US20050238723A1 - Method of producing hydroxyalkyl starch derivatives - Google Patents

Method of producing hydroxyalkyl starch derivatives Download PDF

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US20050238723A1
US20050238723A1 US11078098 US7809805A US2005238723A1 US 20050238723 A1 US20050238723 A1 US 20050238723A1 US 11078098 US11078098 US 11078098 US 7809805 A US7809805 A US 7809805A US 2005238723 A1 US2005238723 A1 US 2005238723A1
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hydroxyalkyl starch
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Norbert Zander
Harald Conradt
Wolfram Eichner
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Fresenius Kabi Deutschland GmbH
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    • C08B31/00Preparation of derivatives of starch
    • C08B31/08Ethers
    • C08B31/12Ethers having alkyl or cycloalkyl radicals substituted by heteroatoms, e.g. hydroxyalkyl or carboxyalkyl starch
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    • C08B31/00Preparation of derivatives of starch
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    • A61K38/00Medicinal preparations containing peptides

Abstract

The present invention relates to methods of producing hydroxyalkyl starch (HAS) derivatives having a structure according to formula (I) comprising reacting HAS of formula (I) at its optionally oxidized reducing end or a HAS derivative, obtainable by reacting HAS of formula (I) at its optionally oxidized reducing end with a compound (D) comprising at least one functional group Z1 capable of being reacted with the optionally oxidized reducing end of the HAS and at least one functional group W, with a compound (L) comprising at least one functional group Z1 capable of being reacted with said HAS, or at least one functional group Z2 capable of being reacted with functional group W comprised in said HAS derivative, and at least one functional group X capable of being reacted with a functional group Y of a further compound. The present invention further relates to HAS derivatives and pharmaceutical compositions comprising them.

Description

  • 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 especially 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 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, 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 (Sommermeyer 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-1,6-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 physicochemical 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 physiological effect. One specific example of these polypeptides is erythropoietin, an acid glycoprotein 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 immunogenicity and antigenicity and circulate in the bloodstream considerably longer than unconjugated 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, CRCS, 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 activity have to be purified from isoforms with a low degree of sialic acids (see EP 0 428 267 B1). 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 hydroxyalkyl 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 US20050238723A1-20051027-C00001
      • 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 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, 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 R1, 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 R1, R2, 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 monoalkyl 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 preferably the same as mentioned above for substituted alkyl residues.
  • Techniques for the esterification of starch are known in the art (see e.g. Klemn D. et al, Comprehensive Cellulose Chemistry Vol. 2, 1998, Whiley-VCH, Weinheim, N.Y., 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 differs from the other with respect to the polymerisation degree, the number and pattern of branching sites, and the substitution pattern. BES 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 molecules. 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 R1, 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, R1, 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 R1, R2 and R3 are independently hydrogen or a linear or branched hydroxyalkyl group with from 1 to 6 carbon atoms.
  • Thus, R1, 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 R1, 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 Z1 with the reducing end where group Z1 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 (IIa)
    Figure US20050238723A1-20051027-C00002

    and/or according to formula (IIb)
    Figure US20050238723A1-20051027-C00003
  • 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 (IIa) and/or (IIb).
  • Although the oxidation may be carried out according to all suitable method or methods resulting in the oxidized reducing end of hydroxyakyl starch, it is preferably carried out using an alkaline iodine solution as described, e.g., in U.S. Pat. No. 196 28 705 A1.
  • 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 US20050238723A1-20051027-C00004
  • 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 Z1 with the optionally oxidized reducing end of the hydroxyalkyl starch. As functional group Z1, 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 Z1 comprises the chemical structure —NH—.
  • Therefore, the present invention also relates to a method as described above wherein the functional group Z1 comprises the structure —NH—.
  • According to one preferred embodiment of the present invention, the functional group Z1 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, Cl or Br may be mentioned. Especially preferred residues R′ are hydrogen, allyl 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 Z1 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 US20050238723A1-20051027-C00005

    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 Z1 is selected from the group consisting of
    Figure US20050238723A1-20051027-C00006

    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, Cl 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 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 US20050238723A1-20051027-C00007

    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 factional group X is selected from the group consisting of
    Figure US20050238723A1-20051027-C00008

    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 US20050238723A1-20051027-C00009

    wherein Hal is Cl, Br or I, preferably Br or I.
  • Therefore, the present invention also relates to a method as described above whereinwherein the functional group Y is —SH and the functional group X is selected from the group consisting of
    Figure US20050238723A1-20051027-C00010

    wherein Hal is Cl, 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 provided 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 structure 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, hydroxylarylamino groups, hydroxylaralkylamino groups, or hydroxalalkarylamino groups;
      • alkoxyamino groups, aryloxyamino groups, araloxyamino groups, or alkaryloxyamino 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 aralkyithio group, or an alkarylthio group;
        • an alkylcarbonyloxy group, an arylcarbonyloxy group, an aralkylcarbonyloxy 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 pentafluorophenyl, paranitrophenyl or trochlorophenyl;
        • wherein Q is absent or NH or a heteroatom such as S or O;
      • —NH—NH2, or —NH—NH—;
      • —NO2;
      • the ntril 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 triflate;
      • —C—C—H;
      • —(C═NH2Cl)—OAlkyl
      • groups —(C═O)—CH2-Hal wherein Hal is Cl, Br, or I;
      • —CH═CH—SO2—;
      • a disulfide group comprising the structure —S—S—;
      • the group
        Figure US20050238723A1-20051027-C00011
      • the group
        Figure US20050238723A1-20051027-C00012
      • 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 US20050238723A1-20051027-C00013

    wherein Hal is Cl, 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 US20050238723A1-20051027-C00014

    wherein Hal is Cl, 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 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, Cl 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 US20050238723A1-20051027-C00015

    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
    Figure US20050238723A1-20051027-C00016

    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 capable 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 US20050238723A1-20051027-C00017

    to give a structure of the following formula
    Figure US20050238723A1-20051027-C00018

    which is, e.g., reactive towards a thio group.
  • Another example for a functional group to be modified prior to the reaction with a fatter compound is a —NH2 group which is modified by the reaction with, e.g., a compound according to the following formula
    Figure US20050238723A1-20051027-C00019

    to give a structure of the following formula
    Figure US20050238723A1-20051027-C00020

    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 Z1-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 Z1-L′-X where L′ is absent:
    Figure US20050238723A1-20051027-C00021
  • 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 Z1-L′-X where L′ is not absent:
    Figure US20050238723A1-20051027-C00022
  • 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 Z1-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 Z1-D′-W where D′ is absent:
    Figure US20050238723A1-20051027-C00023
  • 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 Z1-D′-W where D′ is not absent are:
    Figure US20050238723A1-20051027-C00024
    Figure US20050238723A1-20051027-C00025
  • 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 functional 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:
    Type of compound (L) Functional group X Functional group Z2
    C Iodoalkyl N-succinimide ester
    D Bromoalkyl N-succinimide ester
    E Maleimido N-succinimide ester
    F Pydridyldithio N-succinimide ester
    G Vinylsulfone N-succinimide ester
  • 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:
    Type of compound (L) Functional group X Functional group Z2
    A Hydrazide Maleimido
    B Hydrazide Pyridyldithio
  • In Table 1 at the end of the present description, some preferred examples of compounds (L) according to the types given above are listed.
  • The separating groups L′ and/or D′ may be suitably substituted. Preferred substituents are, e.g, halides such as F, Cl, Br or I.
  • The separating groups L′ and/or D′ may comprise one or more cleavage sites such as
    Figure US20050238723A1-20051027-C00026

    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 compound (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 US20050238723A1-20051027-C00027

    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 Z2 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 US20050238723A1-20051027-C00028

    the compounds (D)
    Figure US20050238723A1-20051027-C00029

    being particularly preferred.
  • Together with the above-mentioned preferred compounds (D), the following compounds (L)
    Figure US20050238723A1-20051027-C00030

    are preferred, the compound (L)
    Figure US20050238723A1-20051027-C00031

    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 Z1 is a group R′—NH—, as described above, the hydroxyalkyl starch derivative may have a constitution according to formula (IIIa)
    Figure US20050238723A1-20051027-C00032
  • If the reaction is carried out in an aqueous system and the functional group Z1 is a group R′—NH— with R′=H, as described above, the hydroxyalkyl starch derivative may have a constitution according to formula (IIIa) or formula (IIIb) or be a mixture of compounds according to formulae (IIIa) and (IIIb)
    Figure US20050238723A1-20051027-C00033
  • 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 (IIIa) 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 (IIIb) 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 (IIIb) or according to formula (IIIb) or according to formulae (IIIa) and (IIIb).
  • In some cases it may be desirable to stabilize the compound according to formula (IIIa). This is especially the case where the compound according to formula (IIIa) is produced and/or used in an aqueous solution. As stabilizing method, acylation of the compound according to formula (IIIa) 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 (IVa)
    Figure US20050238723A1-20051027-C00034
  • 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 (IVa).
  • In other cases it may be desirable to stabilize the compound according to formula (IIIb). This is especially the case where the compound according to formula (IIIb) is produced and/or used in an aqueous solution. As stabilizing method, reduction of the compound according to formula (IIIb) 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 (IVb)
    Figure US20050238723A1-20051027-C00035
  • According to especially preferred embodiments of the present invention, as reduction reagents boro hydrides such as NaCNBH3 or NaBH4 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 according to formula (IVb).
  • The present invention further relates to mixtures of compounds having constitutions according to formulae (IIIa) and (IIIb), (IVa) and (IVb), (IIIa) and (IVa), (IIIa) and (IVb), (IIIb) and (IVa), (IIIb) and (IVb), (IIIa) and (IIIb) and (IVa), (IIIa) and (IIIb) and (IVb), (IVa) and (IVb) and (IIIa), and (IVa) and (IVb) and (IIIb) wherein (IIIa) and/or (IVa) may be independently present in a conformation where the N atom in equatorial or axial position and/or wherein (IIIb) may be present with the C—N double bond in E or 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 certain 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 Z1 is a group R′—NH—, as described above, the hydroxyalkyl starch derivative may have a constitution according to formula (Va)
    Figure US20050238723A1-20051027-C00036
  • 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 (I) 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 compound (L) via the reaction of functional group Z1 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).
  • Another embodiment of the present invention relates to a method as described above wherein hydroxyalkyl starch is reacted with a compound (L) via the reaction of functional group Z1 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).
  • 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 Z1 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 functional 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 Z1 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).
  • 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 compound (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 compound (D) or compound (L) is a hydroxyl amine or a hydrazide, the reaction 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 tie range of from 2 h to 7 d.
  • The pH value for the reaction of hydroxyalkyl starch with compound (D) or compound (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 compound (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 monomethyltrityl 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 functional 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 functional group X available for further 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 functional group X available for reaction with yet a further 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 molecules, i.e. a multiple HAS substituted compound (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 further 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 compound (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 compound 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 further 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 functional group Y may be linked directly to the polypeptide backbone or be part of a side-chain of the backbone. 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 functional 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, interleukin-6 and therapeutic antibodies.
  • According to a preferred embodiment, the polypeptide is an antithrombin (AT), preferably AT III (Levy J H, Weisinger A, Ziomek C A, Echelard Y, Recombinant Antithrombin: Production and Role in Cardiovascular Disorder, Seminars in Thrombosis and Hemostasis 27, 4 (2001) 405-416; Edmunds T, Van Patten S M, Pollock J, Hanson E, Bernasconi R, Higgins E, Manavalan P, Ziomek C, Meade H, McPherson J, Cole E S, Transgenically Produced Human Antithrombin: Structural and Functional Comparison to Human Plasma-Derived Antithrombin, Blood 91, 12 (1998) 4661-4671; Minnema M C, Chang A C K, Jansen P M, Lubbers Y T P, Pratt B M, Whittaker B G, Taylor F B, Hack C E, 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 S M, Hanson E H, Bernasconi R, Zhang K, Manavaln P, Cole E S, McPherson J M, 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 1a (cf. Avonex®, REBIFO) and IFN-beta 1b (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® (Filgrastitn), a recombinant human 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 polypeptides 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 reacted 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 hydroxyaldehydes or hydroxyketones as well as to chemical modifications thereof (see Römpp Chemielexikon, Thieme Verlag Stuttgart, Germany, 9th edition 1990, Volume 9, pages 2281-2285 and the literature cited therein). Furthermore, it also refers to derivatives of 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. Preferably, 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 (I) 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 further reacted with compound (L) is linked to a sialic acid residue of the carbohydrate side chains, preferably the terminal sialic acid residue of the carbohydrate 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 peijodate (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 1 h 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 functional 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 compound (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 functional 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 erythropoietin with high in vivo activity from the urine of anemic patients, Biol. Pharm. Bull. 17(2), 180-4; Miyake, Kung, Goldwasser, 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 “erytliropoietin” 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 another amino acid and which exhibits erythropoietic activity (see e.g. EP 640 619 B1). 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, Erytlropoietin concentrated solution, Pharmaeuropa, 8, 371-377; Fibi, Hermentin, Pauly, Lauffer, Zettlmeissl., 1995, N- and O-glycosylation 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 of 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, Begtrin, 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 glycosylation 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, Masuda, Sasaki, 1991, Effects of site-directed removal of N-glycosylation sites in human erythropoietin on its production and biological properties, J. Biol. Chem., 266(30), 20434-9; Takeuchi, Inoue, Strickland, Kubota, Wada, Shimizu, Hoshi, Kozutsuni, Takasaki, Kobata, 1989, Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese hamster 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 erythropoietin 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 erythropoietic 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. Furthermore, 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 detained 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 baculovirus vector, Blood, 74(2), 652-7; EP 640 619 B1 and EP 668 351 B1.
  • 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 bave been extensively studied in the art (see EP 428 267 B1; EP 640 619 B1; Rush, Derby, Smith, Merry, Rogers, Rohde, Katta, 1995, Microheterogeneity of erythropoietin carbohydrate structure, Anal Chem., 67(8), 1442-52; Takeuchi, Kobata, 1991, Structures and functional 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 preferably 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 quanatitative 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 carbohydrate 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 reaction product of hydroxyalkyl starch and compound (L) or the reaction product of hydroxyalkyl starch and compound (I) 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 (I) which is further 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 further 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 compound (L) or the reaction product of hydroxyalkyl starch and compound (D) which is further reacted with compound (S) 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 compound (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 (I), optionally with compound (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)-ethyl]-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 preferably erythropoietin, can be performed by using known procedures for the purification of natural and recombinant EPO (e.g. size exclusion 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 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 such as, e.g., by a column containing Q-sepharose, HPLC, MPLC, gel filtration and/or lyophilisation. 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 obtainable by a method of producing a hydroxyalkyl starch derivative, said hydroxyalkyl starch having a structure according to formula (I)
    Figure US20050238723A1-20051027-C00037
      • 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 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, or a thio group.
  • According to a preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein R1, 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 R1, R2 and R3 are independently hydrogen or a 2-hydroxyethyl group.
  • According to a further 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 Z1 comprises the structure —NH—.
  • According to an especially preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative as described above wherein Z1 is selected from the group consisting of
    Figure US20050238723A1-20051027-C00038

    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 keto 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 US20050238723A1-20051027-C00039

    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 US20050238723A1-20051027-C00040

    wherein Hal is Cl, 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 —SH and the functional group Z2 or the functional group W is selected from the group consisting of
    Figure US20050238723A1-20051027-C00041

    wherein Hal is Cl, 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 US20050238723A1-20051027-C00042

    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 US20050238723A1-20051027-C00043
  • 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 (IIa)
    Figure US20050238723A1-20051027-C00044
      • and/or according to formula (IIb)
        Figure US20050238723A1-20051027-C00045
  • According to a further preferred embodiment, the present invention relates to a hydroxyalkyl 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 Z1 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).
  • 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 Z1 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 Z1 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.
  • 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 Z1 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).
  • 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 erythropoietin.
  • 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 compound (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 B1), 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 circulation 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, Lurn, Davis, Feldman, Siekman, 1998, Human erythropoietin dhiers 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 preferable 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 comprising, in a therapeutically effective amount, the HAS-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 US20050238723A1-20051027-C00046
      • 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 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, 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 disorders 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 J H, Weisinger A, Ziomek C A, Echelard Y, Recombinant Antithrombin: Production and Role in Cardiovascular Disorder, Seminars in Thrombosis and Hemostasis 27, 4 (2001) 405-416; Edmunds T, Van Patten S M, Pollock J, Hanson E, Bemasconi R, Higgins E, Manavalan P, Ziomek C, Meade H, McPherson J, Cole E S, Transgenically Produced Human Antithrombin: Structural and Functional Comparison to Human Plasma-Derived Antithrombin, Blood 91, 12 (1998) 4661-4671; Minnema M C, Chang A C K, Jansen P M, Lubbers Y T P, Pratt B M, Whittaker B G, Taylor F B, Hack C E, Friedrnan 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 S M, Hanson E H, Bernasconi R, Zhang K, Manavaln P, Cole E S, McPherson J M, Edmunds T, Oxidation of Methionine Residues in Antithrombin, J. Biol. Chemistry 15 274, 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 compound (L) is a compound of general formula Z1-L′-X where the functional group Z1 is selected from the group consisting of
    Figure US20050238723A1-20051027-C00047

    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 US20050238723A1-20051027-C00048

    wherein Hal is Cl, Br or I, and where L′ is an organic chain bridging Z1 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 Z1-L′-X where the functional group Z1 is selected from the group consisting of
    Figure US20050238723A1-20051027-C00049

    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 US20050238723A1-20051027-C00050

    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 Z1 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 Z1 -D′-W, and compound (L) is a compound of general formula Z2-L′-X, where the functional group Z1 is selected from the group consisting of
    Figure US20050238723A1-20051027-C00051

    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 US20050238723A1-20051027-C00052

    wherein Hal is Cl, 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 US20050238723A1-20051027-C00053

    wherein Hal is Cl, 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 US20050238723A1-20051027-C00054

    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 Z1 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 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, compound (D) is a compound of general formula Z1-D′-W, and compound (L) is a compound of general formula Z2-L′-X, where the functional group Z1 is selected from the group consisting of
    Figure US20050238723A1-20051027-C00055

    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 US20050238723A1-20051027-C00056

    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 functional group Z2 or the functional group W is selected from the group consisting of
    Figure US20050238723A1-20051027-C00057

    wherein Hal is Cl, 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 US20050238723A1-20051027-C00058

    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 Z1 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
    Figure US20050238723A1-20051027-C00059

    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 pharmaceutical 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 administration of erythropoietin isoforms 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 of 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/l in the blood. For this, the pharmaceutical composition may be administered in a way that the hemoglobin value increases between from 0.6 mmol/l and 1.6 mmol/l per week. If the hemoglobin value exceeds 8.7 mmol/l, the therapy should be preferably interrupted until the hemoglobin value is below 8.1 mmol/l.
  • The composition of the invention is preferably used in a formulation 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 further 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
  • FIG. 1
  • FIG. 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.
  • FIG. 2
  • FIG. 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.
  • FIG. 3
  • FIG. 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.
  • FIG. 4
  • FIG. 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 PRESTADMD (Carl Roth GmbH+Co, Karisruhe, 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.
  • FIG. 5
  • FIG. 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 Example 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 starting material
  • FIG. 6
  • FIG. 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 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.
  • Lane D: 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.
  • FIG. 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 of EPO after removal of N-glycans is shown (+PNGASE).
  • FIG. 8
  • HPAEC-PAD pattern of oligosaccharides isolated from untreated EPO and from 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 diantennary structure, II=trisialylated 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. The elution area of oligosaccharides structures without, with 1-4 sialic acid is indicated by brackets.
  • FIG. 9
  • HPAEC-PAD of N-linked oligosaccharides after desialylation; the elution position of N-acetylneuraminic acid is shown; numbers 1-9 indicate the elution position of standard oligosaccharides: 1=diantennary; 2=triantennary (24 isomer), 3=triantennary (2-6 isomer); 4=tetraantennary.; S=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.
  • FIG. 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.
  • FIG. 11
  • HPAEC-PAD 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.
  • FIG. 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 hydroxylamine-modified HES derivative X for 30 min, 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 m 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.
  • FIG. 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 I; C=sample m; K=control EPO-GT-1; A1, B1, C1 and K1 indicated the flow-through fraction; A2, B2, C2 and K2 indicates the fraction eluted with high salt concentration.
  • FIG. 14 a
  • 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).
  • FIG. 14 b
  • SDS-PAGE analysis after mild hydrolysis of HES-modified EPO sample A2 (see FIG. 13), control EPO sample K2 and EPO-GT-LA which were untreated or digested in the presence of N-glycosidase in order to remove N-linked oligosaccharides (see FIG. 14 a). 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.
  • FIG. 15
  • HPAEC-PAD 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=trisialylated 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.
  • FIG. 16
  • HPAEC-PAD analysis of N-linked oligosaccharide material liberated from HES-modified sample A, from EPO-GT-LA 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 (24 isomer); 3=trianteimnay (2-6 isomer); 4=tetrtennary; 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.
  • FIGS. 17 to 23
  • FIGS. 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]+) 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.
  • FIG. 17
  • MALDI/TOF spectrum: desialylated oligosaccharides of HES-modified EPO A2.
  • FIG. 18
  • MALDI/TOF spectrum: desialylated oligosaccharides of EPO GT-1-A.
  • FIG. 19
  • MALDI/TOF spectum: desialylated oligosaccharides of EPO K2.
  • FIG. 20
  • MALDI/ROF spectum: desialylated oligosaccharides of EPO-GT-1.
  • FIG. 21
  • MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1 subjected to acid hydrolysis for 5 min.
  • FIG. 22
  • MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1 subjected to acid hydrolysis for 10 min.
  • FIG. 23
  • MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1 subjected to acid hydrolysis for 60 min.
  • FIG. 24
  • FIG. 24 shows an SDS page analysis of two HES-EPO conjugates
  • mw: marker
  • Lane 1: HES-EPO produced according to example protocol 8B: EPO is conjugated to hydrazido-HES 12KD L
  • Lane 2: HES-EPO produced according to example protocol 9B: EPO is conjugated to hydroxylamino HES 12 KD K
  • C: control (unconjugated EPO); the upper band represents EPO dimer
  • FIG. 25
  • FIG. 25 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 polypeptide N-glycosidase
  • Lane 1: HES-EPO produced according to example protocol 8B after digestion with N-glycosidase
  • Lane 2: HES-EPO produced according to example protocol 9B 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, Calif., USA)
  • In the context of the present invention, the degree of substitution, denoted as DS, relates to the molar substitution, as described above (see also Sommermeyer et al., 1987, Krankenhauspharmazie, 8(8), 271-278, in particular p. 273). Throughout the invention, the DS of the HES18/04 when measured according to Sommermeyer et al., 1987, Krankenhauspharmazie, 8(8), 271-278 was 0,5.
  • 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 1,3-diamino-2-hydroxy propane
  • Figure US20050238723A1-20051027-C00060
  • a) To a solution of 200 mg hydroxyethyl starch (HES18/0.4 (MW=18,000 D, DS=0.4)) in 5 ml water, 0.83 mmol 1,3-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 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 1,3-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 1,2-dihydroxy-3-amino propane
  • Figure US20050238723A1-20051027-C00061
  • a) To a solution of 200 mg hydroxyethyl starch (HES18/0.4 (MW=18,000 D, DS=0.4)) in 5 ml water, 0.83 mmol 1,2-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 1,2-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 1,2-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
  • Figure US20050238723A1-20051027-C00062
  • a) To a solution of 200 mg hydroxyethyl starch (HES18/0.4 (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 1-mercapto-2-amino ethane
  • Figure US20050238723A1-20051027-C00063
  • a) To a solution of 200 mg hydroxyethyl starch (HES18/0.4 (MW=18,000 D, DS=0.4)) in 5 ml water, 0.83 mmol 1-mercapto-2-amino ethane (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-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 US20050238723A1-20051027-C00064
  • 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 US20050238723A1-20051027-C00065
  • 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, Frankurt/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 Science Deutschland GmbH, Bonn, D), and lyophilized.
  • Example 2.3 Reaction of Hydroxyethyl Starch with 1,4-phenylene-bis-3-thiosemicarbazide
  • Figure US20050238723A1-20051027-C00066
  • 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 1,4-phenylene-bis-3-thiosemicarbazide (Lancaster Synthesis, Frankfurt/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 centrifugated 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 centrifugated 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
  • Figure US20050238723A1-20051027-C00067
  • 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 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 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 US20050238723A1-20051027-C00068
  • 0.12 mmol Oxo-HES 10/0.4 (MW=10,000 D, DS=0.4, prepared according to DE 196 28 705 A1) 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 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 3.2 Reaction of Hydroxyethyl Starch with 1,4-phenylene-bis-3-thiosemicarbazide
  • Figure US20050238723A1-20051027-C00069
  • 0.12 mmol Oxo-BES 10/0.4 (MW=10,000 D, DS=0.4, prepared according to DE 196 28 705 A1) were dissolved in 3 ml absolute dimethyl sulfoxide (DMSO) and added dropwise under nitrogen to a mixture of 15 mmol of 1,4phenylene-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 according to DE 196 28 705 A1) 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
  • Figure US20050238723A1-20051027-C00070
  • 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 A1) 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-aminooxyethoxy)-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 US20050238723A1-20051027-C00071
  • 1.74 g (15 mmol) adepic dihydraride (Lancaster Synthesis, Frankfurt/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 A1), 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 tubing, 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
  • Figure US20050238723A1-20051027-C00072
  • 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 A1) 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-HESIOKD/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 erythropoietin, 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, Calif., 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 bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • Example 5.2 Reaction of Oxidized Erythropoietin with the Reaction Product of Example 23
  • 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, Calif., 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, Calif., 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, Calif., 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, Calif., 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 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 US20050238723A1-20051027-C00073

    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 bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • Example 7.2 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 23 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. 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 7.6 Reaction of thio-erythropoietin with the Reaction Product of Example 13 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 bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives linked to the protein.
  • Example 7.7 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, Calif., 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, Calif., 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, Calif., 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, Calif., 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, Calif., 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 7.12 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 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, Calif., 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 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 UV 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. Complete 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 supernatants 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 3×500 μl of 80% acetonitrile (v/v) in 0.1% TFA followed by washes with 3×500 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 NH4OH 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 Bruker ULTRAFLEX time-of-flight (TOF/TOF) instrument was used: native desialylated oligosaccharides were analyzed using 2,5-dihydroxybenzoic acid as UV-absorbing material in the positive as well as in the negative ion mode using the reflectron 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 approximate concentration of 1-10 pmol·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 P P et al., 1999, Dorner A J et al., 1984) and the preparations were characterized according to methods described in the Eur. Phar. (Ph. Eur. 4, Monography January 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 Ser126 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 H2SO4 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 centrifuge equipped with a fixed angle rotor. After freezing in liquid nitrogen 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 HPAEC-PAD 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 January 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 Vydac, Calif., 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 Na-phosphate buffer pH 6.8 in the presence of 16 μl catalase (6214 units/200 ml) and 80 μl of galactose oxidase (2250 units/ml from Dactylium dendroides (Sigma-Aldrich, Steinheim, Germany); incubation at 37° C. spas 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 N-morpholine propane sulfonic acid [MOPS/NaOH] 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 (CV) 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 mm/min) and elution was performed by using buffer B (20 mM morpholine ethane sulfonic acid [MES/NaOH], 0.5 M NaCl 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 Amperometric Detection (HPAEC-PAD)
  • Purified native and desialylated oligosaccharides were analyzed by high-pH anionexchange (HPAE) chromatography using a Dionex BioLC system (Dionex, USA) equipped with a CarboPac PA1 column (0.4×25 cm) in combination with a pulsed amperometric detector (PAD) (Schröter et al., 1999; Nimtz et al., 1999). Detector potentials (E) and pulse durations (T) were: E1: +50 mV, T1: 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 PA1 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, Calif.) running in the positive ion EI 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 N-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 N-linked oligosaccharides by incubation with N-glycosidase as shown in FIG. 7. N-linked oligosaccharides were subjected to HPAEC-PAD oligosaccharide mapping (FIG. 8). The untreated EPO-GT-1 contained >90% of N-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. HPAEC-PAD of the desialylated N-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 FIG. 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 HPAEC-PAD analysis a shift of oligosaccharides to earlier elution times (compare FIGS. 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 μL 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 FIG. 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 diffuse 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 N-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 N-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 FIG. 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 concentration. 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 FIG. 11) were compared to periodate oxidized form EPO-GT-1-A. The samples were subjected to N-glycosidase treatment and as is depicted in FIGS. 14 a and 14 b 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 8.11(c)(aa)). This band (and also the heavily HES-modified high mw form of N-glycosylated EPO, see FIGS. 14 a and 14 b) 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 trifuiloacetic 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), 2175triantennary, 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-FIGS. 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% acetonitrile in water containing 0.1% TFA; under these conditions 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 H2O containing 0.1% TFA. The oligosaccharide fractions from the RP-C18 step of N-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 HPAEC-PAD mapping before (see FIGS. 15) and after mild acid treatment under conditions which enabled quantitative removal of sialic acids from glycans (see FIGS. 16).
  • The HPAEC-PAD 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 in FIG. 11 (sample named EPO-GT-1 after mild periodate treatment). The elution profile of oligosaccharides obtained from the control EPO sample (K) yielded the expected pattern (compare profile in FIG. 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 FIGS. 16) with the expected qualitative and quantitative composition 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 N-glycosidase and is acid-labile since it is removed from the N-glycans using mild acid treatment conditions known to desialylate carbohydrates (see FIGS. 14 a+b).
  • (cc) Monosaccharide Compositional Analysis of HES-EPO and HES-EPO N-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 derivatives 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 E P, Conradt H S.
    • Carbohydrate structures of a human tissue plasminogen activator expressed in recombinant Chinese hamster ovary cells.
    • FEBS Lett. Oct. 1, 1990; 271(1-2):14-8
    • Dorner A J, Wasley L C, Kaufman R J.
    • Increased synthesis of secreted proteins induces expression of glucose-regulated proteins in butyrate-treated Chinese hamster ovary cells.
    • J Biol Chem. Dec. 5, 1989; 264 (34):20602-7
    • Mueller P P, Schlenke P, Nimtz M, Conradt H S, Hauser H
    • Recombinant glycoprotein quality in proliferation-controlled BHK-21 cells.
    • Biotechnol Bioeng. Dec. 5, 1999; 65(5):529-36
    • Nimtz M, Martin W, Way V, Kloppel K D, Augustin J, Conradt H S.
    • Structures of sialylated oligosaccharides of human erythropoietin expressed in recobminant BHK-21 cells.
    • Eur J Biochem. Apr. 1, 1993; 213(1):39-56
    • Hermentin P, Witzel R. Vliegenthart J F, Kamerling J P, Nimtz M, Conradt H S.
    • A strategy for the mapping of N-glycans by high-ph anion-exchange chromatography with pulsed amperometric detection.
    • Anal Biochem. June 1992; 203(2):281-9
    • Schroter S, Derr P, Conradt H S, Nimtz M, Hale G, Kirchhoff C.
    • Male specific modification of human CD52.
    • J Biol Chem. Oct. 15, 1999; 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 expression 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-VII in the biosynthesis of Lewis(x) and sialyl Lewis(x) motifs on complex-type N-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 described (Grabenhorst et al.). Three days after transfection, the cells were subcultivated 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 supernatants 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 NeuAc (e.g. 2-8, 4-10, 8-12 NeuAc 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 is 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 are infected at cell density of 2×106 or X107 cells per mL and EPO titers are determined every day in the cell culture supernatants. EPO is purified by Blue sepharose chromatography, ion-exchange chromatography on Q-Sepharose and finally RP-HPLC on C4-Phase.
  • Purity of the product is checked by SDS-PAGE and N-terminal sequencing. Detailed carbohydrate structural analysis (N- and O-glycosylation) may be performed according to published procedures.
  • Literature:
  • Grabenhorst, Hofer, Nimtz, Jager, Conradt, 1993, Biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus-infected Sf21 cells. Characterization of polypeptides 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 baculovirus vector, Blood, 74(2), 652-7
  • Example 10A
  • Formation of Reactive HES Derivatives
  • 1. SH-Reactive HES 1.1 Reaction of EMCH with Oxo-HES12KD to form SH-Reactive HES12KD B
  • 0.144 g (0.012 mmol) of Oxo-HES12KD (Fresenius German Patent DE 196 28 705 A1)
    Figure US20050238723A1-20051027-C00074

    are dissolved in 0.3 mL absolute dimethyl sulfoxide (DMSO) and are 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 is added to 16 mL of a 1:1 mixture of ethanol and acetone. The precipitate is collected by centrifugation, redissolved in 3 mL DMSO and again precipitated as described. The SH-reactiv-HES12KD B is 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 2; marked with an “A”. Furthermore, another group of cross-linkers exhibiting an activated disulfide function instead of a maleimide function could also be used.
  • 1.2 Halogenacetamide-Derivatives of HES Glycosylamines
  • a) Glycosylamine-Formation 1
  • A 1 mg sample of HES12KD is dissolved in 3 mL of saturated ammonium bicarbonate. Additional solid ammonium bicarbonate is then added to maintain saturation of the solution during incubation for 120 h at 30° C. The Amino-HES12KD C is 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 is dissolved in 1 mL of 1 M sodium bicarbonate and cooled on ice. To this is added a crystal of solid chloroacetic acid anhydride (˜5 mg), and the reaction mixture is allowed to warm to room temperature. The pH is monitored and additional base is added if the pH dropped below 7.0. After two hours at room temperature a second aliquot of base and anhydride is added. After six hours the product Chloroacetamide-HES D1 (X═Cl) is desalted by passage over a mixed bed Amberlite MB-3(H)(OH) ion exchange resins.
  • c) Acylation of the Glycosylamine with Bromoacetic Anhydride2
  • Bromoacetic anhydride is prepared as described by Thomas.3 A 1 mg sample of amino-HES12KD C is dissolved in 0.1 mL of dry DMF and cooled on ice and 5 mg bromoacetic anhydride is added. The reaction mixture is brought slowly to room temperature and the solution is stirred for 3 h. The reaction mixture is added to 1 mL of a 1:1 mixture of ethanol and acetone with −20° C. The precipitate is collected by centrifugation, redissolved in 0.1 mL DMF and again precipitated as described. The Bromoacetamide-HES D2 (X═Br) is obtained by centrifugation and drying in vaccuo. The conjugation reaction with Thio-EPO is described in Example 11, 1.2.
    2Black, Kiss, Tull, Withers, 1993, Carbohydr. Res., 250, 195

    3Thomas, 1977, Methodes Enzymol., 46, 362

    d) The corresponding Iodo-derivative D3 (X═I) is synthesized as described for D2. Instead bromoacetic anhydride N-succinimidyl iodoacetate is used and all steps are performed in the dark.
    Figure US20050238723A1-20051027-C00075

    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 acetyl 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 2 with an “D”. For the use as cross-linkers for the ligation of amino-HES with thio-EPO without isolation of the halogenacetamide-HES derivatives see remarks in example 11, 1.2.
  • 1.3 Halogenacetamide-Derivatives of Amino-HES E1
  • a) Reaction of 1,4-Diaminobutane with Oxo-HES12KD to Amino-HES12KD E4
  • 1.44 g (0.12 mmol) of Oxo-HES12KD are dissolved in 3 mL dry dimethyl sulfoxide (DMSO) and are 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 is added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitate Amino-HES12KD E is collected by centrifugation, redissolved in 40 mL of water an dialyzed for 4 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized.
    1Manger, Wong, Rademacher, Dwek, 1992, Biochemistry, 31, 10733-10740; Manger, Rademacher, Dwek, 1992, Biochemistry, 31, 10724-10732

    4S. Frie, Diplomarbeit, Fachhochschule Hamburg, 1998
    Figure US20050238723A1-20051027-C00076

    b) Chloroacetamide-HES12KD F1 is Prepared as Described for Chloroacetamide-HES12KD D1 in 1.3 above.
    c) Bromoacetamide-HES12KD F2 (X═Br) is Prepared as Described for Bromoacetamide-HES12KD D2 in 1.3 above. The Conjugation Reaction with Thio-EPO is Described in Example 11, 1.2.
    d) The corresponding Iodo-derivative F3 (X═I) is not isolated before its reaction 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
  • a) Reaction of Hydrazine with Oxo-HES12KD
    Figure US20050238723A1-20051027-C00077
  • 1.44 g (0.12 mmol) of Oxo-HES12KD are dissolved in 3 mL absolute dimethyl sulfoxide (DMSO) and are 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 is added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitated product J is collected by centrifugation, redissolved in 40 mL of water and dialyzed 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 oxidized Glyco-EPO is described in Example 12, 2.2.
    b) Reaction of Adipic Dihydrazide with Oxo-HES12KD
    Figure US20050238723A1-20051027-C00078
  • 1.74 g (15 mmol) adipic dihydrazide are dissolved in 20 mL absolute dimethyl sulfoxide (DMSO) at 65° C. and 1.44 g (0.12 mmol) of Oxo-HES12KD, dissolved in 3 mL absolute DMSO are added dropwise under nitrogen. After stirring for 68 h at 60° C. the reaction mixture is added to 200 mL of water The solution containing L is dialyzed 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 oxidized Glyco-EPO is described in Example 12, 2.2.
  • Alternatives:
  • Furthermore, derivatives can be used, wherein 2 hydrazide groups are separated by any spacer.
  • 3. Further Amino-HES12KD Derivatives I and H1
  • Ammonolysis of D or F is performed separately by dissolving a 1 mg sample of each halogenacetamide in 0.1 mL of saturated ammonium carbonate. Additional solid ammonium carbonate is then added to maintain saturation of the solution during incubation of 120 h at 30° C. The reaction mixture is added to 1 mL of a 1:1 mixture of ethanol and acetone with −20° C. The precipitate is collected by centrifugation, redissolved in 0.05 mL water and again precipitated as described. The product aminoHES H or I is obtained by centrifugation and drying in vaccuo. The conjugation reaction with oxidized Glyco-EPO is described in Example 12, 4.1.
    1Manger, Wong, Rademacher, Dwek, 1992, Biochemistry, 31, 10733-10740; Manger, Rademacher, Dwek, 1992, Biochemistry, 31, 10724-10732
    Figure US20050238723A1-20051027-C00079
  • 4. Hydroxylamine-Modified HES12KD K
  • Figure US20050238723A1-20051027-C00080
  • O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine is synthesized as described by Boturyn et al in 2 steps from commercially available materials.5 1.44 g (0.12 mmol) of Oxo-HES12KD are dissolved in 3 mL absolute dimethyl sulfoxide (DMSO) and are 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 is added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitated product K is collected by centrifugation, redissolved in 40 mL of water and dialyzed 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 oxidized Glyco-EPO is described in Example 12, 3.1.
    5Boturyn, Boudali, Constant, Defrancq, Lhomme, 1997, Tetrahedron, 53, 5485
  • 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
  • Figure US20050238723A1-20051027-C00081
  • 1.44 g (0.12 mmol) of Oxo-HES12KD are dissolved in 3 mL absolute dimethyl sulfoxide (DMSO) and are added to a mixture of 1.16 g (15 mmol) cysteamine in 15 mL DMSO under nitrogen dropwise. After stirring for 24 h at 40° C. the reaction mixture is added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitated product M is collected by centrifugation, redissolved in 40 mL of water and dialyzed 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 oxidized 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 hydrazide 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 Modification of Amino-HES12KD E, H or I
  • a) Modification with SATA/SATP
  • 1.44 g (0.12 mmol) of Amino-HES12KD E, H or I are dissolved in 3 mL absolute dimethyl sulfoxide (DMSO) and are 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 is added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitated product N is collected by centrifugation, redissolved in 40 mL of water and dialyzed for 2 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized.
  • The deprotection is 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 is purified by dialysis against a 0.1 M sodium acetate buffer pH 5.5, containing 1 mM EDTA. The deprotection reaction is performed immediately before the conjugation reaction which is described in Example 12, 2.1.
    Figure US20050238723A1-20051027-C00082

    b) Modification with SPDP
  • 1.44 g (0.12 mmol) of Amino-HES12KD E, H or I are dissolved in 3 mL absolute dimethyl sulfoxide (DMSO) and are 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 is added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitated product P is collected by centrifugation, redissolved in 40 mL of water and dialyzed for 2 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized.
  • The deprotection is performed in a solution of 12 mg dithiothreitol (DTT) per 0.5 mL 100 mM sodium acetate 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 is 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, several 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 2, marked with an “H”. The derivatives being analogous to SPDP could have an active ester-function and a disulfide-function, separated by any spacer. Further members of these groups are found in table 2, marked with an “F”. Further analogous derivatives could have an active ester-function and a thiol-function, protected as a trityl derivative, separated by any spacer.
  • Example 10B
  • Formation of Reactive HES Derivatives
  • 1. Halogenacetamide-derivatives of Amino-HES E1
  • Reaction of 1,4-diaminobutane with Oxo-HES18KD to amino-HES18KD E6
  • 1.44 g (0.12 mmol) of Oxo-HES18KD 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-HES18KD E was collected by centrifugation, redissolved in 40 mL of water an dialyzed for 4 days against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized.
    1Manger, Wong, Rademacher, Dwek, 1992, Biochemistry, 31, 10733-10740; Manger, Rademacher, Dwek, 1992, Biochemistry, 31, 10724-10732

    6S. Frie, Diplomarbeit, Fachhochschule Hamburg, 1998
    Figure US20050238723A1-20051027-C00083
  • 2. CHO-Reactive HES
  • a. Hydrazide-HES
  • a) Reaction of Hydrazine with Oxo-HES18KD
    Figure US20050238723A1-20051027-C00084
  • 1.44 g (0.12 mmol) of Oxo-HES18KD 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. The precipitated product J was collected by centrifugation, redissolved in 40 mL of water and dialyzed 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 oxidized Glyco-EPO is described in Example 12, 2.2.
    b) Reaction of Adipic Dihydrazide with Oxo-HES18KD
    Figure US20050238723A1-20051027-C00085
  • 1.74 g (15 mmol) adipic dihydrazide were dissolved in 20 mL absolute dimethyl sulfoxide (DMSO) at 65° C. and 1.44 g (0,12 mmol) of Oxo-HES18KD, 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 dialyzed 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 oxidized Glyco-EPO is described in Example 12, 2.2.
  • 3. Hydroxylamine-Modified HES18KD K
  • Figure US20050238723A1-20051027-C00086
  • O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine was synthesized as described by Boturyn et al in 2 steps from commercially available materials.7 1.44 g (0.12 mmol) of Oxo-HES18KD 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 dialyzed 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 oxidized Glyco-EPO is described in Example 12, 3.1.
    7Boturyn, Boudali, Constant, Defrancq, Lhomme, 1997, Tetrahedron, 53, 5485
  • 4. Thio-HES18KD
  • a. Addition to Oxo-HES18KD
    Figure US20050238723A1-20051027-C00087
  • 1.44 g (0.12 mmol) of Oxo-HES18KD were dissolved in 3 mL absolute dimethyl sulfoxide (DMSO) and were added to a mixture of 1.16 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 dialyzed 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 oxidized Glyco-EPO is described in Example 12, 2.1.
  • 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-HES12KD (E, H or I) with a Cross-linker containing a NHS-active-ester and an iodoacetamide group, e.g. SIA.8
  • Materials
  • A. Borate buffer. Composition is 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. AminoHES12KD 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, 2×5 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)
    8Cumber, Forrester, Foxwell, Ross, Thorpe, 1985, Methods Enzymol., 112, 207
  • Method
  • 100 μL SIA solution is added to 400 μL of the aminoHES12KD E solution and is allowed to react with agitation for 0.5 hours at room temperature. The excess crosslinker is removed by centrifuging the sample at 14000×g for 60 minutes using a microconcentrator. After centrifuging the sample is brought up to its original volume in borate buffer and this process is repeated two more times. The residual solution is added to 1 mL of ThioEPO solution and the reaction mixture is incubated for 16 hour at room temperature. Reactivity of the excess iodoacetamide is quenched at the end of the incubation period by the addition of cysteine to a final concentration of 10 mM. The reaction mixture is applied to a desalting column equilibrated with PBS buffer and the protein content of the fractions are monitored with a Coomassie protein assay reagent. All fractions containing the protein conjugate are pooled and the conjugate was obtained by lyophylization 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 2. They are marked with a “C” and are available 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. 9
  • Materials
  • A. Phosphate buffer. Composition is 100 mM sodium phosphate, pH 6.1, 5 mM EDTA.
  • B. PBS, phosphate buffered saline:10 mM 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, 2×5 mL bed volume (Perbio Science Deutschland GmbH, Bonn, Germany)
  • E. Coomassie® Protein Assay Reagent (Perbio Science Deutschland GmbH, Bonn, Germany)
  • F. ThioEPO solution: 5 mg/mL of ThioEPO 1 in phosphate buffer.
    9de Valasco, Merkus, Anderton, Verheul, Lizzio, Van der Zee, van Eden, Hoffmann, Verhoef, Snippe, 1995, Infect. Immun., 63, 961
  • Method
  • 1 mL SH reactiveHES12KD bromoacetamide D2 solution and 1 mL of ThioEPO solution are combined and the reaction mixture is incubated for 48 hours at room temperature. Reactivity of the excess bromoacetamide is quenched at the end of the incubation period by the addition of cysteine to a final concentration of 10 mM. The reaction mixture is applied to a desalting column, equilibrated with PBS buffer. The protein content of the fractions are monitored with a Coomassie protein assay reagent, all fractions containing the protein conjugate are pooled and the conjugate is obtained by lyophylization 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 bromoacetamide function (see 1.1 above). SBAP is a member of this group of cross-linkers and is found in table 2, marked with a “D”.
  • 2. Reaction of Thio-EPO with a Maleimide-Modified SH-Reactive HES 2.1 Example Protocol 3
  • Conjugation of ThioEPO to HES12KD with a cross-linker containing a hydrazide and a maleimide functional group, e.g. M2C2H.
  • Materials
  • A. M2C2H stock: 10 mg/mLM2C2H in DMSO, prepared fresh
  • B. HES12KD: 10 mg/mL in 0.1 M sodium acetate buffer, pH 5.5
  • C. ThioEPO solution: 5 mg/mL of ThioEPO in phosphate/NaCl-buffer
  • D. Phosphate/NaCl: 0.1 M sodium phosphate, 50 mM NaCl, pH 7.0
  • E. Microconcentrator: Microcon YM-3 (amicon, Milipore GmbH, Eschborn, Germany)
  • F. Gel filtration column: for example, Sephadex® G-200 (1.5×45 cm)
  • G. Coomassie® Protein Assay Reagent (Perbio Science Deutschland GmbH, Bonn, Germany)
  • H. PBS, phosphate buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4.
  • Method
  • M2C2H solution is added to 400 μL of the HES12KD solution to a final concentration of 1 mM and is allowed to react with agitation for 2 hours at room temperature. The excess cross-linker is removed by centrifuging the sample at 14000×g for 60 minutes using a microconcentrator. After centrifuging the sample is brought up to its original volume in phosphate/NaCl buffer and this process is repeated two more times. To the M2C2H-modified HES12KD 0.5 mL of ThioEPO solution is added and the reaction mixture is incubated for 2 hours at room temperature. Reactivity of the excess maleimides is quenched at the end of the incubation period by the addition of cysteine to a final concentration of 10 mM. The reaction mixture is applied to Sephadex® G-200 (1.5×45 cm) equilibrated with PBS buffer and 1 mL fractions are collected. The protein content of the fractions is monitored with a Coomassie protein assay reagent. All fractions containing the protein conjugate are pooled and the conjugate was obtained by lyophylization 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 is unnecessary.
  • 2.2 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/NaCl-buffer
  • C. Phosphate/NaCl: 0.1 M sodium phosphate, 50 mM NaCl, pH 7.0
  • D. Gel filtration column: for example, Sephadex® G-200 (1.5×45 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 SH-reactive-HES12KD B solution and 1 mL of ThioEPO 1 solution are combined and the reaction mixture is incubated for 2 hours at room temperature. Reactivity of the excess maleimides is quenched at the end of the incubation period by the addition of cysteine to a final concentration of 10 mM. The reaction mixture is applied to Sephadex® G-200 (1.5×45 cm) equilibrated with PBS buffer and 1 mL fractions are collected. The protein content of the fractions is monitored with a Coomassie protein assay reagent. All fractions containing the protein conjugate are pooled and the conjugate is obtained by lyophylization after dialysis against water over night.
  • 2.3 Example Protocol 12
  • Conjugation of ThioEPO to aminoHES12KD (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. AminoHES12KD 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, 2×5 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 is added and the reaction mixture is allowed to react with agitation for 80 min at room temperature and for 10 min at 46° C. The excess crosslinker is removed by centrifugation of the reaction mixture through a microconcentrator at 14000×g for 60 min. The volume is brought up to 450 μL with PBS buffer and the process is repeated two more times. After the last centrifugation, the residual solution is brought up to 450 μL with PBS and is added to 1 mL of ThioEPO solution and the reaction mixture are incubated for 16 hours at room temperature. Reactivity of the excess maleimide is quenched at the end of the incubation period by the addition of cysteine to a final concentration of 10 mM. The reaction mixture is applied to a desalting column equilibrated with PBS buffer. The protein content of the fractions are monitored with a Coomassie protein assay reagent, all fractions containing the protein conjugate are pooled and the conjugate is obtained by lyophylization 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, 2, marked with an “E”. There is a further group of cross-linkers, which have instead of a maleimide function 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 2 with a “F”. A third group of cross-linkers uses instead of a maleimide function a vinylsulfone function as a SH-reactive group. A member of this group “SVSB” is marked in table 2 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, respectively.
  • 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 oxidation reaction was allowed to proceed for I 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×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 oxidized Glyco-EPO to Thio-HES12KD M, O or Q with a Cross-linker containing a hydrazide and a maleimide functional group, e.g. M2C2H (Perbio Science, Deutschland GmbH, Bonn, Germany).
  • Materials
  • A. M2C2H stock: 10 mg/mL M2C2H in DMSO, prepared fresh
  • B. Oxidized Glyco-EPO solution from 6.1.1: 5 mg/mL of Glyco-EPO in acetate buffer
  • C. Thio-HES12KD 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/NaCl: 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 (1.5×45 cm)
  • H. 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 is added to 1 mL of oxidized Glyco-EPO to a final concentration of 1 mM and is allowed to react with agitation for 2 hours at room temperature. The excess crosslinker is removed by centrifuging the sample at 14000×g for 60 minutes using a microconcentrator. After centrifuging the sample is brought up to its original volume in phosphate/NaCl buffer and this process was repeated two more times. To the M2C2H-modified Glyco-EPO 1 mL of Thio-HES12KD M, O or Q solution is added and the reaction mixture is incubated for 16 hours at room temperature. Reactivity of the excess maleimides is quenched at the end of the incubation period by the addition of cysteine. The reaction mixture is applied to Sephadex® G-200 (1.5×45 cm) equilibrated with PBS and 1 mL fractions are collected. The protein content of the fractions is monitored with a Coomassie protein assay reagent, all fractions containing the protein conjugate are pooled and the conjugate is obtained by lyophylization 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 8A
  • Direct conjugation of oxidized Glyco-EPO to Hydrazido-HES12KD L or J.
  • Materials
  • A. Oxidized Glyco-EPO solution from 6.1.1:5 mg/mL of Glyco-EPO in acetate buffer
  • B. Hydrazido-HES12KD 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 (1.5×45 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 or J solution and 1 mL of oxidized Glyco-EPO solution are combined and the reaction mixture is allowed to react with agitation for 16 hours at room temperature. The reaction mixture is applied to Sephadex® G-200 (1.5×45 cm) equilibrated with PBS and 1 mL fractions are collected. The protein content of the fractions are monitored with a Coomassie protein assay reagent, all fractions containing the protein conjugate are pooled and the conjugate is obtained by lyophylization 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, the hydrazone may be reduced by treatment with 30 mM sodium cyanoborohydride in PBS buffer to a hydrazine. For most applications, this extra step is unnecessary.
  • 2.3 Example Protocol 8B
  • To 100 μl of a 0.5 mg/ml solution of oxidized EPO in a buffer containing 0.1 M sodium acetate and 150 mM sodium chloride at a pH of 5.2, 50 μL of a 20 mg/ ml solution of HES18/0.5 L dissolved in a 0.1 M sodium acetate buffer, pH 5.2 (synthesized corresponding to Example 10 2.1b) were added and the mixture was incubated at 22° C. for 14.5 h. The crude reaction mixture was analyzed by SDS gel electrophoresis and stained with Coomassie. The result of the conjugation is shown in FIG. 24. The observed molecular shift demonstrates that the conjugation was successful. The smear results from the heterogeneity of HES. FIG. 25 demonstrates that HES is conjugated to a carbohydrate moiety of a carbohydrate side chain.
  • 3. Conjugation with Hydroxylamine-Derivatives10 3.1 Example Protocol 9A
  • Conjugation of oxidized Glyco-EPO to Hydroxylamino-HES12KD K
  • Materials
  • A. Oxidized Glyco-EPO solution from 6.1.1: 5 mg/mL of Glyco-EPO in acetate buffer
  • B. Hydroxylamino-HES12KD 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 (1.5×45 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
    10Rose, 1994, Am. Chem. Soc., 116, 30
  • Method
  • 1 mL of Hydroxylamino-HES12KD K solution and 1 mL of oxidized Glyco-EPO solution are combined and the reaction mixture is allowed to react with agitation for 16 hours at room temperature. The reaction mixture is applied to Sephadex® G-200 (1.5×45 cm) equilibrated with PBS and 1 mL fractions were collected. The protein content of the fractions are monitored with a Coomassie protein assay reagent, all fractions containing the protein conjugate are pooled and the conjugate is obtained by lyophylization after dialysis against water over night.
  • 3.2 Example Protocol 9B
  • To 100 μl of a 0.5 mg/ml solution of oxidized EPO in a buffer containing 0.1 M sodium acetate and 150 mM sodium chloride at a pH of 5.2, 50 μL of a 20 mg/ ml solution of HES18/0.5 K dissolved in a 0.1 M sodium acetate buffer, pH 5.2 (synthesized corresponding to Example 10. 4) were added and the mixture was incubated at 22° C. for 14.5 h. The crude reaction mixture was analyzed by SDS gel electrophoresis and stained with Coomassie. The result of the conjugation is shown in FIG. 24. The observed molecular shift demonstrates that the conjugation was successful. The smear results from the heterogeneity of HES. FIG. 25 demonstrates that HES is conjugated to a carbohydrate moiety of a carbohydrate side chain.
  • Example 13
  • Characterization of 2Galactose Oxidase Treated EPO N-Glycans
  • Recombinant EPO or partially desialylated EPO forms (generated by limited mild acid hydrolysis) 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 HPAEC-PAD 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 oxidized galactose residues in individual EPO oligosaccharides was performed by the typical shift observed in HPAEC-PAD and was also verified by MALDI/TOF MS of the oligosaccharide mixtures.
  • Example 14
  • Characterization 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 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 Brilliant Blue. The higher molecular weight of HAS 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 galactose oxidase treated EPO O-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-HULK on a C8-phase before SDS-PAGE analysis. HAS O-glycan modification of EPO was also analyzed 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 employing 20, 40, 80 and 120 μg of the BRP standard EPO reference preparation for calibration.
  • Example 16
  • In-vitro Biological Activity of HES-Modified Recombinant Human EPO:
  • Purified HES-modified EPO is tested for activity using the erythropoietin bioactivity assay as described by Krystal [Krystal, 1984, Exp. Heamatol., 11, 649-660].
  • Anemia is induced in NMRI mice by treatment with phenylhydrazine hydrochloride and spleen cells are collected and used as described in [Fibi et al., 1991, Blood, 77, 1203 ff.]. Dilutions of EPO are incubated with 3×105 cells/well in 96-well microtiter plates. After 24 hours at 37° C. in a humidified atmosphere (5% CO2) cells are labeled for 4 hours with 1 μCi of 3H-thymidine per well. Incorporated radioactivity is determined by liquid scintillation counting. The International reference EPO standard (BRP-standard) is used for comparison.
  • Alternatively, EPO bioactivity may be 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 are washed free of growth factors and are incubated in the presence of serial dilutions of the EPO for further 48 hours. Proliferation of the cells is assessed by using the MTT reduction assay as described by Mosmann [Mosmann, 1983, J. Immunol. Methods, 65, 55-63].
  • Example 17
  • In-vivo Activity Determination of EPO and HAS-Modified EPO Forms: (HCO Fragen)
  • In vivo activity determinations are performed 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 are performed using the BRP EPO standard which is calibrated against the WHO EPO standard in the polycythemic mouse assay. EPO samples are 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) are infected subcutaneously per animal. Blood samples are taken after 4 days after injection and reticulocytes are stained with acridine orange; quantitation of reticulocytes is performed by flow-cytometry by counting a total of 30,000 blood cells within 5 hours after the blood sample was taken (see Ph. Eur, 2000, Erythropoietini solutio concentrata, 1316, pages 780-785) and European Pharmacopoeia (1996/2000, attachment 2002).
  • Example 18
  • In-vivo Half-Life Determinations
  • Rabbits are injected intravenously with specified amounts of unmodified or HAS-modified EPO forms. Blood samples are obtained at specified times, and serum is prepared. Serum erythropoietin levels are determined by in vitro bioassay or by an EPO-specific commercial ELISA.
  • Example 19
  • In vivo Pharmacokinetics
  • In mice: Each animal receive 300 IU EPO/kg subcutaneously. Seven days after the post-treatment hematocrit of each animal is determined. A substantial increase in hematocrit is observed in all animals treated with modified EPO.
  • In rabbits: Rabbits are 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 are analyzed by using a commercial EPO-specific ELISA for determination of plasma concentrations. Mean plasma EPO concentrations are determined and the average initial half-lives (α-phase) and the terminal half-lives (β-phase) are 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 Exhibits Enhanced Biological Properties, J. Biol. Chem., 274, 24773-24778.
  • Example 20
  • Assessment of the in vitro Biological Activity of HES-Modified Recombinant Human IL-2
  • Modified IL2 is recovered by gel filtration on Ultrogel AcA 54. Aliquots of corresponding fraction are sterile filtrated and IL2 bioactivity is determined by using the IL2 dependent murine CTLL-2 cell line [Gillis, Ferm, On, and Smith, 1978, J. Immunol., 120, 2027-2032]. Activity is related to the international reference IL2 standard preparation.
    TABLE 1
    Abreviation Chemical Name Type
    AMAS N-(α-Maleiinidoacetoxy) succinimide ester E
    Figure US20050238723A1-20051027-C00088
    BMPH N-(β-Maleimidopropionic acid) hydrazide.TFA A
    Figure US20050238723A1-20051027-C00089
    BMPS N-(β-Maleimidopropyloxy) succinimide ester E
    Figure US20050238723A1-20051027-C00090
    EMCH N-(ε-Maleimidocaproic acid) hydrazide A
    Figure US20050238723A1-20051027-C00091
    EMCS N-(ε-Maleimidocaproyloxy) succinimide ester E
    Figure US20050238723A1-20051027-C00092
    GMBS N-γ-Maleimidobutyryloxy-succinimide ester E
    Figure US20050238723A1-20051027-C00093
    KMUH N-(κ-Maleimidoundecanoic acid) hydrazide A
    Figure US20050238723A1-20051027-C00094
    LC-SMCC Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxy-(6-amido-caproate) E
    Figure US20050238723A1-20051027-C00095
    LC-SPDP Succinimidyl 6-(3′-[2-pyridyl-dithio]propionamido) hexanoate F
    Figure US20050238723A1-20051027-C00096
    MBS m-Maleimidobenzoyl-N-hydroxysuccinimide ester E
    Figure US20050238723A1-20051027-C00097
    M2C2H 4-(N-Maleimidomethyl)-cyclohexane-1-carboxyl- hydrazide.HCl.½dioxane A
    Figure US20050238723A1-20051027-C00098
    MPBH 4-(4-N-Maleimidophenyl)-butyric acid hydazide.HCl A
    Figure US20050238723A1-20051027-C00099
    SATA N-Succinimidyl S-acetylthio-acetate H
    Figure US20050238723A1-20051027-C00100
    SATP N-Succinimidyl S-acetylthio-propionate H
    Figure US20050238723A1-20051027-C00101
    SBAP Succinimidyl 3-(bromoacetamido) propionate D
    Figure US20050238723A1-20051027-C00102
    SIA N-Succinimidyl iodoacetate C
    Figure US20050238723A1-20051027-C00103
    SIAB N-Succinimidyl(4-iodoacetyl)aminobenzoate C
    Figure US20050238723A1-20051027-C00104
    SMCC Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate E
    Figure US20050238723A1-20051027-C00105
    SMPB Succinimidyl 4-(p-maleimidophenyl)butyrate E
    Figure US20050238723A1-20051027-C00106
    SMPH Succinimidyl-6-(β-maleimidopropionamido) hexanoate E
    Figure US20050238723A1-20051027-C00107
    SMPT 4-Succinimidyloxy-carbonyl-methyl-α-(2- pyridyldithio)toluene F
    Figure US20050238723A1-20051027-C00108
    SPDP N-Succinimidyl 3-(2-pyridyldithio)propionate F
    Figure US20050238723A1-20051027-C00109
    Sulfo-EMCS N-(ε-Maleimidocaproyloxy) sulfosuccinimide ester E
    Figure US20050238723A1-20051027-C00110
    Sulfo-GMBS N-γ-Maleimidobutryloxy-sulfosuccinimide ester E
    Figure US20050238723A1-20051027-C00111
    Sulfo-KMUS N-(κ-Maleimidoundecanoyloxy)-sulfosuccinimide ester E
    Figure US20050238723A1-20051027-C00112
    Sulfo-LC-SPDP Sulfosuccinimidyl 6-(3′-[2-pyridyl- dithio]propionamido) hexanoate F
    Figure US20050238723A1-20051027-C00113
    Sulfo-MBS m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester E
    Figure US20050238723A1-20051027-C00114
    Sulfo-SIAB Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate C
    Figure US20050238723A1-20051027-C00115
    Sulfo-SMCC Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate E
    Figure US20050238723A1-20051027-C00116
    Sulfo-SMPB Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate E
    Figure US20050238723A1-20051027-C00117
    Sulfo-LC-SMPT Suflosuccinimidyl 6-(α-methyl-α-[2-pyridyldithio]- toluamido)hexanoate F
    Figure US20050238723A1-20051027-C00118
    SVSB N-Succinimidyl-(4-vinylsulfonyl)benzoate G
    Figure US20050238723A1-20051027-C00119
  • TABLE 2
    Monosaccharide compositional analysis of glycans
    from HES-modified EPO and control samples
    II. VI.
    I. Glycans III. III. IV. V. Cystein
    Glycans from Glycans Glycans Glycans Glycans modified
    **Mono- from EPO-GT- from from from EPO- from EPO pro-
    saccharide A2 1A K2 A2 GT-1A K2 tein*
    fucose 1,935 3,924 2,602 2,246 4,461 2,601 2,181
    mannose 6,028 11,020 9,198 6,379 11,668 6,117 6,260
    galactose 8,886 19,935 14,427 10,570 16,911 11,555 10,386
    glucose 17,968 21,193 trace trace 33,021
    GlcNAc 7,839 21,310 14,440 11,360 15,953 10,503 10,498
    GlcHe1 5,583 5,926 14,857
    GlcHe2 1,380 1,552 3,775
    NeuNAc 5,461 822 4,504 3,895 4,871 13,562 13,003
    inositol 1,230 2,310 1,620 2,050 1,320 1,134 1,087

    *the equivalent of Cys-HES-modified EPO protein was subjected to compositional analysis; the EPO protein was isolated from the HES-incubation mixture by chromatography on a Q-Sepharose column as described above and was desalted by centrifugation using a Vivaspin 5 separation device.

    **Monosaccharide determinations were performed from single GC runs of the pertrimethylsilylated methylglycosides; the electronical integration values of peaks are given without correction for losses during the derivatisation procedure and recoveries of each compound.
  • TABLE 3
    Calculated specific activity of
    Sample EPO sample (basedon A280 nm
    No. Sample description and RP-HPLC determination)
    850247 1. HES-modified EPO A2 344,000 U/mg
    850248 2. EPO-GT-1-A 82,268 U/mg
    850249 3. Control EPO K2 121,410 U/mg
    850250 4. BRP EPO standard 86,702 U/mg
    850251 1. diluted with 4 309,129 U/mg
    volume of PBS
    850252 2. diluted with 4 94,500 U/mg
    volume of PBS
    850253 3. diluted with 4 114,100 U/mg
    volume of PBS
    850254 4. diluted with 4 81,200 U/mg
    volume of PBS
    850255 1. diluted with 4 230,720 U/mg
    volume of PBS

Claims (59)

  1. 1. A method of producing a hydroxyalkyl starch derivative, said hydroxyalkyl starch having a structure according to formula (I)
    Figure US20050238723A1-20051027-C00120
    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 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 aldehyde group, a keto group, a hemiacetal group, an acetal group, and a thio group,
    wherein 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 Z1 with the optionally oxidized reducing end of the hydroxyalkyl starch,
    and wherein the functional group Z1 comprises the structure —NH—.
  2. 2. A method as claimed in claim 1 wherein R1, R2 and R3 are independently hydrogen or a linear or branched hydroxyalkyl group.
  3. 3. A method as claimed in claim 2 wherein R1, R2 and R3 are independently hydrogen or a 2-hydroxyethyl group.
  4. 4. A method as claimed in claim 1 wherein the hydroxyalkyl starch is hydroxyethyl starch.
  5. 5. A method as claimed in claim 1 wherein Z, is selected from the group consisting of
    Figure US20050238723A1-20051027-C00121
    wherein G is O or S and, if present twice, independently O or S, and R′ is methyl.
  6. 6. A method as claimed in claim 1 wherein the functional group Y is selected from the group consisting of an aldehyde group, a keto group, a hemiacetal group, and an acetal group, and the functional group X comprises the structure —NH—.
  7. 7. A method as claimed in claim 6 wherein X is selected from the group consisting of
    Figure US20050238723A1-20051027-C00122
    wherein G is O or S and, if present twice, independently O or S, and R′ is methyl.
  8. 8. A method as claimed in claim 1 wherein the functional group Y is —SH and the functional group X is selected from the group consisting of
    Figure US20050238723A1-20051027-C00123
    wherein Hal is Cl, Br or I.
  9. 9. A method as claimed in claim 1 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 US20050238723A1-20051027-C00124
    wherein Hal is Cl, Br, or I.
  10. 10. A method as claimed in claim 1 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 group consisting of
    Figure US20050238723A1-20051027-C00125
    wherein G is O or S and, if present twice, independently O or S, and R′ is methyl.
  11. 11. A method as claimed in claim 1 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 US20050238723A1-20051027-C00126
  12. 12. A method as claimed in claim 1 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 (IIa)
    Figure US20050238723A1-20051027-C00127
    and/or according to formula (IIb)
    Figure US20050238723A1-20051027-C00128
  13. 13. A method as claimed in claim 12 wherein the reducing end is oxidized by an alkaline iodine solution.
  14. 14. A method as claimed in claim 1 wherein hydroxyalkyl starch is reacted with a compound (L) via the reaction of functional group Z1 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).
  15. 15. A method as claimed in claim 1 wherein hydroxyalkyl starch is reacted with a compound (L) via the reaction of functional group Z1 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).
  16. 16. A method as claimed in claim 1 wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of the functional group Z1 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.
  17. 17. A method as claimed in claim 16 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).
  18. 18. A method as claimed in claim 1 wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of functional group Z1 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).
  19. 19. A method as claimed in claim 1 wherein the at least one further compound (M) is a polypeptide.
  20. 20. A method as claimed in claim 19 wherein the polypeptide is erythropoietin.
  21. 21. 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 US20050238723A1-20051027-C00129
    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 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 aldehyde group, a keto group, a hemiacetal group, an acetal group, and a thio group,
    wherein 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 Z1 with the optionally oxidized reducing end of the hydroxyalkyl starch,
    and wherein the functional group Z1 comprises the structure —NH—.
  22. 22. A hydroxyalkyl starch derivative as claimed in claim 21 wherein R1, R2 and R3 are independently hydrogen or a linear or branched hydroxyalkyl group.
  23. 23. A hydroxyalkyl starch derivative as claimed in claim 22 wherein R1, R2 and R3 are independently hydrogen or a 2-hydroxyethyl group.
  24. 24. A hydroxyalkyl starch derivative as claimed in claim 21 wherein the hydroxyalkyl starch is hydroxyethyl starch.
  25. 25. A hydroxyalkyl starch derivative as claimed in claim 21 wherein Z1 is selected from the group consisting of
    Figure US20050238723A1-20051027-C00130
    wherein G is O or S and, if present twice, independently O or S, and R′ is methyl.
  26. 26. A hydroxyalkyl starch derivative as claimed in claim 21 wherein the functional group Y is selected from the group consisting of an aldehyde group, a keto group, a hemiacetal group, and an acetal group, and the functional group X comprises the structure —NH—.
  27. 27. A hydroxyalkyl starch derivative as claimed in claim 26 wherein X is selected from the group consisting of
    Figure US20050238723A1-20051027-C00131
    wherein G is O or S and, if present twice, independently O or S, and R′ is methyl.
  28. 28. A hydroxyalkyl starch derivative as claimed in claim 21 wherein the functional group Y is —SH and the functional group X is selected from the group consisting of
    Figure US20050238723A1-20051027-C00132
    wherein Hal is Cl, Br or I.
  29. 29. A hydroxyalkyl starch derivative as claimed in claim 21 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 US20050238723A1-20051027-C00133
    wherein Hal is Cl, Br, or I.
  30. 30. A hydroxyalkyl starch derivative as claimed in claim 21 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 group consisting of
    Figure US20050238723A1-20051027-C00134
    wherein G is O or S and, if present twice, independently O or S, and R′ is methyl.
  31. 31. A hydroxyalkyl starch derivative as claimed in claim 21 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 US20050238723A1-20051027-C00135
  32. 32. A hydroxyalkyl starch derivative as claimed in claim 21 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 (IIa)
    Figure US20050238723A1-20051027-C00136
    and/or according to formula (IIb)
    Figure US20050238723A1-20051027-C00137
  33. 33. A hydroxyalkyl starch derivative as claimed in claim 32 wherein the reducing end is oxidized by an alkaline iodine solution.
  34. 34. A hydroxyalkyl starch derivative as claimed in claim 21 wherein hydroxyalkyl starch is reacted with a compound (L) via the reaction of functional group Z1 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).
  35. 35. A hydroxyalkyl starch derivative as claimed in claim 21 wherein hydroxyalkyl starch is reacted with a compound (L) via the reaction of functional group Z1 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).
  36. 36. A hydroxyalkyl starch derivative as claimed in claim 21 wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of the functional group Z1 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.
  37. 37. A hydroxyalkyl starch derivative as claimed in claim 36 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).
  38. 38. A hydroxyalkyl starch derivative as claimed in claim 21 wherein hydroxyalkyl starch is reacted with a compound (D) via the reaction of functional group Z1 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).
  39. 39. A hydroxyalkyl starch derivative as claimed in claim 21 wherein the at least one further compound (M) is a polypeptide.
  40. 40. A hydroxyalkyl starch derivative claimed in claim 39 wherein the polypeptide is erythropoietin.
  41. 41. 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 US20050238723A1-20051027-C00138
    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 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 aldehyde group, a keto group, a hemiacetal group, an acetal group, and 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,
    wherein 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 Z1 with the optionally oxidized reducing end of the hydroxyalkyl starch,
    and wherein the functional group Z1 comprises the structure —NH—.
  42. 42. A pharmaceutical composition as claimed in claim 41 wherein the polypeptide is AT III.
  43. 43. A pharmaceutical composition as claimed in claim 41 wherein the polypeptide is erythropoietin.
  44. 44. A pharmaceutical composition as claimed in claim 41 wherein the functional group Y is —SH and compound (L) is a compound of general formula Z1-L′-X where the functional group Z1 is selected from the group consisting of
    Figure US20050238723A1-20051027-C00139
    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 US20050238723A1-20051027-C00140
    wherein Hal is Cl, Br or I, and where L′ is an organic chain bridging Z1 and X or where L′ is absent.
  45. 45. A pharmaceutical composition as claimed in claim 41 wherein the functional group Y is selected from the group consisting of an aldehyde group, a keto group, a hemiacetal group, and an acetal group, and compound (L) is a compound of general formula Z1-L′-X where the functional group Z1 is selected from the group consisting of
    Figure US20050238723A1-20051027-C00141
    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 US20050238723A1-20051027-C00142
    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 Z1 and X or where L′ is absent.
  46. 46. A pharmaceutical composition as claimed in claim 41 wherein the functional group Y is —SH, compound (D) is a compound of general formula Z1-D′-W, and compound (L) is a compound of general formula Z2-L′-X, where the functional group Z1 is selected from the group consisting of
    Figure US20050238723A1-20051027-C00143
    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 US20050238723A1-20051027-C00144
    wherein Hal is Cl, 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 US20050238723A1-20051027-C00145
    wherein Hal is Cl, 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 US20050238723A1-20051027-C00146
    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 Z1 and W or where D′ is absent and where L′ is an organic chain bridging Z2 and X or where L′ is absent.
  47. 47. A pharmaceutical composition as claimed in claim 41 wherein the functional group Y is selected from the group consisting of an aldehyde group, a keto group, a hemiacetal group, and an acetal group, compound (D) is a compound of general formula Z1-D′-W, and compound (L) is a compound of general formula Z2-L′-X, where the functional group Z1 is selected from the group consisting of
    Figure US20050238723A1-20051027-C00147
    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 US20050238723A1-20051027-C00148
    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 functional group Z2 or the functional group W is selected from the group consisting of
    Figure US20050238723A1-20051027-C00149
    wherein Hal is Cl, 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 US20050238723A1-20051027-C00150
    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 Z1 and W or where D′ is absent and where L′ is an organic chain bridging Z2 and X or where L′ is absent.
  48. 48. A pharmaceutical composition as claimed in claim 41 wherein hydroxyethyl starch is reacted in an aqueous medium with a compound according to the following formula
    Figure US20050238723A1-20051027-C00151
    and the reaction product is reacted with erythropoietin.
  49. 49. A pharmaceutical composition as claimed in claim 48 wherein the erythropoietin is oxidised with sodium periodate prior to the reaction.
  50. 50. A pharmaceutical composition as claimed in claim 49 wherein the erythropoietin is partially desialylated and subsequently oxidised with sodium periodate prior to the reaction.
  51. 51. A method of producing a hydroxyethyl starch derivative, said hydroxyethyl starch having a structure according to formula (I)
    Figure US20050238723A1-20051027-C00152
    comprising reacting hydroxyethyl starch at its optionally oxidized reducing end with a compound according to the following formula
    Figure US20050238723A1-20051027-C00153
    or a compound of the following formula
    Figure US20050238723A1-20051027-C00154
    and reacting the reaction product with an aldehyde group, a keto group, a hemiacetal group, or an acetal group of a terminal moiety of a carbohydrate side chain of erythropoietin which is oxidised with sodium periodate prior to the reaction or partially desialylated and subsequently oxidised with sodium periodate prior to the reaction.
  52. 52. A method as claimed in claim 51, wherein hydroxyethyl starch is reacted at its reducing end which is not oxidized, with a compound according to the following formula
    Figure US20050238723A1-20051027-C00155
    in an aqueous medium.
  53. 53. A method of producing a hydroxyethyl starch derivative, said hydroxyethyl starch having a structure according to formula (I)
    Figure US20050238723A1-20051027-C00156
    comprising reacting hydroxyethyl starch at its optionally oxidized reducing end with a compound according to the following formula
    Figure US20050238723A1-20051027-C00157
    or a compound according to the following formula
    Figure US20050238723A1-20051027-C00158
    further comprising reacting the reaction product with a compound according to the following formula
    Figure US20050238723A1-20051027-C00159
    and reacting the reaction product with a thio group of erythropoietin.
  54. 54. A hydroxyethyl starch derivative obtainable by a method of producing a hydroxyethyl starch derivative, said hydroxyethyl starch having a structure according to formula (I)
    Figure US20050238723A1-20051027-C00160
    comprising reacting hydroxyethyl starch at its optionally oxidized reducing end with a compound according to the following formula
    Figure US20050238723A1-20051027-C00161
    or a compound according to the following formula
    Figure US20050238723A1-20051027-C00162
    and reacting the reaction product with an aldehyde group, a keto group, a hemiacetal group, or an acetal group of a terminal moiety of a carbohydrate side chain of erythropoietin which is oxidised with sodium periodate prior to the reaction or partially desialylated and subsequently oxidised with sodium periodate prior to the reaction.
  55. 55. A hydroxyethyl starch derivative as claimed in claim 54, wherein hydroxyethyl starch is reacted at its reducing end which is not oxidized, with a compound according to the following formula
    Figure US20050238723A1-20051027-C00163
    in an aqueous medium.
  56. 56. A hydroxyethyl starch derivative obtainable by a method of producing a hydroxyethyl starch derivative, said hydroxyethyl starch having a structure according to formula (I)
    Figure US20050238723A1-20051027-C00164
    comprising reacting hydroxyethyl starch at its optionally oxidized reducing end with a compound according to the following formula
    Figure US20050238723A1-20051027-C00165
    or a compound according to the following formula
    Figure US20050238723A1-20051027-C00166
    further comprising reacting the reaction product with a compound according to the following formula
    Figure US20050238723A1-20051027-C00167
    and reacting the reaction product with a thio group of erythropoietin.
  57. 57. A pharmaceutical composition comprising, in a therapeutically effective amount, a hydroxyethyl starch derivative obtainable by a method of producing a hydroxyethyl starch derivative, said hydroxyethyl starch having a structure according to formula (I)
    Figure US20050238723A1-20051027-C00168
    comprising reacting hydroxyethyl starch at its optionally oxidized reducing end with a compound according to the following formula
    Figure US20050238723A1-20051027-C00169
    or a compound according to the following formula
    Figure US20050238723A1-20051027-C00170
    and reacting the reaction product with an aldehyde group, a keto group, a hemiacetal group, or an acetal group of a terminal moiety of a carbohydrate side chain of erythropoietin which is oxidised with sodium periodate prior to the reaction or partially desialylated and subsequently oxidised with sodium periodate prior to the reaction.
  58. 58. A pharmaceutical composition as claimed in claim 57, wherein hydroxyethyl starch is reacted at its reducing end which is not oxidized, with a compound according to the following formula
    Figure US20050238723A1-20051027-C00171
    in an aqueous medium.
  59. 59. A pharmaceutical composition comprising, in a therapeutically effective amount, a hydroxyethyl starch derivative obtainable by a method of producing a hydroxyethyl starch derivative, said hydroxyethyl starch having a structure according to formula (I)
    Figure US20050238723A1-20051027-C00172
    comprising reacting hydroxyethyl starch at its optionally oxidized reducing end with a compound according to the following formula
    Figure US20050238723A1-20051027-C00173
    or a compound according to the following formula
    Figure US20050238723A1-20051027-C00174
    further comprising reacting the reaction product with a compound according to the following formula
    Figure US20050238723A1-20051027-C00175
    and reacting the reaction product with a thio group of erythropoietin.
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Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050063943A1 (en) * 2001-03-16 2005-03-24 Klaus Sommermeyer Conjugated of hydroxyalkyl starch and an active agent
US20050181985A1 (en) * 2002-03-06 2005-08-18 Jurgen Hemberger Coupling proteins to a modified polysaccharide
US20050234230A1 (en) * 2002-09-11 2005-10-20 Norbert Zander Hydroxyalkyl starch derivatives
US20060052342A1 (en) * 2002-12-04 2006-03-09 Klaus Sommermeyer Aldonic acid esters, methods for producing the same, and methods for producing pharmaceutical active ingredients coupled to polysaccharides or polysaccharide derivatives on free amino groups
US20060100163A1 (en) * 2002-10-08 2006-05-11 Michele Orlando Pharmaceutically active oligosaccharide conjugates
US20060121062A1 (en) * 2002-09-11 2006-06-08 Wolfram Eichner Conjugated hydroxyalkyl starch allergen compounds
US20060217293A1 (en) * 2002-03-06 2006-09-28 Michele Orlando Coupling low-molecular substances to a modified polysaccharide
US20070087961A1 (en) * 2004-03-11 2007-04-19 Wolfram Eichner Conjugates of hydroxyalkyl starch and erythropoietin
US20080206182A1 (en) * 2003-08-08 2008-08-28 Fresenius Kabi Deutschland Gmbh Conjugates of a Polymer and a Protein Linked by an Oxime Group
US20080207562A1 (en) * 2005-09-12 2008-08-28 Fresenius Kabi Deutschland Gmbh Conjugates of Hydroxyalkyl Starch and Active Substance, Prepared by Chemical Ligation Via Thiazolidine
US20080274948A1 (en) * 2003-08-08 2008-11-06 Fresenius Kabi Deutschland Gmbh Conjugates of Hydroxyalkyl Starch and G-Csf
US20090047251A1 (en) * 2004-03-11 2009-02-19 Wolfram Eichner Conjugates of hydroxyalkyl starch and a protein
EP2070950A1 (en) * 2007-12-14 2009-06-17 Fresenius Kabi Deutschland GmbH Hydroxyalkyl starch derivatives and process for their preparation
EP2070951A1 (en) * 2007-12-14 2009-06-17 Fresenius Kabi Deutschland GmbH Method for producing a hydroxyalkyl starch derivatives with two linkers
WO2010011735A2 (en) 2008-07-23 2010-01-28 Ambrx, Inc. Modified bovine g-csf polypeptides and their uses
US20100062973A1 (en) * 2005-03-11 2010-03-11 Fresenius Kabi Deutschland Gmbh Production of bioactive glycoproteins from inactive starting material
US20100311670A1 (en) * 2004-03-11 2010-12-09 Nobert Zander Conjugates of hydroxyalkyl starch and a protein, prepared by native chemical ligation
WO2012004009A1 (en) * 2010-07-09 2012-01-12 Fresenius Kabi Deutschland Gmbh Conjugates comprising hydroxyalkyl starch and a cytotoxic agent and process for their preparation
WO2012004006A1 (en) * 2010-07-09 2012-01-12 Fresenius Kabi Deutschland Gmbh Conjugates comprising hydroxyalkyl starch and a cytotoxic agent and process for their preparation
WO2012004007A1 (en) * 2010-07-09 2012-01-12 Fresenius Kabi Deutschland Gmbh Conjugates comprising hydroxyalkyl starch and a cytotoxic agent and process for their preparation
US8287850B2 (en) 2004-03-11 2012-10-16 Fresenius Kabi Deutschland Gmbh Conjugates of hydroxyalkyl starch and a protein, prepared by reductive amination
EP2805964A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified bovine somatotropin polypeptides and their uses
EP2805965A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified porcine somatotropin polypeptides and their uses

Families Citing this family (68)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2214166T1 (en) * 2002-09-11 2004-09-16 Fresenius Kabi Deutschland Gmbh Polypeptides has-ilados especially eriptropoyetina has-ilada.
RU2370281C2 (en) * 2003-08-08 2009-10-20 Фрезениус Каби Дойчланд Гмбх Hydroxyalkyl starch and g-csf conjugates
US8754031B2 (en) 2004-03-08 2014-06-17 Oncolix, Inc. Use of prolactin receptor antagonists in combination with an agent that inactivates the HER2/neu signaling pathway
US7589063B2 (en) 2004-12-14 2009-09-15 Aplagen Gmbh Molecules which promote hematopoiesis
WO2006094826A3 (en) * 2005-03-11 2007-08-23 Fresenius Kabi De Gmbh Method for coupling enzymatically activated glycoconjugates to a hydroxyalkyl starch
WO2008000517A3 (en) 2006-06-30 2009-04-02 Andre Koltermann Conjugates for innate immunotherapy of cancer
ES2581983T3 (en) 2006-07-25 2016-09-08 Lipoxen Technologies Limited Polysaccharide derivatives of erythropoietin
WO2008065372A3 (en) 2006-11-28 2008-10-23 Nautilus Biotech S A Modified erythropoietin polypeptides and uses thereof for treatment
CN103804489A (en) * 2006-12-15 2014-05-21 巴克斯特国际公司 Factor viia-(poly)sialic acid conjugate having prolonged in vivo half-life
CA2701032A1 (en) 2007-09-27 2009-04-02 Amgen Inc. Pharmaceutical formulations
CN104888264B (en) 2008-01-14 2018-01-09 北京环球利康科技有限公司 Biocompatible hemostatic, anti-adhesion, promoting healing, surgical closure of modified starch
CA2711826C (en) 2008-01-25 2018-02-27 Amgen Inc. Ferroportin antibodies and methods of use
EP2310423A1 (en) * 2008-04-28 2011-04-20 SurModics, Inc. Poly- (1 4)glucopyranose-based matrices with hydrazide crosslinking
EP2294087B1 (en) 2008-05-01 2014-05-14 Amgen, Inc. Anti-hepcidin antibodies and methods of use
EP2166085A1 (en) 2008-07-16 2010-03-24 Suomen Punainen Risti Veripalvelu Divalent modified cells
EP2341942A1 (en) * 2008-09-19 2011-07-13 Nektar Therapeutics Polymer conjugates of therapeutic peptides
US9018372B2 (en) 2009-03-31 2015-04-28 B. Braun Melsungen Ag Bonding products of aminated polysaccharides
WO2010063491A8 (en) 2008-12-06 2011-06-03 B. Braun Melsungen Ag Transport-mediating colloidal pharmaceutical compounds
US8648046B2 (en) 2009-02-26 2014-02-11 Oncolix, Inc. Compositions and methods for visualizing and eliminating cancer stem cells
WO2010099003A3 (en) 2009-02-26 2011-02-17 Oncolix, Inc. Compositions and methods for visualizing and eliminating cancer stem cells
US8642737B2 (en) 2010-07-26 2014-02-04 Baxter International Inc. Nucleophilic catalysts for oxime linkage
US8809501B2 (en) 2009-07-27 2014-08-19 Baxter International Inc. Nucleophilic catalysts for oxime linkage
CA2769244A1 (en) 2009-07-27 2011-02-03 Lipoxen Technologies Limited Glycopolysialylation of non-blood coagulation proteins
CN104530182A (en) * 2009-07-27 2015-04-22 利普森技术有限公司 Glycopolysialylation of non-blood coagulation proteins
KR101832937B1 (en) 2009-07-27 2018-02-28 박스알타 인코퍼레이티드 Blood coagulation protein conjugates
US8945897B2 (en) 2010-07-26 2015-02-03 Baxter International Inc. Materials and methods for conjugating a water soluble fatty acid derivative to a protein
CN104487095A (en) 2012-05-16 2015-04-01 巴克斯特国际公司 Nucleophilic catalysts for oxime linkage
US9662271B2 (en) 2009-10-23 2017-05-30 Amgen Inc. Vial adapter and system
WO2011098095A1 (en) 2010-02-09 2011-08-18 Aplagen Gmbh Peptides binding the tpo receptor
CN103025369B (en) 2010-06-07 2016-07-06 安姆根有限公司 A drug delivery device
US20140073602A9 (en) 2010-07-09 2014-03-13 Fresenius Kabi Deutschland Gmbh Nitric oxide delivering hydroxyalkyl starch derivatives
CN108079311A (en) 2010-07-30 2018-05-29 百深有限责任公司 Nucleophilic catalysts for oxime linkage
CA2831100A1 (en) 2011-03-31 2012-10-04 Mark Dominis Holt Vial adapter and system
WO2012166622A1 (en) 2011-05-27 2012-12-06 Baxter International Inc. Therapeutic proteins with increased half-life and methods of preparing same
US20150044714A1 (en) * 2011-06-07 2015-02-12 Philadelphia Health & Education Corporation D/B/A Drexel University College Of Medicine sPLA2 MONITORING STRIP
WO2013021056A1 (en) 2011-08-10 2013-02-14 Ludwig-Maximilians-Universität München Method for the controlled intracellular delivery of nucleic acids
CN106943645A (en) 2011-10-14 2017-07-14 安姆根有限公司 Injector and method of assembly
WO2013113503A1 (en) 2012-01-31 2013-08-08 Fresenius Kabi Deutschland Gmbh Conjugates of hydroxyalkyl starch and an oligonucleotide
WO2014081780A1 (en) 2012-11-21 2014-05-30 Amgen Inc. Drug delivery device
KR20150102009A (en) * 2012-11-30 2015-09-04 가부시키가이샤 도우사 고가쿠 겐큐쇼 Sugar chain-attached linker, compound containing sugar chain-attached linker and physiologically active substance or salt thereof, and method for producing same
RU2015144105A (en) 2013-03-15 2017-04-24 ИНТРИНСИК ЛАЙФСАЙЕНСИС, ЭлЭлСи Antibodies to hepcidin and their applications
WO2014147175A1 (en) * 2013-03-20 2014-09-25 Fresenius Kabi Deutschland Gmbh Hydroxyalkyl starch derivatives as reactants for coupling to thiol groups
US20160311934A1 (en) * 2013-03-20 2016-10-27 Fresenius Kabi Deutschland Gmbh Hydroxyalkyl starch derivatives as reactants for coupling to thiol groups
CA2907378A1 (en) * 2013-03-20 2014-09-25 Fresenius Kabi Deutschland Gmbh Process for the preparation of thiol functionalized hydroxyalkyl starch derivatives
US20150374919A1 (en) 2013-03-22 2015-12-31 Amgen Inc. Injector and method of assembly
JP2016539756A (en) 2013-10-24 2016-12-22 アムジエン・インコーポレーテツド Injector and assembly of the way
JP2016538096A (en) 2013-10-24 2016-12-08 アムジエン・インコーポレーテツド Drug delivery system with temperature sensing control
US20180193623A1 (en) 2014-02-05 2018-07-12 Amgen Inc. Drug delivery system with electromagnetic field generator
EP3114138A1 (en) 2014-03-05 2017-01-11 Pfizer Inc Improved muteins of clotting factor viii
KR20160149198A (en) 2014-05-07 2016-12-27 암겐 인코포레이티드 Autoinjector with shock reducing elements
WO2015187805A3 (en) 2014-06-03 2016-04-14 Amgen Inc. System and method for supporting patient compliance with a medical treatment regimen
CN104072608A (en) * 2014-06-23 2014-10-01 山东齐都药业有限公司 Bovine serum albumin conjugate modified through carboxyl-containing hydroxyethyl starch derivative and preparation method thereof
EP3206739A2 (en) 2014-10-14 2017-08-23 Amgen Inc. Drug injection device with visual and audio indicators
WO2016100055A1 (en) 2014-12-19 2016-06-23 Amgen Inc. Drug delivery device with live button or user interface field
JP2017538511A (en) 2014-12-19 2017-12-28 アムジエン・インコーポレーテツド Proximity sensor with a drug delivery device
WO2017039786A1 (en) 2015-09-02 2017-03-09 Amgen Inc. Syringe assembly adapter for a syringe
WO2017100501A1 (en) 2015-12-09 2017-06-15 Amgen Inc. Auto-injector with signaling cap
WO2017120178A1 (en) 2016-01-06 2017-07-13 Amgen Inc. Auto-injector with signaling electronics
WO2017160799A1 (en) 2016-03-15 2017-09-21 Amgen Inc. Reducing probability of glass breakage in drug delivery devices
WO2017189089A1 (en) 2016-04-29 2017-11-02 Amgen Inc. Drug delivery device with messaging label
WO2017192287A1 (en) 2016-05-02 2017-11-09 Amgen Inc. Syringe adapter and guide for filling an on-body injector
WO2017197222A1 (en) 2016-05-13 2017-11-16 Amgen Inc. Vial sleeve assembly
WO2017200989A1 (en) 2016-05-16 2017-11-23 Amgen Inc. Data encryption in medical devices with limited computational capability
GB201609083D0 (en) 2016-05-24 2016-07-06 Syntab Therapeutics Gmbh Synthetic compound
WO2017209899A1 (en) 2016-06-03 2017-12-07 Amgen Inc. Impact testing apparatuses and methods for drug delivery devices
WO2018004842A1 (en) 2016-07-01 2018-01-04 Amgen Inc. Drug delivery device having minimized risk of component fracture upon impact events
WO2018034784A1 (en) 2016-08-17 2018-02-22 Amgen Inc. Drug delivery device with placement detection
WO2018081234A1 (en) 2016-10-25 2018-05-03 Amgen Inc. On-body injector

Citations (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3191291A (en) * 1959-01-21 1965-06-29 Continental Can Co Art of producing very thin steel and like sheets in wide strips
US4001200A (en) * 1975-02-27 1977-01-04 Alza Corporation Novel polymerized, cross-linked, stromal-free hemoglobin
US4001401A (en) * 1975-02-02 1977-01-04 Alza Corporation Blood substitute and blood plasma expander comprising polyhemoglobin
US4053590A (en) * 1975-02-27 1977-10-11 Alza Corporation Compositions of matter comprising macromolecular hemoglobin
US4061736A (en) * 1975-02-02 1977-12-06 Alza Corporation Pharmaceutically acceptable intramolecularly cross-linked, stromal-free hemoglobin
US4064118A (en) * 1975-10-22 1977-12-20 Hematech Inc. Blood substitute based on hemoglobin
US4125492A (en) * 1974-05-31 1978-11-14 Pedro Cuatrecasas Affinity chromatography of vibrio cholerae enterotoxin-ganglioside polysaccharide and the biological effects of ganglioside-containing soluble polymers
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4261973A (en) * 1976-08-17 1981-04-14 Pharmacia Ab Allergen-containing substances
US4412989A (en) * 1981-06-10 1983-11-01 Ajinomoto Company Incorporated Oxygen carrier
US4667016A (en) * 1985-06-20 1987-05-19 Kirin-Amgen, Inc. Erythropoietin purification
US4703008A (en) * 1983-12-13 1987-10-27 Kiren-Amgen, Inc. DNA sequences encoding erythropoietin
US4766106A (en) * 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US4847325A (en) * 1988-01-20 1989-07-11 Cetus Corporation Conjugation of polymer to colony stimulating factor-1
US4863964A (en) * 1985-07-02 1989-09-05 Biomedical Frontiers, Inc. Method for the stabilization of deferoxamine to chelate free ions in physiological fluid
US4900780A (en) * 1988-05-25 1990-02-13 Masonic Medical Research Laboratory Acellular resuscitative fluid
US4904584A (en) * 1987-12-23 1990-02-27 Genetics Institute, Inc. Site-specific homogeneous modification of polypeptides
US4925677A (en) * 1988-08-31 1990-05-15 Theratech, Inc. Biodegradable hydrogel matrices for the controlled release of pharmacologically active agents
US4952496A (en) * 1984-03-30 1990-08-28 Associated Universities, Inc. Cloning and expression of the gene for bacteriophage T7 RNA polymerase
US5068321A (en) * 1988-10-27 1991-11-26 Wolff Walsrode Aktiengesellschaft Carbonic acid esters of polysaccharides and a process for their production
US5079337A (en) * 1986-07-02 1992-01-07 Pasteur Merieux Serums Et Vaccins S.A. Macromolecular conjugates of hemoglobin, a procedure for their preparation and their uses
US5214132A (en) * 1986-12-23 1993-05-25 Kyowa Hakko Kogyo Co., Ltd. Polypeptide derivatives of human granulocyte colony stimulating factor
US5218108A (en) * 1989-06-16 1993-06-08 Fresenius Ag Hydroxylethylstarch (hes) as plasma expander and process for preparing hes
US5218092A (en) * 1988-09-29 1993-06-08 Kyowa Hakko Kogyo Co., Ltd. Modified granulocyte-colony stimulating factor polypeptide with added carbohydrate chains
US5217998A (en) * 1985-07-02 1993-06-08 Biomedical Frontiers, Inc. Composition for the stabilization of deferoxamine to chelate free ions in physiological fluid
US5281698A (en) * 1991-07-23 1994-01-25 Cetus Oncology Corporation Preparation of an activated polymer ester for protein conjugation
US5362853A (en) * 1986-12-23 1994-11-08 Kyowa Hakko Kogyo Co., Ltd. Polypeptide derivatives of human granulocyte colony stimulating factor
US5470843A (en) * 1992-12-11 1995-11-28 Hoechst Aktiengesellschaft Carbohydrate-containing polymers, their preparation and use
US5484903A (en) * 1991-09-17 1996-01-16 Wolff Walsrode Aktiengesellschaft Process for the production of polysaccharide carbonates
US5543332A (en) * 1991-07-04 1996-08-06 Immunodex K/S Water-soluble, polymer-based reagents and conjugates comprising moieties derived from divinyl sulfone
US5581476A (en) * 1993-01-28 1996-12-03 Amgen Inc. Computer-based methods and articles of manufacture for preparing G-CSF analogs
US5622718A (en) * 1992-09-25 1997-04-22 Keele University Alginate-bioactive agent conjugates
US5876980A (en) * 1995-04-11 1999-03-02 Cytel Corporation Enzymatic synthesis of oligosaccharides
US5880270A (en) * 1995-06-07 1999-03-09 Cellpro, Incorporated Aminooxy-containing linker compounds for formation of stably-linked conjugates and methods related thereto
US5952347A (en) * 1997-03-13 1999-09-14 Merck & Co., Inc. Quinoline leukotriene antagonists
US6011008A (en) * 1997-01-08 2000-01-04 Yissum Research Developement Company Of The Hebrew University Of Jerusalem Conjugates of biologically active substances
US6083909A (en) * 1996-07-08 2000-07-04 Fresenius Ag Haemoglobin-hydroxyethyl starch conjugates as oxygen carriers
US6261800B1 (en) * 1989-05-05 2001-07-17 Genentech, Inc. Luteinizing hormone/choriogonadotropin (LH/CG) receptor
US6299881B1 (en) * 1997-03-24 2001-10-09 Henry M. Jackson Foundation For The Advancement Of Military Medicine Uronium salts for activating hydroxyls, carboxyls, and polysaccharides, and conjugate vaccines, immunogens, and other useful immunological reagents produced using uronium salts
US6340746B1 (en) * 1997-08-07 2002-01-22 University Of Utah Prodrugs and conjugates of thiol- and selenol- containing compounds and methods of use thereof
US6500930B2 (en) * 1998-03-31 2002-12-31 Hemosol Inc. Hemoglobin-polysaccharide conjugates
US6555660B2 (en) * 2000-01-10 2003-04-29 Maxygen Holdings Ltd. G-CSF conjugates
US6586398B1 (en) * 2000-04-07 2003-07-01 Amgen, Inc. Chemically modified novel erythropoietin stimulating protein compositions and methods
US6596861B1 (en) * 1999-03-12 2003-07-22 Aventis Pasteur S.A. Method for the reductive amination of polysaccharides
US20040043446A1 (en) * 2001-10-19 2004-03-04 Neose Technologies, Inc. Alpha galalctosidase a: remodeling and glycoconjugation of alpha galactosidase A
US20040180858A1 (en) * 2001-06-21 2004-09-16 Klaus Sommermeyer Water-soluble antibiotic comprising an amino sugar, in the form of a polysaccharide conjugate
US20050063943A1 (en) * 2001-03-16 2005-03-24 Klaus Sommermeyer Conjugated of hydroxyalkyl starch and an active agent
US6875594B2 (en) * 1997-11-13 2005-04-05 The Rockefeller University Methods of ligating expressed proteins
US20050181985A1 (en) * 2002-03-06 2005-08-18 Jurgen Hemberger Coupling proteins to a modified polysaccharide
US20060019877A1 (en) * 2002-09-11 2006-01-26 Conradt Harald S Hasylated polypeptides
US20060217293A1 (en) * 2002-03-06 2006-09-28 Michele Orlando Coupling low-molecular substances to a modified polysaccharide
US7285661B2 (en) * 2002-02-20 2007-10-23 Fresenius Kabi Deutschland Gmbh Starch derivatives, starch active substance conjugates, method for the production thereof and their use as medicaments

Family Cites Families (97)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE279486C (en)
NL269801A (en) * 1960-10-04 1964-03-10 Farbwerke Höchst Ag Process for the preparation of water-insoluble dyes
DE2233977A1 (en) 1971-07-14 1973-02-01 Unilever Nv A process for preparing oxidized carbohydrates
GB1419080A (en) 1972-12-29 1975-12-24 Cheminova As Chemical compounds having juvenile hormone activity
DE2616086C2 (en) * 1976-04-13 1986-04-03 Dr. Eduard Fresenius, Chemisch-Pharmazeutische Industrie Kg, 6380 Bad Homburg, De
FR2378094B2 (en) 1977-01-24 1980-06-20 Inst Nat Sante Rech Med
EP0019403B1 (en) * 1979-05-10 1985-07-31 American Hospital Supply Corporation Hydroxyalkyl-starch drug carrier
DE3029307C2 (en) * 1980-08-01 1989-12-07 Fresenius Ag, 6380 Bad Homburg, De
US4454161A (en) * 1981-02-07 1984-06-12 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Process for the production of branching enzyme, and a method for improving the qualities of food products therewith
FI82266C (en) 1982-10-19 1991-02-11 Cetus Corp Foerfarande Foer framstaellning of IL-2 -mutein.
DE3485810T2 (en) 1983-05-27 1992-12-10 Texas A & M Univ Sys A method for producing a recombinant baculovirus expression vector.
FR2553099B1 (en) 1983-10-11 1989-09-08 Fidia Spa Fractions of hyaluronic acid having pharmaceutical activity, processes for their preparation and pharmaceutical compositions containing them
WO1985002610A1 (en) 1983-12-13 1985-06-20 Kirin-Amgen, Inc. Production of erythropoietin
US4496689A (en) * 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
EP0205564B2 (en) 1984-12-04 2000-06-21 Genetics Institute, Inc. Method for the production of erythropoietin
DE3501616A1 (en) 1985-01-17 1986-07-17 Schering Ag Process for the preparation of hydroxylamine derivatives
GB8610551D0 (en) 1986-04-30 1986-06-04 Hoffmann La Roche Polypeptide & protein derivatives
CA1338235C (en) 1986-06-30 1996-04-09 Valle Francesco Della Esters of alginic acid
JP2594123B2 (en) * 1987-09-12 1997-03-26 株式会社林原生物化学研究所 Desensitizing agent
EP0307827A3 (en) 1987-09-15 1989-12-27 Kuraray Co., Ltd. Novel macromolecular complexes, process for producing same and medicinal use of such complexes
JPH0270703A (en) 1987-10-23 1990-03-09 Yissum Res Dev Co Of Hebrew Univ Of Jerusalem Phospholipase a2 inhibiting composition and its use
DK110188D0 (en) 1988-03-02 1988-03-02 Claus Selch Larsen High molecular weight prodrug derivatives of antiinflammatory drugs
FR2630329B1 (en) * 1988-04-20 1991-07-05 Merieux Inst Macromolecular conjugated hemoglobin, their method of preparing and applications
DE68919435T2 (en) 1988-05-13 1995-07-06 Fidia Spa Esters of polysaccharides.
US5420105A (en) * 1988-09-23 1995-05-30 Gustavson; Linda M. Polymeric carriers for non-covalent drug conjugation
EP0401384B1 (en) * 1988-12-22 1996-03-13 Kirin-Amgen, Inc. Chemically modified granulocyte colony stimulating factor
JP2896580B2 (en) 1989-08-25 1999-05-31 チッソ株式会社 Amylose - Lysozyme hybrids with activated sugars and their preparation
JP2838800B2 (en) * 1989-09-02 1998-12-16 株式会社林原生物化学研究所 Desensitizing agent
KR100263845B1 (en) 1989-10-13 2000-08-16 스튜어트 엘.왓트 Erythropoietin isoforms and preparation method thereof and pharmaceutical composition containing it
JP2975632B2 (en) 1990-03-30 1999-11-10 生化学工業株式会社 Glycosaminoglycan-modified protein
US5169784A (en) 1990-09-17 1992-12-08 The Texas A & M University System Baculovirus dual promoter expression vector
US6172208B1 (en) * 1992-07-06 2001-01-09 Genzyme Corporation Oligonucleotides modified with conjugate groups
US5840900A (en) * 1993-10-20 1998-11-24 Enzon, Inc. High molecular weight polymer-based prodrugs
WO1994005332A3 (en) * 1992-09-01 1994-04-14 Berlex Lab Glycolation of glycosylated macromolecules
EP0605963A3 (en) 1992-12-09 1995-11-08 Ortho Pharma Corp Peg hydrazone and peg oxime linkage forming reagents and protein derivatives thereof.
FI935485A (en) * 1992-12-09 1994-06-10 Ortho Pharma Corp PEG and PEG-hydrazone-oxime bond forming reagents and their protein derivatives
CA2110543A1 (en) 1992-12-09 1994-06-10 David E. Wright Peg hydrazone and peg oxime linkage forming reagents and protein derivatives thereof
EP0646130B1 (en) 1993-03-16 1996-12-11 Hemosol Inc. Selective crosslinking of hemoglobins by oxidized, ring-opened saccharides
WO1994028024A1 (en) * 1993-06-01 1994-12-08 Enzon, Inc. Carbohydrate-modified polymer conjugates with erythropoietic activity
CN1057534C (en) 1993-08-17 2000-10-18 柯瑞英-艾格公司 Erythropoietin analogs
JPH07188291A (en) 1993-12-27 1995-07-25 Hayashibara Biochem Lab Inc Protein, its production and use
US6214331B1 (en) * 1995-06-06 2001-04-10 C. R. Bard, Inc. Process for the preparation of aqueous dispersions of particles of water-soluble polymers and the particles obtained
US5736533A (en) * 1995-06-07 1998-04-07 Neose Technologies, Inc. Bacterial inhibition with an oligosaccharide compound
US5981507A (en) * 1995-12-14 1999-11-09 Advanced Magnetics, Inc. Polymeric carriers linked to nucleotide analogues via a phosphoramide bond
US5723589A (en) 1995-12-21 1998-03-03 Icn Pharmaceuticals Carbohydrate conjugated bio-active compounds
EP1683520B1 (en) * 1996-03-12 2013-11-20 PG-TXL Company, L.P. Water-soluble prodrugs
US5696152A (en) 1996-05-07 1997-12-09 Wisconsin Alumni Research Foundation Taxol composition for use as organ preservation and cardioplegic agents
CA2203480C (en) * 1996-05-31 2009-06-30 Pascal Sebastian Bailon Interferon conjugates
US5770645A (en) * 1996-08-02 1998-06-23 Duke University Medical Center Polymers for delivering nitric oxide in vivo
US5851984A (en) * 1996-08-16 1998-12-22 Genentech, Inc. Method of enhancing proliferation or differentiation of hematopoietic stem cells using Wnt polypeptides
US5990237A (en) * 1997-05-21 1999-11-23 Shearwater Polymers, Inc. Poly(ethylene glycol) aldehyde hydrates and related polymers and applications in modifying amines
US5847110A (en) * 1997-08-15 1998-12-08 Biomedical Frontiers, Inc. Method of reducing a schiff base
US6624142B2 (en) 1997-12-30 2003-09-23 Enzon, Inc. Trimethyl lock based tetrapartate prodrugs
DE19808079A1 (en) 1998-02-20 1999-08-26 Schering Ag New hydroxyethyl starch conjugates useful as X-ray, NMR and blood-pool diagnostic agents, e.g. for diagnosis of tumors, cardiovascular disorders and inflammation
WO1999045163A1 (en) 1998-03-05 1999-09-10 Asahi Glass Company Ltd. Sputtering target, transparent conductive film, and method for producing the same
US6153655A (en) * 1998-04-17 2000-11-28 Enzon, Inc. Terminally-branched polymeric linkers and polymeric conjugates containing the same
US6660843B1 (en) * 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
US6261594B1 (en) * 1998-11-25 2001-07-17 The University Of Akron Chitosan-based nitric oxide donor compositions
US6583272B1 (en) 1999-07-02 2003-06-24 Hoffmann-La Roche Inc. Erythropoietin conjugates
US7279176B1 (en) 1999-09-02 2007-10-09 Rice University Nitric oxide-producing hydrogel materials
US20020065410A1 (en) * 1999-12-02 2002-05-30 Antrim Richard L. Branched starches and branched starch hydrolyzates
US6749865B2 (en) * 2000-02-15 2004-06-15 Genzyme Corporation Modification of biopolymers for improved drug delivery
FR2811967B1 (en) 2000-07-24 2002-12-13 Cebal Tube fitted with a fastening head for various closures and various closures provided with fixing means on said tube
DE10041541A1 (en) 2000-08-24 2002-03-14 Michael Duchene New nucleic acid encoding moth allergens, related polypeptides and antibodies, useful in the diagnosis and treatment of arthropod allergies
US6417347B1 (en) * 2000-08-24 2002-07-09 Scimed Life Systems, Inc. High yield S-nitrosylation process
DE60144188D1 (en) 2000-09-08 2011-04-21 Amylin Pharmaceuticals Inc "pseudo"-native chemische ligation
DE10105921A1 (en) 2001-02-09 2002-08-14 Braun Melsungen Ag At colloids bound drug targets
DE10126158A1 (en) 2001-05-30 2002-12-12 Novira Chem Gmbh Mixtures of alpha-hydroxy-omega-alkoxy- and alpha-omega-dialkoxy-polyoxyalkylene containing little or no dihydroxy-polyoxyalkylene, used for coupling and modification of proteins and other bioactive molecules
DE10135694A1 (en) 2001-07-21 2003-02-06 Supramol Parenteral Colloids New amphiphilic conjugate of starch or hydroxyethylstarch, useful as drug carrier, contain e.g. fatty acyl residues, are not taken up by the reticuloendothelial system
US7179617B2 (en) 2001-10-10 2007-02-20 Neose Technologies, Inc. Factor IX: remolding and glycoconjugation of Factor IX
EP2431377B1 (en) * 2001-10-26 2017-04-19 Noxxon Pharma AG Modified L-nucleic acids
US6375846B1 (en) 2001-11-01 2002-04-23 Harry Wellington Jarrett Cyanogen bromide-activation of hydroxyls on silica for high pressure affinity chromatography
DE10155098A1 (en) 2001-11-09 2003-05-22 Supramol Parenteral Colloids Agent for protecting cell and tissue cultures against fungi, comprises water-soluble conjugate of polyene macrolide and polysaccharide
US6916962B2 (en) 2001-12-11 2005-07-12 Sun Bio, Inc. Monofunctional polyethylene glycol aldehydes
WO2003076490A8 (en) 2002-03-13 2004-10-28 Beijing Jiankai Technology Co Hydrophilic polymer derivate with y type branch and preparation method of it medical composite comprising above compound
DE10217994A1 (en) 2002-04-23 2003-11-06 Supramol Parenteral Colloids A coupling product from (sic) chemical compounds of hyperbranched polysaccharide completely catabolized in the body under control of the body enzymes useful for parenteral pharmaceutically active materials
WO2003102018A3 (en) * 2002-06-03 2004-12-09 Inst Systems Biology Methods for quantitative proteome analysis of glycoproteins
EP1591467A1 (en) 2002-09-09 2005-11-02 Nektar Therapeutics Al, Corporation Conjugate between a polyethylene glycol having a terminal alkanal group and a human growth hormone
JP4764630B2 (en) 2002-09-09 2011-09-07 ネクター セラピューティックス Water-soluble polymer alkanal
RU2370281C2 (en) 2003-08-08 2009-10-20 Фрезениус Каби Дойчланд Гмбх Hydroxyalkyl starch and g-csf conjugates
US20080206182A1 (en) * 2003-08-08 2008-08-28 Fresenius Kabi Deutschland Gmbh Conjugates of a Polymer and a Protein Linked by an Oxime Group
ES2214166T1 (en) 2002-09-11 2004-09-16 Fresenius Kabi Deutschland Gmbh Polypeptides has-ilados especially eriptropoyetina has-ilada.
WO2005014655A3 (en) * 2003-08-08 2005-06-16 Harald Conradt Conjugates of hydroxyalkyl starch and a protein
DE10242076A1 (en) * 2002-09-11 2004-03-25 Fresenius Kabi Deutschland Gmbh New covalently bonded conjugates of hydroxyalkyl starch with allergens, useful as modified allergens with depot effect for use in specific immunotherapy for combating allergies, e.g. hay fever
US20080274948A1 (en) 2003-08-08 2008-11-06 Fresenius Kabi Deutschland Gmbh Conjugates of Hydroxyalkyl Starch and G-Csf
ES2314238T3 (en) * 2002-10-08 2009-03-16 Fresenius Kabi Deutschland Gmbh Pharmaceutically active oligosaccharide conjugates.
DE10254745A1 (en) 2002-11-23 2004-06-03 Supramol Parenteral Colloids Gmbh New aldonic acid imidazolides of starch compounds selectively oxidized at the reducing terminal, useful for coupling with amino functions of pharmaceutically active agents, e.g. proteins
US20040101546A1 (en) 2002-11-26 2004-05-27 Gorman Anne Jessica Hemostatic wound dressing containing aldehyde-modified polysaccharide and hemostatic agents
DE10256558A1 (en) 2002-12-04 2004-09-16 Supramol Parenteral Colloids Gmbh Esters of polysaccharide aldonic acids, process for their preparation and use for coupling to active pharmaceutical ingredients
EP1758608A2 (en) * 2004-03-11 2007-03-07 Fresenius Kabi Deutschland GmbH Conjugates of hydroxyethyl starch and erythropoietin
EP1732609B1 (en) * 2004-03-11 2012-07-11 Fresenius Kabi Deutschland GmbH Conjugates of hydroxyalkyl starch and a protein
CA2558738C (en) 2004-03-11 2013-02-05 Fresenius Kabi Deutschland Gmbh Conjugates of hydroxyalkyl starch and a protein, prepared by reductive amination
EP1861124A2 (en) * 2005-03-11 2007-12-05 Fresenius Kabi Deutschland GmbH Production of bioactive glycoproteins from inactive starting materials by conjugation with hydroxyalkylstarch
EP1762250A1 (en) * 2005-09-12 2007-03-14 Fresenius Kabi Deutschland GmbH Conjugates of hydroxyalkyl starch and an active substance, prepared by chemical ligation via thiazolidine
JP2009093397A (en) 2007-10-09 2009-04-30 Panasonic Corp Touch panel and input device using the same
EP2070950A8 (en) * 2007-12-14 2009-08-19 Fresenius Kabi Deutschland GmbH Hydroxyalkyl starch derivatives and process for their preparation
EP2070951A1 (en) * 2007-12-14 2009-06-17 Fresenius Kabi Deutschland GmbH Method for producing a hydroxyalkyl starch derivatives with two linkers

Patent Citations (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3191291A (en) * 1959-01-21 1965-06-29 Continental Can Co Art of producing very thin steel and like sheets in wide strips
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4125492A (en) * 1974-05-31 1978-11-14 Pedro Cuatrecasas Affinity chromatography of vibrio cholerae enterotoxin-ganglioside polysaccharide and the biological effects of ganglioside-containing soluble polymers
US4001401A (en) * 1975-02-02 1977-01-04 Alza Corporation Blood substitute and blood plasma expander comprising polyhemoglobin
US4061736A (en) * 1975-02-02 1977-12-06 Alza Corporation Pharmaceutically acceptable intramolecularly cross-linked, stromal-free hemoglobin
US4053590A (en) * 1975-02-27 1977-10-11 Alza Corporation Compositions of matter comprising macromolecular hemoglobin
US4001200A (en) * 1975-02-27 1977-01-04 Alza Corporation Novel polymerized, cross-linked, stromal-free hemoglobin
US4064118A (en) * 1975-10-22 1977-12-20 Hematech Inc. Blood substitute based on hemoglobin
US4261973A (en) * 1976-08-17 1981-04-14 Pharmacia Ab Allergen-containing substances
US4412989A (en) * 1981-06-10 1983-11-01 Ajinomoto Company Incorporated Oxygen carrier
US4703008A (en) * 1983-12-13 1987-10-27 Kiren-Amgen, Inc. DNA sequences encoding erythropoietin
US4952496A (en) * 1984-03-30 1990-08-28 Associated Universities, Inc. Cloning and expression of the gene for bacteriophage T7 RNA polymerase
US4667016A (en) * 1985-06-20 1987-05-19 Kirin-Amgen, Inc. Erythropoietin purification
US4766106A (en) * 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US4863964A (en) * 1985-07-02 1989-09-05 Biomedical Frontiers, Inc. Method for the stabilization of deferoxamine to chelate free ions in physiological fluid
US5217998A (en) * 1985-07-02 1993-06-08 Biomedical Frontiers, Inc. Composition for the stabilization of deferoxamine to chelate free ions in physiological fluid
US5079337A (en) * 1986-07-02 1992-01-07 Pasteur Merieux Serums Et Vaccins S.A. Macromolecular conjugates of hemoglobin, a procedure for their preparation and their uses
US5214132A (en) * 1986-12-23 1993-05-25 Kyowa Hakko Kogyo Co., Ltd. Polypeptide derivatives of human granulocyte colony stimulating factor
US5362853A (en) * 1986-12-23 1994-11-08 Kyowa Hakko Kogyo Co., Ltd. Polypeptide derivatives of human granulocyte colony stimulating factor
US4904584A (en) * 1987-12-23 1990-02-27 Genetics Institute, Inc. Site-specific homogeneous modification of polypeptides
US4847325A (en) * 1988-01-20 1989-07-11 Cetus Corporation Conjugation of polymer to colony stimulating factor-1
US4900780A (en) * 1988-05-25 1990-02-13 Masonic Medical Research Laboratory Acellular resuscitative fluid
US4925677A (en) * 1988-08-31 1990-05-15 Theratech, Inc. Biodegradable hydrogel matrices for the controlled release of pharmacologically active agents
US5218092A (en) * 1988-09-29 1993-06-08 Kyowa Hakko Kogyo Co., Ltd. Modified granulocyte-colony stimulating factor polypeptide with added carbohydrate chains
US5068321A (en) * 1988-10-27 1991-11-26 Wolff Walsrode Aktiengesellschaft Carbonic acid esters of polysaccharides and a process for their production
US6261800B1 (en) * 1989-05-05 2001-07-17 Genentech, Inc. Luteinizing hormone/choriogonadotropin (LH/CG) receptor
US5218108A (en) * 1989-06-16 1993-06-08 Fresenius Ag Hydroxylethylstarch (hes) as plasma expander and process for preparing hes
US5543332A (en) * 1991-07-04 1996-08-06 Immunodex K/S Water-soluble, polymer-based reagents and conjugates comprising moieties derived from divinyl sulfone
US5281698A (en) * 1991-07-23 1994-01-25 Cetus Oncology Corporation Preparation of an activated polymer ester for protein conjugation
US5484903A (en) * 1991-09-17 1996-01-16 Wolff Walsrode Aktiengesellschaft Process for the production of polysaccharide carbonates
US5622718A (en) * 1992-09-25 1997-04-22 Keele University Alginate-bioactive agent conjugates
US5470843A (en) * 1992-12-11 1995-11-28 Hoechst Aktiengesellschaft Carbohydrate-containing polymers, their preparation and use
US5581476A (en) * 1993-01-28 1996-12-03 Amgen Inc. Computer-based methods and articles of manufacture for preparing G-CSF analogs
US5876980A (en) * 1995-04-11 1999-03-02 Cytel Corporation Enzymatic synthesis of oligosaccharides
US5880270A (en) * 1995-06-07 1999-03-09 Cellpro, Incorporated Aminooxy-containing linker compounds for formation of stably-linked conjugates and methods related thereto
US6083909A (en) * 1996-07-08 2000-07-04 Fresenius Ag Haemoglobin-hydroxyethyl starch conjugates as oxygen carriers
US6011008A (en) * 1997-01-08 2000-01-04 Yissum Research Developement Company Of The Hebrew University Of Jerusalem Conjugates of biologically active substances
US5952347A (en) * 1997-03-13 1999-09-14 Merck & Co., Inc. Quinoline leukotriene antagonists
US6299881B1 (en) * 1997-03-24 2001-10-09 Henry M. Jackson Foundation For The Advancement Of Military Medicine Uronium salts for activating hydroxyls, carboxyls, and polysaccharides, and conjugate vaccines, immunogens, and other useful immunological reagents produced using uronium salts
US6340746B1 (en) * 1997-08-07 2002-01-22 University Of Utah Prodrugs and conjugates of thiol- and selenol- containing compounds and methods of use thereof
US6875594B2 (en) * 1997-11-13 2005-04-05 The Rockefeller University Methods of ligating expressed proteins
US6500930B2 (en) * 1998-03-31 2002-12-31 Hemosol Inc. Hemoglobin-polysaccharide conjugates
US6596861B1 (en) * 1999-03-12 2003-07-22 Aventis Pasteur S.A. Method for the reductive amination of polysaccharides
US6555660B2 (en) * 2000-01-10 2003-04-29 Maxygen Holdings Ltd. G-CSF conjugates
US6586398B1 (en) * 2000-04-07 2003-07-01 Amgen, Inc. Chemically modified novel erythropoietin stimulating protein compositions and methods
US20050063943A1 (en) * 2001-03-16 2005-03-24 Klaus Sommermeyer Conjugated of hydroxyalkyl starch and an active agent
US20040180858A1 (en) * 2001-06-21 2004-09-16 Klaus Sommermeyer Water-soluble antibiotic comprising an amino sugar, in the form of a polysaccharide conjugate
US7115576B2 (en) * 2001-06-21 2006-10-03 Fresenius Kabi Deutschland Gmbh Water-soluble antibiotic comprising an amino sugar, in the form of a polysaccharide conjugate
US20040043446A1 (en) * 2001-10-19 2004-03-04 Neose Technologies, Inc. Alpha galalctosidase a: remodeling and glycoconjugation of alpha galactosidase A
US7285661B2 (en) * 2002-02-20 2007-10-23 Fresenius Kabi Deutschland Gmbh Starch derivatives, starch active substance conjugates, method for the production thereof and their use as medicaments
US20050181985A1 (en) * 2002-03-06 2005-08-18 Jurgen Hemberger Coupling proteins to a modified polysaccharide
US20060217293A1 (en) * 2002-03-06 2006-09-28 Michele Orlando Coupling low-molecular substances to a modified polysaccharide
US20060019877A1 (en) * 2002-09-11 2006-01-26 Conradt Harald S Hasylated polypeptides

Cited By (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060188472A1 (en) * 2001-03-16 2006-08-24 Fresenius Kabi Deutschland Gmbh, A Germany Corporation HAS-active ingredient conjugates
US7816516B2 (en) 2001-03-16 2010-10-19 Fresenius Kabi Deutschland Gmbh Conjugates of hydroxyalkyl starch and an active agent
US20050063943A1 (en) * 2001-03-16 2005-03-24 Klaus Sommermeyer Conjugated of hydroxyalkyl starch and an active agent
US20050181985A1 (en) * 2002-03-06 2005-08-18 Jurgen Hemberger Coupling proteins to a modified polysaccharide
US8466277B2 (en) 2002-03-06 2013-06-18 Fresenius Kabi Deutschland Gmbh Coupling low-molecular substances to a modified polysaccharide
US8916518B2 (en) 2002-03-06 2014-12-23 Fresenius Kabi Deutschland Gmbh Coupling proteins to a modified polysaccharide
US20060217293A1 (en) * 2002-03-06 2006-09-28 Michele Orlando Coupling low-molecular substances to a modified polysaccharide
US7541328B2 (en) * 2002-03-06 2009-06-02 Fresenius Kabi Deutschland Gmbh Coupling proteins to a modified polysaccharide
US20060121062A1 (en) * 2002-09-11 2006-06-08 Wolfram Eichner Conjugated hydroxyalkyl starch allergen compounds
US8618266B2 (en) 2002-09-11 2013-12-31 Fresenius Kabi Deutschland Gmbh Hasylated polypeptides
US8475765B2 (en) 2002-09-11 2013-07-02 Fresenius Kabi Deutschland Gmbh Hydroxyalkyl starch derivatives
US20060019877A1 (en) * 2002-09-11 2006-01-26 Conradt Harald S Hasylated polypeptides
US20050234230A1 (en) * 2002-09-11 2005-10-20 Norbert Zander Hydroxyalkyl starch derivatives
US20110054152A1 (en) * 2002-09-11 2011-03-03 Fresenius Kabi Deutschland Gmbh Hydroxyalkyl Starch Derivatives
US7815893B2 (en) 2002-09-11 2010-10-19 Fresenius Kabi Deutschland Gmbh Hydroxyalkyl starch derivatives
US7538092B2 (en) 2002-10-08 2009-05-26 Fresenius Kabi Deutschland Gmbh Pharmaceutically active oligosaccharide conjugates
US20060100163A1 (en) * 2002-10-08 2006-05-11 Michele Orlando Pharmaceutically active oligosaccharide conjugates
US20060052342A1 (en) * 2002-12-04 2006-03-09 Klaus Sommermeyer Aldonic acid esters, methods for producing the same, and methods for producing pharmaceutical active ingredients coupled to polysaccharides or polysaccharide derivatives on free amino groups
US20080206182A1 (en) * 2003-08-08 2008-08-28 Fresenius Kabi Deutschland Gmbh Conjugates of a Polymer and a Protein Linked by an Oxime Group
US20080274948A1 (en) * 2003-08-08 2008-11-06 Fresenius Kabi Deutschland Gmbh Conjugates of Hydroxyalkyl Starch and G-Csf
US20110200555A1 (en) * 2004-03-11 2011-08-18 Fresenius Kabi Deutschland Gmbh Conjugates of hydroxyalkyl starch and a protein
US20120046235A9 (en) * 2004-03-11 2012-02-23 Nobert Zander Conjugates of hydroxyalkyl starch and a protein, prepared by native chemical ligation
US20100311670A1 (en) * 2004-03-11 2010-12-09 Nobert Zander Conjugates of hydroxyalkyl starch and a protein, prepared by native chemical ligation
US8840879B2 (en) 2004-03-11 2014-09-23 Fresenius Kabi Deutschland Gmbh Conjugates of hydroxyalkyl starch and a protein
US20090047251A1 (en) * 2004-03-11 2009-02-19 Wolfram Eichner Conjugates of hydroxyalkyl starch and a protein
US8287850B2 (en) 2004-03-11 2012-10-16 Fresenius Kabi Deutschland Gmbh Conjugates of hydroxyalkyl starch and a protein, prepared by reductive amination
US20070087961A1 (en) * 2004-03-11 2007-04-19 Wolfram Eichner Conjugates of hydroxyalkyl starch and erythropoietin
US8017739B2 (en) 2004-03-11 2011-09-13 Fresenius Kabi Deutschland Gmbh Conjugates of hydroxyalkyl starch and a protein
US20100062973A1 (en) * 2005-03-11 2010-03-11 Fresenius Kabi Deutschland Gmbh Production of bioactive glycoproteins from inactive starting material
US20080207562A1 (en) * 2005-09-12 2008-08-28 Fresenius Kabi Deutschland Gmbh Conjugates of Hydroxyalkyl Starch and Active Substance, Prepared by Chemical Ligation Via Thiazolidine
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US20100305033A1 (en) * 2007-12-14 2010-12-02 Fresenius Kabi Deutschland Gmbh Hydroxyalkyl starch derivatives and process for their preparation
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KR20100099282A (en) * 2007-12-14 2010-09-10 프레제니우스 카비 도이치란트 게엠베하 Hydroxyalkyl starch derivatives and process for their preparation
KR101578955B1 (en) 2007-12-14 2015-12-21 프레제니우스 카비 도이치란트 게엠베하 Hydroxyalkyl starch derivatives and process for their preparation
WO2009077153A1 (en) * 2007-12-14 2009-06-25 Fresenius Kabi Deutschland Gmbh Method for producing a hydroxyalkyl starch derivative with two linkers
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US8404834B2 (en) 2007-12-14 2013-03-26 Fresenius Kabi Deutschland Gmbh Hydroxyalkyl starch derivatives and process for their preparation
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EP3225248A1 (en) 2008-07-23 2017-10-04 Ambrx, Inc. Modified bovine g-csf polypeptides and their uses
WO2010011735A2 (en) 2008-07-23 2010-01-28 Ambrx, Inc. Modified bovine g-csf polypeptides and their uses
EP2805964A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified bovine somatotropin polypeptides and their uses
EP2805965A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified porcine somatotropin polypeptides and their uses
WO2012004005A1 (en) * 2010-07-09 2012-01-12 Fresenius Kabi Deutschland Gmbh Conjugates comprising hydroxyalkyl starch and a cytotoxic agent and process for their preparation
WO2012004007A1 (en) * 2010-07-09 2012-01-12 Fresenius Kabi Deutschland Gmbh Conjugates comprising hydroxyalkyl starch and a cytotoxic agent and process for their preparation
WO2012004009A1 (en) * 2010-07-09 2012-01-12 Fresenius Kabi Deutschland Gmbh Conjugates comprising hydroxyalkyl starch and a cytotoxic agent and process for their preparation
WO2012004006A1 (en) * 2010-07-09 2012-01-12 Fresenius Kabi Deutschland Gmbh Conjugates comprising hydroxyalkyl starch and a cytotoxic agent and process for their preparation

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