110 to 150 %, most preferably from 110 % to 140 %, if the in-vivo activity of the unconjugated EPO is set as 100 %.
  • the HAS- EPO exhibits preferably at least 50%, more preferably at least 70 %, even more preferably at least 85 % or at least 95 %, at least 150 %, at least 200 % or at least 300 % of the in vivo activity of the highly sialylated EPO if the in-vivo activity of highly sialylated EPO is set as 100 %. Most preferably, it exhibits at least 95 % of the in vivo activity of the highly sialylated EPO.
  • the high in-vivo biological activity of the HAS-EPO conjugate of the invention mainly results from the fact that the HAS-EPO conjugate remains longer in the circu- lation than the unconjugated EPO because it is less recognized by the removal systems of the liver and because renal clearance is reduced due to the higher molecular weight.
  • Methods for the determination of the in-vivo half life time of EPO in the circulation are known in the art (Sytkowski, Lunn, Davis, Feldman, Siekman, 1998, Human erythropoietin dimers with markedly enhanced in vivo activity, Proc. Natl. Acad. Sci. USA, 95(3), 1184-8).
  • HAS-EPO conjugate which may be administered less frequently than the EPO preparations commercially available at present. While standard EPO preparations have to be administered at least every 3 days, the HAS-EPO conjugate of the invention is pref- erable administered twice a week, more preferably once a week.
  • the method of the invention has the advantage that an effective EPO derivative can be produced at reduced costs since the method does not comprise extensive and time consuming purification steps resulting in low final yield, e.g. it is not necessary to purify away under-sialylated EPO forms which are known to exhibit low or no in-vivo biological activity.
  • Example 8.11(d) demonstrates that a HES-EPO produced with few modifications steps exhibits a 3-fold activity over standard BRP EPO.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition compris- ing, in a therapeutically effective amount, the FIAS-polypeptide conjugate, preferably the HAS-EPO conjugate, more preferably the HES-EPO conjugate of the present invention.
  • the pharmaceutical composition comprises further at least one pharmaceutically acceptable diluent, adjuvant and/or carrier useful in erythropoietin therapy.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising, in a therapeutically effective amount, a hydroxyalkyl starch derivative obtainable by a method of producing a hydroxyalkyl starch derivative, said hydroxyalkyl starch having a structure according to formula (I)
  • hydroxyalkyl starch of fonnula (I) at its optionally oxidized reducing end or a hydroxyalkyl starch derivative obtainable by reacting hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end with a compound (D), said compound (D) comprising at least one functional group Zi capable of being reacted with the optionally oxidized reducing end of the hydroxyalkyl starch, and at least one functional group W, with a compound (L) comprising at least one functional group Zi capable of being reacted with said hydroxyalkyl starch, or at least one functional group Z 2 capable of being reacted with functional group W comprised in said hydroxyalkyl starch derivative, and at least one functional group X capable of being reacted with a functional group Y of a further compound (M), wherein said fimctional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group
  • the present invention relates to the use of a hydroxyalkyl starch derivative as described for the preparation of a medicament for the treatment of anemic disorders or hematopoietic dysfunction disoirders or diseases related thereto.
  • the present invention relates to a pharmaceutical composition as described above wherein the polypeptide is an antithrombin (AT), preferably AT III (Levy JH, Weisinger A, Ziomek CA, Echelard Y, Recombinant Antithrombin: Production and Role in Cardiovascular Disorder, Seminars in Thrombosis and Hemostasis 27, 4 (2001) 405-416; Edmunds T, Van Patten SM, Pollock J, Flanson E, Bernasconi R, Higgins E, Manavalan P, Ziomek C, Meade H, McPherson J, Cole ES, Transgenically Produced Human Antithrombin: Structural and Functional Comparison to Human Plasma-Derived Antithrombin, Blood
  • AT antithrombin
  • the present invention relates to pharmaceutical compositions wherein the polypeptide is G-CSF or IFN-beta.
  • the present invention relates to a pharmaceutical composition as described above wherein the polypeptide is erythropoietin.
  • the present invention relates to a pharmaceutical composition as described above wherein the functional group Y is -SH and corn- pound (L) is a compound of general formula Zi-L'-X where the fimctional group Zi is selected from the group consisting of
  • G is O or S and, if present twice, independently O or S, and R' is methyl, and where the functional group X is selected from the group consisting of
  • Hal is CI, Br or I, and where L' is an organic chain bridging Zi and X or where L' is absent.
  • the present invention relates to a pharmaceutical composition as described above wherein the functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group, and compound (L) is a compound of general formula Zi-L'-X where the functional group Zi is selected from the group consisting of
  • G is O or S and, if present twice, independently O or S, and R' is methyl, and where the functional group X is selected from the group consisting of
  • G is O or S and, if present twice, independently O or S, and R' is methyl, and where L' is an organic chain bridging Zi and X or where L' is absent.
  • the present invention relates to a pharmaceutical composition as described above wherein the functional group Y is -SH, compound
  • (D) is a compound of general formula Zi-D'-W
  • compound (L) is a compound of general formula Z 2 -L'-X, where the functional group Zi is selected from the group consisting of
  • G is O or S and, if present twice, independently O or S, and R' is methyl, where the functional group X is selected from the group consisting of
  • Hal is CI, Br or I
  • the functional group W or the functional group Z 2 is -SH and the fimctional group Z 2 or the fimctional group W is selected from the group consisting of wherein Hal is CI, Br, or I, or where the functional group W or the functional group Z 2 is selected from the group consisting of an activated ester, as described above, or a carboxy group which is optionally transformed into an activated ester and the functional group Z 2 or the functional group W is selected from the group consisting of
  • G is O or S and, if present twice, independently O or S, and R' is methyl, and where D' is an organic chain bridging Zi and W or where D' is absent and where L' is an organic chain bridging Z 2 and X or where L' is absent.
  • the present invention relates to a pharmaceu- tical composition as described above wherein the functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group, compound (D) is a compound of general formula Zi-D'-W, and compound (L) is a compound of general formula Z 2 -L'-X, where the fimctional group Zi is selected from the group consisting of
  • G is O or S and, if present twice, independently O or S, and R' is methyl, where the functional group X is selected from the group consisting of
  • G is O or S and, if present twice, independently O or S, and R' is methyl, the functional group W or the functional group Z 2 is -SH and the fimctional group Z 2 or the functional group W is selected from the group consisting of
  • Flal is CI, Br, or I., or where the functional group W or the functional group Z 2 is selected from the group consisting of an activated ester, as described above, or a carboxy group which is optionally transformed into an activated ester and the functional group Z 2 or the functional group W is selected from the group consisting of
  • G is O or S and, if present twice, independently O or S, and R' is methyl, and where D' is an organic chain bridging Zi and W or where D' is absent and where L' is an organic chain bridging Z 2 and X or where L' is absent.
  • the present invention relates to a pharmaceutical composition as described above wherein hydroxyethyl starch is reacted in an aqueous medium with a compound according to the following formula
  • the present invention relates to the aformentioned pharmaceutical composition wherein the erythropoietin is oxidised with sodium periodate prior to the reaction.
  • the present invention relates to phar- maceutical composition as described above wherein the erythropoietin is partially desialylated and subsequently oxidised with sodium periodate prior to the reaction.
  • compositions comprising a hydroxyalkyl starch derivative which are produced on the basis of a completely reduced Thio-EPO according to Example 6 are excluded.
  • the above-mentioned pharmaceutical composition is especially suitable for the treatment of anemic disorders or hematopoietic dysfunction disorders or diseases related thereto.
  • a "therapeutically effective amount” as used herein refers to that amount which provides therapeutic effect for a given condition and administration regimen.
  • the ad- ministration of erythropoietin isofonns is preferably by parenteral routes. The specific route chosen will depend upon the condition being treated.
  • the administration of erythropoietin isoforms is preferably done as part of a formulation containing a suitable carrier, such as human serum albumin, a suitable diluent, such as a buffered saline solution, and/or a suitable adjuvant.
  • the required dosage will be in amounts sufficient to raise the hematocrit of patients and will vary depending upon the severity pf the condition being treated, the method of administration used and the like.
  • the object of the treatment with the pharmaceutical composition of the invention is preferably an increase of the hemoglobin value of more than 6.8 mmol/1 in the blood.
  • the pharmaceutical composition may be administered in a way that the hemoglobin value increases between from 0.6 mmol/1 and 1.6 mmol/1 per week. If the hemoglobin value exceeds 8.7 mmol/1, the therapy should be preferably interrupted until the hemoglobin value is below 8.1 mmol/1.
  • composition of the invention is preferably used in a fonnulation suitable for subcutaneous or intravenous or parenteral injection.
  • suitable excipients and carriers are e.g. sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chlorate, polysorbate 80, HSA and water for injection.
  • the composition may be administered three times a week, preferably two times a week, more preferably once a week, and most preferably every two weeks.
  • the pharmaceutical composition is administered in an amount of 0.01-10 ⁇ g/kg body weight of the patient, more preferably 0,1 to 5 ⁇ g/kg, 0,1 to 1 ⁇ g/kg, or 0.2-0.9 ⁇ g/kg, most preferably 0-3-0.7 ⁇ g/kg, and most preferred 0.4-0.6 ⁇ g/kg body weight.
  • the invention further relates to a HAS-polypeptide according to the present invention for use in method for treatment of the human or animal body.
  • the invention further relates to the use of a HAS-EPO conjugate of the present invention for the preparation of a medicament for the treatment of anemic disorders or hematopoietic dysfunction disorders or diseases related hereto.
  • compound (II) may be present in R conformation or in S conformation or as racemic compound with respect to each chiral center.
  • compound (D) optionally used in the present invention comprises one or more chiral centers
  • compound (D) may be present in R conformation or in S conformation or as racemic compound with respect to each chiral center.
  • Figure 1 shows an SDS page analysis of the HES-EPO conjugate, produced according to example 5.1.
  • Lane A Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co, Düsseldorf, D); molecular weights (in kD) of the protein marker from top to bottom: 245, 123, 77, 42, 30, 25.4, and 17.
  • Lane B Crude product after conjugation according to example 5.1.
  • Lane C EPO starting material.
  • Figure 2 shows an SDS page analysis of the HES-EPO conjugate, produced according to example 5.3.
  • Figure 3 shows an SDS page analysis of the HES-EPO conjugate, produced according to example 5.4 and 5.5.
  • Lane A Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co, Düsseldorf, D); molecular weights (in kD) of the protein marker from top to bottom: 245, 123, 77, 42, 30, 25.4, and 17. Lane B: Crude product after conjugation according to example 5.4. Lane C: Crude product after conjugation according to example 5.5. Lane D: EPO starting material.
  • Figure 4 shows an SDS page analysis of HES-EPO conjugates, produced according to examples 7.1 and 7.4.
  • Lane A Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co, Düsseldorf, D); molecular weights (in kD) of the protein marker from top to bottom: 245, 123, 77, 42, 30, 25.4, and 17.
  • Figure 5 shows an SDS page analysis of HES-EPO conjugates, produced according to examples 7.2, 7.3, 7.5, and 7.6.
  • Lane A Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co, Düsseldorf, D); molecular weights (in kD) of the protein marker from top to bottom: 245, 123, 77, 42, 30, 25.4, and 17. Lane B: Crude product after conjugation according to example 7.6, based on Example 1.3 b).
  • Lane C Crude product after conjugation according to example 7.5, based on Example 1.1 b).
  • Lane D Crude product after conjugation according to example 7.6, based on Ex- ample 1.3 a).
  • Lane E Crude product after conjugation according to example 7.5, based on Example 1.1 a).
  • Lane F Crude product after conjugation according to example 7.2.
  • Lane G Crude product after conjugation according to example 7.3.
  • Lane K EPO startmg material.
  • Figure 6 shows an SDS page analysis of HES-EPO conjugates, produced according to examples 7.7, 7.8, 7.9, 7.10, 7.11, and 7.12.
  • Lane B Crude product after conjugation according to example 7.11.
  • Lane C Crude product after conjugation according to example 7.10.
  • 15 L Laannee DD: Crude product after conjugation according to example 7.7.
  • Lane E Crude product after conjugation according to example 7.8.
  • Lane F Crude product after conjugation according to example 7.12.
  • Lane G EPO starting material.
  • the elution area of oligosaccharides structures without and with 1-4 sialic acid is indicated by brackets 1-5.
  • Figures 17 to 23 represent MALDI/TOF mass spectra of the enzymatically liberated and chemically desialylated N-glycans isolated from HES-modified EPO and control EPO preparations.
  • Major signals at m/z 1809.7, 2174.8, 2539.9, 2905.0 and 3270.1 correspond to di- to tetraantennary complex-type N-glycan structures with no, one or two N-acetyllactosamine repeats accompanied by weak signals due to loss of fucose or galactose which are due to acid hydrolysis conditions employed for the desialylation of samples for MS analysis.
  • MALDI/TOF spectrum desialylated oligosaccharides of HES-modified EPO A2.
  • MALDI/TOF spectrum desialylated oligosaccharides of EPO GT- 1 -A.
  • MALDI/TOF spectrum desialylated oligosaccharides of EPO K2.
  • MALDI/TOF spectrum desialylated oligosaccharides of EPO-GT-1.
  • MALDI/TOF spectrum desialylated oligosaccharides of EPO-GT-1 subjected to acid hydrolysis for 10 min.
  • MALDI/TOF spectrum desialylated oligosaccharides of EPO-GT-1 subjected to acid hydrolysis for 60 min.
  • Figure 24 shows an SDS page analysis of two HES-EPO conjugates
  • Lane 1 HES-EPO produced according to example protocol 8: EPO is conjugated to hydrazido-HES 12KD L
  • Lane 2 HES-EPO produced according to example protocol 9 : EPO is conjugated to hydroxylamino HES 12 KD K C: control (unconjugated EPO); the upper band represents EPO dimer
  • Figure 2 demonstrates that the HES is conjugated to a carbohydrate moiety of a carbohydrate side chain by showing a digestion of HAS modified EPO forms with po- lyppetide N-glycosidase
  • Lane 1 HES-EPO produced according to example protocol 8 after digestion with N-glycosidase
  • Lane 2 HES-EPO produced according to example protocol 9 after digestion with
  • Lane 4 BRP EPO standard after digestion with N-glycosidase mw: marker (Bio-Rad SDS-PAGE Standards Low range Catalog No 161-
  • Example 1 Formation of hydroxyethyl starch derivatives by reductive amination of the non-oxidised reducing end
  • Example 2 Formation of hydroxyethyl starch derivatives by conjugation with the non-oxidised reducing end
  • the precipitated product was collected by centrifugation, re-dissolved in 40 ml water, and centrifu- gated for 15 min at 4,500 rpm.
  • the clear supernatant was dialysed for 3 d against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science GmbH, Bonn, D), and lyophilized.
  • DMSO absolute dimethyl sulfoxide
  • DMSO absolute dimethyl sulfoxide
  • the precipitated product was collected by centrifugation, redissolved in 40 ml of water and dialysed for 4 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science GmbH, Bonn, Germany) and lyophilized.
  • reaction mixture After stirring for 68 h at 60°C the reaction mixture was added to 200 ml of water The solution containing the reaction product was dialysed for 2 days against a 0.5 % (v/v) triethylamine in water solution and for 2 days against water (SnakeSkin dialysis tub- ing, 3.5 KD cut off, Perbio Science GmbH, Bonn, Germany) and lyophilized.
  • the precipitate Amino-HES10KD/0.4 was collected by centrifugation, redissolved in 40 ml of water and dialysed for 4 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science GmbH, Bonn, Germany) and lyophilized.
  • Oxidized erythropoietin was produced as described in Example 8.
  • EPO-GT-1 -A as described in Example 8.11(c) was used (EPO-GT-1 without acid hydroylsis, treated with mild periodate oxidation).
  • Example 5 Conjugation of hydroxyethyl starch derivatives with oxidized erythropoietin of example 4
  • Example 5.1 Reaction of oxidized erythropoietin with the reaction product of example 2.1
  • Oxidized EPO (1.055 ⁇ g/ ⁇ l) in 20 mM PBS buffer was adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2.
  • 18 ⁇ l of a solution of the HES derivate as produced according to example 2.1 (MW 18 kD; 18.7 ⁇ g/ ⁇ l in 0.1 M sodium acetate buffer, pH 5.2) was added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analyzed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Düsseldorf, D) overnight.
  • Fig. 1 The experimental result is shown in Fig. 1.
  • a successful conjugation is indicated by the migration of the protein band to higher molecular weights.
  • the increased band- width is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • Oxidized EPO (1.055 ⁇ g/ ⁇ l) in 20 mM PBS buffer was adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2.
  • 18 ⁇ l of a solution of the HES derivate as produced according to example 2.3 was added, and the mixture was incubated for 16 h at 25 °C.
  • the crude product was analyzed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen.
  • Example 5.3 Reaction of oxidized erythropoietin with the reaction product of example 2.4
  • Oxidized EPO (1.055 ⁇ g/ ⁇ l) in 20 mM PBS buffer was adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2.
  • EPO solution 18 ⁇ l of a solution of the HES derivate as produced according to example 2.4 (MW 18 kD; 18.7 ⁇ g/ ⁇ l in 0.1 M sodium acetate buffer, pH 5.2) was added, and the mixture was incubated for 16 h at 25 °C.
  • Fig. 2 The experimental result is shown in Fig. 2.
  • a successful conjugation is indicated by the migration of the protein band to higher molecular weights.
  • the increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • Oxidized EPO (1.055 ⁇ g/ ⁇ l) in 20 mM PBS buffer was adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2.
  • 18 ⁇ l of a solution of the HES derivate as produced according to example 3.1 was added, and the mixture was incubated for 16 h at 25 °C.
  • the crude product was analyzed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Düsseldorf, D) overnight.
  • Oxidized EPO (1.055 ⁇ g/ ⁇ l) in 20 mM PBS buffer was adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2.
  • 18 ⁇ l of a solution of the HES derivate as produced according to example 3.1 was added, and the mixture was incubated for 16 h at 25 °C.
  • the crude product was analyzed by SDS-Page with NuPAGE 10% Bis-Tris Gels MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Düsseldorf, D) overnight.
  • Fig. 3 The experimental result is shown in Fig. 3. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased band- width is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • EPO-GT-1 erythropoietin
  • a 0.1 M sodium borate buffer 5 mM EDTA, 10 mM DTT (Lancaster, Morcambe, UK), pH 8.3, were incubated for 1 h at 37 °C.
  • the DTT was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 10 KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, subsequent washing 3 times with the borate buffer and twice with a phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2).
  • the gel is stained with Roti-Blue Coomassie staining reagent (Roth, Düsseldorf, D) overnight.
  • Example 7 Conjugation of hydroxyethyl starch derivatives with thio- erythropoietin using a crosslinking compound
  • N-(alpha-maleimidoacetoxy) succinimide ester (AMAS) N-(alpha-maleimidoacetoxy) succinimide ester
  • the experimental result is shown in Fig 5.
  • a successful conjugation is indicated by the migration of the protein band to higher molecular weights.
  • the increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • the experimental result is shown in Fig. 5.
  • a successful conjugation is indicated by the migration of the protein band to higher molecular weights.
  • the increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • Remain- ing AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30 min with the phosphate buffer.
  • the experimental result is shown in Fig. 5.
  • a successful conjugation is indicated by the migration of the protein band to higher molecular weights.
  • the increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • Example 7.6 Reaction of thio-erythropoietin with the reaction product of example 1.3 and the crosslinking compound To 50 nmol HES derivate as produced according to example 1.3, at incubation conditions of 80 °C and 17 h as well as of 25 °C and 3 d, and dissolved in 200 ⁇ l of a 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 ⁇ l of a solution of 2.5 ⁇ mol AMAS (Sigma Aldrich, Tauf Wegn, D) in DMSO were added. The clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C.
  • a 0.1 M sodium phosphate buffer 0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2
  • AMAS Sigma Aldrich, Taufmün, D
  • Remaining AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30 min with the phosphate buffer.
  • the experimental result is shown in Fig 5.
  • a successful conjugation is indicated by the migration of the protein band to higher molecular weights.
  • the increased band- width is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • the experimental result is shown in Fig 6.
  • a successful conjugation is indicated by the migration of the protein band to higher molecular weights.
  • the increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • the experimental result is shown in Fig 6.
  • a successful conjugation is indicated by the migration of the protein band to higher molecular weights.
  • the increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • Example 7.10 Reaction of thio-erythropoietin with the reaction product of example 3.4 and the crosslinking compound To 50 nmol HES derivate, produced according to Example 3.4 and dissolved in 200 ⁇ l phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 ⁇ l of a solution of 2.5 ⁇ mol AMAS (Sigma Aldrich, Tauf Wegn, D) in DMSO was added, and the clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C.
  • phosphate buffer 0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2
  • the AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, Germany) at 13,000 rpm and washing 4 times for 30 min with the phosphate buffer.
  • the experimental result is shown in Fig 6.
  • a successful conjugation is indicated by the migration of the protein band to higher molecular weights.
  • the increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • the experimental result is shown in Fig 6.
  • a successful conjugation is indicated by the migration of the protein band to higher molecular weights.
  • the increased band- width is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • the experimental result is shown in Fig 6.
  • a successful conjugation is indicated by the migration of the protein band to higher molecular weights.
  • the increased band- width is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • HES-EPO conjugates were synthesized by coupling of HES derivatives (average mw of 18,000 Dalton; hydroxyethyl substitution degree of 0.4) to the partially (mild periodate) oxidized sialic acid residues on the oligosaccharide chains of recombinant human EPO. Based on carbohydrate structural analysis the modifications introduced did not affect the structural integrity of the core oligosaccharide chains since MALDI/TOF-MS of the mild acid treated HES-modified glycans revealed intact neutral N-acetyllactosamine-type chains which were indistinguishable from those observed in unmodified EPO product.
  • the results obtained indicate that at least 3 modified HES-residues are attached per EPO molecule in the case of the EPO preparation which was subjected to modification without prior partial sialic acid removal.
  • An EPO variant lacking about 50% of the sialic acid residues of the former protein showed a similar apparent high molecular weight mobility in SDS-PAGE (60-110 KDa vs 40 KDa for the BRP EPO standard).
  • the HES modified EPO is stable under standard ion-exchange chromatography conditions at room temperature at pH 3-10.
  • the EPO-bioassay in the normocythaemic mouse system indicates that the HES- modified EPO has 2.5-3.0 fold higher specific activity (IU/mg) in this assay when compared to the International BRP EPO reference standard based on protein determination using the UN absorption value from the European Pharmacopeia and an RP-HPLC EPO protein determination method calibrated against the BRP EPO standard preparation.
  • the oligosaccharides in the pooled supematants were dried in a vacuum centrifuge (Speed Vac concentrator, Savant Instruments Inc., USA).
  • the glycan samples were desalted using Hypercarb cartridges (25 mg or 100 mg of HyperCarb) as follows prior to use: the columns were washed with 3 x 500 ⁇ l of 80% acetonitrile (v/v) in 0.1% TFA followed by washes with 3 x 500 ⁇ l of water.
  • the samples were diluted with water to a final volume of 300 ⁇ l — 600 ⁇ l before loading onto the cartridge which then was rigorously washed with water.
  • Oligosaccharides were eluted with 1.2 ml (25 mg cartridges; 1.8 ml in the case of 100 mg cartridges) 25% acetonitrile in water containing 0.1% trifluoroacetic acid (v/v).
  • the eluted oligosaccharides were neutralized with 2 M NH OH and were dried in a Speed Vac concentrator.
  • desalting of N-glycosidase released oligosaccharides was performed by adsorption of the digestion mixture from samples ⁇ 100 ⁇ g of total (glyco)protein onto 100 mg Hypercarb cartridges.
  • a Broker ULTRAFLEX time-of-flight (TOF/TOF) instrument was used: native desialylated oligosaccharides were analyzed using 2,5-dihydroxybenzoic acid as UN- absorbing material in the positive as well as in the negative ion mode using the re- flectron in both cases.
  • selected parent ions were subjected to laser induced dissociation (LID) and the resulting fragment ions separated by the second TOF stage (LIFT) of the instrument.
  • Sample solutions of 1 ⁇ l and an ap- proximate concentration of 1-10 p ol- ⁇ l '1 were mixed with equal amounts of the respective matrix. This mixture was spotted onto a stainless steel target and dried at room temperature before analysis.
  • EPO was expressed from recombinant CHO cells as described (Mueller PP et al., 1999, Dorner AJ et al., 1984) and the preparations were characterized according to methods described in the Eur. Phar. (Ph. Eur. 4, Monography 01/2002:1316: Erythropoietin concentrated solution).
  • the final product had a sialic acid content of 12 nMol (+/- 1.5 nMol) per nMol of protein.
  • the structures of N-linked oligosaccharides were determined by HPAEC-PAD and by MALDI/TOF-MS as described (Nimtz et al., 1999, Grabenhorst, 1999).
  • the EPO preparations that were obtained contained di-, tri- and tetrasialylated oligosaccharides (2-12%, 15-28% and 60-80%, respectively, sulphated and pentasialylated chains were present in small amounts).
  • the overall glycosylation characteristics of EPO preparations were similar to that of the international BRP EPO standard preparation.
  • the isoelectric focusing pattern of the recombinant EPO was comparable to that of the international BRP Reference EPO standard preparation showing the corresponding isoforms. 25% of the EPO protein lacked O-glycosylation at Ser 1 6 of the polypeptide chain.
  • EPO GT-1 protein (2.84 mg/ml) was heated to 80°C in 20 mM Na-phosphate buffer pH 7.0 and then 100 ⁇ l of 1 N H SO was added per 1 ml of the EPO solution; incubation was continued for 5 min, 10 min and 60 min, respectively, yielding EPO preparations of different degree of sialylation. Quantitation of oligosaccharides with 0-4 sialic acids was performed after liberation of oligosaccharides with polypeptide N-glycosidase and isolation of N-linked chains was performed by desalting using Hypercarb cartridges (25 mg HyperSep Hypercarb; ThermoHypersil-Keystone, UK). EPO preparations were neutralized by addition of 1 N NaOH and were frozen in liquid N 2 and were stored at -20°C until further use.
  • the partially oxidized EPO forms were separated from reagents by desalting using VIVASPIN concentrators (10,000 MWCO, PES Vivascience AG, Hannover, Germany) according to manufacturer's recommendation at 3000 rpm in a laboratory cen- trifuge equipped with a fixed angle rotor. After freezing in liquid mtrogen the EPO preparations were stored in a final volume of 4 ml at -20°C.
  • oligosaccharides were isolated using Hypercarb cartridges as described. Oligosaccharides were desialylated by mild acid treatment and were analyzed by HP AEC-P AD and their retention times were compared to those of authentic standard oligosaccharides as described (Nimtz et al., 1990 and 1993).
  • EPO-GT-1 5 mg was incubated in 5 ml of 0.1 M Tris/HCl buffer pH 8.1 in the presence of 30 mM dithioerythreitol (DTT) at 37°C for 60 minutes; removal of DTT was achieved by using a Vivaspin concentrator at 4 °C, 4 cycles of buffer exchange.
  • DTT dithioerythreitol
  • EPO protein determination Quantitative determination of EPO protein was performed by measuring UV absorption at 280 nm according to the Eur. Phar. (European Pharmacopeia 4, Monography 01/2002: 1316: erythropoietin concentrated solution) in a cuvette with 1 cm path length.
  • EPO was quantitated by applying a RP-HPLC method using a RP-C4 column (Vydac Protein C4, Cat 214TP5410, Grace Nydac, Ca, US); the HPLC method was calibrated using the erythropoietin BRP 1 reference standard (European Pharmacopeia, Riverside de l'Europe B.P. 907-F67029, France Cedex
  • EPO incubation mixtures (approximately 5 mg of EPO protein) were diluted 1:10 with buffer A (20 mM ⁇ -morpholine propane sulfonic acid [MOPS/ ⁇ aOH] in H 2 O bidest, pH 8.0) and were applied to a column containing 3 ml Q-Sepharose HP (Pharmacia Code no. 17-1014-03, Lot no. 220211) equilibrated with 10 column volumes (CN) of buffer A by using a flow rate of 0.5 ml/min. The column was.
  • buffer A 20 mM ⁇ -morpholine propane sulfonic acid [MOPS/ ⁇ aOH] in H 2 O bidest, pH 8.0
  • buffer A 20 mM ⁇ -morpholine propane sulfonic acid [MOPS/ ⁇ aOH] in H 2 O bidest, pH 8.0
  • Q-Sepharose HP Pharmacia Code no. 17-1014-03, Lot no. 220211
  • buffer B 20 mM morpholine ethane sulfonic acid [MES/ ⁇ aOH], 0.5 M ⁇ aCl in H 2 O bidest, pH 6.5
  • EPO was detected by UV absorption at 280 nm and eluted in about 6 ml.
  • Buffer exchange of EPO eluates obtained from the Q-Sepharose step was performed using Vivaspin concentrators and phosphate buffered saline (PBS) with each 3 centrifugation cycles per sample; samples were adjusted to 2 ml with PBS and were stored at -20°C.
  • PBS phosphate buffered saline
  • oligosaccharides were analyzed by high-pH anion- exchange (HPAE) chromatography using a Dionex BioLC system (Dionex, USA) equipped with a CarboPac PAl column (0.4 x 25 cm) in combination with a pulsed amperometric detector (PAD) (Schr ⁇ ter et al., 1999; Ni tz et al., 1999).
  • Detector potentials (E) and pulse durations (T) were: El: +50 mV, TI: 480 ms; E2: +500 mV,
  • solvent C 0.1 M NaOH in bidistilled H 2 O
  • elution flow rate: 1 ml-min "1
  • Solvent D consisted of 0.6 M NaAc in solvent C.
  • Monosaccharides were identified by their retention time and characteristic fragmentation pattern. The uncorrected results of electronic peak integration were used for quantification. Monosaccharides yielding more than one peak due to anomericity and/or the presence of furanoid and pyranoid forms were quantified by adding all major peaks. 0.5 ⁇ g of myo-inositol was used as an internal standard compound.
  • EPO-GT-1 preparations subjected to mild acid treatment for 5, 10 or 60 min. were analyzed by SDS-PAGE before and after liberation of ⁇ -linked oligosaccharides by incubation with ⁇ -glycosidase as shown in Figure 7.
  • ⁇ -linked. oligosaccharides were subjected to HP AEC-P AD oligosaccharide mapping ( Figure 8).
  • the untreated EPO- GT-1 contained >90% of ⁇ -linked oligosaccharides with 3 or 4 sialic acid residues whereas after 5 min. of incubation in the presence of mild acid ⁇ 40% of carbohydrate chains had 3 or 4 sialic acid residues.
  • HP AEC-P AD of the desialylated ⁇ -glycans revealed that the ratio of neutral oligosaccharides that were detected for the untreated EPO-GT-1 and remained stable in the preparations subjected to acid treatment for 5, 10 or 60 min.
  • MALDI/TOF-MS of the desialylated glycans revealed that ⁇ 90% of the proximal fucose was present after mild acid treatment of the protein.
  • hydroxylamine-modified HES derivative X 400 ⁇ g was added to 20 ⁇ g of EPO- GT-1 -A (mild periodate oxidized EPO, not acid hydrolyzed prior to mild periodate oxidation) in 20 ⁇ of 0.5 M NaOAc buffer pH 5.5 and the reaction was stopped after 30 min, 2, 4, and 17 hours, respectively, by freezing samples in liquid nitrogen. Subsequently samples were stored at -20°C until further analysis.
  • HES-EPO conjugates I originating from EPO-GT-1 after mild periodate oxidation, i.e. from EPO-GT-1-A
  • II resulting from EPO-GT-1 subjected to 5 min acid hydrolysis and mild periodate oxidation
  • III resulting from EPO-GT-1 subjected to 10 min acid hydrolysis and mild periodate oxidation
  • K was included containing unmodified EPO-GT-1 under the same buffer conditions to which an equivalent amount of unmodified HES was added. The incubation mixtures were subjected to further purification for subsequent biochemical analysis of the HES-EPO derivatives.
  • HES-modified EPO sample A and K were compared to periodate oxidized form EPO-GT-1 -A.
  • the samples were subjected to N-glycosidase treatment and as is depicted in Figures 14a and 14b the release of N-glycans resulted in the two low molecular weight bands at the position of the O-glycosylated and nonglycosylated EPO forms of the standard EPO preparation.
  • oligosaccharide fractions from the RP-C18 step of ⁇ - glycosidase-treated sample A, EPO GT-1 -A and sample K were neutralized and subjected to desalting using Hypercarb cartridges as described before.
  • the isolated oligosaccharides were subjected to HP AEC-P AD mapping before (see Figures 15) and after mild acid treatment under conditions which enabled quantitative removal of sialic acids from glycans (see Figures 16).
  • the HP AEC-P AD profile for the native material obtained from the HES-modified sample A showed only neglectable signals for oligosaccharides whereas EPO GT-1- A-derived oligosaccharides exhibited the same glycan profile as the one shown hi Fig. 11 (sample named EPO-GT-1 after mild periodate treatment).
  • the elution pro- file of oligosaccharides obtained from the control EPO sample ( ) yielded the expected pattern (compare profile in Figure 8).
  • the native oligosaccharide profile of the international BRP -EPO standard is included for comparison and as reference standard.
  • the EPO-bioassay in the normocythaemic mouse system indicates was performed according to the procedures described in the European Pharmacopeia; the laboratory that carried out the EPO assay was using the International BRP EPO reference standard preparation.
  • the HES-modified EPO A2 preparation a mean value for the specific activity of 294,600 units per mg EPO of protein was determined indicating an approximately 3-fold higher specific activity when compared to the International BRP EPO reference standard preparation that was included in the samples sent for activity assays.
  • Recombinant EPO was produced in CHO cells as follows:
  • a plasmid harbouring the human EPO cDNA was cloned into the eukaryotic expres- sion vector (pCR3 and named afterwards pCREPO).
  • Site directed mutagenesis was performed using standard procedures as described (Grabenhorst, Nimtz, Costa et al., 1998, In vivo specificity of human alpha 1,3/4-fucosyltransferases III-NII in the biosynthesis of Lewis(x) and sialyl Lewis(x) motifs on complex-type ⁇ -glycans - Coexpression studies from BHK-21 cells together with human beta-trace protein, J. Biol. Chem., 273(47), 30985-30994).
  • CHO cells stably expressing human EPO or amino acid variants e.g. Cys- 29->Ser/Ala, or Cys-33- ⁇ Ser/Ala , Ser-126- Ala etc.
  • human EPO or amino acid variants e.g. Cys- 29->Ser/Ala, or Cys-33- ⁇ Ser/Ala , Ser-126- Ala etc.
  • G418-sulfate as de- scribed
  • EPO was produced from stable subclones in spinner flasks or in 21 perfusion reactors. Different glycoforms of EPO with different amounts of ⁇ euAc (e.g. 2-8, 4-10, 8-12 ⁇ euAc residues) were isolated according to published protocols using combinations various chromatographic procedures as described below.
  • Recombinant human EPO was produced from insect cell lines SF9 and SF 21 after infection of cells with recombinant baculovirus vector containing the human EPO cDNA under control of the polyhedrin promoter as described in the literature.
  • EPO was purified by Blue sepharose chromatography, ion-exchange chromatography on Q-Sepharose and finally RP-HPLC on C -Phase.
  • Oxo-HES12KD (Fresenius German Patent DE 196 28 705 Al) were dissolved in 0.3 mL absolute dimethyl sulfoxide (DMSO) and were added dropwise under nitrogen to a mixture of 34 mg (0.15 mmol) EMCH (Perbio Science, GmbH, Bonn, Germany) in 1.5 mL DMSO. After stirring for 19 h at 60°C the reaction mixture was added to 16 mL of a 1:1 mixture of ethanol and acetone. The precipitate was collected by centrifugation, redissolved in 3 mL DMSO and again precipitated as described. The SH-reactiv-HES12KD B was obtained by centrifugation and drying in vaccuo. The conjugation reaction with Thio-EPO is described in Example 11, 2.2.
  • cross-linkers In this reaction, all cross-linkers can be used, which exhibit a hydrazide- and a maleimide function, separated by a spacer. Further examples for molecules of that group, available from Perbio Science, Kunststoff GmbH, Bonn, Germany, are shown in Table 1; marked with an "A". Furthermore, another group of cross-linkers exhibiting an activated disulfide function instead of a maleimide fimcion could also be used.
  • HES12KD A 1 mg sample of HES12KD was dissolved in 3 mL of saturated ammonium bicarbonate. Additional solid ammonium bicarbonate was then added to maintain saturation of the solution during incubation for 120 h at 30°C. The Amino-HES12KD C was desalted by direct lyophilization of the reaction mixture.
  • acylation of amino groups other activated forms of halogen acidic acids can be used, e.g.
  • esters e.g. N-hydroxysuccinimide ester, esters with substituted phenoles (p- nitrophenole, pentafluorophenole, trichlorophenole etc)
  • cross-linkers having an amino reactive group and a halogen ace- tyl function, separated by a spacer, could be used.
  • An example thereof is SBAP. This molecule and others are available from Perbio Science GmbH,
  • Oxo-HES 12KD 1.44 g (0.12 mmol) of Oxo-HES 12KD were dissolved in 3 mL dry dimethyl sulfoxide (DMSO) and were added dropwise under nitrogen to a mixture of 1.51 mL (15 mmol) 1,4-diaminobutane in 15 mL DMSO. After stirring for 19 h at 40°C the reaction mixture was added to 160 mL of a 1 :1 mixture of ethanol and acetone.
  • DMSO dry dimethyl sulfoxide
  • the precipitate Amino-HES 12KD E was collected by centrifugation, redissolved in 40 mL of water an dialysed for 4 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science GmbH, Bonn, Germany) and lyophilized.
  • Chloroacetamide-HES12KD FI was prepared as described for Chloroaceta- mide-HES12KD DI in 1.3 above.
  • the precipitated product J was collected by centrifugation, redissolved in 40 mL of water and dialysed for 2 days against a 0.5 % (v/v) triethylamine in water solution and for 2 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Per- bio Science GmbH, Bonn, Germany) and lyophilized.
  • the conjugation reaction with oxidised Glyco-EPO is described in Example 12, 2.2.
  • derivatives can be used, wherein 2 hydrazid groups are separated by any spacer.
  • O-[2-(2-aminooxy-ethoxy)-ethyl] -hydroxylamine was synthesized as described by Boturyn et al in 2 steps from commercially available materials. 5 1,44 g (0.12 mmol) of Oxo-HES 12KD were dissolved in 3 mL absolute dimethyl sulfoxide (DMSO) and were added dropwise under nitrogen to a mixture of 2.04 g (15 mmol) O-[2-(2- aminooxy-ethoxy)-ethyl]-hydroxylamine in 15 mL DMSO. After stirring for 48 h at 65°C the reaction mixture was added to 160 mL of a 1 :1 mixture of ethanol and acetone.
  • DMSO dimethyl sulfoxide
  • the precipitated product K was collected by centrifugation, redissolved in 40 mL of water and dialysed for 4 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science GmbH, Bonn, Germany) and lyophilized.
  • the conjugation reaction with oxidised Glyco-EPO is described in Example 12, 3.1.
  • the precipitated product M was collected by centrifugation, redissolved in 40 mL of water and dialysed for 2 days against a 0.5 % (v/v) triethylamine in water solution and for 2 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science GmbH, Bonn, Germany) and lyophilized.
  • the conjugation reaction with oxidised Glyco-EPO is described in Example 12, 2.1.
  • Derivatives could be used, wherein the amino group and the thio-function are separated by any spacer. Furthermore, the amino group in the derivatives could be replaced by a hydrazine, a hydrazid or a hydroxylamine.
  • the thio-function could be protected in the form of e.g. a disulfide or a trityl-derivative. However, in this case, a further deprotection step must be preformed before the conjugation, which would release a component being analogous to M.
  • reaction mixture was added to 160 mL of a 1 :1 mixture of ethanol and acetone.
  • the precipitated product N was collected by centrifugation, redissolved in 40 mL of water and dialysed for 2 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science GmbH, Bonn, Germany) and lyophilized.
  • the deprotection was performed in a 50 mM sodium phosphate buffer, containing 25 mM EDTA and 0.5M hydroxylamine, pH7.5 for 2 hours at room temperature and the product O was purified by dialysis against a 0.1 M sodium acetate buffer pH 5.5, containing 1 mM EDTA. The deprotection reaction was performed immediately before the conjugation reaction which is de-
  • the deprotection was performed in a solution of 12 mg dithiothreitol (DTT) per 0.5 mL 100 mM sodiumacetate buffer, containing 100 mM sodium chloride at pH 4.5 for 30 min at room temperature and the product Q was purified by dialysis against a 0.1 M sodium acetate buffer pH 5.5, containing 1 mM EDTA.
  • DTT dithiothreitol
  • thio-HES derivatives in a protected form may be useful.
  • SATA being a further member of this group
  • SPDP could have an acitve ester-function and a disulfide-function, separated by any spacer.
  • Further members of these groups are found in Table 1, marked with an "F”.
  • Further analogous derivatives could have an active ester-function and a thiol-fimction, protected as a trityl derivative, separated by any spacer.
  • Composition was 50 mM sodium borate, pH 8.3, 5 mM EDTA.
  • PBS phosphate buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4.
  • SIA solution 100 ⁇ L SIA solution was added to 400 ⁇ L of the aminoHES 12KD E solution and was allowed to react with agitation for 0.5 hours at room temperature.
  • the excess crosslinker was removed by centrifuging the sample at 14000 x g for 60 minutes using a microconcentrator. After centrifuging the sample was brought up to its o- riginal volume in borate buffer and this process was repeated two more times.
  • the residual solution was added to 1 mL of ThioEPO solution and the reaction mixture was incubated for 16 hour at room temperature. Reactivity of the excess iodoacetamide was quenched at the end of the incubation period by the addition of cysteine to a final concentration of 10 mM.
  • reaction mixture was applied to a desalting column equilibrated with PBS buffer and the protein content of the fractions were monitored with a Coomassie protein assay reagent. All fractions containing the protein conjugate were pooled and the the conjugate was obtained by lyophylisation after dialysis against water over night.
  • cross-linkers which have a succinimide- or a sulfosuccinimide function and a iodoacetamide function separated by a spacer.
  • A. Phosphate buffer. Composition was 100 mM sodium phosphate, pH 6.1, 5 mM EDTA.
  • the reaction mixture was applied to a desalting column, equilibrated with PBS buffer.
  • the protein content of the fractions were monitored with a Coomassie protein assay reagent, all fractions containing the protein conjugate were pooled and the the conjugate was obtained by lyophylisation after dialysis against water over night.
  • amino HES12KD (E, H, I) could be linked with a cross-linker via a succinimide- and a bromoacetamid function (see 1.1 above).
  • SBAP is a member of this group of cross-linkers and is found in Table 1, marked with a "D".
  • Microconcentrator Microcon YM-10 (amicon, Milipore GmbH, Eschborn, Germany).
  • PBS phosphate buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4.
  • D. SMCC solution 1 mg SMCC were dissolved in 50 ⁇ L DMSO E. D-SaltTM Dextran Desalting Columns, 2 x 5 mL bed volume (Perbio Science GmbH, Bonn, Germany)
  • Reactivity of the excess maleimide was quenched at the end of the incubation period by the addition of cysteine to a final concentration of 10 mM.
  • the reaction mixture was applied to a desalting column equilibrated with PBS buffer.
  • the protein content of the fractions were monitored with a Coomassie protein assay reagent, all fractions containing the protein conjugate were pooled and the conjugate was obtained by lyophylisation after dialysis against water over night.
  • cross-linkers In this reaction, all cross-linkers could be used which have a succinimide- or a sulfosuccinimide function and a maleimide-function, separated by a spacer. Further examples for this group of molecules, available from Perbio Science GmbH, Bonn, Germany, are found in Table 1, marked with an "E”. There is a fiirther group of cross-linkers, which have instead of a maleimide fiinc- tion an activated disulfide function. These cross-linkers could also be used for the conjugation. However, the disulfide bond of the conjugate is cleavable under reductive conditions. Members of this group are marked in Table 1 with a "F”. A third group of cross-linkers uses instead of a maleimide function a vinylsulfon function as a SH-reactive group. A member of this group "SNSB" is marked in Table 1 with a "G”.
  • Glyco-EPO solution 10 mg/mL of Glyco-EPO in acetate buffer
  • Sodium meta-periodate solution 10 mM or 100 mM sodium periodate in acetate buffer, prepared fresh. Keep in dark. Using these solutions, the final concentration of sodium periodate in the oxidation mixture is 1 mM or 10 mM, respec- tively.
  • Microconcentrator Microcon YM-3 (amicon, Milipore GmbH, Eschborn, Germany)
  • M 2 C 2 H stock 10 mg/mL M 2 C 2 H in DMSO, prepared fresh
  • Acetate buffer 0.1 M sodium acetate buffer, pH 5.5
  • Phosphate/NaCI 0.1 M sodium phosphate, 50 mM NaCl, pH 7.0
  • Microconcentrator Microcon YM-3 (amicon, Milipore GmbH, Eschborn, Germany)
  • Gel filtration column for example, Sephadex® G-200 (1.5 x 45 cm)
  • M 2 C 2 H stock solution was added to 1 mL of oxidized Glyco-EPO to a final con- centration of 1 mM and was allowed to react with agitation for 2 hours at room temperature.
  • the excess crosslinker was removed by centrifuging the sample at .14000 x g for 60 minutes using a microconcentrator..After centrifuging the sample was brought up to its original volume in phosphate/NaCI buffer and this process was repeated two more times.
  • O or Q solution was added and the reaction mixture was incubated for 16 hours at room temperature.
  • Reactivity of the excess maleimides was quenched at the end of the incubation period by the addition of cysteine.
  • the reaction mixture was applied to Sephadex® G-200 (1.5 x 45 cm) equilibrated with PBS and 1 mL fractions were collected.
  • the protein content of the fractions were monitored with a Coomassie protein assay reagent, all fractions containing the protein conjugate were pooled and the conjugate was obtained by lyophylisation after dialysis against water over night.
  • the hydrazone adduct is slightly less stable at extremes of pH. For applications that may involve treatment at low pH, we reduced the hydrazone by treatment with 30 mM sodium cyanoborohydride in PBS buffer to a hydrazine. For most applications, this extra step was unnecessary.
  • Acetate buffer 0.1 M sodium acetate buffer, pH 5.5
  • PBS phosphate buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4
  • the hydrazone adduct is slightly less stable at extremes of pH. For applications that may involve treatment at low pH, we reduced the hydrazone by treatment with 30 mM sodium cyanoborohydride in PBS buffer to a hydrazine. For most applications, this extra step was unnecessary.
  • PBS phosphate buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4
  • EPO or partially desialylated EPO forms were incubated with galactose oxidase in the presence of catalase at 37°C from 30 min - 4 hours at 37°C in 0.05 M Na-phosphate buffer pH 7.0. Progress of the reaction was monitored by removal of 50 ⁇ g aliquots of the EPO and subsequent treatment of the protein with polypeptide N-glycanase.
  • HAS modified EPO forms from nonreacted EPO and HAS-precursor molecules was achieved by gel filtration using e.g. Ultrogel AcA 44 / 54 or similar gel filtration media.
  • nonreacted HAS was removed by immuno affinity
  • HAS modified EPOs were identified by SDS-PAGE analysis (using 12.5 or 10% acrylamide gels) through detection of their higher molecular weight compared to unmodified EPO upon staining of gels with Coomassie Brillant Blue.
  • the higher molecular weight of FIAS modified EPO polypeptides was also identified by Western Blot analysis of samples using a polyclonal antibody raised against recombinant human EPO.
  • N-glycan modification of EPO forms was demonstrated by their successful removal from the EPO protein with polypeptide N-glycanase (recombinant N-glycosidase from Roche, Germany employing 25 units / mg EPO protein at 37°C for 16 hours); analysis by SDS-PAGE resulted in a typical shift of the EPO protein to a migration position of the N-glycosidase treated unmodified EPO of approximately 20 KDa.
  • Modification of the single desialylated and glacatose oxidase treated EPO Q-glycan at Ser 126 was demonstrated by SDS-PAGE migration of the de-N-glycosylated product by detection of its migration position compared to nonreacted de-N- glycosylated EPO. If required, modified EPO was fractionated by RP-HPLC on a C8-phase before SDS-PAGE analysis. FIAS O-glycan modification of EPO was also analysed by ⁇ -elimination of the O-glycan and detection of the de-O-glycosylated form of EPO in Western blots using a polyclonal antibody raised against recombinant human EPO.
  • EPO and modified EPO forms EPO forms where quantitated by UV measurements as described in Ph.Eur (2000, Erythropoietini solutio concentrata, 1316, 780-785) and compared to the international BRP reference EPO standard.
  • EPO concentrations were determined by a RP-HPLC assay using a RP-C4-column and absorption at 254 nm em- ploying 20, 40 , 80 and 120 ⁇ g of the BRP standard EPO reference preparation for calibration.
  • Anemia was induced in NMRI mice by treatment with phenylhydrazine hydrochloride and spleen cells were collected and used as described in [Fibi et al, 1991, Blood, 77, 1203 ff.]. Dilutions of EPO were incubated with 3x10 5 cells/well in 96-well mi- crotiter plates. After 24 hours at 37° C in a humified atmosphere (5% CO 2 ) cells were labelled for 4 hours with 1 ⁇ Ci of 3 H-thymidine per well. Incorporated radioactivity was determined by liquid scintillation counting. The International reference EPO standard (BRP-standard) was used for comparison .
  • EPO bioactivity was measured by an in vitro assay using the EPO- sensitive cell line TF-1 (Kitamura et. al., [J. cell Phys., 140. 323-334]. Exponentially, growing cells were washed free of growth factors and were incubated in the presence of serial dilutions of the EPO for further 48 hours. Proliferation of the cells was assessed by using the MTT reduction assay as described by Mosmann [Mosman, 1983, J.Immunol. Methods, 65, 55-63],
  • In vivo activity determinations were perfonned in normocythemic mice by measuring the increase of reticulocytes after 4 days after animals received the foreseen dose of EPO or modified EPO forms. Assays were performed using the BRP EPO standard which was calibrated against the WHO EPO standard in the polycythemic mouse assay. EPO samples were diluted in phosphate buffered saline containing 1 mg/ml of bovine serum albumin (Sigma).
  • EPO test solution 0.5 ml of the EPO test solution in Dulbecco's buffered saline (corresponding to an EPO protein equivalent of a 100, 80, 40 or 20 IU/ml of the BRP standard EPO) were infected subcutaneously per animal. Blood samples were taken after 4 days after injection and reticulocytes were stained with acridine orange; quantitation of reticulo- cytes was performed by flow-cytometry by coimting a total of 30,000 blood cells within 5 hours after the blood sample was taken (see Ph. Eur, 2000, Erytliropoietini solutio concentrata, 1316, pages 780-785) and European Pharmacopoeia (1996/2000, attachment 2002).
  • Rabbits were injected intravenously with specified amounts of unmodified or HAS- modified EPO forms. Blood samples were obtained at specified times, and serum was prepared. Serum erythropoietin levels were determined by in vitro bioassay or by an EPO-specific commercial ELISA.
  • mice Each animal received 300 IU EPO/kg subcutaneously. Seven days after the post-treatment hematocrit of each animal was determined. A substantial increase in hematocrit was observed 9in all animals treated with modified EPO, an expected result in view o the relatively short half- life of untreated EPO. The mean change in hematocrit of the modified EPO-treated group was significantly different from that of the untreated EPO group and that of the control group.
  • Rabbits were treated with a single dose of unmodified or HAS-modified EPO corresponding to 200 or up to 800 ng/kg body weight. After 2, 6, 16, 24 and 48 hours blood samples were analyzed by using a commercial EPO-specific ELISA for determination of plasma concentrations. Mean plasma EPO concentrations were determined and the average initial half-lives ( ⁇ -phase) and the terminal half-lives ( ⁇ - phase) were calculated from the ELISA values as described: (Zettlmissl et al., 1989, J. Biol. Chem., 264, 21153-21159).
  • Modified IL2 was recovered by gelfiltration on Ultrogel AcA 54. Aliquots of corre- sponding fraction were sterile filtrated and IL2 bioactivity was determined by using the IL2 dependent murine CTLL-2 cell line [Gillis, Ferm, On, and Smith, 1978, J.Immunol., 120, 2027-2032]. Activity was related to the international reference IL2 standard preparation.

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Abstract

The present invention relates to a method of producing a hydroxyalkyl starch derivative, said hydroxyalkyl starch having a structure according to formula (I) comprising reacting - hydroxyalkyl starch o formula (I) at its optionally oxidized reducing end or - a hydroxyalkyl starch derivative, obtainable by reacting hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end with a compound (D), said compound (D) comprising - at least one functional group Z1 capable of being reacted with the optionally oxidized reducing end of the hydroxyalkyl starch, and at least one functional group W, with a compound (L) comprising at least one functional group Z1 capable of being reacted with said hydroxyalkyl starch, or at least one functional group Z2 capable of being reacted with functional group W comprised in said hydroxyalkyl starch derivative, and at least one functional group X capable of being reacted with a functional group Y of a further compound (M), wherein said functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, an acetal group, and a thio group. The present invention further relates to the hydroxyalkyl starch derivatives as such ans a pharmaceutical composition comprising the hydroxyalkyl starch derivatives.

Description

Method of Producing Hydroxyalkyl Starch Derivatives
The present invention relates to hydroxyalkyl starch derivates, particularly hydroxyalkyl starch derivatives obtainable by a process in which hydroxyalkyl starch is reacted with a primary or secondary amino group of a crosslinking compound or with two crosslinking compounds wherein the resulting hydroxaylkyl starch derivative has at least one functional group X which is capable of being reacted with a functional group Y of a further compound and wherein this group Y is an aldehyd group, a keto group, a hemiacetal group, an acetal group, or a thio group. According to an espe- cially preferred embodiment, the present invention relates to hydroxyalkyl starch derivatives obtainable by a process according to which hydroxyalkyl starch is reacted with a primary or secondary amino group of a crosslinking compound, the resulting reaction product optionally being further reacted with a second crosslinking compound, wherein the resulting hydroxaylkyl starch derivative has at least one func- tional group X which is capable of being reacted with a functional group Y of a further compound and wherein this group Y is an aldehyd group, a keto group, a hemiacetal group, an acetal group, or a thio group, and the resulting reaction product is reacted with a polypeptide, preferably with a polypeptide such as AT III, IFN-beta or erythropoietin and especially preferably with erythropoietin, which comprises at least one of these functional groups Y. A hydroxyalkyl starch which is especially preferred is hydroxyethyl starch. According to the present invention, the hydroxyalkyl starch and preferably the hydroxyl ethyl starch is reacted with the linker compound at its reducing end which is optionally oxidized prior to said reaction.
Hydroxyethyl starch (HES) is a derivative of naturally occurring amylopectin and is degraded by alpha-amylase in the body. HES is a substituted derivative of the carbohydrate polymer amylopectin, which is present in corn starch at a concentration of up to 95 % by weight. HES exhibits advantageous biological properties and is used as a blood volume replacement agent and in hemodilution therapy in the clinics (Som- mermeyer et al, 1987, Krankenhauspharmazie, 8(8), 271-278; and Weidler et al., 1991, Arzneim.-Forschung Drug Res., 41, 494-498).
Amylopectin consists of glucose moieties, wherein in the main chain alpha- 1,4- glycosidic bonds are present and at the branching sites alpha-l,6-glycosidic bonds are found. The physical-chemical properties of this molecule are mainly determined by the type of glycosidic bonds. Due to the nicked alpha- 1,4-glycosidic bond, helical structures with about six glucose-monomers per turn are produced. The physico- chemical as well as the biochemical properties of the polymer can be modified via substitution. The introduction of a hydroxyethyl group can be achieved via alkaline hydroxyethylation. By adapting the reaction conditions it is possible to exploit the different reactivity of the respective hydroxy group in the unsubstituted glucose monomer with respect to a hydroxyethylation. Owing to this fact, the skilled person is able to influence the substitution pattern to a limited extent-
Some ways of producing a hydroxyethyl starch derivative are described in the art.
DE 26 16 086 discloses the conjugation of hemoglobin to hydroxyethyl starch wherein, in a first step, a cross-linking agent, e.g. bromocyane, is bound to hydroxyethyl starch and subsequently hemoglobin is linked to the intermediate product.
One important field in which HES is used is the stabilisation of polypeptides which are applied, e.g., to the circulatory system in order to obtain a particular physiologi- cal effect. One specific example of these polypeptides is erythropoietin, an acid gly- coprotein of approximately 34,000 kD which is essential in regulating the level of red blood cells in the circulation.
A well-known problem with the application of polypeptides and enzymes is that these proteins often exhibit an unsatisfactory stability. Especially erythropoietin has a relatively short plasma half live (Spivak and Hogans, 1989, Blood 73, 90; McMahon et al., 1990, Blood 76, 1718). This means that therapeutic plasma levels are rapidly lost and repeated intravenous administrations must be carried out. Furthermore, in certain circumstances an immune response against the peptides is observed.
It is generally accepted that the stability of polypeptides can be improved and the immune response against these polypeptides is reduced when the polypeptides are coupled to polymeric molecules. WO 94/28024 discloses that physiologically active polypeptides modified with polyethyleneglycol (PEG) exhibit reduced immunogenic- ity and antigenicity and circulate in the bloodstream considerably longer than uncon- jugated proteins, i.e. have a longer clearance rate. However, PEG-drug conjugates exhibit several disadvantages, e.g. they do not exhibit a natural structure which can be recognized by elements of in vivo degradation pathways. Therefore, apart from PEG-conjugates, other conjugates and protein polymerates have been produced. A plurality of methods for the cross-linking of different proteins and macromolecules such as polymerase have been described in the literature (see e.g. Wong, Chemistry of protein conjugation and cross-linking, 1993, CR.CS, Inc.).
In summary, there is still a need for further improved polypeptides with improved stability and/or bioactivity. This applies especially to erythropoietin where isoforms with a high degree of sialic acids and therefore high actvity have to be purified from isoforms with a low degree of sialic acids (see EP 0 428 267 Bl). Therefore, it would be highly advantageous if production methods were available which provide highly active polypeptides without requiring extensive purification. Unfortunately, the production of polypeptides in bacteria or insect cells is often difficult, because the polypeptides are often not produced in a properly folded, native confirmation and lack proper glycosylation.
WO 02/08079 A2 discloses compounds comprising a conjugate of an active agent and a hydroxyalkyl starch wherein active agent and hydroxyalykl starch are either linked directly or via a linker compound. As far as the direct linkage is concerned, the reaction of active agent and hydroxyalkyl starch is carried out in an aqueous medium which comprises at least 10 wt.-% of water. No examples are given which are directed to a hydroxyalkyl starch derivative which is linked to a carbonyl group comprised in the active reagent, neither an aldehyd or keto group nor a an acetal or a hemiacetal group.
Consequently, it is an object of the present invention to provide hydroxyalkyl starch derivatives which are capable of forming a chemical linkage to a further compound, e.g. a polypeptide, which comprises, as functional group, a thio group or an aldehyd group, a keto group, a hemiacetal group, or an acetal group. Preferably, the aldehyd group, the keto group, the hemiacetal group, or the acetal group are comprised in a carbohydrate moiety of the further compound.
Therefore, the present invention relates to a method of producing a hydroxyalkyl starch derivative, said hydroxyalkyl starch having a structure according to formula (I)
Figure imgf000005_0001
comprising reacting - hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end or a hydroxyalkyl starch derivative, obtainable by reacting hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end with a compound (D), said compound (D) comprising at least one functional group Z\ capable of being reacted with the option- ally oxidized reducing end of the hydroxyalkyl starch, and at least one functional group W, with a compound (L) comprising at least one functional group Z\ capable of being reacted with said hydroxyalkyl starch, or at least one functional group Z2 capable of being reacted with functional group W comprised in said hydroxyalkyl starch derivative, and at least one functional group X capable of being reacted with a functional group Y of a further compound (M), wherein said functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, an acetal group, or a thio group.
In the context of the present invention, the term "hydroxyalkyl starch" (HAS) refers to a starch derivative which has been substituted by at least one hydroxyalkyl group. Therefore, the term hydroxyalkyl starch as used in the present invention is not limited to compounds where the terminal carbohydrate moiety comprises hydroxyalkyl groups Ri, R2, and or R3 as depicted, for the sake of brevity, in formula (I), but also refers to compounds in which at least one hydroxy group present anywhere, either in the terminal carbohydrate moiety and/or in the remaining part of the starch molecule, HAS', is substituted by a hydroxyalkyl group i, R , or R3.
In this context, the alkyl group may be a linear or branched alkyl group which may be suitably substituted. Preferably, the hydroxyalkyl group contains 1 to 10 carbon atoms, more preferably from 1 to 6 carbon atoms, more preferably from 1 to 4 carbon atoms, and even more preferably 2-4 carbon atoms. "Hydroxyalkyl starch" therefore preferably comprises hydroxyethyl starch, hydroxypropyl starch and hydroxybutyl starch, wherein hydroxyethyl starch and hydroxypropyl starch are particularly preferred.
Hydroxyalkyl starch comprising two or more different hydroxyalkyl groups is also comprised in the present invention..
The at least one hydroxyalkyl group comprised in HAS may contain two or more hydroxy groups. According to a preferred embodiment, the at least one hydroxyalkyl group comprised HAS contains one hydroxy group.
The expression "hydroxyalkyl starch" also includes derivatives wherein the alkyl group is mono- or polysubstituted. In this context, it is preferred that the alkyl group is substituted with a halogen, especially fluorine, or with an aryl group, provided that the HAS remains soluble in water. Furthermore, the terminal hydroxy group a of hydroxyalkyl group may be esterified or etherified.
Furthermore, instead of alkyl, also linear or branched substituted or unsubstituted alkene groups may be used.
Hydroxyalkyl starch is an ether derivative of starch. Besides of said ether derivatives, also other starch derivatives can be used in the context of the present invention. For example, derivatives are useful which comprise esterified hydroxy groups. These derivatives may be, e.g., derivatives of unsubstituted mono- or dicarboxylic acids with 2-12 carbon atoms or of substituted derivatives thereof. Especially useful are derivatives of unsubstituted monocarboxylic acids with 2-6 carbon atoms, especially derivatives of acetic acid. In this context, acetyl starch, butyl starch and propyl starch are preferred.
Furthermore, derivatives of unsubstituted dicarboxylic acids with 2-6 carbon atoms are preferred.
In the case of derivatives of dicarboxylic acids, it is useful that the second carboxy group of the dicarboxylic acid is also esterified. Furthermore, derivatives of monoal- kyl esters of dicarboxylic acids are also suitable in the context of the present invention.
For the substituted mono- or dicarboxylic acids, the substitute groups may be pref- erably the same as mentioned above for substituted alkyl residues.
Techniques, for the esterification of starch are known in the art (see e.g. Klemm D. et al, Comprehensive Cellulose Chemistry NoL 2, 1998, Whiley-NCH, Weinheim, New York, especially chapter 4.4, Esterification of Cellulose (ISBN 3-527-29489-9).
Hydroxyethyl starch (HES) is most preferred for all embodiments of the present invention. Therefore, the present invention also relates to a method as described above wherein the hydroxyalkyl starch is hydroxyethyl starch.
HES is mainly characterized by the molecular weight distribution and the degree of substitution. There are two possibilities of describing the substitution degree:
1. The substitution degree can be described relatively to the portion of substituted glucose monomers with respect to all glucose moieties (DS).
2. The substitution degree can be described as the "molar substitution" (MS), wherein the number of hydroxyethyl groups per glucose moiety are described.
HES solutions are present as polydisperse compositions, wherein each molecule dif- fers from the other with respect to the polymerisation degree, the number and pattern of branching sites, and the substitution pattern. HES is therefore a mixture of compounds with different molecular weight. Consequently, a particular HES solution is determined by average molecular weight with the help of statistical means. In this context, Mn is calculated as the arithmetic mean depending on the number of mole- cules. Alternatively, Mw, the weight mean, represents a unit which depends on the mass of the HES.
In the context of the present invention, hydroxyethyl starch may have a mean molecular weight (weight mean) of from 1 to 300 kDa, wherein a mean molecular weight of from 5 to 100 kDa is more preferred. Hydroxyethyl starch can further exhibit a molar degree of substitution of from 0.1 to 0.8 and a ratio between C2 : C6 substitution in the range of from 2 to 20 with respect to the hydroxyethyl groups.
As far as the residues Ri, R2 and R3 according to formula (I) are concerned there are no specific limitations given that compound (I) remains capable of being reacted with a compound (D) or a compound (L). According to a preferred embodiment, Rls
R2 and R3 are independently hydrogen or a hydroxyalkyl group, a hydroxyaryl group, a hydroxyaralkly group or a hydroxyalkarly group having of from 1 to 10 carbon atoms. Hydrogen and hydroxyalkyl groups having of from 1 to 6 carbon atoms are preferred. The alkyl, aryl, aralkyl and/or alkaryl group may be linear or branched and suitably substituted.
Therefore, the present invention also related to a method as described above wherein Ri, R and R3 are independently hydrogen or a linear or branched hydroxyalkyl group with from 1 to 6 carbon atoms.
Thus, Ri, R2 and R3 may be hydroxyhexyl, hydroxypentyl, hydroxybutyl, hydroxypropyl such as 1 -hydroxypropyl, 2-hydroxypropyl, 3 -hydroxypropyl, 1- hydroxyisopropyl, 2-hydroxyisopropyl, hydroxyethyl such as 1 -hydroxyethyl, 2- hydroxyethyl, or hydroxymethyl. Hydrogen and hydroxyethyl groups are preferred, hydrogen and the 2-hydroxyethyl group being especially preferred.
Therefore, the present invention also relates to a method as described above wherein Ri, R2 and R3 are independently hydrogen or a 2-hydroxyethyl group.
According to the present invention either compound (D) or compound (L) is reacted with the reducing end of the hydroxyalkyl starch via the reaction of the functional group Zi with the reducing end where group Zi is comprised in compound (D) or compound (L).
According to a first preferred embodiment of the present invention, compound (D) or compound (L) is reacted with the reducing end of the hydroxyalkyl starch and where the reducing end is oxidized prior to the reaction.
This oxidation of the reducing end leads to hydroxyalkyl starch in which the terminal carbohydrate group comprises a lactone group, or in which the terminal carbohydrate group, depending of the chemical reaction conditions and/or the oxidizing agents, has a non-cyclic structure comprising a carboxy group. According to one embodiment of the present invention, the hydroxyalkyl starch which is oxidized at its reducing end is present as a mixture of a compound comprising the lactone group and a compound comprising the carboxy group. In the mixture, the respective compounds may be present at any conceivable ratio.
Therefore, the present invention also relates to a method as described above wherein he reducing end of the hydroxyalkyl starch is oxidized prior to the reaction with compound (D) or compound (L), said hydroxyalkyl starch thus having a structure according to formula (Ila)
Figure imgf000010_0001
and/or according to formula (lib)
Figure imgf000010_0002
The oxidation of the reducing end of the hydroxyalkyl starch may be carried out according to each method or combination of methods which result compounds having the above-mentioned structures (Ila) and/or (lib).
Although the oxidation may be carried out according to all suitable method or methods resulting in the oxidized reducing end of hydroxyalkyl starch, it is preferably carried out using an alkaline iodine solution as described, e.g., in 196 28 705 Al. Therefore, the present invention also relates to a method as mentioned above wherein the reducing end is oxidized by an alkaline iodine solution.
According to a second preferred embodiment of the present invention, compound (D) or compound (L) is reacted with the reducing end of the hydroxyalkyl starch and where the reducing end is not oxidized prior to the reaction.
Therefore, the present invention also relates to a method as mentioned above wherein the reducing end of the hydroxyalkyl starch is not oxidized prior to the reaction with compound (D) or compound (L), said hydroxyalkyl starch thus having a structure according to formula (I)
Figure imgf000011_0001
The formation of a chemical linkage between either compound (L) and hydroxyalkyl starch or compound (D) and hydroxyalkyl starch is achieved by reaction of the functional group Zi with the optionally oxidized reducing end of the hydroxyalkyl starch.
As functional group Zi, each functional group may be used which is capable of forming a chemical linkage with the optionally oxidized reducing end of the hydroxyalkyl starch.
According to a preferred embodiment of the present invention, the functional group Zi comprises the chemical structure -NH-. Therefore, the present invention also relates to a method as described above wherein the functional group Zi comprises the structure -NH-.
According to one preferred embodiment of the present invention, the functional group Zi is a group having the structure R'-NH- where R' is hydrogen or a alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue where the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directly to the NH group or, according to another embodiment, may be linked by an oxygen bridge to the NH group. The alkyl, cycloalkyl, aryl, aralkyl, arylcy- cloalkyl, alkaryl, or cycloalkylaryl residues may be suitably substituted. As preferred substituents, halogenes such as F, CI or Br may be mentioned. Especially preferred residues R' are hydrogen, alkyl and alkoxy groups, and even more preferred are hydrogen and unsubstituted alkyl and alkoxy groups.
Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4, 5, or 6 C atoms are preferred. More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups. Especially preferred are methyl, ethyl, methoxy, ethoxy, and particular preference is given to methyl or methoxy.
Therefore, the present invention also relates to a method as described above wherein R' is hydrogen or a methyl or a methoxy group.
According to another preferred embodiment of the present invention, the functional group Zi has the structure R'-NH-R"- where R"" preferably comprises the structure unit -NH- and/or the structure unit -(C=G)- where G is O or S, and/or the structure unit -SO2-. According to more preferred embodiments, the functional group R" is selected from the group consisting of
Figure imgf000012_0001
H H
and G where, if G is present twice, it is independently O or S.
Therefore, the present invention also relates to a method as mentioned above wherein the fuctional group Zi is selected from the group consisting of
H9N— H /O. k /
H2N ^ 2 H
/NL / H2Ns II hLN f N-S—
2 II H II
G O
Figure imgf000013_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl.
It is an object of the present invention to provide a hydroxyalkyl starch derivative which comprises a functional group X which is capable to react with the functional group Y of a further compound (M) to give as reaction product a hydroxyalkyl starch derivative which comprises the hydroxyalkyl starch, compound (L), optionally compound (D), and the further compound.
As to functional group X, there are no specific restrictions provided that a chemical linkage can be formed with the functional group Y which is comprised in the further compound (M).
If the functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group, the functional group X, the functional group X preferably comprises the chemical structure -NH-. Therefore, the present invention also relates to a method as described above wherein the functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group, and the functional group X comprises the structure -NH-.
According to one preferred embodiment of the present invention, the functional group X is a group having the structure R'-NH- where R' is hydrogen or a alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue where the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directly to the NH group or, according to another embodiment, may be linked by an oxygen bridge to the NH group. The alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl, or cycloalkylaryl residues may be suitably substituted. As preferred substituents, halogenes such as F, CI or Br may be mentioned. Especially preferred residues R' are hydrogen, alkyl and alkoxy groups, and even more preferred are hy- drogen and unsubstituted alkyl and alkoxy groups.
Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4, 5, or 6 C atoms are preferred. More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups. Especially preferred are methyl, ethyl, methoxy, eth- oxy, and particular preference is given to methyl or methoxy.
Therefore, the present invention also relates to a method as described above wherein R' is hydrogen or a methyl or a methoxy group.
According to another preferred embodiment of the present invention, the functional group X has the structure R'-NH-R"- where R"" preferably comprises the structure unit -NH- and/or the structure unit -(C=G)- where G is O or S, and/or the structure unit -SO2-. According to more preferred embodiments, the functional group R" is selected from the group consisting of
Figure imgf000014_0001
where, if G is present twice, it is independently O or S.
Therefore, the present invention also relates to a method as mentioned above wherein the fuctional group X is selected from the group consisting of
Figure imgf000015_0002
H
H2NN ?\ H2N" Y N-S— H II G 0
Figure imgf000015_0003
wherein G is O or S and, if present twice, independently O or S, and R is methyl.
If the functional group Y is a thio group, the functional group X is preferably selected from the groups consisting of
Figure imgf000015_0004
wherein Hal is CI, Br or I, preferably Br or I.
Therefore, the present invention also relates to a method as described above where- inwherein the functional group Y is -SH and the functional group X is selected from the group consisting of
Figure imgf000016_0001
wherein Hal is CI, Br or I.
According to one embodiment of the present invention, hydroxyalkyl starch is reacted with a compound (D) and the resulting reaction product is further reacted with compound (L) where the chemical linkage between compound (L) and the reaction product is formed by reaction of functional group Z2 comprised in compound (L) and functional group W comprised in compound (D) being part of the reaction product.
Regarding the functional groups Z2 and W, there are generally no restrictions pro- vided that the desired chemical linkage is formed.
As possible functional groups W or Z2, the following functional groups are to be mentioned, among others:
C-C-double bonds or C-C-triple bonds or aromatic C-C-bonds; - the thio group or the hydroxy groups; alkyl sulfonic acid hydrazide, aryl sulfonic acid hydrazide;
1,2-dioles;
1 ,2-aminoalcohols; the amino group -NH2 or derivatives of the amino groups comprising the struc- ture unit -NH- such as aminoalkyl groups, aminoaryl group, aminoaralkyl groups, or alkarlyaminogroups; the hydroxylamino group -O-NH2, or derivatives of the hydroxylamino group comprising the structure unit -O-NH-, such as hydroxylalkylamino groups, hy- droxylarylamino groups, hydroxylaralkylamino groups, or hydroxalal- karylamino groups; alkoxyamino groups, aryloxyamino groups, aralkyloxyamino groups, or alkary- loxyamino groups, each comprising the structure unit -NH-O-; residues having a carbonyl group, -Q-C(=G)-M, wherein G is O or S, and M is, for example,
~ -OH or -SH; an alkoxy group, an aryloxy group, an aralkyloxy group, or an alkaryloxy group; an alkylthio group, an arylthio group, an aralkylthio group, or an alkaryl- thio group; an alkylcarbonyloxy group, an arylcarbonyloxy group, an aralkylcarbon- yloxy group, an alkarylcarbonyloxy group; — activated esters such as esters of hydroxylamines having imid structure such as N-hydroxysuccinimide or having a structure unit O-N where N is part of a heteroaryl compound or, with G = O and Q absent, such as aryloxy compounds with a substituted aryl residue such as pentafluoro- phenyl, paranitrophenyl or trochlorophenyl; wherein Q is absent or NH or a heteroatom such as S or O;
-NH-NH2, or -NH-NH-; - -NO2; the nitril group; carbonyl groups such as the aldehyde group or the keto group; - the carboxy group; the -N=C=O group or the -N=C=S group; vinyl halide groups such as the vinyl iodide or the vinyl bromide group or tri- ' flate; -C≡C-H; - -(C=NH2Cl)-OAlkyl groups -(C=O)-CH -Hal wherein Hal is CI, Br, or I; -CH=CH-SO2-; a disulfide group comprising the structure -S-S-;
the group
Figure imgf000017_0001
the group
Figure imgf000018_0001
where Z2 and W, respectively, is a group capable of forming a chemical linkage with one of the above-mentioned groups.
According to preferred embodiments of the present invention, both W and Z2 are groups from the list of groups given above.
According to a first especially preferred embodiment of the present invention, Z2 or W is a thio group. In this particular case, the functional group W is preferably selected from the group consisting of
Figure imgf000018_0002
wherein Hal is CI, Br, or I, preferably Br or I.
Therefore, the present invention also relates to a method as described above wherein the functional group W or the functional group Z2 is -SH and the functional group Z2 or the functional group W is selected from the group consisting of
Figure imgf000018_0003
wherein Hal is CI, Br, or I.
According to a second especially preferred embodiment of the present invention, Z2 or W is selected from the group consisting of an activated ester, as described above, or a carboxy group which is optionally transformed into an activated ester. In this particular case, the functional group W or Z2, respectively, comprises the chemical structure -NH-.
Therefore, the present invention also relates to a method as described above wherein Z2 or W is selected from the group consisting of an activated ester, as described above, or a carboxy group which is optionally transformed into an activated ester, and the functional group W or Z2., respectively, comprises the chemical structure - NH-.
According to one preferred embodiment of the present invention, the functional group W or Z2 comprising the structure -NH- is a group having the structure R'-NH- where R' is hydrogen or a alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue where the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directly to the NH group or, according to an- other embodiment, may be linked by an oxygen bridge to the NH group. The alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl, or cycloalkylaryl residues may be suitably substituted. As preferred substituents, halogenes such as F, CI or Br may be mentioned. Especially preferred residues R' are hydrogen, alkyl and alkoxy groups, and even more preferred are hydrogen and unsubstituted alkyl and alkoxy groups.
Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4, 5, or 6 C atoms are preferred. More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups. Especially preferred are methyl, ethyl, methoxy, ethoxy, and particular preference is given to methyl or methoxy.
Therefore, the present invention also relates to a method as described above wherein W or Z2 is selected from the group consisting of an activated ester, as described above, or a carboxy group which is optionally transformed into an activated ester, and the functional group W or Z2, respectively, is R'-NH- wherein R' is hydrogen or a methyl or a methoxy group. According to another preferred embodiment of the present invention, the functional group W or Z2 has the structure R'-NH-R"- where R" preferably comprises the structure unit -NH- and/or the structure unit -(C=G)- where G is O or S, and or the structure unit -SO2-. According to more preferred embodiments, the functional group R" is selected from the group consisting of
Figure imgf000020_0001
H H
and G where, if G is present twice, it is independently O or S.
Therefore, the present invention also relates to a method as mentioned above wherein the functional group W or Z2 is selected from the group consisting of
H9N — H /O. /O^ H M ^ R'^ N
H2N ^ 2 H
Figure imgf000020_0002
wherein G is O or S and, if present twice, independently O or S, and R' is methyl.
According to yet another aspect of the present invention, the at least one functional group X, Z2 and or W may be a group which is not capable of reacting directly with a given -further compound, but which may be chemically modified in order to be capa- ble of reacting in the desired way. As an example of a functional group to be modified prior to the reaction with a further compound, a 1,2-amino alcohol or a 1,2-diol may be mentioned which is modified, e.g., by oxidation to form an aldehyd or a keto group.
Another example for a functional group to be modified prior to the reaction with a further compound is a -NH2 group which is modified by the reaction with, e.g., a compound according to the following formula
Figure imgf000021_0001
to give a structure of the following formula
Figure imgf000021_0002
which is, e.g., reactive towards a thio group.
Another example for a functional group to be modified prior to the reaction with a further compound is a -NH2 group which is modified by the reaction with, e.g., a compound according to the following formula
Figure imgf000021_0003
to give a structure of the following formula
Figure imgf000022_0001
which is, e.g., reactive towards a thio group.
Yet another example for a functional group to be modified prior to the reaction with a further compound is an amino group which is reacted with bromoacetic anhydride or N-succinimidyl iodo acetate.
According to a preferred embodiment of the present invention, a compound (L) has the structure Zi-L'-X or Z2-L'-X, L' being an organic residue separating the functional groups and being optionally absent, the structure depending on whether a compound (D) is reacted with the hydroxyalkyl starch or not.
According to a first preferred embodiment, no compound (D) is involved and Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group.
In this particular case, the following compounds, among others, are preferred as compound (L) having the structure Zi-L'-X where L' is absent:
Figure imgf000022_0002
If, in this particular case, L' is not absent, L' may be a linear or branched alkyl or cycloalkyl or aryl or or aralkyl or arylcycloalkyl or alkaryl or cycloalkylaryl group, wherein L' may comprise at least one heteroatom such as N, O, S, and wherein L' may be suitably substituted. The size of the group L' may be adapted to the specific needs. Generally, the separating group L' generally has from 1 to 60, preferably from 1 to 40, more preferably from 1 to 20, more preferably from 1 to 10, more preferably from 1 to 6 and especially preferably from 1 to 4 carbon atoms. If heteroatoms are present, the separating group comprises generally from 1 to 20, preferably from 1 to 8 and especially preferably from 1 to 4 heteroatoms. According to particularly preferred embodiments of the present invention, the separating group L' comprises 1 to 4 oxygen atoms. The separating group L' may comprise an optionally branched alkyl chain or an aryl group or a cycloalkyl group having, e.g., from 5 to 7 carbon atoms, or be a aralkyl group, an alkaryl group where the alkyl part may be a linear and/or cyclic alkyl group. According to an even more preferred embodiment, the separating group is an alkyl chain of from 1 to 20, preferably from 1 to 8, more preferably from 1 to 6, more preferably from 1 to 4 and especially preferably from 2 to 4 carbon atoms. In case heteroatoms are present, a chain comprising 1 to 4 oxygen atoms is particularly preferred.
As to this particular case where Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group, the following compounds, among others, are preferred as compound (L) having the structure Zi-L'-X where L' is not absent:
Figure imgf000023_0001
Figure imgf000024_0001
XX ,0. H H
N' O' N' .NL H H O' O' O'
According to a second preferred embodiment, a compound (D) is involved.
According to a further preferred embodiment of the present invention, a compound (D) has the structure Zi-D'-W, D' being an organic residue separating the functional groups and being optionally absent.
In this particular case, the following compounds, among others, are preferred as compound (D) having the structure Zi-D'-W where D' is absent:
Figure imgf000024_0002
A specific example of a compound D where D' is absent is NH3.
If, in this particular case, D' is not absent, D' may be a linear or branched alkyl or cycloalkyl or aryl or or aralkyl or arylcycloalkyl or alkaryl or cycloalkylaryl group, wherein D' may comprise at least one heteroatom such as N, O, S, and wherein D' may be suitably substituted. The size of the group D' may be adapted to the specific needs. Generally, the separating group D' generally has from 1 to 60, preferably from 1 to 40, more preferably from 1 to 20, more preferably from 1 to 10, more preferably from 1 to 6 and especially preferably from 1 to 4 carbon atoms. If heteroatoms are present, the separating group comprises generally from 1 to 20, preferably from 1 to 8 and especially preferably from 1 to 4 heteroatoms. According to particularly preferred embodiments of the present invention, the separating group D' comprises 1 to 4 oxygen atoms. The separating group D! may comprise an optionally branched alkyl chain or an aryl group or a cycloalkyl group having, e.g., from 5 to 7 carbon atoms, or be an aralkyl group, an alkaryl group where the alkyl part may be a linear and/or cyclic alkyl group. According to an even more preferred embodiment, the separating group is an alkyl chain of from 1 to 20, preferably from 1 to 8, more preferably from 1 to 6, more preferably from 1 to 4 and especially preferably from 2 to 4 carbon atoms. In case heteroatoms are present, a chain comprising 1 to 4 oxygen atoms is particularly preferred.
As to this particular case, preferred compounds (D) having the structure Zi-D'-W where D' is not absent are:
Figure imgf000025_0001
Figure imgf000026_0001
ONH-
HS' ONH, HS'
Figure imgf000026_0002
\ /O. .0. H H N O' ■ N' ,N. -N- H H O' O'
Figure imgf000026_0003
Depending on the chemical nature of the functional group W comprised in compound (D) and the functional group Y, specific compounds (L) may be used according to the specific needs.
If, e.g., the fimctional group Y is a thio group and the functional group W comprises the structure -NH-, as described above in detail, the following types of compounds (L) are, among others, preferred:
Figure imgf000027_0002
If, e.g., the functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group, and the functional group W is a thio group, the following types of compounds (L) are, among others, preferred:
Figure imgf000027_0003
In Table 1 at the end of the present description, some preferred examples of com- pounds (L) according to the types given above are listed.
The separating groups L' and/or D' may be suitably substituted. Prefeixed substituents are, e.g, halides such as F, CI, Br or I.
The separating groups L' and/or D' may comprise one or more cleavage sites such as
Figure imgf000027_0001
which allow for an easy cleavage of a resulting compound at a pre-determined site.
Especially preferred examples of compounds (L) which may be linked to hydroxyalkyl starch wherein the resulting hydroxyalkyl starch derivative comprises the functional group X capable of being reacted with a functional group Y comprised in a further compoimd (M) and wherein said functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, an acetal group, are
Figure imgf000028_0001
the compounds (L)
Figure imgf000028_0002
being particularly preferred.
Especially preferred examples of compounds (D) which may be linked to hydroxyalkyl starch wherein the resulting hydroxyalkyl starch derivative comprises the functional group W capable of being reacted with a functional group Z comprised in a compound (L) wherein the resulting hydroxyalkyl starch derivative which comprises hydroxyalkyl starch, compound (D) and compound (L), is capable of being reacted with the functional group Y of a further compound (M) and wherein said functional group Y is a thio group, are
Figure imgf000028_0003
Figure imgf000029_0001
the compounds (D)
Figure imgf000029_0002
being particularly preferred.
Together with the above-mentioned preferred compounds (D), the following com- pounds (L)
Figure imgf000029_0003
and are preferced, the compound (L)
Figure imgf000029_0004
being especially preferred.
According to a first preferred embodiment of the present invention, a compound (D) or a compound (L) is reacted with the reducing end of the hydroxyalkyl starch which is not oxidised.
Depending on the reaction conditions such as the solvent or solvent mixture used, the temperature, pressure or pH of the reaction mixture, the reaction product of a compound (D) or a compound (L) is reacted with the reducing end of the hydroxyalkyl starch which is not oxidised may have different constitutions. According to a preferred embodiment of the present invention, this reaction is carried out in an aqueous system.
The term "aqueous system" as used in the context of the present invention refers to a solvent or a a mixture of solvents comprising water in the range of from at least 10 % per weight, preferably at least 50 % per weight, more preferably at least 80 % per weight, even more preferably at least 90 % per weight or up to 100 % per weight, based on the weight of the solvents involved. As additional solvents, solvents such as DMSO, DMF, ethanol or methanol may be mentioned.
If the reaction is carried out in an aqueous system and the functional group Zi is a group R'-NH-, as described above, the hydroxyalkyl starch derivative may have a constitution according to formula (Ilia)
Figure imgf000030_0001
If the reaction is carried out in an aqueous system and the functional group Zi is a group R'-NH- with R' = H, as described above, the hydroxyalkyl starch derivative may have a constitution according to formula (Ilia) or formula (Illb) or be a mixture of compounds according to formulae (Ilia) and (Illb)
Figure imgf000030_0002
Figure imgf000031_0001
Depending on the reaction conditions and/or the chemical nature of compounds (L) or compound (D) used for the reaction, the compounds according to formula (Ilia) may be present with the N atom in equatorial or axial position where also a mixture of both forms may be present having a certain equilibrium distribution.
Depending on the reaction conditions and/or the chemical nature of compounds (L) or (D) used for the reaction, the compounds according to formula (Illb) may be present with the C-N double bond in E or Z conformation where also a mixture of both forms may be present having a certain equilibrium distribution.
Therefore, the present invention also relates to a hydroxyalkyl starch derivative as described above having a constitution according to formula (Illb) or according to fonnula (Illb) or according to formulae (Ilia) and (Illb).
In some cases it may be desirable to stabilize the compound according to formula (Ilia). This is especially the case where the compound according to formula (Ilia) is produced and/or used in an aqueous solution. As stabilizing method, acylation of the compound according to formula (Ilia) is particularly preferred, especially in the case where R' is hydrogen. As acylation reagent, all suitable reagents may be used which result in the desired hydroxyalkyl starch derivative according to formula (INa)
Figure imgf000032_0001
According to especially preferred embodiments of the present invention, the residue Ra being part of the acylation reagent is methyl. As acylation reagents, carboxylic acid anhydrides, carboxylic acid halides, and carboxylic acid active esters are preferably used.
The acylation is carried at a temperature in the range of from 0 to 30 °C, preferably in the range of from 2 to 20 °C and especially preferably in the range of from 4 to 10 °C.
Therefore, the present invention also relates to a hydroxyalkyl starch derivate obtainable by a method as described above wherein said derivative has a constitution according to formula (IV a).
In other cases it may be desirable to stabilize the compound according to formula (Illb). This is especially the case where the compoimd according to fonnula (Illb) is produced and/or used in an aqueous solution. As stabilizing method, reduction of the compound according to formula (Illb) is particularly preferred, especially in the case where R' is hydrogen. As reduction reagent, all suitable reagents may be used which result in the desired hydroxyalkyl starch derivative according to formula (INb) JA
Figure imgf000033_0001
According to especially preferred embodiments of the present invention, as reduction reagents boro hydrides such as ΝaCΝBH3 or NaBH are used.
The reduction is carried at a temperature in the range of from 4 to 100 °C, preferably in the range of from 10 to 90 °C and especially preferably in the range of from 25 to 80 °C.
Therefore, the present invention also relates to a hydroxyalkyl starch derivate obtainable by a method as described above wherein said derivative has a constitution ac- cording to formula (IVb).
The present invention further relates to mixtures of compounds having constitutions according to formulae (ma) and (Illb), (IVa) and (INb), (Ilia) and (IVa), (Ilia) and (IVb), (Illb) and (IVa), (Illb) and (IVb), (Ilia) and (Illb) and (IVa), (Ilia) and (Illb) and (IVb), (IVa) and (IVb) and (Ilia), and (IVa) and (IVb) and (Illb) wherein (Ilia) and/or (IVa) may be independently present in a conformation where the Ν atom .in equatorial or axial position and/or wherein (Illb) may be present with the C-Ν double bond in E ox Z conformation.
According to a second preferred embodiment of the present invention, a compound (D) or a compound (L) is reacted with the reducing end of the hydroxyalkyl starch which is oxidised.
In this case, preferably polar aprotic solvents are used which may also contain a cer- tain amount of water, such as up to 10 wt.-%. Preferred aprotic solvents are, among others, DMSO or DMF. An example of a preferred reaction temperature range is from room 20 to 65 °C, and the reaction times are generally in the range of 1 minute to several hours and up to several days, depending on the chemical nature of the functional group which is reacted with the oxidized reducing end og the hydroxyalkyl starch and the other reaction conditions.
If, in this case, the functional group Zi is a group R-NPI-, as described above, the hydroxyalkyl starch derivative may have a constitution according to formula (Va)
Figure imgf000034_0001
Therefore, the present invention also relates to a hydroxyalkyl starch derivate obtainable by a method as described above wherein said derivative has a constitution according to formula (Va).
As far as the reactions of hydroxyalkyl starch with compound (D) and/or compound (L) as well as compound (M) are concerned, all possible sequences are comprised by the present invention.
A preferred embodiment of the present invention relates to a method as described above wherein hydroxyalkyl starch is reacted with a compoimd (L) via the reaction of functional group Zi with the optionally oxidized reducing end of the hydroxyalkyl starch and the resulting reaction product is reacted with a further compound (M) via the reaction of the functional group X comprised in compound (L) with the fimctional group Y comprised in compound (M).
Another embodiment of the present invention relates to a method as described above wherein hydroxyalkyl starch is reacted with a compoimd (L) via the reaction of func- tional group Zi with the optionally oxidized reducing end of the hydroxyalkyl starch, where compound (L), prior to the reaction with hydroxyalkyl starch, is reacted with a further compound (M) via the reaction of fimctional group X comprised in compound (L) with the functional group Y comprised in compound (M).
Still another embodiment of the present invention relates to a method as described above wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of the functional group Zi comprised in compound (D), with the optionally oxidized reducing end of the hydroxyalkyl starch to give a first hydroxyalkyl starch derivative, and where the first hydroxyalkyl starch derivative is reacted with a compound (L) via the reaction of functional group Z2 comprised in compound (L) with the functional group W comprised in compound (D) to give a second hydroxyalkyl starch derivative.
Yet another embodiment of the present invention relates to the latter method wherein the second hydroxyalkyl starch derivative is reacted with a further compound (M) via the reaction of functional group X comprised in compound (L) with the fimctional group Y comprised in compound (M).
Still yet another embodiment of the present invention relates to a method as described above wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of functional group Zi comprised in compound (D) with the optionally oxidized reducing end of the hydroxyalkyl starch to give a first hydroxyalkyl starch derivative, and where the first hydroxyalkyl starch derivative is reacted, via the reaction of the functional group W, comprised in compound (D), and the functional group Z2, comprised in compound (L), with compoimd (L), where compound (L), prior to the reaction with the first hydroxyalkyl starch derivative, is reacted with a further compound (M) via the reaction of functional group X comprised in compound (L) with the functional group Y comprised in compound (M).
As far as the reaction conditions of each of the above-described reactions steps are concerned, all parameters such as temperature, pressure, pH, or solvent or solvent mixture may be adapted to the specific needs and the chemical nature of compounds to be reacted.
According to an especially preferred embodiment of the present invention, water is used as solvent, either alone or in combination with at least one other solvent. As at least one other solvent, DMSO, DMF, methanol and ethanol may be mentioned. Preferred solvents other than water are DMSO, DMF, methanol and ethanol. In this embodiment, hydroxylalkyl starch is preferably reacted via the non-oxidized reducing end.
If hydroxyalkyl starch is reacted with compound (D) or compound (L) in an aqueous medium and compoimd (D) or compound (L) is a hydroxylamine or a hydrazide, the temperature of the reaction is preferably in the range of from 5 to 45 °C, more preferably in the range of from 10 to 30 °C and especially preferably in the range of from 15 to 25 °C.
If hydroxyalkyl starch is reacted with compound (D) or compound (L) in an aqueous medium and the reaction being a reductive amination, the temperature is preferably in the range of up to 100 °C, more preferably in the range of from 70 to 90 °C and especially preferably in the range of from 75 to 85 °C.
During the course of the reaction the temperature may be varied, preferably in the above-given ranges, or held essentially constant.
The reaction time for the reaction of hydroxyalkyl starch with compound (D) or compound (L) may be adapted to the specific needs and is generally in the range of from 1 h to 7 d.
In case compoimd (D) or compound (L) is a hydroxylamine or a hydrazide, the reac- tion time is preferably in the range of from 1 h to 3 d and more preferably of from 2 h to 48 h. In case the reaction of hydroxyalkyl starch with compound (D) or compound (L) is a reductive amination, the reaction time is preferably in the range of from 2 h to 7 d.
The pH value for the reaction of hydroxyalkyl starch with compound (D) or com- pound (L) may be adapted to the specific needs such as the chemical nature of the reactants.
In case compound (D) or compound (L) is a hydroxylamine or a hydrazide, the pH value is preferably in the range of from 4.5 to 6.5.
In case the reaction of hydroxyalkyl starch with compound (D) or compound (L) is a reductive amination, the pH value is preferably in the range of from 8 to 12.
The suitable pH value of the reaction mixture may be adjusted, for each reaction step, by adding at least one suitable buffer. Among the preferred buffers, sodium acetate buffer, phosphate or borate buffers may be mentioned.
If necessary, the at least one functional group X may be protected with at least one suitable protecting group prior to the reaction of hydroxyalkyl starch with compound (L) or prior to the reaction of compound (D) with compoimd (L) or prior to the reaction of compound (L) with the reaction product of the reaction of hydroxyalkyl starch with compound (D). In this respect, all conceivable protecting groups are possible which prevent the protected compound (L) from reacting via the at least one functional group X. Hence, the protecting group may be chosen depending from the chemical nature of the functional group X to be protected, from, e.g., the solvent the reaction is carried out in or the pH of the reaction mixture. Preferred protecting groups are, among others, the benzyloxycarbonyl group, the tert-butoxycarbonyl group, the methoxyphenyl group, the 2,4-dimethoxyphenyl group, triarly methyl groups, trityl, the monomethoxytrityl group, the dimethoxytrityl group, the mono- methyltrityl group, the dimethyltrityl group, the trifluoracetyl group, phthalimin compounds, 2-(trialkylsilyl)ethoxy carbonyl compounds, Fmoc, the tert-butyl group, or trialkyl silyl groups. If two or more different functional groups X are present in compound (L), at least one group may be protected whereas at least one other group may be left unprotected.
After the reaction of compound (L), the at least one protecting group may be left in the reaction product or removed by suitable methods such as conventional methods known to the person skilled in the art. If two different fimctional groups X are protected by suitable protecting groups, it is possible to remove at least one protecting group so as to make at least one fimctional group X available for fiirther reaction with at least one further compound (M), and leave at least one other functional group protected until the reaction product comprising compound (L) is reacted with the further compound (M). Afterwards, the protecting group of the functional group still protected may be removed to make the remaining fimctional group X available for reaction with yet a fiirther compound (M).
The use of at least one protecting group may be important for preventing the reaction from resulting in a hydroxyalkyl starch derivative comprising a compound (L) or compound (D) which has been reacted with two or more hydroxyalkyl starch mole- cules, i.e. a multiple HAS substituted compoimd (L) or (D). The same result, however, may be achieved by reacting hydroxyalkyl starch with an excess of compound (L) or (D). If an excess amount of compound (L) or (D) is used in the process of the present invention, the molar ratio of compound (L) or (D) to hydroxyalkyl starch is preferably in the range of from 2 to 100.
Once the reaction product of the respective reaction step, as described above, is formed, it may be isolated from the reaction mixture by at least one suitable method. If necessary, the reaction product may be precipitated prior to the isolation by at least one suitable method.
If the reaction product is precipitated first, it is possible, e.g., to contact the reaction mixture with at least one solvent or solvent mixture other than the solvent or solvent mixture present in the reaction mixture at suitable temperatures. According to a particularly preferred embodiment of the present invention where an aqueous system is used as solvent, the reaction mixture is contacted with a mixture of ethanol and acetone, preferably a 1:1 mixture, indicating equal volumes of said compounds, at a temperature, preferably in the range of from -20 to +50 °C and especially preferably in the range of from 0 to 25 °C.
Isolation of the reaction product may be carried out by a suitable process which may comprise one or more steps. According to a preferred embodiment of the present invention, the reaction product is first separated off the reaction mixture or the mixture of the reaction mixture with, e.g., the ethanol-acetone mixture, by a suitable method such as centrifugation or filtration. In a second step, the separated reaction product may be subjected to a further treatment such as an after-treatment like dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography, HPLC, MPLC, gel filtration and/or lyophilisation. According to an even more preferred embodiment, the separated reaction product is first dialysed, preferably against water, and then lyophilized until the solvent content of the reaction product is sufficiently low according to the desired specifications of the product. Lyophilisation may be carried out at temperature of from 20 to 35 °C, preferably of from 25 to 30 °C.
According to preferred embodiments of the present invention, the hydroxyalkyl starch derivative comprising hydroxyalkyl starch and compound (L) or comprising hydroxyalkyl starch, compound (D) and compound (L) is fiirther reacted with the further compound (M) which comprises at least one functional group Y.
Generally, there are no limitations regarding compound (M). Preferably, a polypeptide is used as compoimd (M) in the context of the present invention. However, other compounds (M) are also possible, either polymers or oligomers or monomolecular compounds or mixtures of two or more thereof.
The term "polypeptide" as used in the context of the present invention refers to a compoimd which comprises at least 2 amino acids which are linked via a peptide bond, i.e. a bond with structure -(C=O)-NH-. The polypeptide may be a naturally occuring compound or a polypeptide which does not occur naturally, the latter comprising naturally occuring amino acids and/or at least one amino acid which does not naturally occur. The backbone of the polypeptide, the polypeptide chain, may be fur- ther substituted with at least one suitable substituent thus having at least one side- chain. The at least one functional group Y may be part of the polypeptide backbone or of at least one substituent of the backbone wherein embodiments are possible comprising at least one functional group being part of the polypeptide backbone and at least one functional group being part of at least one substituent of the polypeptide backbone.
As far as the polypeptide is concerned, there exist no restrictions, given that the polypeptide comprises at least one functional group Y. Said fimctional group Y may be linked directly to the polypeptide backbone or be part of a side-chain of the back- bone. Either side-chain or functional group Y or both may be part of a naturally occuring polypeptide or may be introduced into a naturally occuring polypeptide or into a polypeptide which, at least partially, does not occur naturally, prior to the reaction with the fimctional group X.
Moreover, the polypeptide can be, at least partly, of any human or animal source. In a preferred embodiment, the polypeptide is of human source.
The polypeptide may be a cytokine, especially erythropoietin, an antithrombin (AT) such as AT III, an interleukin, especially interleukin-2, IFN-beta, IFN-alpha, G-CSF, CSF, inteι eukin-6 and therapeutic antibodies.
According to a preferred embodiment, the polypeptide is an antithrombin (AT), preferably AT III (Levy JH, Weisinger A, Ziomek CA, Echelard Y, Recombinant Antithrombin: Production and Role in Cardiovascular Disorder, Seminars in Thrombosis and Hemostasis 27, 4 (2001) 405-416; Edmunds T, Van Patten SM, Pollock J, Hanson E, Bernasconi R, Higgins E, Manavalan P, Ziomek C, Meade H, McPherson J, Cole ES, Transgenically Produced Human Antithrombin: Structural and Functional Comparison to Human Plasma-Derived Antithrombin, Blood 91, 12 (1998) 4661- 4671; Minnema MC, Chang ACK, Jansen PM, Lubbers YTP, Pratt BM, Whittaker BG, Taylor FB, Hack CE, Friedman B, Recombinant human antithrombin III improves survival and attenuates inflammatory responses in baboons lethally chal- lenged with Escherichia coli, Blood 95, 4 (2000) 1117-1123; Van Patten SM, Hanson EH, Bernasconi R, Zhang K, Manavaln P, Cole ES, McPherson JM, Edmunds T, Oxidation of Methionine Residues in Antithrombin, J. Biol. Chemistry 274, 15 (1999) 10268-10276).
According to another preferred embodiment, the polypeptide is human IFN-beta, in particular IFN-beta la (cf. Avonex®, REBIF®) and IFN-beta lb (cf. BETASERON®).
A further preferred polypeptide is human G-CSF (granulocyte colony stimulating factor). See, e.g., Nagata et al., The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor, EMBO J. 5: 575-581, 1986; Souza et al., Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells, Science 232 (1986) 61-65; and Herman et al., Characterization, formulation, and stability of Neupogen® (Filgrastim), a recombinant hu- man granulocyte-colony stimulating factor, in: Formulalion, characterization, and stability of protein drugs, Rodney Pearlman and Y. John Wang, eds., Plenum Press, New York, 1996, 303-328.
If a mixture of at least two different polypeptides is used, the at least two polypep- tides may differ, e.g., in the molecular mass, the number and/or sequence of amino acids, the number and/or chemical nature of the substituents or the number of polypeptide chains linked by suitable chemical bonds such as disulfide bridges.
According to a preferred embodiment of the present invention, the reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) is isolated, preferably according to at least one of the above-mentioned processes, and then re- acted with a polypeptide having at least one functional group Y. According to a preferred embodiment of the present invention, the functional group Y is comprised in a carbohydrate moiety of the polypeptide.
In the context of the present invention, the term "carbohydrate moiety" refers to hy- droxyaldehydes or hydroxyketones as well as to chemical modifications thereof (see Rompp Chemielexikon, Thieme Verlag Stuttgart, Germany, 9th edition 1990, Volume 9, pages 2281-2285 and the literature cited therein). Furthermore, it also refers to derivatives pf naturally occuring carbohydrate moieties like glucose, galactose, mannose, sialic acid and the like. The term also includes chemically oxidized, naturally occuring carbohydrate moieties. The structure of the oxidized carbohydrate moiety may be cyclic or linear.
The carbohydrate moiety may be linked directly to the polypeptide backbone. Pref- erably, the carbohydrate moiety is part of a carbohydrate side chain. More preferably, the carbohydrate moiety is the terminal moiety of the carbohydrate side chain.
In an even more preferred embodiment, the carbohydrate moiety is a galactose residue of the carbohydrate side chain, preferably the terminal galactose residue of the carbohydrate side chain. This galactose residue can be made available for reaction with the functional group X comprised in the reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L), by removal of terminal sialic acids, followed by oxidation, as described hereinunder.
In a still further preferred embodiment, the reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is fiirther reacted with compound (L) is linked to a sialic acid residue of the carbohydrate side chains, preferably the terminal sialic acid residue of the carbohy- drate side chain. Oxidation of terminal carbohydrate moieties can be performed either chemically or enzymatically.
Methods for the chemical oxidation of carbohydrate moieties of polypeptides are known in the art and include the treatment with perjodate (Chamow et al., 1992, J. Biol. Chem., 267, 15916-15922).
By chemically oxidizing, it is in principle possible to oxidize any carbohydrate moiety, being terminally positioned or not. However, by choosing mild conditions (1 mM periodate, 0 °C in contrast to harsh conditions: 10 mM periodate lh at room temperature), it is possible to preferably oxidize the terminal sialic acid of a carbohydrate side chain.
Alternatively, the carbohydrate moiety may be oxidized enzymatically. Enzymes for the oxidation of the individual carbohydrate moieties are known in the art, e.g. in the case of galactose the enzyme is galactose oxidase. If it is intended to oxidize terminal galactose moieties, it will be eventually necessary to remove terminal sialic acids (partially or completely) if the polypeptide has been produced in cells capable of attaching sialic acids to carbohydrate chains, e.g. in mammalian cells or in cells which have been genetically modified to be capable of attaching sialic acids to carbohydrate chains. Chemical or enzymatic methods for the removal of sialic acids are known in the art (Chaplin and Kennedy (eds.), 1996, Carbohydrate Analysis: a practical approach, especially Chapter 5 Montreuill, Glycoproteins, pages 175-177; IRL Press Practical approach series (ISBN 0-947946-44-3)).
According to another preferred embodiment of the present invention, the fimctional group of the polypeptide is the thio group. Therefore, the reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) may be linked to the polypeptide via a thioether group wherein the S atom can be derived from any thio group comprised in the polypeptide. In the context of this embodiment, it is particularly preferred to react the polypeptide with a reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L).
Therefore, the present invention also relates to a method as described above wherein the reaction product of hydroxyalkyl starch and compound (D) is further reacted with compound (L) is reacted with the polypeptide via a thio group comprised in the polypeptide.
Therefore, the present invention also relates to a method as described above wherein the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) is reacted with the polypeptide via an oxidized carbohydrate moiety and a thio group comprised in the polypeptide.
The thio group may be present in the polypeptide as such. Moreover, it is possible to introduce a thio group into the polyeptide according to a suitbale method. Among others, chemical methods may be mentioned. If a disulfide bridge is present in the polypeptide, it is possible to reduce the -S-S- structure to get a thio group. It is also possible to transform an amino group present in the polypeptide into a SH group by reaction the polypeptide via the amino group with a compound which has at least two different functional groups, one of which is capable of being reacted with the amino group and the other is an SH group or a precursor of an SH group. This modification of an amino group may be regarded as an example where the protein is first reacted with a compoimd (L) which has at least two different functional groups, one of which is capable of being reacted with the amino group and the other is an SH group, and the resulting reaction product is then reacted with, e.g., a HAS derivative comprising HAS and a compound (D), said derivative comprising a functional group being capable of reacting with the SH group. It is also possible to introduce an SH group by mutation of the polypeptide such as by introducing a cystein or a suitable SH func- tional amino acid into the polypeptide or such as removing a cystein from the polypeptide so as to disable another cystein in the polypeptide to form a disulfide bridge. As an especially preferred polypeptide, erythropoietin (EPO) is used.
Therefore, the present invention also relates to a method as described above wherein the polypeptide is erythropoietin.
The EPO can be of any human (see e.g. Inoue, Wada, Takeuchi, 1994, An improved method for the purification of human erythiOpoietin with high in vivo activity from the urine of anemic patients, Biol. Pharm. Bull. 17(2), 180-4; Miyake, Kung, Gold- wasser, 1977, Purification of human erythropoietin., J. Biol. Chem., 252(15), 5558- 64) or another mammalian source and can be obtained by purification from naturally occurring sources like human kidney, embryonic human liver or animal, preferably monkey kidney. Furthermore, the expression "erythropoietin" or "EPO" encompasses also an EPO variant wherein one or more amino acids (e.g. 1 to 25, preferably 1 to 10, more preferred 1 to 5, most preferred 1 or 2) have been exchanged by an- other amino acid and which exhibits erythropoietic activity (see e.g. EP 640 619 Bl). The measurement of erythropoietic activity is described in the art (for measurement of activity in vitro see e.g. Fibi et al.,1991, Blood, 77, 1203 ff; Kitamura et al, 1989, J. Cell Phys., 140, 323-334; for measurement of EPO activity in vivo see Ph. Eur. 2001, 911-917; Ph. Eur. 2000, 1316 Erythropoietini solutio concentrata, 780- 785; European Pharmacopoeia (1996/2000); European Pharmacopoeia, 1996, Erythropoietin concentrated solution, Pharmaeuropa., 8, 371-377; Fibi, Hermentin, Pauly, Lauf- fer, ZettlmeissL, 1995, N- and O-glycosylatipn muteins of recombinant human erythropoietin secreted from BHK-21 cells, Blood, 85(5), 1229-36; (EPO and modified EPO forms were injected into female NMRI mice (equal amounts of protein 50 ng/mouse) at day 1, 2 and 3 blood samples were taken at day 4 and reticulocytes were determined)). Further publications where tests for the measurement pf the activity of EPO are Barbone, Aparicio, Anderson, Natarajan, Ritchie, 1994, Reticulocytes measurements as a bioassay for erythropoietin, J. Pharm. Biomed. Anal., 12(4), 515- 22; Bowen, Culligan, Beguin, Kendall, Villis, 1994, Estimation of effective and total erythropoiesis in myelodysplasia using serum transferrin receptor and erythropoietin concentrations, with automated reticulocyte parameters, Leukemi, 8(1), 151-5; Delorme, Lorenzini, Giffin, Martin, Jacobsen, Boone, Elliott, 1992, Role of glycosy- lation on the secretion and biological activity of erythropoietin, Biochemistry, 31(41), 9871-6; Higuchi, Oheda, Kuboniwa, Tomonoh, Shimonaka, Ochi, 1992 ;Role of sugar chains in the expression of the biological activity of human erythropoietin, J. Biol. Chem., 267(11), 7703-9; Yamaguchi, Akai, Kawanishi, Ueda, Ma- suda, Sasaki, 1991, Effects of site-directed removal of N-glycosylation sites in human erythropoietin pn its production and biological properties, J. Biol. Chem., 266(30), 20434-9; Takeuchi, Inoue, Strickland, Kubota, Wada, Shimizu, Hoshi, Ko- zutsumi, Takasaki, Kobata, 1989, Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese ham- ster ovary cells, Proc. Natl. Acad. Sci. USA, 85(20), 7819-22; Kurtz, Eckardt, 1989, Assay methods for erythropoietin, Nephron., 51(1), 11-4 (German); Zucali, Sulkowski, 1985, Purification of human urinary erythiOpoietin on controlled-pore glass and silicic acid, Exp. Hematol., 13(3), 833-7; Krystal, 1983, Physical and biological characterization of erythroblast enhancing factor (EEF), a late acting erythro- poietic stimulator in serum distinct from erythropoietin, Exp. Hematol., 11(1), 18-31.
Preferably, the EPO is recombinantly produced. This includes the production in eukaryotic or prokaryotic cells, preferably mammalian, insect, yeast, bacterial cells or in any other cell type which is convenient for the recombinant production of EPO. Furthemiore, the EPO may be expressed in transgenic animals (e.g. in body fluids like milk, blood, etc.), in eggs of transgenic birds, especially poultry, preferred chicken, or in transgenic plants.
The recombinant production of a polypeptide is known in the art. In general, this includes the transfection of host cells with an appropriate expression vector, the cultivation of the host cells under conditions which enable the production of the polypeptide and the purification of the polypeptide from the host cells. For detailled information see e.g. Krystal, Pankratz, Farber, Smart, 1986, Purification of human erythropoietin to homogeneity by a rapid five-step procedure, Blood, 67(1), 71-9; Quelle, Caslake, Burkert, Wojchowski, 1989, High-level expression and purification of a recombinant human erythropoietin produced using a baculovims vector, Blood, 74(2), 652-7; EP 640 619 Bl and EP 668 351 Bl. In a preferred embodiment, the EPO has the amino acid sequence of human EPO (see EP 148 605 B2).
The EPO may comprise one or more carbohydrate side chains, preferably 1 to 12, more preferably 1 to 9, even more preferably 1 to 6 and particularly 1 to 4, especially preferably 4 carbohydrate side chains, attached to the EPO via N- and/ or O-linked glycosylation, i.e. the EPO is glycosylated. Usually, when EPO is produced in eukaryotic cells, the polypeptide is posttranslationally glycosylated. Consequently, the carbohydrate side chains may have been attached to the EPO during biosynthesis in mammalian, especially human, insect or yeast cells. The structure and properties of glycosylated EPO have been extensively studied in the art (see EP 428 267 Bl; EP 640 619 Bl; Rush, Derby, Smith, Merry, Rogers, Rohde, Katta, 1995, Microhetero- geneity of erythropoietin carbohydrate structure, Anal Chem., 67(8), 1442-52; Ta- keuclii, Kobata, 1991, Structures and fimctional roles of the sugar chains of human erythropoietins, Glycobiology, 1(4), 337-46 (Review).
Therefore, the hydroxyalkyl starch derivative according to the present invention may comprise at least one, preferably 1 to 12, more preferably 1 to 9, even more prefera- bly 1 to 6 and particularly preferably 1 to 4 HAS molecules per EPO molecule. The number of HAS-molecules per EPO molecule can be determined by quantitative carbohydrate compositional analysis using GC-MS after hydrolysis of the product and derivatisation of the resulting monosaccharides (see Chaplin and Kennedy (eds.), 1986, Carbohydrate Analysis: a practical approach, IRL Press Practical approach series (ISBN 0-947946-44-3), especially Chapter 1, Monosaccharides, page 1-36; Chapter 2, Oligosaccharides, page 37-53, Chapter 3, Neutral Polysaccharides, page 55-96).
According to an especially preferred embodiment of the present invention, the car- bohydrate moiety linked to EPO, is part of a carbohydrate side chain. More preferably, the carbohydrate moiety is the terminal moiety of the carbohydrate side chain. In an even more preferred embodiment, the carbohydrate moiety is a galactose residue of the carbohydrate side chain, preferably the terminal galactose residue of the carbohydrate side chain. This galactose residue can be made available for reaction with the reaction product of compound (I) and compound (II) by removal of terminal sialic acids, followed by oxidation, as described hereinunder. In a further preferred embodiment, the reaction product of compound (I) and (II) is linked to a sialic acid residue of the carbohydrate side chains, preferably the terminal sialic acid residue of the carbohydrate side chain. The sialic acid is oxidized as described hereinunder.
Particularly preferably this galactose residue is made available for reaction with the reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) via the functional group X by removal of terminal sialic acid followed by oxidation.
More preferably, this galactose residue is made available for reaction with the reac- tion product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) via the functional group X by oxidation wherein terminal sialic acid is not removed.
As mentioned above, the reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) be reacted with a thio group comprised in EPO.
It is also possible to react the reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is fur- ther reacted with compound (L) with a thio group as well as with a carbohydrate moiety, each of them comprised in the at least one further compound, preferably a polypeptide, more preferably erythropoietin.
According to a preferred embodiment, this SH group may be linked to a preferably oxidized carbohydrate moiety, e.g. by using a hydroxylamine derivative, e.g. 2-
(aminooxy)ethylmercaptan hydrochloride (Bauer L. et al., 1965, J. Org. Chem., 30, 949) or by using a hydrazide derivative, e.g. thioglycolic acid hydrazide (Whitesides et al., 1977, J. Org. Chem., 42, 332.)
According to a fiirther preferred embodiment, the thio group is preferably introduced in an oxidized carbohydrate moiety of EPO, more preferably an oxidized carbohydrate moiety which is part of a carbohydrate side chain of EPO.
Preferably, the thio group is derived from a naturally occurring cysteine or from an added cysteine. More preferably, the EPO has the amino acid sequence of human EPO and the naturally occurring cysteines are cysteine 29 and/or 33. In a more preferred embodiment, t the reaction product of hydroxyalkyl starch and compoimd (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (L) is reacted with cysteine 29 whereas cysteine 33 is replaced by another amino acid. Alternatively, the reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compoimd (D) which is further reacted with compound (L) is reacted with cysteine 33 whereas cysteine 29 is replaced by another amino acid.
In the context of the present invention, the term "added cysteines" indicates that the polypeptides, preferably EPO, comprise a cysteine residue which is not present in the wild-type polypeptide.
In the context of this aspect of the invention, the cysteine may be an additional amino acid added at the N- or C-terminal end of EPO.
Furthermore, the added cysteine may have been added by replacing a naturally occuring amino acid by cysteine or a suitably substituted cysteine. Preferably, in the context of this aspect of the invention, the EPO is human EPO and the replaced amino acid residue is serine 126.
As to the reaction conditions regarding the reaction of the reaction product of hydroxyalkyl starch and compound (L), optionally with compoimd (D), with the further compound (M), no specific limitations exist, and the reaction conditions may be adjusted to the specific needs. According to an especially preferred embodiment of the present invention, water is used as solvent, either alone or in combination with at least one other solvent. As at least one other solvent, DMSO, DMF, methanol or ethanol may be mentioned. Preferred solvents other than water are methanol and ethanol. According to other preferred embodiments, DMSO or DMF or methanol or ethanol or a mixture of two or more thereof is used as solvent.
If, e.g., hydroxyalkyl starch is reacted with compound (L) in an aqueous system, as it is the case, e.g., when hydroxyethyl starch is reacted with a hydroxyamine such as O- [2-(2-aminooxy-ethoxy)-etlιyl] -hydroxyl amine, via reaction of the non-oxidised reducing end of the starch, and the reaction product is further reacted with a polypeptide, preferably erythropoietin, via an aldehyde, keto, acetal or hemiacetale group, the reaction temperatur is preferably preferably in the range of from 4 to 37 °C, more preferably of from 10 to 30 °C and especially preferably of from 15 to 25 °C.
Isolation of the reaction product comprising the further compound (M), preferably the polypeptide and especially pererably erythropoietin, can be performed by using known procedures for the purification of natural and recombinant EPO (e.g.size ex- elusion chromatography, ion-exchange chromatography, RP-HPLC, hydroxyapatite chromatography, hydrophobic interaction chromatography or combinations thereof). Isolation of the reaction product may be carried out by a suitable process which may comprise one or more steps. According to a preferred embodiment of the present invention, the reaction product is first separated off the reaction mixture or the mix- ture of the reaction mixture with, e.g., the ethanol-acetone mixture, by a suitable method such as centrifugation or filtration. In a second step, the separated reaction product may be subjected to a further treatment such as an after-treatment like dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography such as, e.g., by a column containing Q-sepharose, HPLC, MPLC, gel filtration and/or ly- ophilisation. According to one preferred embodiment, the separated reaction product is first dialysed, preferably against water, and then lyophilized until the solvent content of the reaction product is sufficiently low according to the desired specifications of the product. Lyophilisation may be carried out at temperature of from 20 to 35 °C, preferably of from 25 to 30 °C. According to another preferred embodiment, the reaction mixture comprising the reaction product is applied to a column containing Q- Sepharose to give an eluate which is concentrated, e.g. by centrifugal filtration.
It is another object of the present invention to provide hydroxyalkyl starch derivatives which are produced by one or more of the aforesaid methods.
Therefore, the present invention relates to a hydroxyalkyl starch derivative obtain- able by a method of producing a hydroxyalkyl starch derivative, said hydroxyalkyl starch having a structure according to fonnula (I)
Figure imgf000051_0001
comprising reacting hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end or a hydroxyalkyl starch derivative, obtainable by reacting hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end with a compound (D), said compound (D) comprising at least one functional group Zi capable of being reacted with the optionally oxidized reducing end of the hydroxyalkyl starch, and at least one functional group W, with a compound (L) comprising at least one functional group Zi capable of being reacted with said hydroxyalkyl starch, or at least one functional group Z2 capable of being reacted with functional group W comprised in said hydroxyalkyl starch derivative, and at least one functional group X capable of being reacted with a functional group Y of a further compound (M), wherein said functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, an acetal group, or a thio group.
According to a preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein Ri, R2 and R3 are independently hydrogen or a linear or branched hydroxyalkyl group.
According to an even more preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein Ri, R2 and R3 are inde- pendently hydrogen or a 2-hydroxyethyl group.
According to a fiirther preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described wherein the hydroxyalkyl starch is hydroxyethyl starch.
According to a further preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein the functional group Zi comprises the structure -NH-.
According to an especially preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein Zi is selected from the group consisting of
Figure imgf000052_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl. According to a further preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein the functional group Y is selected from the group consisting of an aldehyd group, a eto group, a hemiacetal group, and an acetal group, and the functional group X comprises the structure -NH-.
According to an especially preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above X is selected from the group consisting of
Figure imgf000053_0001
wherein G is O or S and, if present twice,' independently O or S, and R is methyl.
According to a further preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein the functional group Y is - SH and the -functional group X is selected from the group consisting of
Figure imgf000053_0002
wherein Hal is CI, Br or I.
According to a further preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein the functional group W or the functional group Z2 is -SFI and the functional group Z2 or the functional group W is selected from the group consisting of
Figure imgf000054_0001
wherein Hal is CI, Br, or I.
According to a further preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein the functional group W or the functional group Z2 is selected from the group consisting of an activated ester, as described above, or a carboxy group which is optionally transformed into an activated ester and the functional group Z2 or the functional group W is selected from the group consisting of
Figure imgf000054_0002
wherein G is O or S and, if present twice, independently O or S, and R is methyl.
According to an especially preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein the reducing end of the hydroxyalkyl starch is not oxidized prior to the reaction with compound (D) or compound (L), said hydroxyalkyl starch thus having a structure according to formula (I)
Figure imgf000055_0001
According to another especially preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein the reducing end of the hydroxyalkyl starch is oxidized prior to the reaction with compound (D) or compound (L), said hydroxyalkyl starch thus having a structure according to formula (Ila)
Figure imgf000055_0002
and/or according to formula (lib)
Figure imgf000055_0003
According to a further preferred embodiment, the present invention relates to a hy- droxyalkyl starch derivative as described above wherein the reducing end is oxidized by an alkaline iodine solution. According to a further preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein hydroxyalkyl starch is reacted with a compound (L) via the reaction of functional group Zi with the optionally oxidized reducing end of the hydroxyalkyl starch and the resulting reaction product is reacted with a further compound (M) via the reaction of the functional group X comprised in compoimd (L) with the functional group Y comprised in compound (M).
According to yet a further preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above hydroxyalkyl starch is reacted with a compound (L) via the reaction of functional group Zi with the optionally oxidized reducing end of the hydroxyalkyl starch, where compound (L), prior to the reaction with hydroxyalkyl starch, is reacted with a further compound (M) via the reaction of functional group X comprised in compound (L) with the functional group Y comprised in compound (M).
According to still a further preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of the functional group Zi comprised in compound (D), with the optionally oxidized reducing end of the hydroxyalkyl starch to give a first hydroxyalkyl starch derivative, and where the first hydroxyalkyl starch derivative is reacted with a compound (L) via the reaction of fimctional group Z2 comprised in compound (L) with the functional group W comprised in compound (D) to give a second hydroxyalkyl starch derivative.
According to an especially preferred embodiment, the present invention relates to the aforesaid hydroxyalkyl starch derivative wherein the second hydroxyalkyl starch derivative is reacted with a further compound (M) via the reaction of functional group X comprised in compound (L) with the functional group Y comprised in compound (M). According to a further preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of functional group Zi comprised in compound (D) with the optionally oxidized reducing end of the hydroxyalkyl starch to give a first hydroxyalkyl starch derivative, and where the first hydroxyalkyl starch derivative is reacted, via the reaction of the functional group W, comprised in compound (D), and the functional group Z2, comprised in compound (L), with compound (L), where compound (L), prior to the reaction with the first hydroxyalkyl starch derivative, is reacted with a further compound (M) via the reaction of fimctional group X comprised in compound (L) with the functional group Y comprised in compound CM).
According to an especially preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein the at least one further compound (M) is a polypeptide.
According to a particularly preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein the polypeptide is erythiOpoietin.
The hydroxyalkyl starch derivative which in the following is referred to as HAS- EPO conjugate and which is formed by reaction of hydroxyalkyl starch with compound (L) and optionally compoimd (D) and erythrpoietin, has the advantage that it exhibits an improved biological stability when compared to the erythropoietin before conjugation. Furthermore, it exhibits a higher biological activity than standard BRP EPO. This is mainly due to the fact that this hydroxyalkyl starch derivative is less or even not recognized by the removal systems of the liver and kidney and therefore persists in the circulatory system for a longer period of time. Furthermore, since the HAS is attached site-specifically, the risk of destroying the in- vivo biological activity of EPO by conjugation of HAS to EPO is minimized. The HAS-EPO conjugate of the invention may exhibit essentially the same in- vitro . biological activity as recombinant native EPO, since the in-vitro biological activity only measures binding affinity to the EPO receptor. Methods for determining the in- vitro biological activity are known in the art.
Furthermore, the HAS-EPO exhibits a greater in-vivo activity than the EPO used as a starting material for conjugation (unconjugated EPO). Methods for determining the in vivo biological activity are known in the art.
The HAS-EPO conjugate may exhibit an in vivo activity of from 110 % to 500 %, preferably of from 300 to 400 %, or preferably of from 110 to 300 %, more preferably from 110 % to 200 %, more preferably from 110 % to 180 % or from 110 to 150 %, most preferably from 110 % to 140 %, if the in-vivo activity of the unconjugated EPO is set as 100 %.
Compared to the highly sialylated EPO of Amgen (see EP 428 267 Bl), the HAS- EPO exhibits preferably at least 50%, more preferably at least 70 %, even more preferably at least 85 % or at least 95 %, at least 150 %, at least 200 % or at least 300 % of the in vivo activity of the highly sialylated EPO if the in-vivo activity of highly sialylated EPO is set as 100 %. Most preferably, it exhibits at least 95 % of the in vivo activity of the highly sialylated EPO.
The high in-vivo biological activity of the HAS-EPO conjugate of the invention mainly results from the fact that the HAS-EPO conjugate remains longer in the circu- lation than the unconjugated EPO because it is less recognized by the removal systems of the liver and because renal clearance is reduced due to the higher molecular weight. Methods for the determination of the in-vivo half life time of EPO in the circulation are known in the art (Sytkowski, Lunn, Davis, Feldman, Siekman, 1998, Human erythropoietin dimers with markedly enhanced in vivo activity, Proc. Natl. Acad. Sci. USA, 95(3), 1184-8). Consequently, it is a great advantage of the present invention that a HAS-EPO conjugate is provided which may be administered less frequently than the EPO preparations commercially available at present. While standard EPO preparations have to be administered at least every 3 days, the HAS-EPO conjugate of the invention is pref- erable administered twice a week, more preferably once a week.
Furthermore, the method of the invention has the advantage that an effective EPO derivative can be produced at reduced costs since the method does not comprise extensive and time consuming purification steps resulting in low final yield, e.g. it is not necessary to purify away under-sialylated EPO forms which are known to exhibit low or no in-vivo biological activity. Especially Example 8.11(d) demonstrates that a HES-EPO produced with few modifications steps exhibits a 3-fold activity over standard BRP EPO.
It is yet another object of the present invention to provide a pharmaceutical composition which comprises, in a therapeutically effective amount, the HAS-EPO conjugate of the present invention.
Furthermore, the present invention relates to a pharmaceutical composition compris- ing, in a therapeutically effective amount, the FIAS-polypeptide conjugate, preferably the HAS-EPO conjugate, more preferably the HES-EPO conjugate of the present invention. In a , preferred embodiment, the pharmaceutical composition comprises further at least one pharmaceutically acceptable diluent, adjuvant and/or carrier useful in erythropoietin therapy.
Therefore, the present invention also relates to a pharmaceutical composition comprising, in a therapeutically effective amount, a hydroxyalkyl starch derivative obtainable by a method of producing a hydroxyalkyl starch derivative, said hydroxyalkyl starch having a structure according to formula (I)
Figure imgf000060_0001
comprising reacting, hydroxyalkyl starch of fonnula (I) at its optionally oxidized reducing end or a hydroxyalkyl starch derivative, obtainable by reacting hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end with a compound (D), said compound (D) comprising at least one functional group Zi capable of being reacted with the optionally oxidized reducing end of the hydroxyalkyl starch, and at least one functional group W, with a compound (L) comprising at least one functional group Zi capable of being reacted with said hydroxyalkyl starch, or at least one functional group Z2 capable of being reacted with functional group W comprised in said hydroxyalkyl starch derivative, and at least one functional group X capable of being reacted with a functional group Y of a further compound (M), wherein said fimctional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, an acetal group, or a thio group, said method of producing a hydroxyalkyl starch derivative further comprising reacting the reaction product comprising hydroxyalkyl starch, compound (L) and optionally compound (D) with a further compound (M) wherein the at least one further compound is a polypeptide.
Moreover, the present invention relates to the use of a hydroxyalkyl starch derivative as described for the preparation of a medicament for the treatment of anemic disorders or hematopoietic dysfunction disoirders or diseases related thereto. . According to a preferred embodiment, the present invention relates to a pharmaceutical composition as described above wherein the polypeptide is an antithrombin (AT), preferably AT III (Levy JH, Weisinger A, Ziomek CA, Echelard Y, Recombinant Antithrombin: Production and Role in Cardiovascular Disorder, Seminars in Thrombosis and Hemostasis 27, 4 (2001) 405-416; Edmunds T, Van Patten SM, Pollock J, Flanson E, Bernasconi R, Higgins E, Manavalan P, Ziomek C, Meade H, McPherson J, Cole ES, Transgenically Produced Human Antithrombin: Structural and Functional Comparison to Human Plasma-Derived Antithrombin, Blood 91, 12 (1998) 4661-4671; Minnema MC, Chang ACK, Jansen PM, Lubbers YTP, Pratt BM, Whittaker BG, Taylor FB, Hack CE, Friedman B, Recombinant human antithrombin III improves survival and attenuates inflammatory responses in baboons lethally challenged with Escherichia coli, Blood 95, 4 (2000) 1117-1123; Van Patten SM, Hanson EFl, Bernasconi R, Zhang K, Manavaln P, Cole ES, McPherson JM, Edmunds T, Oxidation of Methionine Residues in Antithrombin, J. Biol. Chemistry 274, 15 (1999) 10268-10276).
According to other preferred embodiments, the present invention relates to pharmaceutical compositions wherein the polypeptide is G-CSF or IFN-beta.
According to an especially preferred embodiment, the present invention relates to a pharmaceutical composition as described above wherein the polypeptide is erythropoietin.
According to a further embodiment, the present invention relates to a pharmaceutical composition as described above wherein the functional group Y is -SH and corn- pound (L) is a compound of general formula Zi-L'-X where the fimctional group Zi is selected from the group consisting of
Figure imgf000061_0001
Figure imgf000062_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, and where the functional group X is selected from the group consisting of
Figure imgf000062_0002
wherein Hal is CI, Br or I, and where L' is an organic chain bridging Zi and X or where L' is absent.
According to a preferred embodiment, the present invention relates to a pharmaceutical composition as described above wherein the functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group, and compound (L) is a compound of general formula Zi-L'-X where the functional group Zi is selected from the group consisting of
Figure imgf000062_0003
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, and where the functional group X is selected from the group consisting of
Figure imgf000062_0004
/ H2N ft
H2N Y N-S-
G O
Figure imgf000063_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, and where L' is an organic chain bridging Zi and X or where L' is absent.
According to another embodiment, the present invention relates to a pharmaceutical composition as described above wherein the functional group Y is -SH, compound
(D) is a compound of general formula Zi-D'-W, and compound (L) is a compound of general formula Z2-L'-X, where the functional group Zi is selected from the group consisting of
Figure imgf000063_0002
/ H2 ft H?N f N-S—
2 II H II
G O
Figure imgf000063_0003
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, where the functional group X is selected from the group consisting of
Figure imgf000063_0004
wherein Hal is CI, Br or I, where the functional group W or the functional group Z2 is -SH and the fimctional group Z2 or the fimctional group W is selected from the group consisting of
Figure imgf000064_0001
wherein Hal is CI, Br, or I, or where the functional group W or the functional group Z2 is selected from the group consisting of an activated ester, as described above, or a carboxy group which is optionally transformed into an activated ester and the functional group Z2 or the functional group W is selected from the group consisting of
Figure imgf000064_0002
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, and where D' is an organic chain bridging Zi and W or where D' is absent and where L' is an organic chain bridging Z2 and X or where L' is absent.
According to yet another embodiment, the present invention relates to a pharmaceu- tical composition as described above wherein the functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group, compound (D) is a compound of general formula Zi-D'-W, and compound (L) is a compound of general formula Z2-L'-X, where the fimctional group Zi is selected from the group consisting of
Figure imgf000064_0003
Figure imgf000065_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, where the functional group X is selected from the group consisting of
Figure imgf000065_0002
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, the functional group W or the functional group Z2 is -SH and the fimctional group Z2 or the functional group W is selected from the group consisting of
Figure imgf000065_0003
wherein Flal is CI, Br, or I., or where the functional group W or the functional group Z2 is selected from the group consisting of an activated ester, as described above, or a carboxy group which is optionally transformed into an activated ester and the functional group Z2 or the functional group W is selected from the group consisting of
Figure imgf000065_0004
Figure imgf000066_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, and where D' is an organic chain bridging Zi and W or where D' is absent and where L' is an organic chain bridging Z2 and X or where L' is absent.
According to a particularly preferred embodiment, the present invention relates to a pharmaceutical composition as described above wherein hydroxyethyl starch is reacted in an aqueous medium with a compound according to the following formula
/O. //\ ^^ /O H2N ^-^ XT ^ NH2 and the reaction product is reacted with erythropoietin.
According to an even more preferred embodiment, the present invention relates to the aformentioned pharmaceutical composition wherein the erythropoietin is oxidised with sodium periodate prior to the reaction.
According to a further preferred embodiment, the present invention relates to phar- maceutical composition as described above wherein the erythropoietin is partially desialylated and subsequently oxidised with sodium periodate prior to the reaction.
According to a further preferred embodiment of the present invention, pharmaceutical compositions comprising a hydroxyalkyl starch derivative which are produced on the basis of a completely reduced Thio-EPO according to Example 6 are excluded.
The above-mentioned pharmaceutical composition is especially suitable for the treatment of anemic disorders or hematopoietic dysfunction disorders or diseases related thereto.
A "therapeutically effective amount" as used herein refers to that amount which provides therapeutic effect for a given condition and administration regimen. The ad- ministration of erythropoietin isofonns is preferably by parenteral routes. The specific route chosen will depend upon the condition being treated. The administration of erythropoietin isoforms is preferably done as part of a formulation containing a suitable carrier, such as human serum albumin, a suitable diluent, such as a buffered saline solution, and/or a suitable adjuvant. The required dosage will be in amounts sufficient to raise the hematocrit of patients and will vary depending upon the severity pf the condition being treated, the method of administration used and the like.
The object of the treatment with the pharmaceutical composition of the invention is preferably an increase of the hemoglobin value of more than 6.8 mmol/1 in the blood. For this, the pharmaceutical composition may be administered in a way that the hemoglobin value increases between from 0.6 mmol/1 and 1.6 mmol/1 per week. If the hemoglobin value exceeds 8.7 mmol/1, the therapy should be preferably interrupted until the hemoglobin value is below 8.1 mmol/1.
The composition of the invention is preferably used in a fonnulation suitable for subcutaneous or intravenous or parenteral injection. For this, suitable excipients and carriers are e.g. sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chlorate, polysorbate 80, HSA and water for injection. The composition may be administered three times a week, preferably two times a week, more preferably once a week, and most preferably every two weeks.
Preferably, the pharmaceutical composition is administered in an amount of 0.01-10 μg/kg body weight of the patient, more preferably 0,1 to 5 μg/kg, 0,1 to 1 μg/kg, or 0.2-0.9 μg/kg, most preferably 0-3-0.7 μg/kg, and most preferred 0.4-0.6 μg/kg body weight.
In general, preferably between 10 μg and 200 μg, preferably between 15 μg and 100 μg are administered per dosis.
The invention further relates to a HAS-polypeptide according to the present invention for use in method for treatment of the human or animal body. The invention further relates to the use of a HAS-EPO conjugate of the present invention for the preparation of a medicament for the treatment of anemic disorders or hematopoietic dysfunction disorders or diseases related hereto.
In case compound (L) used in according to the present invention comprises one or more chiral centers, compound (II) may be present in R conformation or in S conformation or as racemic compound with respect to each chiral center.
In case compound (D) optionally used in the present invention comprises one or more chiral centers, compound (D) may be present in R conformation or in S conformation or as racemic compound with respect to each chiral center.
The invention is fiirther illustrated by the following examples, tables an figures which are in no way intended to restrict the scope of the present invention.
Short description of the Figures
Figure 1
Figure 1 shows an SDS page analysis of the HES-EPO conjugate, produced according to example 5.1.
Lane A: Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D); molecular weights (in kD) of the protein marker from top to bottom: 245, 123, 77, 42, 30, 25.4, and 17.
Lane B: Crude product after conjugation according to example 5.1. Lane C: EPO starting material.
Figure 2
Figure 2 shows an SDS page analysis of the HES-EPO conjugate, produced according to example 5.3.
Lane A Crude product after conjugation according to example 5.3. Lane B EPO starting material.
Lane C Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co,
Karlsruhe, D); molecular weights (in kD) of the protein marker from top to. bottom: 245, 123, 77, 42, 30, 25.4, and 17.
Figure 3
Figure 3 shows an SDS page analysis of the HES-EPO conjugate, produced according to example 5.4 and 5.5.
Lane A: Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D); molecular weights (in kD) of the protein marker from top to bottom: 245, 123, 77, 42, 30, 25.4, and 17. Lane B: Crude product after conjugation according to example 5.4. Lane C: Crude product after conjugation according to example 5.5. Lane D: EPO starting material.
Figure 4
Figure 4 shows an SDS page analysis of HES-EPO conjugates, produced according to examples 7.1 and 7.4.
Lane A: Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D); molecular weights (in kD) of the protein marker from top to bottom: 245, 123, 77, 42, 30, 25.4, and 17.
Lane B Crude product after conjugation according to example 7.4.
Lane C Crude product after conjugation according to example 7.1. Lane D EPO starting material.
Figure 5
Figure 5 shows an SDS page analysis of HES-EPO conjugates, produced according to examples 7.2, 7.3, 7.5, and 7.6.
Lane A: Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D); molecular weights (in kD) of the protein marker from top to bottom: 245, 123, 77, 42, 30, 25.4, and 17. Lane B: Crude product after conjugation according to example 7.6, based on Example 1.3 b).
Lane C: Crude product after conjugation according to example 7.5, based on Example 1.1 b).
Lane D: Crude product after conjugation according to example 7.6, based on Ex- ample 1.3 a).
Lane E: Crude product after conjugation according to example 7.5, based on Example 1.1 a). Lane F: Crude product after conjugation according to example 7.2. Lane G: Crude product after conjugation according to example 7.3. Lane K: EPO startmg material.
5 Figure 6
Figure 6 shows an SDS page analysis of HES-EPO conjugates, produced according to examples 7.7, 7.8, 7.9, 7.10, 7.11, and 7.12.
10 Lane A; Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co,
Karlsruhe, D); molecular weights (in kD) of the protein marker from top to bottom: 245, 123, 77, 42, 30, 25.4, and 17.
Lane B: Crude product after conjugation according to example 7.11.
Lane C: Crude product after conjugation according to example 7.10. 15 L Laannee DD:: Crude product after conjugation according to example 7.7.
Lane E: Crude product after conjugation according to example 7.8.
Lane F: Crude product after conjugation according to example 7.12.
Lane G: EPO starting material.
Lane K: Crude product after conjugation according to example 7.9. 20
Figure 7
SDS-PAGE analyses of EPO-GT-1 subjected to mild acid treatment for 5 min. = lane 2; 10 min. = lane 3; 60 min. = lane 4 and untreated EPO = lane 1; the mobility shift 25 of EPO after removal of N-glycans is shown (+PNGASE).
Figure 8
HP AEC-P AD pattern of oKgosaccharides isolated from untreated EPO and from 3.0 EPO incubated for 5 min., 10 min. and 60 min. under mild acid hydrolysis conditions. Roman numbers I-V indicate the elution position of I = desialylated dianten- nary structure, II = trisialylated triantennary structures (two isomers), III = tetrasia- lylated tetraantennary structure + 2 N-acetyllactosamine repeats, IV = tetrasialylated tetraantennary structure + 1 N-acetyllactosamine repeat; V = tetrasialylated tetraantennary structure + without N-acetyllactosamine repeat. The elution area of oligosac- charides structures without, with 1-4 sialic acid is indicated by brackets.
Figure 9
HP AEC-P AD of N-linked ohgosaccharides after desialylation; the elution position of N-acetylneuraminic acid is shown; numbers 1-9 indicate the elution position of stan- dard oligosaccharides: 1 = diantennary; 2 = triantennary (2-4 isomer), 3 = trianten- nary (2-6 isomer); 4 = tetraantennary; 5 = triantennary plus 1 repeat; 6 = tetraantennary plus 1 repeat; 7 = triantennary plus 2 repeats; 8 = tetraantennary plus 2 repeats and 9 = tetraantennary plus 3 repeats.
Figure 10
SDS-PAGE analysis of mild treated and untreated EPO which were subjected to periodate oxidation of sialic acid residues. 1 = periodate oxidized without acid treatment; 2 = periodate oxidized 5 min. acid treatment; 3 = periodate oxidized and acid treatment 10 min.; 4 = periodate oxidized without acid treatment; 5 = BRP EPO standard without periodate and without acid treatment.
Figure 11
HP AEC-P AD pattern of native oligosaccharides isolated from untreated EPO and from EPO incubated for 5 min and 10 min under mild acid hydrolysis conditions and subsequent periodate treatment. The elution area of oligosaccharides structures without and with 1-4 sialic acid is indicated by brackets 1-5.
Figure 12 SDS-PAGE analysis of the time course of HES-modification of EPO-GT-1-A: 20 μg aliquots of EPO-GT-1-A were reacted with hydroxylamϊne-modified HES derivative X for 30 rnin, 2, 4 and 17 hours. Lane 1 = 30 min reaction time; land 2 = 2 hour reaction time; land 3 = 4 hours reaction time; lane 4 = 17 hours reaction time; lane 5 = EPO-GT-1-A without HES-modification. Left figure shows the shift in mobility of EPO-GT-1-A with increasing incubation time in the presence of the with hydroxylamine-modified HES derivative (flow rate: 1 ml-min"1) X: Lane 1 = 30 min reaction time; lane 2 = 2 hours reaction time; lane 3 = 4 hours reaction time, land 4 = 17 hours reaction time; lane 5 = EPO-GT-1-A with HES modification. The figure on the right shows analysis of the same samples after their treatment with N- glycosidase.
Figure 13
SDS-PAGE analysis of Q-Sepharose fractions of HES-EPO conjugates. Each 1% of the flow-through and 1% of the fraction eluting at high salt concentrations were concentrated in a Speed Vac concentrator and were loaded onto the gels in sample buffer. EPO protein was stained by Coomassie Blue. A = sample I; B = sample II; C = sample III; K = control EPO-GT-1; Al, Bl, CI and Kl indicated the flow-through fraction; A2, B2, C2 and K2 indicates the fraction eluted with high salt concentration.
Figure 14a
SDS-PAGE analysis of HES-modified EPO sample A2 (see Fig. 13), control EPO sample K2 and EPO-GT-1-A EPO preparation were digested in the presence of N- glycosidase in order to remove N-linked oligosaccharides. All EPO samples showed the mobility shift towards low molecular weight forms lacking or containing O- glycan. A lower ratio of the O-glycosylated and nonglycosylated protein band was observed for the HES-modified EPO sample A2 after de-N-glycosylation and a diffuse protein band was detected around 30 KDa, presumably representing HES- modification at the sialic acid of O-glycan residue (see arrow marked by an asterisk). Figure 14b
SDS-PAGE analysis after mild hydrolysis of HES-modified EPO sample A2 (see Fig. 13), control EPO sample K2 and EPO-GT-1 A which were untreated or digested in the presence of N-glycosidase in order to remove N-linked oligosaccharides (see Figure 14a). Both high molecular weight form of A2 before and A after N.glycosidase treatment (see brackets with and without arrow) disappeared upon acid treatment of the samples. The BRP EPO standard which was run for comparison was not subjected to mild acid treatment.
Figure 15
HP AEC-P AD analysis of N-linked oligosaccharide material liberated from HES- modified sample A, from EPO-GT-1 -A and from a control EPO sample incubated with unmodified HES (K). Roman numbers I-V indicate the elution position of I = disialylated diantennary structure, II = τrisialylated triantennary structures (two isomers), III = tetrasialylated tetraantennary structure + 2 N-acetyllactosamine repeats, IV = tetrasialylated tetraantennary structure + 1 N-acetyllactosamine repeat, V = tetrasialylated tetraantennary structure + without N-acetyllactosamine repeat; brackets indicate the elution area of di-, tri- and tetrasialylated N-glycans as reported in the legends of Figs. 8 and 11.
Figure 16
HP AEC-P AD analysis of N-linked oligosaccharide material liberated from HES- modified sample A, from EPO-GT-1 A and from a control EPO sample (K) incubated with unmodified HES. The retention times of a mixture of standard oligosaccharides is shown: numbers 1-9 indicate the elution position of standard oligosaccharides: 1 = diantennary; 2 = triantennary (2-4 isomer); 3 = triantennary (2-6 isomer); 4 = tetraantennary; 5 = triantennary plus 1 repeat; 6 = tetraantennary plus 1 repeat; 7 = trianten- nary plus 2 repeats; 8 = tetraantennary plus 2 repeats and 9 = tetraantennary plus 3 repeats.
Figures 17 to 23
Figures 17 to 23 represent MALDI/TOF mass spectra of the enzymatically liberated and chemically desialylated N-glycans isolated from HES-modified EPO and control EPO preparations. Major signals at m/z 1809.7, 2174.8, 2539.9, 2905.0 and 3270.1 ([M+Na]4) correspond to di- to tetraantennary complex-type N-glycan structures with no, one or two N-acetyllactosamine repeats accompanied by weak signals due to loss of fucose or galactose which are due to acid hydrolysis conditions employed for the desialylation of samples for MS analysis.
Figure 17
MALDI/TOF spectrum: desialylated oligosaccharides of HES-modified EPO A2.
Figure 18
MALDI/TOF spectrum: desialylated oligosaccharides of EPO GT- 1 -A.
Figure 19
MALDI/TOF spectrum: desialylated oligosaccharides of EPO K2.
Figure 20
MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1.
Figure 21 MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1 subjected to acid hydrolysis for 5 min.
Figure 22
MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1 subjected to acid hydrolysis for 10 min.
Figure 23
MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1 subjected to acid hydrolysis for 60 min.
Figure 24
Figure 24 shows an SDS page analysis of two HES-EPO conjugates
mw: marker
Lane 1: HES-EPO produced according to example protocol 8: EPO is conjugated to hydrazido-HES 12KD L
Lane 2: HES-EPO produced according to example protocol 9 : EPO is conjugated to hydroxylamino HES 12 KD K C: control (unconjugated EPO); the upper band represents EPO dimer
Figure 25
Figure 2 demonstrates that the HES is conjugated to a carbohydrate moiety of a carbohydrate side chain by showing a digestion of HAS modified EPO forms with po- lyppetide N-glycosidase
Lane 1 : HES-EPO produced according to example protocol 8 after digestion with N-glycosidase Lane 2: HES-EPO produced according to example protocol 9 after digestion with
N-glycosidase Lane 3 : BRP EPO standard
Lane 4: BRP EPO standard after digestion with N-glycosidase mw: marker (Bio-Rad SDS-PAGE Standards Low range Catalog No 161-
0305, Bio-Rad Laboratories, Hercules, CA, USA)
Examples
Example 1: Formation of hydroxyethyl starch derivatives by reductive amination of the non-oxidised reducing end
Example 1.1 Reaction of hydroxyethyl starch with l,3-diamino-2-hydroxy propane
Figure imgf000078_0001
a) To a solution of 200 mg hydroxyethyl starch (HES 18/0.4 (MW = 18,000 D, DS=0.4)) in 5 ml water, 0.83 mmol l,3-diamino-2-hydroxy propane (Sigma
Aldrich, Taufkirchen, D) and 50 mg sodium cyanoborohydrate NaCNBH3 were added. The resulting mixture was incubated at 80 °C for 17 h. The reaction mixture was added to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The precipitate was collected by centrifiigation and dialysed for 4 d against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science
Deutschland GmbH, Bonn, D), and lyophilized.
b) Incubation of the mixture resulting from adding 0.83 mmol l,3-diamino-2- hydroxy propane and 50 mg sodium cyanoborohydrate NaCNBH3 to the solution of 200 mg hydroxyethyl starch was also possible and carried out at 25 °C for 3 d.
Example 1.2 Reaction of hydroxyethyl starch with l,2-dihydroxy-3-amino propane
Figure imgf000078_0002
a) To a solution of 200 mg hydroxyethyl starch (HES 18/0.4 (MW = 18,000 D, DS=0.4)) in 5 ml water, 0.83 mmol l,2-dihydroxy-3 -amino propane (Sigma Aldrich, Taufkirchen, D) and 50 mg sodium cyanoborohydrate NaCNBH3 were added. The resulting mixture was incubated at 80 °C for 17 h. The reaction mixture was added to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The precipitate was collected by centrifugation and dialysed for 4 d against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science
Deutschland GmbH, Bonn, D), and lyophilized.
b) Incubation of the mixture resulting from adding 0.83 mmol l,2-dihydroxy-3- amino propane and 50 mg sodium cyanoborohydrate NaCNBH3 to the solution of 200 mg hydroxyethyl starch was also possible and carried out at 25 °C for 3 d.
The reaction of l,2-dihydroxy-3 -amino propane with HES was confirmed indirectly by quantification of formaldehyde, resulting from the oxidative cleavage of the 1,2- diole in the reaction product by periodate as described by G. Avigad, Anal. Biochem. 134 (1983) 449-504.
Example 1.3 Reaction of hydroxyethyl starch with 1,4-diamino butane
,NH
H2N'
a) To a solution of 200 mg hydroxyethyl starch (HES 18/0.4 (MW = 18,000 D, DS=0.4)) in 5 ml water, 0.83 mmol 1,4-diamino butane (Sigma Aldrich, Taufkirchen, D) and 50 mg sodium cyanoborohydrate NaCNBH3 were added. The resulting mixture was incubated at 80 °C for 17 h. The reaction mixture was added to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The precipitate was collected by centrifugation and dialysed for 4 d against water
(SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, D), and lyophilized. b) Incubation of the mixture resulting from adding 0.83 mmol 1,4-diamino butane and 50 mg sodium cyanoborohydrate NaCNBH3 to the solution of 200 mg hydroxyethyl starch was also possible and carried out at 25 °C for 3 d.
Example 1.4 Reaction of hydroxyethyl starch with l-mercapto-2-amino ethane
,NH„
HS
a) To a solution of 200 mg hydroxyethyl starch (HES 18/0.4 (MW = 18,000 D, DS=0.4)) in 5 ml water, 0.83 mmol l-mercapto-2-amino ethane (Sigma Al- drich, Taufkirchen, D) and 50 mg sodium cyanoborohydrate NaCNBH3 were added. The resulting mixture was incubated at 80 °C for 17 h. The reaction mixture was added to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The precipitate was collected by centrifugation and dialysed for 4 d against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, D), and lyophilized.
b) Incubation of the mixture resulting from adding 0.83 mmol l-mercapto-2- amino ethane and 50 mg sodium cyanoborohydrate NaCNBH3 to the solution of 200 mg hydroxyethyl starch was also possible and carried out at 25 °C for 3 d.
Example 2: Formation of hydroxyethyl starch derivatives by conjugation with the non-oxidised reducing end
Example 2.1: Reaction of hydroxyethyl starch with carbohydrazide
Figure imgf000080_0001
0.96 g of HES18/0.4 (MW = 18,000 D, DS=0.4) were dissolved in 8 ml aqueous 0.1 M sodium acetate buffer, pH 5.2, and 8 mmol carbohydrazide (Sigma Aldrich, Taufkirchen, D) were added. After stirring for 18 h at 25 °C, the reaction mixture was added to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The precipitated product was collected by centrifugation, re-dissolved in 40 ml water, and dialysed for 3 d against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, D), and lyophilized.
Example 2.2: Reaction of hydroxyethyl starch with adepic dihydrazide
Figure imgf000081_0001
0.96 g of HES18/0.4 (MW = 18,000 D, DS=0.4) were dissolved in 8 ml aqueous 0.1 M sodium acetate buffer, pH 5.2, and 8 mmol adepic dihydrazide (Lancaster Synthesis, Fran-kfiirt/Main, D) were added. After stirring for 18 h at 25 °C, the reaction mixture was added to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The precipitated product was collected by centrifugation, re-dissolved in 40 ml water,. and dialysed for 3 d against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Sci- ence Deutschland GmbH, Bonn, D), and lyophilized.
Example 2.3: Reaction of hydroxyethyl starch with l,4-phenylene-bis-3- thiosemicarbazide
Figure imgf000081_0002
0.96 g of HES18/0.4 (MW = 18,000 D, DS=0.4) were dissolved in 8 ml aqueous 0.1 M sodium acetate buffer, pH 5.2, and 8 mmol ls4-phenylene-bis-3-thiosemicarbazide (Lancaster Synthesis, Fran-kfiirt/Main, D) were added. After stirring for 18 h at 25 °C, 8 ml water was added to the reaction mixture, and the suspension was centrifu- gated for 15 min at 4,500 rpm. The clear supernatant was decanted and subsequently added to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The precipitated product was collected by centrifugation, re-dissolved in 40 ml water, and centrifu- gated for 15 min at 4,500 rpm. The clear supernatant was dialysed for 3 d against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, D), and lyophilized.
Example 2.4: Reaction of hydroxyethyl starch with O-[2-(2-aminooxy- ethoxy)-ethyl] -hydroxyl amine
X /-\ /\ ^O,
H2N N
O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine was synthesized as described in Boturyn et al. Tetrahedron 53 (1997) p. 5485-5492 in 2 steps from commercially available materials.
0.96 g of HES 18/0.4 (MW = 18,000 D, DS=0.4) were dissolved in 8 ml aqueous 0.1 M sodium acetate buffer, pH 5.2, and 8 mmol O-[2-(2-aminooxy-ethoxy)-ethyl]- hydroxyl amine were added. After stirring for 18 h at 25 °C, the reaction mixture was added to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The precipitated product was collected by centrifugation, re-dissolved in 40 ml water, and dialysed for 3 d against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, D), and lyophilized.
Example 3 Formation of hydroxyethyl starch derivatives by reaction with the oxidised reducing end
Example 3.1 Reaction of hydroxyethyl starch with carbohydrazide
Figure imgf000082_0001
0.12 mmol Oxo-HES 10/0.4 (MW = 10,000 D, DS=0.4, prepared according to DE 196 28 705 Al) were dissolved in 3 ml absolute dimethyl sulfoxide (DMSO) and added dropwise under nitrogen to a mixture of 15 mmol of carbohydrazide (Sigma Aldrich, Taufkirchen, D) in 15 ml DMSO. After stirring for 88 h at 65 °C, the reac- tion mixture was added to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The precipitate was collected by centrifugation and was dialysed for 4 d against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, D) and lyophilized.
Example 3.2 Reaction of hydroxyethyl starch with l,4-phenylene-bis-3- thiosemicarbazide
Figure imgf000083_0001
0.12 mmol Oxo-HES 10/0.4 (MW = 10,000 D, DS=0.4, prepared according to DE 196 28 705 Al) were dissolved in 3 ml absolute dimethyl sulfoxide (DMSO) and added dropwise under nitrogen to a mixture of 15 mmol of l,4-phenylene-bis-3- thiosemicarbazide (Lancaster Synthesis, Frankfurt/Main, D) in 15 ml DMSO. After stirring for 88 h at 65 °C, the reaction mixture was added to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The precipitate was collected by centrifugation and was dialysed for 4 d against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, D) and lyophilized.
Example 3.3 Reaction of hydroxyethyl starch with hydrazine
H2N-NH2
1,44 g (0.12 mmol) of Oxo-HES 10/0.4 (MW = 10,000 D, DS=0.4, prepared accord- ing to DE 196 28 705 Al) were dissolved in 3 ml absolute dimethyl sulfoxide
(DMSO) and were added dropwise under nitrogen to a mixture of 0.47 ml (15 mmol) hydrazine in 15 ml DMSO. After stirring for 19 h at 40°C the reaction mixture was added to 160 ml of a 1:1 mixture of ethanol and acetone (v/v). The precipitated product was collected by centrifugation, redissolved in 40 mL of water and dialysed for 2 days against a 0.5 % (v/v) triethylamine in water solution and for 2 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized.
Example 3.4 Reaction of hydroxyethyl starch with hydroxylamine
,
H2N'
O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine was synthesized as described by Boturyn et al in 2 steps from commercially available materials (Boturyn, Boudali, Constant, Defrancq, Lhomme, 1997, Tetrahedron, 53, 5485).
1.44 g (0.12 mmol) of Oxo-HES 10/0.4 (MW = 10,000 D, DS=0.4, prepared according to DE 196 28 705 Al) were dissolved in 3 ml absolute dimethyl sulfoxide (DMSO) and were added dropwise under nitrogen to a mixture of 2.04 g (15 mmol) O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine in 15 ml DMSO. After stirring for 48 h at 65°C the reaction mixture was added to 160 ml of a 1:1 mixture of ethanol and acetone (v/v). The precipitated product was collected by centrifugation, redissolved in 40 ml of water and dialysed for 4 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized.
Example 3.5 Reaction of hydroxyethyl starch with adepic dihydrazide
Figure imgf000084_0001
1.74 g (15 mmol) adepic dihydrazide (Lancaster Synthesis, Fran-kfiirt/Main, D) were dissolved in 20 ml absolute dimethyl sulfoxide (DMSO) at 65°C and 1.44 g (0,12 mmol) of Oxo-HES 10/0.4 (MW = 10,000 D, DS=0.4, prepared according to DE 196 28 705 Al), dissolved in 3 ml absolute DMSO were added dropwise under nitrogen. After stirring for 68 h at 60°C the reaction mixture was added to 200 ml of water The solution containing the reaction product was dialysed for 2 days against a 0.5 % (v/v) triethylamine in water solution and for 2 days against water (SnakeSkin dialysis tub- ing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized.
Example 3.6 Reaction of hydroxyethyl starch with 1,4-diamino butane
.NH
H2N'
1.44 g (0.12 mmol) of Oxo-HES 10/0.4 (MW = 10,000 D, DS=0.4, prepared according, to DE 196 28 705 Al) were dissolved in 3 ml dry dimethyl sulfoxide (DMSO) and were added dropwise under nitrogen to a mixture of 1.51 ml (15 mmol) 1,4- diaminobutane (Sigma Aldrich, Taufkirchen, D) in 15 ml DMSO. After stirring for 19 h at 40°C the reaction mixture was added to 160 ml of a 1:1 mixture of ethanol and acetone (v/v). The precipitate Amino-HES10KD/0.4 was collected by centrifugation, redissolved in 40 ml of water and dialysed for 4 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized.
Example 4 Oxidation of erythropoietin
Oxidized erythropoietin was produced as described in Example 8. As oxidised e-rythropoietin, EPO-GT-1 -A as described in Example 8.11(c) was used (EPO-GT-1 without acid hydroylsis, treated with mild periodate oxidation).
Example 5: Conjugation of hydroxyethyl starch derivatives with oxidized erythropoietin of example 4 Example 5.1 Reaction of oxidized erythropoietin with the reaction product of example 2.1
Oxidized EPO (1.055 μg/μl) in 20 mM PBS buffer was adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2. To 19 μl of the EPO solution, 18 μl of a solution of the HES derivate as produced according to example 2.1 (MW 18 kD; 18.7 μg/μl in 0.1 M sodium acetate buffer, pH 5.2) was added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analyzed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig. 1. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased band- width is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 5.2 Reaction of oxidized erythropoietin with the reaction product of example 2.3
Oxidized EPO (1.055 μg/μl) in 20 mM PBS buffer was adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2. To 19 μl of the EPO solution, 18 μl of a solution of the HES derivate as produced according to example 2.3 (MW 18 kD; 18.7 μg/μl in 0.1 M sodium acetate buffer, pH 5.2) was added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analyzed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen.
Example 5.3 Reaction of oxidized erythropoietin with the reaction product of example 2.4 Oxidized EPO (1.055 μg/μl) in 20 mM PBS buffer was adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2. To 19 μl of the EPO solution, 18 μl of a solution of the HES derivate as produced according to example 2.4 (MW 18 kD; 18.7 μg/μl in 0.1 M sodium acetate buffer, pH 5.2) was added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analyzed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig. 2. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 5.4 Reaction of oxidized erythropoietin with the reaction product of example 3.1
Oxidized EPO (1.055 μg/μl) in 20 mM PBS buffer was adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2. To 19 μl of the EPO solution, 18 μl of a solution of the HES derivate as produced according to example 3.1 (MW 10 kD; 18.7 μg/μl in 0.1 M sodium acetate buffer, pH 5.2) was added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analyzed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig. 3. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein. Example 5.5 Reaction of oxidized erythropoietin with the reaction product of example 3.2
Oxidized EPO (1.055 μg/μl) in 20 mM PBS buffer was adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2. To 19 μl of the EPO solution, 18 μl of a solution of the HES derivate as produced according to example 3.1 (MW 10 kD; 18.7 μg/μl in 0.1 M sodium acetate buffer, pH 5.2) was added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analyzed by SDS-Page with NuPAGE 10% Bis-Tris Gels MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig. 3. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased band- width is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 6 Formation of Thio-EPO by reduction of erythropoietin
241.5 μg erythropoietin (EPO-GT-1, see Example 8) in 500 μl of a 0.1 M sodium borate buffer, 5 mM EDTA, 10 mM DTT (Lancaster, Morcambe, UK), pH 8.3, were incubated for 1 h at 37 °C. The DTT was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 10 KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, subsequent washing 3 times with the borate buffer and twice with a phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2). The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
Example 7: Conjugation of hydroxyethyl starch derivatives with thio- erythropoietin using a crosslinking compound In each of the following examples, N-(alpha-maleimidoacetoxy) succinimide ester (AMAS)
Figure imgf000089_0001
was used as crosslinking compound.
Example 7.1 Reaction of thio-erythropoietin with the reaction product of example 2.1 and the crosslinking compound
To 50 nmol HES derivate as produced according to example 2.1 and dissolved in 200 μl of a 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO were added. The clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C. Remaining AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30 min with the phosphate buffer.
To the residual solution, 15 μg of ThioEPO as produced according to example 5 (1 μg/μl in phosphate buffer) were added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig. 4. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased band- width is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein. Example 7.2 Reaction of thio-erythropoietin with the reaction product of example 2.2 and the crosslinking compound
To 50 nmol_HES derivate as produced according to example 2.2 and dissolved in 200 μl of a 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO were added. The clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C. Remaining AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30 min with the phosphate buffer.
To the residual solution, 15 μg of ThioEPO as produced according to example 5 (1 μg/μl in phosphate buffer) were added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig 5. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 7.3 Reaction of thio-erythropoietin with the reaction product of example 2.3 and the crosslinking compound
To 50 nmol HES derivate as produced according to example 2.3 and dissolved in 200 μl of a 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO were added. The clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C. Remaining AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30 min with the phosphate buffer.
To the residual solution, 15 μg of ThioEPO as produced according to example 5 (1 μg/μl in phosphate buffer) were added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig. 5. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 7.4 Reaction of thio-erythropoietin with the reaction product of example 2.4 and the crosslinking compound
To 50 nmol HES derivate as produced according to example 2.4 and dissolved in 200 μl of a 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO were added. The clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C. Remaining AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30 min with the phosphate buffer.
To the residual solution, 15 μg of ThioEPO as produced according to example 5 (1 μg/μl in phosphate buffer) were added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight. The experimental result is shown in Fig 4. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 7.5 Reaction of thio-erythropoietin with the reaction product of example 1.1 and the crosslinking compound
To 50 nmol HES derivate as produced according to example 1.1 , at incubation conditions of 80 °C and 17 h as well as of 25 °C and 3 d, and dissolved in 200 μl of a 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO were added. The clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C. Remain- ing AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30 min with the phosphate buffer.
- To the residual solution, 15 μg of ThioEPO as produced according to example 5 (1 μg/μl in phosphate buffer) were added,- and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig. 5. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 7.6 Reaction of thio-erythropoietin with the reaction product of example 1.3 and the crosslinking compound To 50 nmol HES derivate as produced according to example 1.3, at incubation conditions of 80 °C and 17 h as well as of 25 °C and 3 d, and dissolved in 200 μl of a 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO were added. The clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C. Remaining AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30 min with the phosphate buffer.
To the residual solution, 15 μg of ThioEPO as produced according to example 5 (1 μg/μl in phosphate buffer) were added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig 5. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased band- width is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 7.7 Reaction of thio-erythropoietin with the reaction product of example 3.1 and the crosslinking compound
To 50 nmol HES derivate, produced according to Example 3.1 and dissolved in 200 μl phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO was added, and the clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C. The AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, Germany) at 13,000 rpm and washing 4 times for 30 min with the phosphate buffer. To the residual solution, 15 μg Thio-EPO (1 μg/μl in phosphate buffer) were added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analysed by SDS-Page with NuPAGE 10 % Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig 6. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 7.8 Reaction of thio-erythropoietin with the reaction product of example 3.2 and the crosslinking compound
To 50 nmol HES derivate, produced according to Example 3.2 and dissolved in 200 μl phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO was added, and the clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C. The AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, Germany) at 13,000 rpm and washing 4 times for 30 min with the phosphate buffer.
To the residual solution, 15 μg Thio-EPO (1 μg/μl in phosphate buffer) were added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analysed by SDS-Page with NuPAGE 10 % Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight. The experimental result is shown in Fig 6. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 7.9 Reaction of thio-erythropoietin with the reaction product of example 3.3 and the crosslinking compound
To 50 nmol HES derivate, produced according to Example 3.3 and dissolved in 200 μl phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO was added, and the clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C. The AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, Germany) at 13,000 rpm and washing 4 times for 30 min with the phosphate buffer.
To the residual solution, 15 μg Thio-EPO (1 μg/μl in phosphate buffer) were added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analysed by SDS-Page with NuPAGE 10 % Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig 6. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 7.10 Reaction of thio-erythropoietin with the reaction product of example 3.4 and the crosslinking compound To 50 nmol HES derivate, produced according to Example 3.4 and dissolved in 200 μl phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO was added, and the clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C. The AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, Germany) at 13,000 rpm and washing 4 times for 30 min with the phosphate buffer.
To the residual solution, 15 μg Thio-EPO (1 μg/μl in phosphate buffer) were added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analysed by SDS-Page with NuPAGE 10 % Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig 6. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 7.11 Reaction of thio-erythropoietin with the reaction product of example 3.5 and the crosslinking compound
To 50 nmol HES derivate, produced according to Example 3.5 and dissolved in 200 μl phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO was added, and the clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C. The AMAS was removed by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, Germany) at 13,000 rpm and washing 4 times for 30 min with the phosphate buffer. To the residual solution, 15 μg Thio-EPO (1 μg/μl in phosphate buffer) were added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analysed by SDS-Page with NuPAGE 10 % Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig 6. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased band- width is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 7.12 Reaction of thio-erythropoietin with the reaction product of example 3.6 and the crosslinking compound
To 50 nmol HES derivate, produced according to Example 36 and dissolved in 200 μl phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in. DMSO was added, and the clear solution was incubated for 80 min at 25 °C and 20 min at 40 °C. The AMAS was removed by centrifiigal filtration with a VINASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, Germany) at 13,000 rpm and washing 4 times for 30 min with the phosphate buffer.
To the residual solution, 15 μg Thio-EPO (1 μg/μl in phosphate buffer) were added, and the mixture was incubated for 16 h at 25 °C. After lyophilisation, the crude product was analysed by SDS-Page with NuPAGE 10 % Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions given by Invitrogen. The gel is stained with Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
The experimental result is shown in Fig 6. A successful conjugation is indicated by the migration of the protein band to higher molecular weights. The increased band- width is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
Example 8 Preparative production of HES-EPO conjugates
Summary
HES-EPO conjugates were synthesized by coupling of HES derivatives (average mw of 18,000 Dalton; hydroxyethyl substitution degree of 0.4) to the partially (mild periodate) oxidized sialic acid residues on the oligosaccharide chains of recombinant human EPO. Based on carbohydrate structural analysis the modifications introduced did not affect the structural integrity of the core oligosaccharide chains since MALDI/TOF-MS of the mild acid treated HES-modified glycans revealed intact neutral N-acetyllactosamine-type chains which were indistinguishable from those observed in unmodified EPO product. The results obtained indicate that at least 3 modified HES-residues are attached per EPO molecule in the case of the EPO preparation which was subjected to modification without prior partial sialic acid removal. An EPO variant lacking about 50% of the sialic acid residues of the former protein showed a similar apparent high molecular weight mobility in SDS-PAGE (60-110 KDa vs 40 KDa for the BRP EPO standard). The HES modified EPO is stable under standard ion-exchange chromatography conditions at room temperature at pH 3-10.
The EPO-bioassay in the normocythaemic mouse system indicates that the HES- modified EPO has 2.5-3.0 fold higher specific activity (IU/mg) in this assay when compared to the International BRP EPO reference standard based on protein determination using the UN absorption value from the European Pharmacopeia and an RP-HPLC EPO protein determination method calibrated against the BRP EPO standard preparation.
Example 8.1 Materials and methods (a) Liberation of N-linked oligosaccharides by digestion with N-glycosidase
Samples were incubated with 25 units (according to manufacturer's specification, Roche Diagnostics, Germany) of recombinant PNGase F over night at 37°C. Com- plete digestion was monitored by the specific mobility shift of the protein in SDS- PAGE. The released N-glycans were separated from the polypeptide by addition of 3 volumes of cold 100% ethanol and incubation at -20°C for at least 2 hours (Schroeter S et al., 1999). The precipitated protein was removed by centrifugation for 10 minutes at 4°C at 13000 rpm. The pellet was then subjected to two additional washes with 500 μl of ice-cold 75% ethanol. The oligosaccharides in the pooled supematants were dried in a vacuum centrifuge (Speed Vac concentrator, Savant Instruments Inc., USA). The glycan samples were desalted using Hypercarb cartridges (25 mg or 100 mg of HyperCarb) as follows prior to use: the columns were washed with 3 x 500 μl of 80% acetonitrile (v/v) in 0.1% TFA followed by washes with 3 x 500 μl of water. The samples were diluted with water to a final volume of 300 μl — 600 μl before loading onto the cartridge which then was rigorously washed with water. Oligosaccharides were eluted with 1.2 ml (25 mg cartridges; 1.8 ml in the case of 100 mg cartridges) 25% acetonitrile in water containing 0.1% trifluoroacetic acid (v/v). The eluted oligosaccharides were neutralized with 2 M NH OH and were dried in a Speed Vac concentrator. In some cases desalting of N-glycosidase released oligosaccharides was performed by adsorption of the digestion mixture from samples < 100 μg of total (glyco)protein onto 100 mg Hypercarb cartridges.
(b) Analysis of oligosaccharides by matrix-assisted laser desorption/ ionization time-of-flight mass-spectrometry (MALDI TOF/TOF-MS)
A Broker ULTRAFLEX time-of-flight (TOF/TOF) instrument was used: native desialylated oligosaccharides were analyzed using 2,5-dihydroxybenzoic acid as UN- absorbing material in the positive as well as in the negative ion mode using the re- flectron in both cases. For MS-MS analyses, selected parent ions were subjected to laser induced dissociation (LID) and the resulting fragment ions separated by the second TOF stage (LIFT) of the instrument. Sample solutions of 1 μl and an ap- proximate concentration of 1-10 p ol-μl'1 were mixed with equal amounts of the respective matrix. This mixture was spotted onto a stainless steel target and dried at room temperature before analysis.
Example 8.2 Preparation and characterization of recombinant human EPO
(EPO-GT-1)
EPO was expressed from recombinant CHO cells as described (Mueller PP et al., 1999, Dorner AJ et al., 1984) and the preparations were characterized according to methods described in the Eur. Phar. (Ph. Eur. 4, Monography 01/2002:1316: Erythropoietin concentrated solution). The final product had a sialic acid content of 12 nMol (+/- 1.5 nMol) per nMol of protein. The structures of N-linked oligosaccharides were determined by HPAEC-PAD and by MALDI/TOF-MS as described (Nimtz et al., 1999, Grabenhorst, 1999). The EPO preparations that were obtained contained di-, tri- and tetrasialylated oligosaccharides (2-12%, 15-28% and 60-80%, respectively, sulphated and pentasialylated chains were present in small amounts). The overall glycosylation characteristics of EPO preparations were similar to that of the international BRP EPO standard preparation.
The isoelectric focusing pattern of the recombinant EPO was comparable to that of the international BRP Reference EPO standard preparation showing the corresponding isoforms. 25% of the EPO protein lacked O-glycosylation at Ser1 6 of the polypeptide chain.
Example 8.3 Preparation of partially desialylated EPO forms
EPO GT-1 protein (2.84 mg/ml) was heated to 80°C in 20 mM Na-phosphate buffer pH 7.0 and then 100 μl of 1 N H SO was added per 1 ml of the EPO solution; incubation was continued for 5 min, 10 min and 60 min, respectively, yielding EPO preparations of different degree of sialylation. Quantitation of oligosaccharides with 0-4 sialic acids was performed after liberation of oligosaccharides with polypeptide N-glycosidase and isolation of N-linked chains was performed by desalting using Hypercarb cartridges (25 mg HyperSep Hypercarb; ThermoHypersil-Keystone, UK). EPO preparations were neutralized by addition of 1 N NaOH and were frozen in liquid N2 and were stored at -20°C until further use.
Example 8.4 Periodate oxidation of sialylated EPO forms
To 10 mg of untreated or mild acid treated EPO dissolved in 3.5 ml of 20 mM Na- phosphate buffer pH 7.0 was added 1.5 ml of 0.1 M Na-acetate buffer pH 5.5 and the mixture was cooled to 0°C in an ice-bath; 500 μl of 10 mM Na-periodate was added and the reaction mixture was kept in the dark for 60 min at 0°C. Then 10 μl of glycerol was added and incubation was continued for further 10 min in the dark. The partially oxidized EPO forms were separated from reagents by desalting using VIVASPIN concentrators (10,000 MWCO, PES Vivascience AG, Hannover, Germany) according to manufacturer's recommendation at 3000 rpm in a laboratory cen- trifuge equipped with a fixed angle rotor. After freezing in liquid mtrogen the EPO preparations were stored in a final volume of 4 ml at -20°C.
100 μg aliquots of the partially oxidized EPO preparation were subjected to N- glycosidase treatment and oligosaccharides were isolated using Hypercarb cartridges as described. Oligosaccharides were desialylated by mild acid treatment and were analyzed by HP AEC-P AD and their retention times were compared to those of authentic standard oligosaccharides as described (Nimtz et al., 1990 and 1993).
Example 8.5 Reduction of EPO disulfides with dithioerythreitol
5 mg of EPO-GT-1 was incubated in 5 ml of 0.1 M Tris/HCl buffer pH 8.1 in the presence of 30 mM dithioerythreitol (DTT) at 37°C for 60 minutes; removal of DTT was achieved by using a Vivaspin concentrator at 4 °C, 4 cycles of buffer exchange. The final reduced EPO preparation was frozen in liquid nitrogen and stored at -20°C in 50 mM Na-acetate buffer pH 5.5.
Example 8.6 EPO protein determination Quantitative determination of EPO protein was performed by measuring UV absorption at 280 nm according to the Eur. Phar. (European Pharmacopeia 4, Monography 01/2002: 1316: erythropoietin concentrated solution) in a cuvette with 1 cm path length. In addition, EPO was quantitated by applying a RP-HPLC method using a RP-C4 column (Vydac Protein C4, Cat 214TP5410, Grace Nydac, Ca, US); the HPLC method was calibrated using the erythropoietin BRP 1 reference standard (European Pharmacopeia, Conseil de l'Europe B.P. 907-F67029, Strasbourg Cedex
1).
Example 8.7 Oxidation of desialylated EPO with galactose oxidase
4.485 mg of completely desialylated EPO was incubated in 20 mM Νa-phosphate buffer pH 6.8 in the presence of 16 μl catalase (6214 units/200 ml) and 80 μl of ga- lactose oxidase (2250 units/ml from Dactylium dendroides (Sigma-Aldrich, Stein- heim, Germany); incubation at 37°C was over night; 2 times 20 μl of galactose oxidase was added after 4 hours and after 8 hours after starting of the incubation.
Example 8.8 Preparation of EPO samples for bioassays
Purification of EPO from incubations of periodate- or galactose-oxidase-oxidized EPO protein preparations with activated HES
Purification of EPO samples (removal of unreacted HES derivatives) was carried out at room temperature. The EPO incubation mixtures (approximately 5 mg of EPO protein) were diluted 1:10 with buffer A (20 mM Ν-morpholine propane sulfonic acid [MOPS/ΝaOH] in H2O bidest, pH 8.0) and were applied to a column containing 3 ml Q-Sepharose HP (Pharmacia Code no. 17-1014-03, Lot no. 220211) equilibrated with 10 column volumes (CN) of buffer A by using a flow rate of 0.5 ml/min. The column was. washed with 6-8 CV of buffer A (flow rate = 0.8 ml/min) and elution was performed by using buffer B (20 mM morpholine ethane sulfonic acid [MES/ΝaOH], 0.5 M ΝaCl in H2O bidest, pH 6.5) at a flow rate of 0.5 ml/min. EPO was detected by UV absorption at 280 nm and eluted in about 6 ml. The column was regenerated by using 3 CV of buffer C (20 mM MES, 1.5 M NaCl in H2O adjusted to pH 6.5) and was re-equilibrated by using 10 CV of buffer A (flow rate = 0.7 ml/min).
Buffer exchange of EPO eluates obtained from the Q-Sepharose step was performed using Vivaspin concentrators and phosphate buffered saline (PBS) with each 3 centrifugation cycles per sample; samples were adjusted to 2 ml with PBS and were stored at -20°C.
Only <25% of the partially desialylated and subsequently mild periodate oxidized EPO forms that were subjected to HES-modification were obtained from the Q- Sepharose eluate since under the conditions employed the basic EPO forms did not bind Q-Sepharose and were found in the flow-through together with nonreacted HES derivatives.
Example 8.9 High-pH anion-exchange chromatography with pulsed am- perometrϊc detection (HPAEC-PAD)
Purified native and desialylated oligosaccharides were analyzed by high-pH anion- exchange (HPAE) chromatography using a Dionex BioLC system (Dionex, USA) equipped with a CarboPac PAl column (0.4 x 25 cm) in combination with a pulsed amperometric detector (PAD) (Schrδter et al., 1999; Ni tz et al., 1999). Detector potentials (E) and pulse durations (T) were: El: +50 mV, TI: 480 ms; E2: +500 mV,
T2: 120 ms; E3: -500 mV, T3: 60 ms, and the output range was 500-1500 nA. The oligosaccharides were then injected onto the CarboPac PAl column which was equilibrated with 100% solvent A. For desialylated oligosaccharides elution (flow rate: 1 ml-min"1) was performed by applying a linear gradient (0-20%) of solvent B over a period of 40 min followed by a linear increase from 20-100% solvent B over 5 min. Solvent A was 0.2 M NaOH in bidistilled H2O, solvent B consisted of 0.6 M NaOAc in solvent A. For native oligosaccharides the column was equilibrated with
100% solvent C (0.1 M NaOH in bidistilled H2O) and elution (flow rate: 1 ml-min"1) was performed by applying a linear gradient (0-35%) of solvent D over a period of 48 min followed by a linear increase from 35-100% solvent D over 10 min. Solvent D consisted of 0.6 M NaAc in solvent C.
Example 8.10 Monosaccharide compositional analysis of N-glycans, HES- modified N-glycans and EPO protein by GC-MS
Monosaccharides were analyzed as the corresponding methyl glycosides after methanolysis, N-reacetylation and trimethylsilylation by GC/MS [Chaplin, M.F. (1982) A rapid and sensitive method for the analysis of carbohydrate. Anal. Biochem. 123, 336-341]. The analyses were performed on a Finnigan GCQ ion trap mass spectrometer (Finnigan MAT corp., San Jose, CA) running in the positive ion El mode equipped with a 30 m DB5 capillary column. Temperature program: 2 min isotherm at 80°C, then 10 degrees min"1 to 300°C.
Monosaccharides were identified by their retention time and characteristic fragmentation pattern. The uncorrected results of electronic peak integration were used for quantification. Monosaccharides yielding more than one peak due to anomericity and/or the presence of furanoid and pyranoid forms were quantified by adding all major peaks. 0.5 μg of myo-inositol was used as an internal standard compound.
Example 8.11 Results
Example 8.11(a) Characterization of Ν-glycans of mild acid treated (partially desialylated) EPO-GT-1
EPO-GT-1 preparations subjected to mild acid treatment for 5, 10 or 60 min. were analyzed by SDS-PAGE before and after liberation of Ν-linked oligosaccharides by incubation with Ν-glycosidase as shown in Figure 7. Ν-linked. oligosaccharides were subjected to HP AEC-P AD oligosaccharide mapping (Figure 8). The untreated EPO- GT-1 contained >90% of Ν-linked oligosaccharides with 3 or 4 sialic acid residues whereas after 5 min. of incubation in the presence of mild acid <40% of carbohydrate chains had 3 or 4 sialic acid residues. HP AEC-P AD of the desialylated Ν-glycans revealed that the ratio of neutral oligosaccharides that were detected for the untreated EPO-GT-1 and remained stable in the preparations subjected to acid treatment for 5, 10 or 60 min. MALDI/TOF-MS of the desialylated glycans revealed that <90% of the proximal fucose was present after mild acid treatment of the protein.
Example 8.11(b) Characterization of periodate treated EPO-GT-1
SDS-PAGE mobility of mild periodate treated EPO forms that were previously subjected to a 5 and 10 minute treatment with acid or were not treated are compared in Figure 10. The conditions used for periodate oxidation of sialic acids did not change the SDS-PAGE pattern of EPO preparations (compare Fig. 7). Oxidation of sialic acids resulted in HP AEC-P AD analysis a shift of oligosaccharides to earlier elution times (compare Figures 8 and 11).
Example 8.11(c) Characterization of HES-modified EPO derivatives
(aa) Time course of HES modification of EPO-GT-1-A with hydroxylamine- modified HES derivative X, produced according to Example 2.4
400 μg of hydroxylamine-modified HES derivative X was added to 20 μg of EPO- GT-1 -A (mild periodate oxidized EPO, not acid hydrolyzed prior to mild periodate oxidation) in 20 μ of 0.5 M NaOAc buffer pH 5.5 and the reaction was stopped after 30 min, 2, 4, and 17 hours, respectively, by freezing samples in liquid nitrogen. Subsequently samples were stored at -20°C until further analysis.
SDS-PAGE sample buffer was added and the samples were heated to 90°C and applied onto SDS-gels. As shown in Figure 12, increasing incubation times resulted in an increased shift towards higher molecular weight of the protein. After 17 hours of incubation in the presence of the hydroxylamine-modified HES derivative X a dif- fuse Coomassie stained protein band was detected migrating in an area between 60 and 11 KDa, based on the position of molecular weight standards (see left part of Fig. 12). Upon treatment with N-glycosidase most of the protein was shifted towards the position of de-N-glycosylated EPO (see Fig. 12, right gel; arrow A indicates migration position of Ν-glycosidase, arrow B indicates migration position of de-N- glycosylated EPO; the diffuse protein band visible in the region between the 28 KDa and 36 KDa molecular weight standards presumably represents EPO-forms which are modified by HES and the O-glycosylation site of the molecule. In view of the specificity of Ν-glycosidase we conclude from this result that in fact HES- modification occurs at the periodate oxidized sialic acid residues of glycans of the EPO protein.
(bb) Characterization of HES-EPO conjugates
HES-EPO conjugates I (originating from EPO-GT-1 after mild periodate oxidation, i.e. from EPO-GT-1-A), II (resulting from EPO-GT-1 subjected to 5 min acid hydrolysis and mild periodate oxidation), III (resulting from EPO-GT-1 subjected to 10 min acid hydrolysis and mild periodate oxidation) were synthesized as described before. A control incubation (K) was included containing unmodified EPO-GT-1 under the same buffer conditions to which an equivalent amount of unmodified HES was added. The incubation mixtures were subjected to further purification for subsequent biochemical analysis of the HES-EPO derivatives.
Incubations HES-EPO conjugates I, II and III as well as the control incubation K were subjected to a Q-Sepharose purification step as described under "Material and Methods" (Example 8.8) in order to remove the excess of nonreacted HES-reagent which was expected in flow through of the ion-exchange column. Due to the high amounts of basic EPO forms contained in previously acid treated samples II and III we expected considerable amounts of modified EPO product from these incubations in the flow through. As is shown in Figure 13, almost all of the EPO material from samples I was retained by Q-Sepharose column whereas only approximately 20-30% of the samples III and II was recovered in the fraction eluting with high salt concen- tration. All of the protein material from the incubations with HES derivative X, both in the flow-through and the fractions eluting with high salt, had apparent higher molecular weight in SDS-PAGE when compared to the control EPO. In order to characterize in more detail the HES-modified EPO sample A and K (see Figure 11) were compared to periodate oxidized form EPO-GT-1 -A. The samples were subjected to N-glycosidase treatment and as is depicted in Figures 14a and 14b the release of N-glycans resulted in the two low molecular weight bands at the position of the O-glycosylated and nonglycosylated EPO forms of the standard EPO preparation. In the case of sample A a further band migrating at the position of the 28 KDa mw standard was detected suggesting HES-modification at the O-glycan of this EPO variant (cf. Example 8-ll(c)(aa)). This band (and also the heavily HES- modified high mw form of N-glycosylated EPO, see Figs. 14a and 14b) disappeared after subjecting the samples to mild hydrolysis which is in agreement with the view that HES modification was achieved at the periodate oxidised sialic acid residues of erythropoietin.
Aliquots of the N-glycosidase incubation mixtures were hydrolyzed using conditions enabling the complete removal of sialic acids residues (and also the sialic acid linked HES derivative) from oligosaccharides; after neutralization, the mixtures were then absorbed onto small Hypercarb columns for their desalting. The columns were washed rigorously with water followed by elution of bound neutral oligosaccharides with 40% acetonitrile in H2O containing 0.1% of trifuloacetic acid. The resulting oligosaccharides were subjected to MALDI/TOF-MS. The spectra of the desialylated oligosaccharide fractions from sample A, EPO-GT-1 -A and sample K showed identical masses for complex type oligosaccharides at m z = 1810 Da (diantennary), 2175 = triantennary, 2540 = tetraantennary, 2906 = tetraantennary plus 1 N- acetyllactosamine repeat and 3271 = tetraantennary plus 2 N-acetyllactosamine repeats; small signals corresponding to lack of fucose (-146) and galactose (minus 162) were detected which are attributable to the acid hydrolysis conditions applied for sialic acid removal (see MALDI-Figures 17, 18 and 19).
In a parallel experiment the N-glycosidase digestion mixture was absorbed onto 1 ml RP-C18 cartridge (without prior acid hydrolysis of oligosaccharides) and elution was performed with 5% acetomtrile in water containing 0.1% TFA; under these condi- tions the EPO protein was completely retained onto the RP -material and oligosaccharides were washed off from the column with 5% acetonitrile in H2O containing 0.1% TFA. The de-N-glycosylated EPO protein was eluted with 70% acetonitrile in H O containing 0.1% TFA. The oligosaccharide fractions from the RP-C18 step of Ν- glycosidase-treated sample A, EPO GT-1 -A and sample K were neutralized and subjected to desalting using Hypercarb cartridges as described before. The isolated oligosaccharides were subjected to HP AEC-P AD mapping before (see Figures 15) and after mild acid treatment under conditions which enabled quantitative removal of sialic acids from glycans (see Figures 16).
The HP AEC-P AD profile for the native material obtained from the HES-modified sample A showed only neglectable signals for oligosaccharides whereas EPO GT-1- A-derived oligosaccharides exhibited the same glycan profile as the one shown hi Fig. 11 (sample named EPO-GT-1 after mild periodate treatment). The elution pro- file of oligosaccharides obtained from the control EPO sample ( ) yielded the expected pattern (compare profile in Figure 8). For comparison, the native oligosaccharide profile of the international BRP -EPO standard is included for comparison and as reference standard.
After mild acid hydrolysis, all oligosaccharide preparations showed an identical elution profile of neutral oligosaccharide structures (see Figures 16) with the expected qualitative and quantitative compositon of di-, tri- and tetraantennary complex-type carbohydrate chains as described in the methods section for the EPO preparation which was used as a starting material in the present study. This result demonstrates that the HES-modification of the EPO sample results in a covalent linkage of the HES derivative which is detached from the EPO-protein by Ν-glycosidase and is acid-labile since it is removed from the Ν-glycans using mild acid treatment conditions known to desialylate carbohydrates (see Figures 14a+b).
(cc) Monosaccharide compositional analysis of HES-EPO and HES-EPO Ν- glycans by GC-MS In order to further confirm HES-modification of EPO at the N-glycans of the molecule, EPO samples were digested with N-glycosidase and the EPO protein was adsorbed onto RP-C18 cartridges whereas oligosaccharide material was washed off as described above. As shown in Table 2, glucose and hydroxyethylated glucose deriva- tives were detected only in the EPO protein which was subjected to HES- modification at cysteine residues and in oligosaccharide fractions of EPO sample A2.
Example 8.11(d) In-vivo assay of the biological activity of HES-modified EPO
The EPO-bioassay in the normocythaemic mouse system indicates was performed according to the procedures described in the European Pharmacopeia; the laboratory that carried out the EPO assay was using the International BRP EPO reference standard preparation. For the HES-modified EPO A2 preparation a mean value for the specific activity of 294,600 units per mg EPO of protein was determined indicating an approximately 3-fold higher specific activity when compared to the International BRP EPO reference standard preparation that was included in the samples sent for activity assays.
The results of the study are summarized in Table 3.
References for examples 1 to 8:
Nimtz M, Noll G, Paques EP, Conradt HS.
Carbohydrate structures of a human tissue plasminogen activator expressed in re- combinant Chinese hamster ovary cells. FEBS Lett. 1990 Oct. 1; 271(l-2):14-8
Dorner AJ, Wasley LC, Kaufman RJ.
Increased synthesis of secreted proteins induces expression of glucose-regulated pro- teins in butyrate-treated Chinese hamster ovary cells. J Biol Chem. 1989 Dec 5; 264 (34):20602-7
Mueller PP, Schlenke P, Nimtz M, Conradt HS, Hauser H Recombinant glycoprotein quality in proliferation-controlled BHK-21 cells. Biotechnol Bioeng. 1999 Dec 5; 65(5):529-36
Nimtz M, Martin W, Wray N, Kloppel KD, Augustin J, Conradt HS. Structures of sialylated oligosaccharides of human erythropoietin expressed in re- cobminant BHK-21 cells. Eur J Biochem. 1993. Apr. 1; 213(l):39-56
Hermentin P, Witzel R, Nliegenthart JF, Kamerling JP, Νimtz M, Conradt HS. A strategy for the mapping of Ν-glycans by high-ph anion-exchange chromatography with pulsed amperometric detection. Anal Biochem. 1992 Jun; 203(2):281 -9
Schroter S, Derr P, Conradt HS, Νimtz M, Hale G, Kirchhoff C. Male specific modification of human CD52. J Biol Chem. 1999 Oct. 15;274(42):29862-73 Example 9
Production of recombinant EPO
A) Production in mammalian cells
Recombinant EPO was produced in CHO cells as follows:
A plasmid harbouring the human EPO cDNA was cloned into the eukaryotic expres- sion vector (pCR3 and named afterwards pCREPO). Site directed mutagenesis was performed using standard procedures as described (Grabenhorst, Nimtz, Costa et al., 1998, In vivo specificity of human alpha 1,3/4-fucosyltransferases III-NII in the biosynthesis of Lewis(x) and sialyl Lewis(x) motifs on complex-type Ν-glycans - Coexpression studies from BHK-21 cells together with human beta-trace protein, J. Biol. Chem., 273(47), 30985-30994).
CHO cells stably expressing human EPO or amino acid variants (e.g. Cys- 29->Ser/Ala, or Cys-33-→Ser/Ala , Ser-126- Ala etc.) thereof were generated with the calcium phosphate precipitation method and selected with G418-sulfate as de- scribed (Grabenhorst et al.). Three days after transfection, the cells were subculti- vated 1:5 and selected in DMEM containing 10% FBS and 1.5 g/liter G418 sulfate.
Using this selection procedure, usually 100-500 clones survived and where propagated in selection medium for a further time period of 2-3 weeks. Cell culture super- natants of confluently growing monolayers were then analyzed for EPO expression levels by Western blot analysis and by IEF/Western Blot analysis.
EPO was produced from stable subclones in spinner flasks or in 21 perfusion reactors. Different glycoforms of EPO with different amounts of ΝeuAc (e.g. 2-8, 4-10, 8-12 ΝeuAc residues) were isolated according to published protocols using combinations various chromatographic procedures as described below. Literature:
Grabenhorst, Conradt, 1999, The cytoplasmic, transmembrane, and stem regions of glycosyltransferases specify their in vivo functional sublocalization and stability in- the Golgi., J Biol Chem., 274(51), 36107-16; Grabenhorst, Schlenke, Pohl, Nimtz, Conradt, 1999, Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells, Glycoconj J., 16(2), 81-97; Mueller, Schlenke, Nimtz, Conradt, Hauser, 1999, Recombinant glycoprotein product quality in proliferation-controlled BHK-21 cells, Biotechnology and bioengineering, 65(5), 529- 536; Schlenke, Grabenhorst, Nimtz, Conradt, 1999, Construction and characterization of stably transfected BHK-21 cells with human-type sialylation characteristic, Cytotechnology, 30(1-3), 17-25.
B) Production in insect cells
Recombinant human EPO was produced from insect cell lines SF9 and SF 21 after infection of cells with recombinant baculovirus vector containing the human EPO cDNA under control of the polyhedrin promoter as described in the literature.
Cells grown in serum-free culture medium were infected at cell density of 2x10 or XI 07 cells per mL and EPO titers were determined every day in the cell culture supematants. EPO was purified by Blue sepharose chromatography, ion-exchange chromatography on Q-Sepharose and finally RP-HPLC on C -Phase.
Purity of the product was checked by SDS-PAGE and N-terminal sequencing . Detained carbohydrate structural analysis (N- and O-glycosylation) was performed according to published procedures.
Literature:
Grabenhorst, Hofer, Nimtz, Jager, Conradt, 1993, Biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculo virus-infected Sf21 cells. Char- acterization ofpolypeptides and posttranslational modifications, Eur J Biochem., 215(1), 189-97; Quelle, Caslake, Burkert, Wojchowski, 1989, High-level expression and purification of a recombinant human erythropoietin produced using a baculovi- rus vector, Blood, 74(2), 652-7
Example 10
Formation of reactive HES derivatives
1. SH-reactive HES
1.1 Reaction of EMCH with Oxo-HES12KD to form SH-reactive HES12KD B
Figure imgf000113_0001
0.144 g (0.012 mmol) of Oxo-HES12KD (Fresenius German Patent DE 196 28 705 Al) were dissolved in 0.3 mL absolute dimethyl sulfoxide (DMSO) and were added dropwise under nitrogen to a mixture of 34 mg (0.15 mmol) EMCH (Perbio Science, Deutschland GmbH, Bonn, Germany) in 1.5 mL DMSO. After stirring for 19 h at 60°C the reaction mixture was added to 16 mL of a 1:1 mixture of ethanol and acetone. The precipitate was collected by centrifugation, redissolved in 3 mL DMSO and again precipitated as described. The SH-reactiv-HES12KD B was obtained by centrifugation and drying in vaccuo. The conjugation reaction with Thio-EPO is described in Example 11, 2.2.
Alternatives: In this reaction, all cross-linkers can be used, which exhibit a hydrazide- and a maleimide function, separated by a spacer. Further examples for molecules of that group, available from Perbio Science, Deutschland GmbH, Bonn, Germany, are shown in Table 1; marked with an "A". Furthermore, another group of cross-linkers exhibiting an activated disulfide function instead of a maleimide fimcion could also be used.
1.2 Halogenacetamide-derivatives of HES glycosylamines
a) Glycosylamine-formation l
A 1 mg sample of HES12KD was dissolved in 3 mL of saturated ammonium bicarbonate. Additional solid ammonium bicarbonate was then added to maintain saturation of the solution during incubation for 120 h at 30°C. The Amino-HES12KD C was desalted by direct lyophilization of the reaction mixture.
b) Acylation of the glycosylamine C with chloroacetic acid anhydride
A 1 mg sample of Amino-HES12KD C was dissolved in 1 mL of 1 M sodium bicarbonate and cooled on ice. To this was added a crystal of solid chloroacetic acid anhydride (~5 mg), and the reaction mixture was allowed to warm to room temperature. The pH was monitored and additional base was added if the pH dropped below 7.0. After two hours at room temperature a second ali- quot of base and anhydride was added. After six hours the product Chloroace- tamide-HES DI (X = CI) was desalted by passage over a mixed bed Amberli- te MB-3(H)(OH) ion exchange resins.
c) Acylation of the glycosylamine with bromoacetic anhydride2
Manger, Wong, Rademacher, Dwek, 1992, Biochemistry, 31, 10733-10740; Manger, Rademacher,
Dwek, 1992, Biochemistry, 31, 10724-10732 2 Black, Kiss, Tull, Withers, 1993,Carbohydr. Res., 250, 195 Bromoacetic anhydride was prepared as described by Thomas.3 A 1 mg sample of amino-HES12KD C was dissolved in 0.1 mL of dry DMF and cooled on ice and 5 mg bromoacetic anhydride was added. The reaction mixture was brought slowly to room temperature and the solution was stirred for 3 h. The reaction mixture was added to 1 mL of a 1:1 mixture of ethanol and acetone with -20 °C. The precipitate was collected by centrifugation, redissolved in 0.1 mL DMF and again precipitated as described. The Bromoacetamide-HES D2 (X = Br) was obtained by centrifugation and drying in vaccuo. The conju- gation reaction with Thio-EPO is described in Example 11 , 1.2.
d) The corresponding lodo-derivative D3 (X = I) was synthesised as described for D2. Instead bromoacetic anhydride N-succinimidyl iodoacetate was used
Figure imgf000115_0001
and all steps were performed in the dark.
Alternatives:
For acylation of amino groups, other activated forms of halogen acidic acids can be used, e.g.
- -bromides or -chlorides - esters, e.g. N-hydroxysuccinimide ester, esters with substituted phenoles (p- nitrophenole, pentafluorophenole, trichlorophenole etc)
Furthermore, all cross-linkers having an amino reactive group and a halogen ace- tyl function, separated by a spacer, could be used. An example thereof is SBAP. This molecule and others are available from Perbio Science Deutschland GmbH,
Bonn, Germany. They are marked in Table 1 with an "D". For the use as cross-
,
Thomas, 1977, Methodes Enzymol., 46, 362 linkers for the ligation of amino-HES with thio-EPO without isolation of the ha- logenacetamid-HES derivatives see remarks in example 11, 1.2.
1.3 Haloffenacetamide-derivatives of Amino-HES E 1
a) Reaction of 1,4-diaminobutane with Oxo-HES12KD to amino-HES12KD E
1.44 g (0.12 mmol) of Oxo-HES 12KD were dissolved in 3 mL dry dimethyl sulfoxide (DMSO) and were added dropwise under nitrogen to a mixture of 1.51 mL (15 mmol) 1,4-diaminobutane in 15 mL DMSO. After stirring for 19 h at 40°C the reaction mixture was added to 160 mL of a 1 :1 mixture of ethanol and acetone. The precipitate Amino-HES 12KD E was collected by centrifugation, redissolved in 40 mL of water an dialysed for 4 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized.
Figure imgf000116_0001
b) Chloroacetamide-HES12KD FI was prepared as described for Chloroaceta- mide-HES12KD DI in 1.3 above.
c) Bromoacetamide-HES12KD F2 (X = Br) was prepared as described for Bro- moacetamide-HES12KD D2 in 1.3 above. The conjugation reaction with Thio-EPO is described in Example 11, 1.2.
d) The corresponding lodo-derivative F3 (X = I) was not isolated before its re- action with Thio-EPO. The experiment is described in Example 11, 1.1.
Alternatives: See 1 ,2 above
2. CHO-Reactive HES
2.1 Hydrazide-HES
Figure imgf000117_0001
a) Reaction of hydrazine with Oxo-HES 12KD
1,44 g (0.12 mmol) of Oxo-HES 12KD were dissolved in 3 mL absolute dimethyl sulfoxide (DMSO) and were added dropwise under nitrogen to a mixture of 0.47 mL (15 mmol) hydrazine in 15 mL DMSO. After stirring for 19 h at 40°C the reaction mixture was added to 160 mL of a l.T mixture of ethanol and acetone. The precipitated product J was collected by centrifugation, redissolved in 40 mL of water and dialysed for 2 days against a 0.5 % (v/v) triethylamine in water solution and for 2 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Per- bio Science Deutschland GmbH, Bonn, Germany) and lyophilized. The conjugation reaction with oxidised Glyco-EPO is described in Example 12, 2.2.
Figure imgf000117_0002
b) Reaction of adipic dihydrazide with Oxo-HES 12KD
S. Frie, Diplomarbeit, Fachhochschule Hamburg, 1998 1.74 g (15 mmol)' adepic dihydrazide were dissolved in 20 mL absolute dimethyl sulfoxide (DMSO) at 65°C and 1,44 g (0,12 mmol) of Oxo-HES 12KD, dissolved in 3 mL absolute DMSO were added dropwise under nitrogen. After stirring for 68 h at 60°C the reaction mixture was added to 200 mL of water The solution containing L was dialysed for 2 days against a 0.5 % (v/v) triethylamine in water solution and for 2 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized. The conjugation reaction with oxidised Glyco-EPO is described in Example 12, 2.2.
Alternatives:
Furthermore, derivatives can be used, wherein 2 hydrazid groups are separated by any spacer.
3. Further Amino-HES12KD derivatives I and H
Ammonolysis of D or F was performed separately by dissolving a 1 mg sample of each halogeneacetamide in 0.1 mL of saturated ammonium carbonate. Additional solid ammonium carbonate was then added to maintain saturation of the solution during incubation of 120 h at 30°C. The reaction mixture was added to 1 mL of a 1:1 mixture of ethanol and acetone with —20 °C. The precipitate was collected by centrifugation, redissolved in 0.05 L water and again precipitated as described. The product aminoHES H or I was obtained by centrifugation and drying in vaccuo. The conjugation reaction with oxidised Glyco-EPO is described in Example 12, 4.1.
Figure imgf000118_0001
Figure imgf000119_0001
4. Hydroxylamine-modified HES12KD K
O-[2-(2-aminooxy-ethoxy)-ethyl] -hydroxylamine was synthesized as described by Boturyn et al in 2 steps from commercially available materials.5 1,44 g (0.12 mmol) of Oxo-HES 12KD were dissolved in 3 mL absolute dimethyl sulfoxide (DMSO) and were added dropwise under nitrogen to a mixture of 2.04 g (15 mmol) O-[2-(2- aminooxy-ethoxy)-ethyl]-hydroxylamine in 15 mL DMSO. After stirring for 48 h at 65°C the reaction mixture was added to 160 mL of a 1 :1 mixture of ethanol and acetone. The precipitated product K was collected by centrifugation, redissolved in 40 mL of water and dialysed for 4 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized. The conjugation reaction with oxidised Glyco-EPO is described in Example 12, 3.1.
Alternatives: Furthermore, derivatives could be used, wherein the two hydroxylamine groups are separated by any spacer.
5. Thio-HES12KD
5.1 Addition to Oxo-HES12KD
Boturyn, Boudali, Constant, Defrancq, Lhomme, 1997, Tetrahedron, 53, 5485
Figure imgf000120_0001
WI
1,44 g (0.12 mmol) of Oxo-HES12KD were dissolved in 3 mL absolute dimethyl sulfoxide (DMSO) and were added to a mixture of l.l6 g (15 mmol) cysteamine in 15 mL DMSO under nitrogen dropwise. After stirring for 24 h at 40° C the reaction mixture was added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitated product M was collected by centrifugation, redissolved in 40 mL of water and dialysed for 2 days against a 0.5 % (v/v) triethylamine in water solution and for 2 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized. The conjugation reaction with oxidised Glyco-EPO is described in Example 12, 2.1.
Alternatives:
Derivatives could be used, wherein the amino group and the thio-function are separated by any spacer. Furthermore, the amino group in the derivatives could be replaced by a hydrazine, a hydrazid or a hydroxylamine. The thio-function could be protected in the form of e.g. a disulfide or a trityl-derivative. However, in this case, a further deprotection step must be preformed before the conjugation, which would release a component being analogous to M.
5.2 Modifikation of Amino-HES12KD E, H or I
a) Modification with SATA/S ATP
1,44 g (0.12 mmol) of Amino-HES12KD E, H or I were dissolved in 3 mL absolute dimethyl sulfoxide (DMSO) and were added to a mixture of 139 mg (0.6 mmol) SATA in 5 mL DMSO under nitrogen dropwise. After stirring for
24 h at room temperature the reaction mixture was added to 160 mL of a 1 :1 mixture of ethanol and acetone. The precipitated product N was collected by centrifugation, redissolved in 40 mL of water and dialysed for 2 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized.
The deprotection was performed in a 50 mM sodium phosphate buffer, containing 25 mM EDTA and 0.5M hydroxylamine, pH7.5 for 2 hours at room temperature and the product O was purified by dialysis against a 0.1 M sodium acetate buffer pH 5.5, containing 1 mM EDTA. The deprotection reaction was performed immediately before the conjugation reaction which is de-
Figure imgf000121_0001
scribed in Example 12, 2.1.
Modification with SPDP
1,44 g (0.12 mmol) of Amino-HES 12KD E, H or I were dissolved in 3 mL absolute dimethyl sulfoxide (DMSO) and were dropwise added to a mixture of 187 mg (0.6 mmol) SPDP in 5 mL DMSO under nitrogen. After stirring for 24 h at room temperature the reaction mixture was added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitated product P was collected by centrifugation, redissolved in 40 mL of water and dialysed for 2 days a- gainst water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized.
The deprotection was performed in a solution of 12 mg dithiothreitol (DTT) per 0.5 mL 100 mM sodiumacetate buffer, containing 100 mM sodium chloride at pH 4.5 for 30 min at room temperature and the product Q was purified by dialysis against a 0.1 M sodium acetate buffer pH 5.5, containing 1 mM EDTA. The deprotection reaction was performed immediately before the conjugation reaction which is described in Example 12, 2.1.
Alternatives:
For the conversion of amino- to thiol-groups, either in free form or protected, sev- eral reagants are available. After the modification, the products could be isolated.
Alternatively, as accepted for the use of cross-linkers, they could be directly used for the conjugation reaction, preferably after a purification step. For the isolation and storage of thio-HES derivatives, the synthesis of thio-HES derivatives in a protected form may be useful. For this, all derivatives being analogous to SATA could be used, which have an active ester-function and a thioester-function, separated by any spacer. SATP, being a further member of this group, is found in Table 1, marked with an "H". The derivatives being analogous to SPDP could have an acitve ester-function and a disulfide-function, separated by any spacer. Further members of these groups are found in Table 1, marked with an "F". Further analogous derivatives could have an active ester-function and a thiol-fimction, protected as a trityl derivative, separated by any spacer.
Example 11
Conjugation reactions with Thio-EPO 1. Reaction of Thio-EPO with a halogenacetamide-modified SH-reactive HES
1.1 Example Protocol 1
Conjugation of ThioEPO to Amino-HES 12KD (E, II or I) with a Cross-linker containing a NHS -active-ester and an iodoacetamide group, e.g. SIA.
Materials
A. Borate buffer. Composition was 50 mM sodium borate, pH 8.3, 5 mM EDTA.
B. PBS, phosphate buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4.
C. AminoHES 12KD E, H or I. Prepared at 1 mg/mL in borate buffer.
D. Crosslinker stock solution: 14 mg SIA were dissolved in 1 mL DMSO E. D-Salt™ Dextran Desalting Columns, 2 x 5 mL bed volume (Perbio Science
Deutschland GmbH, Bonn, Germany)
F. Coomassie® Protein Assay Reagent (Perbio Science Deutschland GmbH, Bonn, Germany)
G. ThioEPO solution: 5 mg/mL of ThioEPO 1 in borate buffer. H. Microconcentrator: Microcon YM-3 (amicon, Milipore GmbH, Eschborn,
Germany)
Method
100 μL SIA solution was added to 400 μL of the aminoHES 12KD E solution and was allowed to react with agitation for 0.5 hours at room temperature. The excess crosslinker was removed by centrifuging the sample at 14000 x g for 60 minutes using a microconcentrator. After centrifuging the sample was brought up to its o- riginal volume in borate buffer and this process was repeated two more times. The residual solution was added to 1 mL of ThioEPO solution and the reaction mixture was incubated for 16 hour at room temperature. Reactivity of the excess iodoacetamide was quenched at the end of the incubation period by the addition of cysteine to a final concentration of 10 mM. The reaction mixture was applied to a desalting column equilibrated with PBS buffer and the protein content of the fractions were monitored with a Coomassie protein assay reagent. All fractions containing the protein conjugate were pooled and the the conjugate was obtained by lyophylisation after dialysis against water over night.
Alternatives:
In this reaction, all cross-linkers could be used, which have a succinimide- or a sulfosuccinimide function and a iodoacetamide function separated by a spacer.
Further examples are found in Table 1. They are marked with a "C" and are avi- alable from Perbio Science Deutschland GmbH, Bonn, Germany.
1.2 Example Protocol 2
Conjugation of ThioEPO 1 to SH reactiveHES12KD bromoacetamide D2, F2 or iodoacetamide D3.
Materials
A. Phosphate buffer. Composition was 100 mM sodium phosphate, pH 6.1, 5 mM EDTA.
B. PBS, phosphate buffered saline: 10 M sodium phosphate, 150 mM NaCl, pH 7.4. C. SH reactiveHES12KD bromoacetamide D2. Prepared at 10 mg/mL in phosphate buffer.
D. D-Salt™ Dextran Desalting Columns, 2 x 5 mL bed volume (Perbio Science Deutschland GmbH, Bonn, Germany)
6Cumber, Forrester, Foxwell, Ross, Thorpe, 1985, Methods Enzymol, 112, 207 - de Valasco, Merkus, Anderton, Verheul, Lizzio, Van der Zee, van Eden, Hoffmann, Verhoef, Snippe,
1995, Infect. Immun., 63, 961 E. Coomassie® Protein Assay Reagent (Perbio Science Deutschland GmbH, Bonn, Germany)
F. ThioEPO solution: 5 mg/mL of ThioEPO 1 in phosphate buffer.
Method
1 mL SH reactivePIES12KD bromoacetamide D2 solution and 1 mL of ThioEPO solution were combined and the reaction mixture was incubated for 48 hours at room temperature. Reactivity of the excess bromoacetamide was quenched at the end of the incubation period by the addition of cysteine to a final concentration of
10 mM. The reaction mixture was applied to a desalting column, equilibrated with PBS buffer. The protein content of the fractions were monitored with a Coomassie protein assay reagent, all fractions containing the protein conjugate were pooled and the the conjugate was obtained by lyophylisation after dialysis against water over night.
Alternatives:
Instead of the isolation of the SH reactive HES12KD-bromoacetamid D2, amino HES12KD (E, H, I) could be linked with a cross-linker via a succinimide- and a bromoacetamid function (see 1.1 above). SBAP is a member of this group of cross-linkers and is found in Table 1, marked with a "D".
2. Reaction of Thio-EPO with a maleimide-modified SH-reactive HES
2.1 Example Protocol 4
Conjugation of ThioEPO to Maleimido-HES12KD B.
Materials
A. Maleimido-HES12KD B: 10 mg/mL in 0.1 M sodium acetate buffer, pH 5.5
B. ThioEPO solution: 5 mg/mL of ThioEPO in phosphate/NaCI-buffer C. Phosphate/NaCI: 0.1 M sodium phosphate, 50 mM NaCl, pH 7.0
D. Gel filtration column: for example, Sephadex® G-200 (1.5 x 45 cm)
E. Coomassie® Protein Assay Reagent (Perbio Science Deutschland GmbH, Bonn, Germany) F. PBS, phosphate buffered saline: 10 mM sodium phosphate, 150 M NaCl, pH
7.4.
Method
1 mL SH-reactive-HES12KD B solution and 1 mL of ThioFiPO 1 solution were combined and the reaction mixture was incubated for 2 hours at room temperature. Reactivity of the excess maleimides was quenched at the end of the incubation period by the addition of cysteine to a final concentration of 10 mM. The reaction mixture was applied to Sephadex® G-200 (1.5 x 45 cm) equilibrated with PBS buffer and 1 mL fractions were collected. The protein content of the fractions were monitored with a Coomassie protein assay reagent. All fractions containing the protein conjugate were pooled and the the conjugate was obtained by lyophylisation after dialysis against water over night.
2.2 Example Protocol 12
Conjugation of ThioEPO to aminoHES 12KD (E, H, I) with a Cross-linker containing a NHS -active-ester and a maleimide group, e.g. SMCC
Materials
A: Microconcentrator: Microcon YM-10 (amicon, Milipore GmbH, Eschborn, Germany).
B. PBS, phosphate buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4.
C. AminoHES 12KD E, H or I. Prepared at 10 mg/mL in PBS buffer.
D. SMCC solution: 1 mg SMCC were dissolved in 50 μL DMSO E. D-Salt™ Dextran Desalting Columns, 2 x 5 mL bed volume (Perbio Science Deutschland GmbH, Bonn, Germany)
F. Coomassie® Protein Assay Reagent (Perbio Science Deutschland GmbH, Bonn, Germany) G. ThioEPO 1 solution: 5 mg/mL of ThioEPO 1 in PBS buffer.
Method
To 50 μL SMCC solution 400 μL of the aminoHES12KD E solution was added and the reaction mixture was allowed to react with agitation for 80 min at room temperature and for 10 min at 46°C. The excess crosslinker was removed by centrifugation of the reaction mixture through a microconcentrator at 14000 x g for 60 min. The volume was brought up to 450 μL with PBS buffer and the process was repeated two more times. After the last centrifugation, the residual solution was brought up to 450 μL with PBS and was added to 1 mL of ThioEPO solution and the reaction mixture was incubated for 16 hours at room temperature. Reactivity of the excess maleimide was quenched at the end of the incubation period by the addition of cysteine to a final concentration of 10 mM. The reaction mixture was applied to a desalting column equilibrated with PBS buffer. The protein content of the fractions were monitored with a Coomassie protein assay reagent, all fractions containing the protein conjugate were pooled and the conjugate was obtained by lyophylisation after dialysis against water over night.
Alternatives: In this reaction, all cross-linkers could be used which have a succinimide- or a sulfosuccinimide function and a maleimide-function, separated by a spacer. Further examples for this group of molecules, available from Perbio Science Deutschland GmbH, Bonn, Germany, are found in Table 1, marked with an "E". There is a fiirther group of cross-linkers, which have instead of a maleimide fiinc- tion an activated disulfide function. These cross-linkers could also be used for the conjugation. However, the disulfide bond of the conjugate is cleavable under reductive conditions. Members of this group are marked in Table 1 with a "F". A third group of cross-linkers uses instead of a maleimide function a vinylsulfon function as a SH-reactive group. A member of this group "SNSB" is marked in Table 1 with a "G".
Example 12
Conjugation reactions with oxidized EPO
1. Oxidation of Glyco-EPO
1.1 Oxidation of Glyco-EPO with sodium meta-periodate: Example Protocol 5
Materials
A. Glyco-EPO solution: 10 mg/mL of Glyco-EPO in acetate buffer
B. Sodium meta-periodate solution: 10 mM or 100 mM sodium periodate in acetate buffer, prepared fresh. Keep in dark. Using these solutions, the final concentration of sodium periodate in the oxidation mixture is 1 mM or 10 mM, respec- tively.
C. acetate buffer: 0.1 M sodium acetate buffer, pH 5.5
D. Glycerol
E. Microconcentrator: Microcon YM-3 (amicon, Milipore GmbH, Eschborn, Germany)
Method
All steps were performed in the dark.
To 1 mL of cold Glyco-EPO solution 0.1 mL of cold sodium meta-periodate solution were added and the the oxidation reaction was allowed to proceed for 1 hour in the dark. If the Glyco-EPO to be oxidized contained sialic acid residues, then the oxidation conditions were 1 mM sodium periodate, 0°C. Otherwise, 10 mM sodium periodate at room temperature was used. To stop the oxidation glycerol was added to a final concentration of 15 mM and incubated for 5 minutes at 0°C. The excess reagents and by-products were remove by centrifuging of the product at 14000 x g for 60 minutes using a microconcentrator. After centrifuging, sample was brought up to its original volume in the buffer used in the next modification step, e.g. in the acetate buffer. This process was repeated two more times.
1.2 Enzymatic oxidation of Glyco-EPO: Example Protocol 6
The enzymatic oxidation of EPO is described elsewhere (Chamow et al., 1992, J. Biol. Chem., 267, 15916-15922).
2. Conjugation with Hydrazine/Hydrazide-Derivatives
2.1 Example Protocol 7
Conjugation of oxidised Glyco-EPO to Thio-HES 12KD M, O or Q with a Cross- linker containing a hydrazide and a maleimide fimctional group, e.g.M2C2H (Perbio Science, Deutschland GmbH, Bonn, Germany).
Materials
A. M2C2H stock: 10 mg/mL M2C2H in DMSO, prepared fresh
B. Oxidised Glyco-EPO solution from 6.1.1: 5 mg/mL of Glyco-EPO in acetate buffer
C. Thio-HES 12KD M, O or Q: 10 mg/mL in phosphate/NaCl buffer
D. Acetate buffer: 0.1 M sodium acetate buffer, pH 5.5 E. Phosphate/NaCI: 0.1 M sodium phosphate, 50 mM NaCl, pH 7.0
F. Microconcentrator: Microcon YM-3 (amicon, Milipore GmbH, Eschborn, Germany) G. Gel filtration column: for example, Sephadex® G-200 (1.5 x 45 cm)
FI. Coomassie® Protein Assay Reagent (Perbio Science Deutschland GmbH,
Bonn, Germany)
I. PBS, phosphate buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4
Method
M2C2H stock solution was added to 1 mL of oxidized Glyco-EPO to a final con- centration of 1 mM and was allowed to react with agitation for 2 hours at room temperature. The excess crosslinker was removed by centrifuging the sample at .14000 x g for 60 minutes using a microconcentrator..After centrifuging the sample was brought up to its original volume in phosphate/NaCI buffer and this process was repeated two more times. To the M2C H-modified Glyco-EPO 1 mL of Thio-HES 12KD M, O or Q solution was added and the reaction mixture was incubated for 16 hours at room temperature. Reactivity of the excess maleimides was quenched at the end of the incubation period by the addition of cysteine. The reaction mixture was applied to Sephadex® G-200 (1.5 x 45 cm) equilibrated with PBS and 1 mL fractions were collected. The protein content of the fractions were monitored with a Coomassie protein assay reagent, all fractions containing the protein conjugate were pooled and the conjugate was obtained by lyophylisation after dialysis against water over night.
Procedural Notes
The hydrazone adduct is slightly less stable at extremes of pH. For applications that may involve treatment at low pH, we reduced the hydrazone by treatment with 30 mM sodium cyanoborohydride in PBS buffer to a hydrazine. For most applications, this extra step was unnecessary.
2.2 Example Protocol 8 Direct conjugation of oxidised Glyco-EPO to Hydrazido-HES12KD L.
Materials
A. Oxidised Glyco-EPO solution from 6.1.1 : 5 mg/mL of Glyco-EPO in acetate buffer
B. Hydrazido-HES18KD L or J: 10 mg/mL in acetate buffer
C. Acetate buffer: 0.1 M sodium acetate buffer, pH 5.5
D. Gel filtration column: for example, Sephadex® G-200 (1.5 x 45 cm) E. Coomassie® Protein Assay Reagent (Perbio Science Deutschland GmbH,
Bonn, Germany)
F. PBS, phosphate buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4
Method
1 mL of Hydrazido-HES12KD L solution and 1 mL of oxidized Glyco-EPO solution were combined and the reaction mixture was allowed to react with agitation for 16 hours at room temperature. The reaction mixture was applied to Sephadex® G-200 (1.5 x 45 cm) equilibrated with PBS and 1 mL fractions were collected. The protein content of the fractions were monitored with a Coomassie protein assay reagent, all fractions containing the protein conjugate were pooled and the the conjugate was obtained by lyophylisation after dialysis against water over night. The result of the conjugation is shown in Figure 24. The observed mo- lecular shift demonstrates that the conjugation was successful. The smear results from the heterogenity of HES. Figure 25 demonstrates that HES is conjugated to a carbohydrate moiety of a carbohydrate side chain.
Procedural Notes
The hydrazone adduct is slightly less stable at extremes of pH. For applications that may involve treatment at low pH, we reduced the hydrazone by treatment with 30 mM sodium cyanoborohydride in PBS buffer to a hydrazine. For most applications, this extra step was unnecessary.
3. Conjugation with Hydroxylamine-Derivatives8
3.1 Example Protocol 9
• Conjugation of oxidized Glyco-EPO to Hydroxylamino-HES 12KD K
Materials
A. Oxidised Glyco-EPO solution from 6.1.1: 5 mg/mL of Glyco-EPO in acetate buffer
B. Hydroxylamino-ITES12KD K: 10 mg/mL in acetate buffer C. Acetate buffer: 0.1 M sodium acetate buffer, pH 5.5
D. Gel filtration column: for example, Sephadex® G-200 (1.5 x 45 cm)
E. Coomassie® Protein Assay Reagent (Perbio Science Deutschland GmbH, Bonn, Germany)
F. PBS, phosphate buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4
Method
1 mL of Hydroxylamino-HES 12KD K solution and 1 mL of oxidized Glyco-EPO solution were combined and the reaction mixture was allowed to react with agitation for 16 hours at room temperature. The reaction mixture was applied to Sephadex® G-200 (1.5 x 45 cm) equilibrated with PBS and 1 mL fractions were collected. The protein content of the fractions were monitored with a Coomassie protein assay reagent, all fractions containing the protein conjugate were pooled and the conjugate was obtained by lyophylisation after dialysis against water over night. The result of the conjugation is shown in Figure 24. The observed molecu- lar shift in lane 2 demonstrates that the conjugation was successful. The smear results from the heterogenity of HES. Figure 25 demonstrates that HES is conjugated to a carbohydrate moiety of a carbohydrate side chain.
Example 13
Characterisation of galactose oxidase treated EPO N-glycans
Recombinant EPO or partially desialylated EPO forms (generated by limited mild acid hydroysis) were incubated with galactose oxidase in the presence of catalase at 37°C from 30 min - 4 hours at 37°C in 0.05 M Na-phosphate buffer pH 7.0. Progress of the reaction was monitored by removal of 50 μg aliquots of the EPO and subsequent treatment of the protein with polypeptide N-glycanase.
Liberated N-linked oligosaccharides (monitored by SDS-PAGE detection of the de- N-glycosylated polypeptide) were subjected to HP AEC-P AD mapping as described ( Grabenhorst et al., 1999, Nimtz et al, 1993/1994; Schlenke et al., 1999) before and after removal of sialic acids. Quantitation of oxidised galactose residues in individual EPO oligosaccharides was performed by the typical shift observed in HP AEC-P AD and was also verified by MALDI/TOF MS of the oligosaccharide mixtures.
Example 14
Characterisation of HAS modified EPO
Separation of HAS modified EPO forms from nonreacted EPO and HAS-precursor molecules was achieved by gel filtration using e.g. Ultrogel AcA 44 / 54 or similar gel filtration media. Alternatively, nonreacted HAS was removed by immuno affinity
Rose, 1994, Am. Chem. Soc, 116, 30 isolation of EPO on a 4 mL column containing a monoclonal antibody coupled to Affigel (BioRad) and subsequent separation of unmodified EPO by gel filtration (e.g. using a matrix enabling the separation of globular proteins of a relative molecular mass between 20 kDa and 200 kDa ).
HAS modified EPOs were identified by SDS-PAGE analysis (using 12.5 or 10% acrylamide gels) through detection of their higher molecular weight compared to unmodified EPO upon staining of gels with Coomassie Brillant Blue. The higher molecular weight of FIAS modified EPO polypeptides was also identified by Western Blot analysis of samples using a polyclonal antibody raised against recombinant human EPO.
N-glycan modification of EPO forms was demonstrated by their successful removal from the EPO protein with polypeptide N-glycanase (recombinant N-glycosidase from Roche, Germany employing 25 units / mg EPO protein at 37°C for 16 hours); analysis by SDS-PAGE resulted in a typical shift of the EPO protein to a migration position of the N-glycosidase treated unmodified EPO of approximately 20 KDa.
Modification of the single desialylated and glacatose oxidase treated EPO Q-glycan at Ser 126 was demonstrated by SDS-PAGE migration of the de-N-glycosylated product by detection of its migration position compared to nonreacted de-N- glycosylated EPO. If required, modified EPO was fractionated by RP-HPLC on a C8-phase before SDS-PAGE analysis. FIAS O-glycan modification of EPO was also analysed by β-elimination of the O-glycan and detection of the de-O-glycosylated form of EPO in Western blots using a polyclonal antibody raised against recombinant human EPO.
Example 15
Quantitation of EPO and modified EPO forms EPO forms where quantitated by UV measurements as described in Ph.Eur (2000, Erythropoietini solutio concentrata, 1316, 780-785) and compared to the international BRP reference EPO standard. Alternatively, EPO concentrations were determined by a RP-HPLC assay using a RP-C4-column and absorption at 254 nm em- ploying 20, 40 , 80 and 120 μg of the BRP standard EPO reference preparation for calibration.
Example 16
In-vitro biological actvity of HES-modified recombinant human EPO:
Purified HES-modified EPO was tested for activity using the erythropoietin bioactivity assay as described by Krystal [Krystal, 1984, Exp. Fleamatol, 11, 649-660].
Anemia was induced in NMRI mice by treatment with phenylhydrazine hydrochloride and spleen cells were collected and used as described in [Fibi et al, 1991, Blood, 77, 1203 ff.]. Dilutions of EPO were incubated with 3x105 cells/well in 96-well mi- crotiter plates. After 24 hours at 37° C in a humified atmosphere (5% CO2) cells were labelled for 4 hours with 1 μCi of 3H-thymidine per well. Incorporated radioactivity was determined by liquid scintillation counting. The International reference EPO standard (BRP-standard) was used for comparison .
Alternatively, EPO bioactivity was measured by an in vitro assay using the EPO- sensitive cell line TF-1 (Kitamura et. al., [J. cell Phys., 140. 323-334]. Exponentially, growing cells were washed free of growth factors and were incubated in the presence of serial dilutions of the EPO for further 48 hours. Proliferation of the cells was assessed by using the MTT reduction assay as described by Mosmann [Mosman, 1983, J.Immunol. Methods, 65, 55-63],
Example 17 In-vivo activity determination of EPO and HAS-modified EPO forms:
In vivo activity determinations were perfonned in normocythemic mice by measuring the increase of reticulocytes after 4 days after animals received the foreseen dose of EPO or modified EPO forms. Assays were performed using the BRP EPO standard which was calibrated against the WHO EPO standard in the polycythemic mouse assay. EPO samples were diluted in phosphate buffered saline containing 1 mg/ml of bovine serum albumin (Sigma).
0.5 ml of the EPO test solution in Dulbecco's buffered saline (corresponding to an EPO protein equivalent of a 100, 80, 40 or 20 IU/ml of the BRP standard EPO) were infected subcutaneously per animal. Blood samples were taken after 4 days after injection and reticulocytes were stained with acridine orange; quantitation of reticulo- cytes was performed by flow-cytometry by coimting a total of 30,000 blood cells within 5 hours after the blood sample was taken (see Ph. Eur, 2000, Erytliropoietini solutio concentrata, 1316, pages 780-785) and European Pharmacopoeia (1996/2000, attachment 2002).
Example 18
In-vivo half-life Determinations
Rabbits were injected intravenously with specified amounts of unmodified or HAS- modified EPO forms. Blood samples were obtained at specified times, and serum was prepared. Serum erythropoietin levels were determined by in vitro bioassay or by an EPO-specific commercial ELISA.
Example 19 In vivo pharmakokinetics
In mice: Each animal received 300 IU EPO/kg subcutaneously. Seven days after the post-treatment hematocrit of each animal was determined. A substantial increase in hematocrit was observed 9in all animals treated with modified EPO, an expected result in view o the relatively short half- life of untreated EPO. The mean change in hematocrit of the modified EPO-treated group was significantly different from that of the untreated EPO group and that of the control group.
In rabbits: Rabbits were treated with a single dose of unmodified or HAS-modified EPO corresponding to 200 or up to 800 ng/kg body weight. After 2, 6, 16, 24 and 48 hours blood samples were analyzed by using a commercial EPO-specific ELISA for determination of plasma concentrations. Mean plasma EPO concentrations were determined and the average initial half-lives (α-phase) and the terminal half-lives (β- phase) were calculated from the ELISA values as described: (Zettlmissl et al., 1989, J. Biol. Chem., 264, 21153-21159).
Literature:
Sytkowski, Lunn, Risinger, and Davis, 1999, An Erythropoietin Fusion Protein Comprised of Identical Repeating Domains Exhibitis Enhanced Biological Prop- erites, J. Biol. Chem., 274, 24773-24778.
Example 20
Assessment of the in vitro biological activity of HES-modified recombinant hu- man IL-2
Modified IL2 was recovered by gelfiltration on Ultrogel AcA 54. Aliquots of corre- sponding fraction were sterile filtrated and IL2 bioactivity was determined by using the IL2 dependent murine CTLL-2 cell line [Gillis, Ferm, On, and Smith, 1978, J.Immunol., 120, 2027-2032]. Activity was related to the international reference IL2 standard preparation.
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Table 2 Monosaccharide compositional analysis of glycans from HES-modified EPO and control sam les
Figure imgf000145_0001
Table 3
Figure imgf000145_0002

Claims

Claims
A method of producing a hydroxyalkyl starch derivative, said hydroxyalkyl starch having a structure according to formula (I)
Figure imgf000146_0001
comprising reacting hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end or a hydroxyalkyl starch derivative, obtainable by reacting hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end with a compound (D), said compound (D) comprising at least one functional group Zi capable of being reacted with the optionally oxidized reducing end of the hydroxyalkyl starch, and at least one functional group W, with a compound (L) comprising at least one functional group Z\ capable of being reacted with said hydroxyalkyl starch, or at least one functional group Z2 capable of being reacted with functional group W comprised in said hydroxyalkyl starch derivative, and at least one functional group X capable of being reacted with a functional group Y of a further compound (M), wherein said functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, an acetal group, and a thio group.
2. A method as claimed in claim 1 wherein Ri, R2 and R are independently hydrogen or a linear or branched hydroxyalkyl group.
3. A method as claimed in claim 2 wherein Ri, R2 and R3 are independently hydro- gen or a 2-hydroxyethyl group.
4. A method as claimed in any of claims 1 to 3 wherein the hydroxyalkyl starch is hydroxyethyl starch.
5. A method as claimed in any of claims 1 to 4 wherein the functional group Zi comprises the structure -NH-.
6. A method as claimed in claim 5 wherein Z\ is selected from the group consisting of
Figure imgf000147_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl.
7. A method as claimed in any of claims 1 to 6 wherein the fimctional group Y is selected from the group consisting of an aldehyd group, a keto group, a he- miacetal group, and an acetal group, and the functional group X comprises the structure -NH-.
8. A method as claimed in claim 7 wherein X is selected from the group consisting of
Figure imgf000148_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl.
A method as claimed in any of claims 1 to 6 wherein the functional group Y is - SH and the functional group X is selected from the group consisting of
Figure imgf000148_0002
wherein Hal is CI, Br or I.
10. A method as claimed in any of claims 1 to 9 wherein the functional group W or the functional group Z2 is -SH and the functional group Z2 or the functional group is selected from the group consisting of
Figure imgf000148_0003
wherein Hal is CI, Br, or I.
11. A method as claimed in any of claims 1 to 9 wherein the functional group W or the functional group Z is selected from the group consisting of an activated es- ter or a carboxy group which is optionally transformed into an activated ester and the functional group Z2 or the functional group W is selected from the group consisting of
Figure imgf000149_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl.
12. A method as claimed in any of claims 1 to 11 wherein the reducing end of the hydroxyalkyl starch is not oxidized prior to the reaction with compound (D) or compound (L), said hydroxyalkyl starch thus having a structure according to formula (I)
Figure imgf000149_0002
13. A method as claimed in any of claims 1 to 11 wherein the reducing end of the hydroxyalkyl starch is oxidized prior to the reaction with compound (D) or compound (L), said hydroxyalkyl starch thus having a structure according to formula (Ila)
Figure imgf000150_0001
and/or according to formula (lib)
Figure imgf000150_0002
14. A method as claimed in claim 13 wherein the reducing end is oxidized by an alkaline iodine solution.
15. A method as claimed in any of claims 1 to 9 and 12 to 14 wherein hydroxyalkyl starch is reacted with a compound (L) via the reaction of functional group Zi with the optionally oxidized reducing end of the hydroxyalkyl starch and the resulting reaction product is reacted with a further compound (M) via the reaction of the functional group X comprised in compound (L) with the functional group Y comprised in compoimd (M).
16. A method as claimed in any of claims 1 to 9 and 12 to 14 wherein hydroxyalkyl Starch is reacted with a compound (L) via the reaction of functional group Zi with the optionally oxidized reducing end of the hydroxyalkyl starch, where compound (L), prior to the reaction with hydroxyalkyl starch, is reacted with a further compound (M) via the reaction of functional group X comprised in compound (L) with the functional group Y comprised in compound (M).
17. A method as claimed in any of claims 1 to 14 wherein hydroxyalkyl starch is reacted widi a compound (D) via the reaction of the functional group Z\ comprised in compound (D), with the optionally oxidized reducing end of the hydroxyalkyl starch to give a first hydroxyalkyl starch derivative, and where the first hydroxyalkyl starch derivative is reacted with a compound (L) via the reaction of functional group Z2 comprised in compound (L) with the functional group comprised in compoimd (D) to give a second hydroxyalkyl starch derivative.
18. A method as claimed in claim 17 wherein the second hydroxyalkyl starch derivative is reacted with a further compoimd (M) via the reaction of fimctional group X comprised in compound (L) with the functional group Y comprised in compoimd (M).
19. A method as claimed in any of claims 1 to 18 wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of functional group Zi comprised in compound (D) with the optionally oxidized reducing end of the hydroxyalkyl starch to give a first hydroxyalkyl starch derivative, and where the first hydroxyalkyl starch derivative is reacted, via the reaction of the functional group W, comprised in compound (D), and the functional group Z2, comprised in compound (L), with compound (L), where compound (L), prior to the reaction with the first hydroxyalkyl starch derivative, is reacted with a further compound (M) via the reaction of functional group X comprised in compound (L) with the functional group Y comprised in compound (M).
20. A method as claimed in any of claims 1 to 19 wherein the at least one further compound (M) is a polypeptide. .
21. A method as claimed in claim 20 wherein the polypeptide is erythropoietin.
22. A hydroxyalkyl starch derivative obtainable by a method of producing a hydroxyalkyl starch derivative, said hydroxyalkyl starch having a structure according to formula (I)
Figure imgf000152_0001
comprising reacting hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end or a hydroxyalkyl starch derivative, obtainable by reacting hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end with a compound (D), said compound (D) comprising at least one functional group Zi capable of being reacted with the optionally oxidized reducing end of the hydroxyalkyl starch, and at least one functional group W, with a compound (L) comprising at least one functional group Zi capable of being reacted with said hydroxyalkyl starch, or at least one functional group Z2 capable of being reacted with functional group W comprised in said hydroxyalkyl starch derivative, and at least one functional group X capable of being reacted with a functional group Y of a further compound (M), wherein said functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, an acetal group, or a thio group.
23. A hydroxyalkyl starch derivative as claimed in claim 22 wherein i, R2 and R3 are independently hydrogen or a linear or branched hydroxyalkyl group.
24. A hydroxyalkyl starch derivative as claimed in claim 23 wherein Rj, R2 and R3 are independently hydrogen or a 2-hydroxyethyl group.
25. A hydroxyalkyl starch derivative as claimed in any of claims 22 to 24 wherein the hydroxyalkyl starch is hydroxyethyl starch.
26. A hydroxyalkyl starch derivative as claimed in any of claims 22 to 25 wherein the functional group Zi comprises the structure -NH-.
27. A hydroxyalkyl starch derivative as claimed in claim 26 wherein Z\ is selected from the group consisting of
Figure imgf000153_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl.
28. A hydroxyalkyl starch derivative as claimed in any of claims 22 to 27 wherein the functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group, and the functional group X comprises the structure -NH-.
29. A hydroxyalkyl starch derivative as claimed in claim 28 wherein X is selected from the group consisting of
Figure imgf000153_0002
Figure imgf000154_0001
wherein G is O or S and, if present twice, independently O or S, and R1 is methyl.
30. A hydroxyalkyl starch derivative as claimed in any of claims 22 to 27 wherein the fimctional group Y is -SH and the fimctional group X is selected from the group consisting of
Figure imgf000154_0002
wherein Hal is CI, Br or I.
31. A hydroxyalkyl starch derivative as claimed in any of claims 22 to 30 wherein the functional group W or the functional group Z2 is -SH and the functional group Z or the functional group W is selected from the group consisting of
Figure imgf000154_0003
wherein Hal is CI, Br, or I.
32. A hydroxyalkyl starch derivative as claimed in any of claims 22 to 30 wherein the functional group W or the functional group Z2 is selected from the group consisting of an activated ester or a carboxy group which is optionally transformed into an activated ester and the functional group Z2 or the functional group W is selected from the grpup consisting of
Figure imgf000154_0004
Figure imgf000155_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl.
33. A hydroxyalkyl starch derivative as claimed in any of claims 22 to 32 wherein the reducing qnd of the hydroxyalkyl starch is not oxidized prior to the reaction with compound (D) or compound (L), said hydroxyalkyl starch thus having a structure according to formula (I)
Figure imgf000155_0002
34. A hydroxyalkyl starch derivative as claimed in any of claims 22 to 32 wherein the reducing end of the hydroxyalkyl starch is oxidized prior to the reaction with compound (D) or compound (L), said hydroxyalkyl starch thus having a structure according to formula (Ila)
Figure imgf000155_0003
and/or according to formula (lib)
Figure imgf000156_0001
35. A hydroxyalkyl starch derivative as claimed in claim 34 wherein the reducing end is oxidized by an alkaline iodine solution.
36. A hydroxyalkyl starch derivative as claimed in any of claims 22 to 30 and 33 to 35 wherein hydroxyalkyl starch is reacted with a compound (L) via the reaction of functional group Zj with the optionally oxidized reducing end of the hydroxyalkyl starch and the resulting reaction product is reacted with a further compound (M) via the reaction of the functional group X comprised in compound (L) with the functional group Y comprised in compound (M).
37. A hydroxyalkyl starch derivative as claimed in any of claims 22 to 30 and 33 to 35 wherein hydroxyalkyl starch is reacted with a compound (L) via the reaction of functional group i with the optionally oxidized reducing end of the hy- droxyalkyl starch, where compound (L), prior to the reaction with hydroxyalkyl starch, is reacted with a further compound (M) via the reaction of functional group X comprised in compound (L) with the functional group Y comprised in compound (M).
38. A hydroxyalkyl starch derivative as claimed in any of claims 22 to 35 wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of the functional group Zi comprised in compound (D), with the optionally oxidized reducing end of the hydroxyalkyl starch to give a first hydroxyalkyl starch derivative, and where the first hydroxyalkyl starch derivative is reacted with a compound (L) via the reaction of fimctional group Z2 comprised in compound (L) with the functional group W comprised in compound (D) to give a second hydroxyalkyl starch derivative.
39. A hydroxyalkyl starch derivative as claimed in claim 38 wherein the second hy- droxyalkyl starch derivative is reacted with a fiirther compound (M) via the reaction of functional group X comprised in compound (L) with the functional group Y comprised in compound (M).
40. A hydroxyalkyl starch derivative as claimed in any of claims 22 to 35 wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of functional group Zi comprised in compound (D) with the optionally oxidized reducing end of the hydroxyalkyl starch to give a first hydroxyalkyl starch derivative, and where the first hydroxyalkyl starch derivative is reacted, via the reaction of the functional group W, comprised in compound (D), and the fimctional group Z2, comprised in compound (L), with compound (L), where compound (L), prior to the reaction with the first hydroxyalkyl starch derivative, is reacted with a further compound (M) via the reaction of functional group X comprised in compound (L) with the functional group Y comprised in compound (M).
41. A hydroxyalkyl starch derivative as claimed in any of claims 22 to 40 wherein the at least one further compound (M) is a polypeptide.
42. A hydroxyalkyl starch derivative claimed in claim 41 wherein the polypeptide is erythropoietin.
43. A pharmaceutical composition comprising, in a therapeutically effective amount, a hydroxyalkyl starch derivative obtainable by a method of producing a hydroxyalkyl starch derivative, said hydroxyalkyl starch having a structure according to formula (I)
Figure imgf000158_0001
comprising reacting, hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end or a hydroxyalkyl starch derivative, obtainable by reacting hydroxyalkyl starch of formula (I) at its optionally oxidized reducing end with a compound (D), said compound (D) comprising at least one functional group Z\ capable of being reacted with the optionally oxidized reducing end of the hydroxyalkyl starch, and at least one functional group W, with a compound (L) comprising at least one functional group Zi capable of being reacted with said hydroxyalkyl starch, or at least one functional group Z2 capable of being reacted with functional group W comprised in said hydroxyalkyl starch derivative, and at least one functional group X capable of being reacted with a functional group Y of a further compound (M), wherein said functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, an acetal group, or a thio group, said method of producing a hydroxyalkyl starch derivative further comprising reacting the reaction product comprising hydroxyalkyl starch, compound (L) and optionally compound (D) with a further compound (M) wherein the at least one further compound is a polypeptide.
44. A pharmaceutical composition as claimed in claim 43 wherein the polypeptide is AT III.
45. A pharmaceutical composition as claimed in claim 43 wherein the polypeptide is erythropoietin.
46. A pharmaceutical composition as claimed in any of claims 43 to 45 wherein the functional group Y is -SH and compound (L) is a compound of general formula Zi-L'-X where the functional group Zi is selected from the group consisting of
H2N- H .0
H- \ R' N
H2N H
Figure imgf000159_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, and where the functional group X is selected from the group consisting of
Figure imgf000159_0002
wherein Hal is CI, Br or I, and where L1 is an organic chain bridging Zi and X or where L' is absent.
47. A pharmaceutical composition as claimed in any of claims 43 to 45 wherein the functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group, and compound (L) is a compound of general formula Zj-L'-X where the functional group Zi is selected from the group consisting of
Figure imgf000159_0003
Figure imgf000160_0001
wherein G is O or S and, if present twice, independently O or S, and R is alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl, or cycloalkylaryl, and where the functional group X is selected from the group consisting of
Figure imgf000160_0002
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, and where L' is an organic chain bridging Zi and X or where L' is absent.
48. A pharmaceutical composition as claimed in any of claims 43 to 45 wherein the functional group Y is -SH, compound (D) is a compound of general formula ZΪ- D'-W, and compound (L) is a compound of general formula Z2-L'-X, where the fimctional group Zi is selected from the group consisting of
Figure imgf000160_0003
Figure imgf000161_0001
wherein G is O or S and, if present twice, independently O or S, and R is methyl, where the functional group X is selected from the group consisting of
Figure imgf000161_0002
wherein Hal is CI, Br or I, where the functional group W or the functional group Z2 is -SH and the functional group Z2 or the functional group W is selected from the group consisting of
Figure imgf000161_0003
wherein Hal is CI, Br, or I., or where the functional group W or the functional group Z2 is selected from the group consisting of an activated ester or a carboxy group which is optionally transformed into an activated ester and the functional group Z2 or the functional group W is selected from the group consisting of
Figure imgf000161_0004
H H2NN ?ι
H2NT Y N-S— H II
G O
Figure imgf000161_0005
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, and where D' is an organic chain bridging Zi and W or where D' is absent and where L' is an organic chain bridging Z2 and X or where L' is absent.
9. A pharmaceutical composition as claimed in any of claims 43 to 45 wherein the functional group Y is selected from the group consisting of an aldehyd group, a keto group, a hemiacetal group, and an acetal group, compound (D) is a compound of general formula Zi-D'-W, and compound (L) is a compound of general formula Z -L'-X, where the functional group Zi is selected from the group consisting of
Figure imgf000162_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, where the functional group X is selected from the group consisting of
Figure imgf000162_0002
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, where the functional group W or the functional group Z2 is -SH and the fimctional group Z2 or the fimctional group W is selected from the group consisting of
Figure imgf000162_0003
wherein Hal is CI, Br, or I., or where the functional group W or the functional group Z2 is selected from the group consisting of an activated ester or a carboxy group which is optionally transformed into an activated ester and the functional group Z2 or the functional group W is selected from the group consisting of
Figure imgf000163_0001
wherein G is O or S and, if present twice, independently O or S, and R' is methyl, , and where D' is an organic chain bridging Zi and W or where D' is absent and where L' is an organic chain bridging Z2 and X or where L' is absent.
50. A pharmaceutical composition as claimed in claim 43 wherein hydroxyethyl starch is reacted in an aqueous medium with a compound according to the following formula
H2 ^ O ^ \^ "NH2 and the reaction product is reacted with erythropoietin.
51. A pharmaceutical composition as claimed in claim 50 wherein the erythropoietin is oxidised with sodium periodate prior to the reaction.
52. A pharmaceutical composition as claimed in claim 50 wherein the erythropoietin is partially desialylated and subsequently oxidised with sodium periodate prior to the reaction.
PCT/EP2003/008829 2002-09-11 2003-08-08 Method of producing hydroxyalkyl starch derivatives WO2004024776A1 (en)

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JP2004535070A JP4688494B2 (en) 2002-09-11 2003-08-08 Process for producing hydroxyalkyl starch derivatives
BR0314107-1A BR0314107A (en) 2002-09-11 2003-08-08 Production method of hydroxyalkyl starch derivatives
CA2496317A CA2496317C (en) 2002-09-11 2003-08-08 Method of producing hydroxyalkyl starch derivatives
MXPA05002593A MXPA05002593A (en) 2002-09-11 2003-08-08 Method of producing hydroxyalkyl starch derivatives.
AU2003260393A AU2003260393B2 (en) 2002-09-11 2003-08-08 Method of producing hydroxyalkyl starch derivatives
EP10011836.3A EP2272865A3 (en) 2002-09-11 2003-09-11 Method of producing hydroxyalkyl starch derivatives
ES03020424T ES2213506T3 (en) 2002-09-11 2003-09-11 METHOD OF PRODUCING DERIVATIVES OF HIDROXIALQUIL-ALMIDON.
DK03020424T DK1398327T3 (en) 2002-09-11 2003-09-11 Process for the preparation of hydroxyalkyl starch derivatives
DE60323192T DE60323192D1 (en) 2002-09-11 2003-09-11 Process for the preparation of Hydroxyalkylstärkederivaten
DE0001398327T DE03020424T1 (en) 2002-09-11 2003-09-11 Process for the preparation of hydroxyalkyl starch derivatives
PT03020424T PT1398327E (en) 2002-09-11 2003-09-11 Method of producing hydroxyalkyl starch derivatives
DE20321836U DE20321836U1 (en) 2002-09-11 2003-09-11 hydroxyalkyl starch derivatives
EP08012908A EP2017287A3 (en) 2002-09-11 2003-09-11 Method of producing hydroxyalkyl starch derivatives
AT03020424T ATE406387T1 (en) 2002-09-11 2003-09-11 METHOD FOR PRODUCING HYDROXYALKYL STARCH DERIVATIVES
SI200331431T SI1398327T1 (en) 2002-09-11 2003-09-11 Method of producing hydroxyalkyl starch derivatives
EP03020424A EP1398327B1 (en) 2002-09-11 2003-09-11 Method of producing hydroxyalkyl starch derivatives
JP2006522324A JP2007501870A (en) 2003-08-08 2004-08-06 Complex of hydroxyalkyl starch and G-CSF
EP04763853A EP1660134B1 (en) 2003-08-08 2004-08-06 Conjugates of hydroxyalkyl starch and g-csf
SG200806055-0A SG145746A1 (en) 2003-08-08 2004-08-06 Conjugates of hydroxyalkyl starch and g-csf
BRPI0413450-8A BRPI0413450A (en) 2003-08-08 2004-08-06 hydroxyalkyl starch and g-csf conjugates
MXPA06001359A MXPA06001359A (en) 2003-08-08 2004-08-06 Conjugates of a polymer and a protein linked by an oxime linking group.
DE602004030805T DE602004030805D1 (en) 2003-08-08 2004-08-06 CONJUGATES OF HYDROXYALKYL STARCH AND G-CSF
KR1020067002747A KR101154343B1 (en) 2003-08-08 2004-08-06 Conjugates of hydroxyalkyl starch and g-csf
CNA2004800295229A CN1863549A (en) 2003-08-08 2004-08-06 Conjugates of a polymer and a protein linked by an oxime linking group
EP04763855A EP1653991A2 (en) 2003-08-08 2004-08-06 Conjugates of a polymer and a protein linked by an oxime linking group
AT04763853T ATE493150T1 (en) 2003-08-08 2004-08-06 CONJUGATES OF HYDROXYALKYL STARCH AND G-CSF
CA002534412A CA2534412A1 (en) 2003-08-08 2004-08-06 Conjugates of a polymer and a protein linked by an oxime linking group
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AU2004262921A AU2004262921B2 (en) 2003-08-08 2004-08-06 Conjugates of hydroxyalkyl starch and G-CSF
SI200431617T SI1660134T1 (en) 2003-08-08 2004-08-06 Conjugates of hydroxyalkyl starch and g-csf
RU2006106926/15A RU2370281C2 (en) 2003-08-08 2004-08-06 Hydroxyalkyl starch and g-csf conjugates
TW093123553A TWI357337B (en) 2003-08-08 2004-08-06 Conjugates of hydroxyalkyl starch and g-csf
BRPI0412671-8A BRPI0412671A (en) 2003-08-08 2004-08-06 conjugates of a polymer and a protein linked by an oxime linking group
PCT/EP2004/008818 WO2005014050A2 (en) 2003-08-08 2004-08-06 Conjugates of hydroxyalkyl starch and g-csf
CN2004800226500A CN1832762B (en) 2003-08-08 2004-08-06 Conjugates of hydroxyalkyl starch and g-csf
PL04763853T PL1660134T3 (en) 2003-08-08 2004-08-06 Conjugates of hydroxyalkyl starch and g-csf
PCT/EP2004/008821 WO2005014655A2 (en) 2003-08-08 2004-08-06 Conjugates of hydroxyalkyl starch and a protein
ZA200600651A ZA200600651B (en) 2002-09-11 2004-08-06 Conjugates of hydroxyalkyl starch and G-CSF
PCT/EP2004/008820 WO2005014024A2 (en) 2003-08-08 2004-08-06 Conjugates of a polymer and a protein linked by an oxime linking group
US10/567,266 US20080274948A1 (en) 2003-08-08 2004-08-06 Conjugates of Hydroxyalkyl Starch and G-Csf
US10/567,265 US20080206182A1 (en) 2003-08-08 2004-08-06 Conjugates of a Polymer and a Protein Linked by an Oxime Group
MXPA06001358A MXPA06001358A (en) 2003-08-08 2004-08-06 Conjugates of hydroxyalkyl starch and g-csf.
CA002534418A CA2534418A1 (en) 2003-08-08 2004-08-06 Conjugates of hydroxyalkyl starch and g-csf
TW093123647A TW200519128A (en) 2003-08-08 2004-08-06 Conjugates of hydroxyalkyl starch and a protein
TW093123642A TWI356065B (en) 2003-08-08 2004-08-06 Method of producing hydroxyalkyl starch derivative
ARP040102853A AR045450A1 (en) 2003-08-08 2004-08-09 CONJUGATES OF ALMIDON DE HIDROXIALQUILO AND A PROTEIN
ARP040102852A AR045236A1 (en) 2003-08-08 2004-08-09 CONJUGATES OF HYDROXIALQUIL ALMIDON AND G-CSF
ARP040102851A AR045235A1 (en) 2002-09-11 2004-08-09 METHOD TO PRODUCE DERIVATIVES OF THE HYDROXIALQUIL ALMIDON AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM.
HK04106173A HK1063477A1 (en) 2002-09-11 2004-08-18 Method of producing hydroxyalkyl strach derivatives
IL166930A IL166930A (en) 2002-09-11 2005-02-16 Method of producing hydroxyalkyl starch derivatives and pharmaceutical compositions comprising them
US11/078,098 US20050238723A1 (en) 2002-09-11 2005-03-11 Method of producing hydroxyalkyl starch derivatives
NO20051427A NO20051427L (en) 2002-09-11 2005-03-17 Process for the production of hydroxyalkyl starch derivatives
IL173187A IL173187A (en) 2003-08-08 2006-01-17 Method for preparing a conjugate comprising a granulocyte colony stimulating factor protein and a hydroxyalkyl starch polymer derivative and various aspects related thereto
NO20061121A NO20061121L (en) 2003-08-08 2006-03-08 Hydroxyalkyl starch conjugates and G-CSF
HK06110272.2A HK1089683A1 (en) 2003-08-08 2006-09-15 Conjugates of hydroxyalkyl starch and g-csf
US12/824,618 US20120046240A9 (en) 2002-09-11 2010-06-28 Method of Producing Hydroxyalkyl Starch Derivatives
HR20110056T HRP20110056T1 (en) 2003-08-08 2011-01-24 Conjugates of hydroxyalkyl starch and g-csf
JP2012130094A JP2012211329A (en) 2003-08-08 2012-06-07 Conjugate of hydroxyalkyl starch and g-csf

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ATE323723T1 (en) 2006-05-15
KR20050061466A (en) 2005-06-22
ZA200600651B (en) 2007-04-25
KR20050075336A (en) 2005-07-20
AU2003255406B2 (en) 2009-09-10
CN1681844A (en) 2005-10-12
EP2143736A1 (en) 2010-01-13
MXPA05002591A (en) 2005-09-20
JP2010265464A (en) 2010-11-25
JP2006516534A (en) 2006-07-06
IL166930A (en) 2012-08-30
RU2005110412A (en) 2006-01-20
EP2143736B1 (en) 2012-11-07
US8475765B2 (en) 2013-07-02
HK1136226A1 (en) 2010-06-25
EP1398322B1 (en) 2006-04-19
RU2326891C2 (en) 2008-06-20
PL216545B1 (en) 2014-04-30
DK1398328T3 (en) 2010-03-08
JP2010248528A (en) 2010-11-04
JP2011089125A (en) 2011-05-06
IL200150A0 (en) 2011-07-31
EP2154160A1 (en) 2010-02-17
WO2004024761A1 (en) 2004-03-25

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