EP1480682A1

EP1480682A1 - Coupling proteins to a modified polysaccharide

Info

Publication number: EP1480682A1
Application number: EP03743359A
Authority: EP
Inventors: Jürgen Hemberger; Michele Orlando
Original assignee: Fresenius Kabi Deutschland GmbH; Biotechnologie Gesellschaft Mittelhessen mbH
Current assignee: Fresenius Kabi Deutschland GmbH
Priority date: 2002-03-06
Filing date: 2003-02-28
Publication date: 2004-12-01
Also published as: US7541328B2; US8916518B2; KR20040098001A; US20050181985A1; CA2478478A1; IL163702A0; US20090233847A1; WO2003074087A1; CA2478478C; CN1638808A; PL372481A1; AU2003215617A1; DE10209821A1; JP2005528349A

Abstract

The invention relates to a method for coupling proteins to a starch-derived modified polysaccharide. The binding interaction between the modified polysaccharide and the protein is based on a covalent bond which is the result of a coupling reaction between the terminal aldehyde group or a functional group of the modified polysaccharide molecule resulting from the chemical reaction of this aldehyde group and a functional group of the protein which reacts with the aldehyde group or with the resulting functional group of the polysaccharide molecule. The bond directly resulting from the coupling reaction can be optionally modified by a further reaction to the aforementioned covalent bond. The invention further relates to pharmaceutical compositions that comprise conjugates formed in this coupling process and to the use of said conjugates and compositions for the prophylaxis or therapy of the human or animal body.

Description

03 02 083

1

Coupling of proteins to a modified polysaccharide

The rapid development of genetic engineering in recent decades has led to the identification of a large number of genes for proteins with potential therapeutic benefit and the corresponding gene products to be produced in large quantities with ease using biological expression systems.

However, it has been found that the practical use of such proteins, e.g. difficulties often arise in diagnostics, therapy and biotransformations, since their stability and solubility properties, especially at physiological pH values, are often unsatisfactory. Two examples of such proteins are the tumor necrosis factor TNF-α or interleukin-2.

Solubility problems also occur very frequently in the expression of glycoproteins in prokaryotic systems such as E. coli, since these are then expressed without natural glycosylation, which in some cases results in a considerably reduced solubility. This may require the use of much more expensive eukaryotic expression systems.

When used therapeutically in the body, many proteins are quickly removed from the bloodstream or broken down. Systemically applied proteins with a molecular weight of more than about 70 kD can be withdrawn from the circulation by the reticuloendothelial system or specific interactions with cellular receptors. Smaller proteins with a molecular weight of less than about 70 kD can also be removed to a large extent by glomerular filtration in the kidney (cut-off limit about 70 kD).

A recent approach to remedying the problems described has been to couple such problematic proteins with readily water-soluble biocompatible polymers, such as, for example, polyethylene glycol and dextran. The coupling allows the molecular weight to be increased beyond the threshold of 70 kD, so that the plasma residence time of smaller proteins is drastic can be increased, on the other hand, the solubility in the aqueous environment can be improved by the hydrophilic polymer content.

Further, mostly favorable effects, which can be associated with the coupling of proteins with such polymers, are based on the masking of protease recognition sites and antigenic determinants on the protein molecule by the bound polymer. As a result, the therapeutic proteins can largely be removed from proteolytic degradation, and secondly, the triggering of allergenic reactions by the foreign therapeutic protein is largely suppressed. In addition to increasing the molecular weight, the presence of a polymer protects proteins from enzymatic degradation and often also against thermal denaturation. In many cases this significantly increases the stability and the v / Vo half-life of the proteins or decreases the immunogenicity and antigenicity.

So far, most modifications have been made with polyethylene glycol or dextran, with PEG being generally preferred because it results in simpler products.

Dextran couplings have only been described for a few proteins, e.g. Streptokinase, plasmin, hemoglobin or aprotinin. However, dextran conjugates often show high allergenicity, presumably caused by degradation products of dextran, low metabolic stability and in many cases low yields in the coupling reactions. As a result, none of these dextran coupling products has been approved for therapeutic use in humans or animals.

Derivatizations with PEG were carried out much more frequently, so that this method can today be regarded as the standard for the molecular weight increase of proteins. Some of these derivatives are in various phases of clinical trials or have already been approved in the United States. Phase III contains PEG hemoglobin and a PEG adduct of superoxide dismutase (SOD), which is the best-studied protein in terms of polymer coupling. PEG-coupled asparaginase is already being used in acute therapy T EP03 / 02083

3 lymphocytic leukemia used. In 2001, PEG-interferon-α was approved for the treatment of hepatitis C patients.

However, when using these PEG conjugates, unpleasant to dangerous side effects such as itching, hypersensitivity reactions and pancreatitis have also been reported. Furthermore, the biological activity of the proteins after PEG coupling is often very low and the metabolism of the breakdown products of PEG conjugates is still largely unknown and may pose a health risk.

WO 99/49897 describes conjugates of hemoglobin which are formed by reacting the aldehyde groups of oxidatively ring-opened polysaccharides such as hydroxyethyl starch or dextran with primary amine groups of the protein. However, the polysaccharides used act as polyfunctional reagents, which result in a very heterogeneous product mixture with properties that are difficult to adjust.

US Patent 6,083,909 describes a method for coupling selectively oxidized hydroxyethyl starch to hemoglobin in DMSO. However, our investigations showed that the desired product is not obtained under the specified conditions, since hemoglobin denatures in DMSO and thus loses its biological activity.

There is therefore still a need for physiologically well-tolerated alternatives to dextran or PEG-coupled proteins, with which the solubility of proteins can be improved or the residence time of the proteins in the plasma can be increased.

The object of the invention is therefore to provide such alternatives and to develop simple and efficient methods for producing such alternative protein derivatives.

According to the invention, this object is achieved by hydroxyalkyl starch-protein conjugates, which are characterized in that the binding interaction between the hydroxyalkyl starch molecule and the protein is based on a covalent bond, which is the result of a coupling reaction between the terminal aldehyde group or a functional group of the hydroxyalkyl starch molecule resulting from this aldehyde group by chemical reaction and a functional group of the hydroxyalkyl starch molecule reactive with this aldehyde group or resulting therefrom Is protein, and the bond resulting directly from the coupling reaction can optionally be modified by a further reaction to form the above-mentioned covalent bond.

The invention further encompasses pharmaceutical compositions containing these conjugates, the use of these conjugates and compositions for the prophylactic or therapeutic treatment of the human or animal body, and methods for producing these conjugates and compositions.

Surprisingly, it has been found that the reactions described above can be carried out in aqueous solution with a suitable choice of the conditions, as a result of which the biological activity of the proteins can in many cases be wholly or partly retained.

The aqueous reaction medium for the coupling reaction is preferably water or a mixture of water and an organic solvent, the water content in the mixture being at least about 70% by weight, preferably at least about 80% by weight, more preferably at least about 90% by weight. -%.

The molar ratio of hydroxyalkyl starch (HAS) to protein in the coupling reaction is usually about 20: 1 to 1: 1, preferably about 5: 1 to 1: 1.

The residual biological activity of the hydroxyalkyl starch-protein conjugates according to the invention, based on the starting activity of the protein, is generally at least 40%, preferably at least 50%, more preferably at least 70%, more preferably at least 90%, most preferably at least 95%. The hydroxyalkyl starch (HAS) used according to the invention can be prepared using a known method, for example hydroxyalkylation of starch at the C ₂ - and / or C ₆ ~ position of the anhydroglucose units with alkylene oxide or 2-chloroalkanol, for example 2-chloroethanol (see, for example, US 5,218,108 for Hydroxyethylation of starch), with various desired molecular weight ranges and degrees of substitution. Any of the commercially available preparations can also be used. The definition of the alkyl grouping in "hydroxyalkyl starch" as used here includes methyl, ethyl, isopropyl and n-propyl, with ethyl being particularly preferred. A major advantage of the HES is that it is already officially approved as a biocompatible plasma expander and in clinically.

The average molecular weight of the hydroxyalkyl starch can range from about 3 kD to several million Daltons, preferably from about 4 kD to about 1000 kD, more preferably from about 4 kD to about 50 kD, or from about 70 kD to about 1000 kD preferably about 130 kD. For the coupling to small proteins, the average molecular weight of the hydroxyalkyl starch is preferably chosen so that the above-mentioned threshold of 70 kD for the conjugates is exceeded, while for the coupling to large proteins the molecular weight of the hydroxyalkyl starch will preferably be in the lower range of the spectrum mentioned , Since coupling at several sites of a protein is possible, it may also be advantageous to couple several small polymer chains instead of one high molecular one. The degree of substitution (ratio of the number of modified anhydroglucose units to the number of total anhydroglucose units) can also vary and will often range from about 0.2 to 0.8, preferably about 0.3 to 0.7, more preferably about 0.5 , (Note: The numbers refer to the "Degree of Substitution", which is between 0 and 1). The ratio of C ₂ to C ₆ substitution is usually in the range from 4 to 16, preferably in the range from 8 to 12.

These parameters can be set using known methods. Experience with the use of hydroxyethyl starch (HES) as a blood substitute has shown that the residence time of HES in plasma depends on the molecular weight and the Degree of substitution and type of substitution (C ₂ substitution or C ₆ substitution) depends, a higher molecular weight, a higher degree of substitution and a higher proportion of the C ₂ substitution increasing the residence time.

These relationships also apply to the hydroxyalkyl starch-protein conjugates according to the invention, so that the residence time of a specific conjugate in the plasma can be set via the polysaccharide portion.

Hydroxyethyl starch preparations with an average molecular weight of 130 kD and a degree of substitution of 0.5 or with an average molecular weight of 200 kD and a degree of substitution of 0.25 have already been used clinically as blood substitutes and are also suitable for use in the present invention ,

In principle, any protein which has the required functional group, e.g. has a free amino group, thiol group or carboxyl group for reaction with the functional group of the HAS molecule.

To introduce a desired functional group, the protein can also be reacted with a suitable, physiologically compatible, bifunctional linker molecule. The remaining reactive functional group of the coupled linker molecule is then also regarded as “reactive functional group of the protein” for the purposes of the present invention.

Suitable linker molecules contain at one end a group which can form a covalent bond with a reactive functional group of the protein, for example an amino, thiol or carboxyl group, and at the other end a group which is through with the terminal aldehyde group or one of them chemical reaction resulting functional group, for example a carboxyl group, activated carboxyl group, amino or thiol group, can enter into a covalent bond. Between the two functional groups of the linker molecule there is a more suitable biologically compatible bridge molecule Length, for example a group derived from an alkane, an (oligo) alkylene glycol group or another suitable oligomer group. Preferred groups which can react with amino groups are, for example, N-hydroxysuccinimide esters, sulfo-N-hydroxysuccinimide esters, imido esters and other activated carboxyl groups; preferred groups that can react with thiol groups are, for example, maleimide and carboxyl groups; preferred groups which can react with aldehyde or carboxyl groups are, for example, amino or thiol groups.

Exemplary linker molecules for linking SH and NH functions are:

AMAS (N-α (maleimidoacetoxy) succinimide ester)

BMPS (N-ß (Maleimidopropyloxy) succinimide ester)

GMBS (N-γ (Maleimidobutyryloxy) succinimide ester)

EMCS (N-ε (maleimidocaproyloxy) succinimide ester)

MBS (m- (maleimidobenzoyl) -N-hydroxysuccinimide ester)

SMCC (succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate)

SMPB (succinimidyl 4- (p-maleimidophenyl) butyrate)

SPDP (succinimidyl-3- (2-pyridyldithio) proprionate)

Sulfo-GMBS (N-γ (Maleimidobutyryloxy) sulfosuccinimide ester)

Sulfo-EMCS (N-ε (maleimidocaproyloxy) sulfosuccinimide ester).

Exemplary linker molecules for linking SH and SH functions are:

BMB (1.4-bis-maleimidobutane)

BMDB (1.4-bis-maleimido-2.3-dihydroxybutane)

BMH (bis-maleimidohexane)

BMOE (bis-maleimidoethane)

DTME (dithio-bis-maleimidoethane)

HBVS (1,6-hexane-bis-vinyl sulfone)

BM (PEO) ₃ (1.8-bis-maleimidotriethylene glycol)

BM (PEO) ₄ (1.11-bis-maleimidotetraethylene glycol).

Exemplary linker molecules for linking NH and NH functions are: BSOCOES (bis (2- (succinimidyloxycarbonyloxy) ethyl) sulfone BS ³ (bis (sulfosuccinimidyl) suberate)

DFDNB (1.5-difluoro-2.4-dinitrobenzene)

DMA (dimethyl adipimidate HCI))

DSG (disuccinimidyl glutarate)

DSS (disuccinimidyl suberate)

EGS (ethylene glycol bis (succinimidyl succinate)

Exemplary linker molecules for linking SH and CHO functions are:

BMPH (N- (ß-maleimidopropionic acid) hydrazide TFA)

EMCA (N- (ε-maleimidocaproic acid) hydrazide)

KMUH (N- (κ-maleimidoundecanoic acid) hydrazide)

M ₂ C ₂ H (4- (N-maleimidomethyl) cyclohexane-1-carboxylhydrazide HCl)

MPBH (4- (4-N-maleimidophenyl) butyric acid hydrazide HCl)

PDPH (3- (2-pyridyldithio) propionylhydrazide).

An exemplary linker molecule for linking SH and OH functions is PMPI (N- (p-maleimidophenyl) isocyanate).

Exemplary linker molecules for converting an SH function into a COOH

Function are:

BMPA (N-ß-maleimidopropionic acid)

EMCH (N-ß-maleimidocaproic acid)

KMUA (N-κ-maleimidoundecanoic acid).

Exemplary linker molecules for converting an NH function into a COOH function are MSA (methyl-N-succinimidyl adipate) or longer-chain homologs thereof or corresponding derivatives of ethylene glycol.

Exemplary linker molecules for converting a COOH function into an NH function are DAB (1,4-diaminobutane) or longer-chain homologs thereof or corresponding derivatives of ethylene glycol. ",

PCT / EP03 / 02083 9

An exemplary linker molecule that reacts with an amino group of a molecule and provides a protected amino group at a greater distance from this molecule to avoid steric hindrance is TFCS (N-ε (trifluoroacetylcaproyloxy) succinimide ester).

Other suitable linker molecules are known to those skilled in the art and are commercially available or can be designed as required and depending on the available and desired functional groups of the HAS and the protein to be coupled and can be prepared by known processes.

The term “protein” for the purposes of the present invention is intended to include any amino acid sequence which comprises at least 9-12 amino acids, preferably at least 15 amino acids, more preferably at least 25 amino acids, particularly preferably at least 50 amino acids, and also natural derivatives, for example pre- or pro Forms, glycoproteins, phosphoproteins, or synthetic modified derivatives, eg fusion proteins, neoglycoproteins, or proteins modified by genetic engineering processes, eg fusion proteins, proteins with amino acid exchanges for introducing preferred coupling sites.

For the prophylactic or therapeutic treatment of the human or animal body, the protein in question will perform a certain desired function in the body. The protein therefore preferably has e.g. a regulatory or catalytic function, a signaling or transport function or a function in the immune response or triggering an immune response.

The protein can, for example, be selected from the group consisting of enzymes, antibodies, antigens, transport proteins, bioadhesion proteins, hormones, growth factors, cytokines, receptors, suppressors, activators, inhibitors or a functional derivative or fragment thereof. “Functional derivative or fragment” in this context means a derivative or fragment that wholly or partly, for example, at least 10-30%, more preferably more than 50%, even more preferably more than 70%, of a desired biological property or activity of the parent molecule. , most preferably more than 90% ",

PCT / EP03 / 02083 10 has. Antibody fragments are particularly preferred examples of such a fragment.

Specific examples are α-, β- or γ-interferon, interleukins, e.g. IL-1 to IL-18, growth factors, e.g. epidermal growth factor (EGF), platelet growth factor (PDGF), fibroblast growth factor (FGF), brain-derived growth factor ( BDGF), nerve growth factor (NGF), B-cell growth factor (BCGF), brain-derived neurotrophic growth factor (BDNF), ciliary neurotrophic factor (CNTF), transforming growth factors, e.g. B. TGF-α or TGF-ß, colony-stimulating factors (CSF), for example GM-CSF, G-CSF, BMP ("bone morphogenic proteins"), growth hormones, for example human growth hormone, tumor necrosis factors, for example TNF-α or TNF-ß, somatostatin, somatotropin, somatomedine, serum proteins, e.g. the coagulation factors II-XIII, albumin, erythropoietin, myoglobin, hemoglobin, plasminogen activators, e.g. tissue plasminogen activator, hormones or prohormones, e.g. insulin, gonadotropin, stimulating hormone (melanocyte) melanocyte MSH), triptorelin, hypothalamic hormones, e.g. antidiuretic hormones (ADH) and oxytocin, as well as liberins and statins, parathyroid hormone, thyroid hormones, e.g. thyroxine, thyrotropin, thyroliberin, prolactin, calcitonin, glucagon, glucagon-like peptides (GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, GLP-1, etc. ), Exendins, e.g. exendin-4, leptin, vasopressin, gastrin, secretin, integrins, glycoprotein hormones (e.g. LH, FSH etc.), pigment hormones, lipoproteins and apo-lipoproteins, e.g. apo-B, apo-E, apo-L _a , Immunoglobulins, eg IgG, IgE, IgM, IgA, IgD or he a fragment thereof, hirudin, "tissue pathway" inhibitor, plant proteins, for example lectin or ricin, bee venom, snake venom, immunotoxins, antigen E, butroxobina, alpha-proteinase inhibitor, ragweed allergen, melanin, oligolysin proteins, RGD Proteins or, if appropriate, corresponding receptors for one of these proteins; or a functional derivative or fragment of one of these proteins or receptors.

Suitable enzymes can be selected, for example, from the groups of carbohydrate-specific enzymes, proteolytic enzymes, oxidases, oxidoreductases, transferases, hydrolases, lyases, isomerases, kinases and ligases. Specific, non-limiting examples are asparaginase, arginase, arginine deaminase, Adenosine deaminase, glutaminase, glutaminase-asparaginase, phenylalanine ammonium lyase, tryptophanase, tyrosinase, superoxide dismutase, an endotoxinase, a catalase, peroxidase, kallikrein, trypsin, chymotrypsin, elastase, thermolysin, a lipase, phosphinase, a uriclase, a uriclase Bilirubin oxidase, a glucose oxidase, glucodase, gluconate oxidase, galactosidase, glucocerebrosidase, glucuronidase, hyaluronidase, tissue factor, a tissue plasminogen activator, streptokinase, urokinase, MAP kinases, DNAsen, RNAsen, and functionally derived or lactoferrin,.

As mentioned above, the functional group of the HAS molecule involved in the coupling reaction is the terminal aldehyde group or a group resulting therefrom by chemical reaction.

An example of such a chemical reaction is the selective oxidation of this aldehyde group with a gentle oxidizing agent, e.g. iodine, bromine or some metal ions, or also by means of electrochemical oxidation to a carboxyl group or activated carboxyl group, e.g. an ester, lactone, amide, the carboxyl group optionally being converted into the activated derivative in a second reaction. This carboxyl group or activated carboxyl group can then be coupled to a primary amino or thiol group of the protein to form an amide bond or thioester bond.

In a particularly preferred production process, this aldehyde group is selectively oxidized with a molar excess of iodine, preferably in a molar ratio of iodine to HAS of 2: 1 to 20: 1, particularly preferably about 5: 1 to 6: 1, in aqueous basic solution. According to the optimized process described in Example 1, an amount of hydroxyalkyl starch is first dissolved in warm distilled water and a little less than 1 mol equivalent of aqueous iodine solution, preferably in a concentration of about 0.05-0.5 N, particularly preferably about 0.1 N , admitted. Thereafter, an aqueous NaOH solution in a molar concentration that is about 5-15 times, preferably about 10 times, the iodine solution is slowly added dropwise at intervals of several minutes to the reaction solution until the solution starts after the addition PT / EP03 / 02083

Be 12. A little less than 1 molar equivalent of the above aqueous iodine solution is again added to the reaction solution, the dropping of the NaOH solution is resumed and the addition of iodine and NaOH is repeated until about 5.5-6 molar equivalents of iodine solution and 11-12 molar equivalents of NaOH Solution, based on the hydroxyalkyl starch, have been added. Then the reaction is stopped, the reaction solution desalted, e.g. by dialysis or ultrafiltration, subjected to a cation exchange chromatography and the reaction product obtained by lyophilization. In this process, almost quantitative yields are achieved regardless of the molecular weight of the HAS.

In a further particularly preferred embodiment, the selective oxidation with alkaline stabilized solutions of metal ions, for example Cu ^** or Ag ⁺ , likewise takes place in approximately quantitative yield (example 2). An approximately 3-1 times molar excess of the oxidizing agent is preferably used.

The selectively oxidized hydroxyalkyl starch (ox-HAS) which is formed is then reacted with a free amino group of the desired protein in the presence of an activation reagent to form an amide bond. Suitable activation reagents are, for example, N-hydroxysuccinimide, N-hydroxyphthalimide, thiophenol, p-nitrophenol, o, p-dinitrophenol, trichlorophenol, trifluorophenol, pentachlorophenol, pentafluorophenol, 1-hydroxy-1 H-benzotriazole (HOBtNSA, HOOBt Hydroxypyridine, 3-hydroxypyridine, 3,4-dihydro-4-oxobenzotriazin-3-ol, 4-hydroxy-2,5-diphenyl-3 (2H) -thiophenone-1,1-dioxide, 3-phenyl-1- ( p-nitrophenyl) -2-pyrazoiin-5-one), [1-benzotriazolyl-N-oxy-tris (dimethylamino) phosphonium hexafluorophosphate] (BOP), [1-benzotriazolyloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), [O- (benzotriazole -1-yl) -N, N, N \ N etramethyluronium hexafluorophosphate (HBTU), [O- (benzotriazol-1-yl) -N, N, N \ N etramethyluronium tetrafluoroborate (TBTU), [O- (benzotriazole -1 -yl) -N, N, N \ -bis (pentamethylene) uronium hexafluorophosphate, [0- (benzotriazol-1-yl) -N, N, N \ N ^Λ -bis (tetramethylene) uronium hexafluorophosphate, carbonyldiimidazole (CDI), or preferably carbodiimides, e.g. B. 1 - (3-Dimethyaminopropyl) -3-ethylcarbodiimide (EDC), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPC), particularly preferably EDC. In contrast to the common methods described in the literature for similar coupling reactions PT / EP03 / 02083

13

it was surprisingly found that when using a carbodiimide, the use of otherwise mandatory other activators such as triazoles, e.g. B. HOBt, is not required or even the yields deteriorated. When ox-HES was coupled to various model compounds according to the invention in the presence of EDC and the absence of HOBt, on the other hand, high yields could be achieved largely independently of the molecular weight of the HES (see examples).

Instead of the reaction of the carboxyl group or activated carboxyl group with a free primary amino group of the protein (e.g. a lysine or arginine residue), an analogous reaction with a thiol group (a cysteine) of the protein is also possible in principle. However, it must be taken into account that cysteines are usually involved in S-S bridges and are therefore not available for a coupling reaction. However, if free cysteines are present, they often play an important role in catalysis or are involved in the contact point of subunits. Modification of these cysteines will then result in partial or complete loss of biological activity. To solve this problem, free cysteines could be removed using common genetic engineering methods such as directed mutagenesis or chemical peptide synthesis are introduced at those sites in the protein that are known to play no role in activity. In this way, optimal control of the coupling location is possible. In the same way, other reactive amino acids, e.g. Lys, His, Arg, Asp, Glu, are specifically introduced into the protein.

The reactive group of the hydroxyalkyl starch molecule can also be an amino or thiol group formed by chemical reaction of the terminal aldehyde group. For example, reductive amination of the aldehyde group can be carried out by reaction with ammonia in the presence of hydrogen and a catalyst or in the presence of sodium cyanoborohydride. The resulting amino or thiol group can then react with a free carboxyl group of the protein (for example an optionally activated glutamic or aspartic acid) to form an amide or thioester bond. Furthermore, the terminal aldehyde group of the hydroxyalkyl starch molecule or a functional group resulting therefrom by chemical reaction can also be reacted with a suitable physiologically compatible, bifunctional linker molecule. In this case, for the coupling reaction, the “functional group resulting from the terminal aldehyde group of the hydroxyalkyl starch molecule by chemical reaction” is the remaining reactive functional group of the bifunctional linker molecule with which the terminal aldehyde group or the functional group resulting therefrom has been reacted. In this way, the terminal aldehyde group can also be converted into a desired functional group.

Suitable linker molecules contain at one end a group which is linked to the terminal aldehyde group or a functional group resulting therefrom by chemical reaction, e.g. a carboxyl group, activated carboxyl group, amino or thiol group, can form a covalent bond, and at the other end a group which can be linked to a reactive functional group of the protein, e.g. an amino, thiol or carboxyl group, can form a covalent bond. Between the two functional groups of the linker molecule there is a biologically compatible bridge molecule of suitable length, e.g. a group derived from an alkane, an (oligo) alkylene glycol group or another suitable oligomer group. Preferred groups that can react with amino groups are e.g. N-hydroxy succinimide esters, sulfo-N-hydroxysuccinimide esters, imido esters or other activated carboxyl groups; preferred groups that can react with thiol groups are e.g. Maleimide and carboxyl groups; preferred groups that can react with aldehyde or carboxyl groups are e.g. Amino or thiol groups.

A number of specific, non-limiting examples of suitable linker molecules have already been given above with reference to the conjugation of linker molecules to the protein. In an alternative coupling method of the present invention, the terminal aldehyde group is reacted directly with a primary amino group (eg, a lysine or arginine residue or the N-terminus) of the protein to form a Schiff base. Subsequently or in parallel to this, the Schiff base formed is reduced by reaction with a suitable reducing agent, as a result of which a stable bond between protein and HAS is formed in the aqueous environment. Preferred reducing agents are sodium borohydride, sodium cyanoborohydride, organic boron complexes, for example a 4- (dimethylamino) pyridine boron complex, N-ethyldiisopropylamine boron complex, N-ethylmorpholine boron complex, N-methylmorpholine boron complex, N-phenylmorpholine boron complex, lut -Bor complex, triethylamine-boron complex, trimethylamine-boron complex; Suitable stereoselective reducing agents are, for example, sodium triacetate borohydride, sodium triethyl borohydride, sodium trimethoxy borohydride, potassium tri-sec-butyl borohydride (K-Selectride), sodium tri-sec-butyl borohydride (N-Selectride), lithium tri-sec-butyl borohydride (L-Selectride) , Potassium triamyl borohydride (KS-Selectride) and lithium triamyl borohydride (LS-Selectride).

The yields can be improved by suitable variation of the reaction conditions. The parameters for such optimization attempts are the pH of the reaction mixture (possible protein degradation by alkaline borohydride), the temperature and duration of the incubation, and the type of reducing agent for the one-pot reaction. Another alternative is the possibility of carrying out the reaction in two steps, wherein an immobilized reducing agent can be used for the reduction step.

The reaction products of the coupling can be examined using known methods and the coupling efficiency can be determined. For example, the free primary amino groups in the protein can be determined before and after coupling with trinitrobenzenesulfonic acid (Habeeb, ASAF, Anal. Biochem. 14, 328-336 (1966)). The coupling yield of reactions involving primary amino acids could also be determined by derivatizing the unreacted amines with fluorescamine and determining the fluorescence. The molecular weight distribution can be determined by SDS-PAGE and gel permeation. The protein content in the 0 02083

16

Conjugate can be detected by SDS-PAGE and subsequent silver staining, while the saccharide content can be determined by glycan-specific staining of the bands separated by SDS-PAGE after blotting on a membrane. A quantitative glycan determination is also possible. A precise identification of the coupling site on the protein is possible by peptide mapping and / or MALDI-TOF mass spectroscopy or electrospray ionization mass spectroscopy. In this way, the coupling can be optimized and the molecular weight distribution and possibly (e.g. if the reactive groups on the protein react differently) even the coupling point of the products can be predetermined.

The conjugates of the present invention can optionally be used as such or in the form of a pharmaceutical composition for the prophylactic or therapeutic treatment of the human or animal body.

Such compositions comprise a pharmaceutically effective amount of a conjugate according to the invention as an active ingredient as well as a pharmaceutically suitable carrier and optionally other therapeutic or galenic ingredients or auxiliaries. Auxiliaries can e.g. Include diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants) and substances which serve to make the formulation isotonic with the blood of the intended recipient. A pharmaceutically effective amount is that amount which is sufficient to have a desired positive effect when administered once or several times as part of a treatment for the relief, healing or prevention of a disease state. A pharmaceutically acceptable carrier is a carrier that is compatible with both the drug ingredient and the patient's body.

The form of the composition will vary depending on the desired or appropriate route of administration. A preferred route is parenteral administration, for example subcutaneous, intramuscular, intravenous, intraarterial, intra-articular, intra- 83

17 thecal, extradural injection or, if necessary, infusion. Intranasal, intratracheal or topical application is also possible. A topical application of growth factors conjugated according to the invention could accelerate wound healing, for example. The pharmaceutical compositions may conveniently be presented in unit dosage form and prepared by any of the methods well known in the pharmaceutical arts.

The conjugates of the present invention can also be used in all other fields in which other protein-polymer conjugates, e.g. B. PEG-protein conjugates were used. Some specific, non-limiting examples are the use of a HAS-protein conjugate as an immobilized catalyst or reaction partner for a reaction in a heterogeneous phase or as a column material for (immune) affinity chromatography. Other possible uses will be readily apparent to those skilled in the art, knowing the properties of the HAS-protein conjugates according to the invention disclosed here. The following examples are intended to explain the invention in more detail, but without restricting it thereto. In particular, analogous reactions can also be carried out with hydroxymethyl starch and hydroxypropyl starch and similar results can be achieved.

EXAMPLE 1

Selective oxidation of hydroxyethyl starch (HES) with iodine

In a round bottom flask, 10 g of HES-130 kD were dissolved in 12 ml of deionized water while heating. 2 ml of an I ₂ solution (0.1 N) were added to this solution. A pipette with 2 ml of 1.0 N NaOH was connected to the flask via a 2-way connector and the NaOH solution was added dropwise at about 1 drop per 4 minutes. After adding approximately 0.2 ml of the NaOH solution, the solution was decolorized, at which point a second portion of 2 ml of 0.1 N iodine solution was added. The reaction was complete after the addition of a total of 14 ml of iodine solution and 2.8 ml of NaOH solution. The reaction mixture was then dialyzed against deionized water. lactonization:

The partially desalted solution was subjected to chromatography on a cation exchange column (Amberiite IR-120, H ⁺ form) in order to convert the aldonate groups into aldonic acid groups. The water was then removed by lyophilization and the lactone form thus obtained. -

Determination of the degree of oxidation:

1 ml of alkaline copper reagent (3.5 g of Na ₂ P0 ₄ , 4.0 g of K-Na tatrat in 50 ml of H ₂ O, plus 10 ml of 1 N NaOH, 8.0 ml of 10% strength (wt ./Vol.) CuS0 ₄ solution and 0.089 g K-iodate in 10 ml H ₂ O, after adding 18 g Na sulfate make up to 100 ml). It is heated at 100 ° C for 45 minutes. After cooling, 0.2 ml of 2.5% Kl solution and 0.15 ml of 1 MH ₂ SO _{4 are} added. After 5 minutes, 1 drop of phenol red indicator solution (1% by volume) is added and the mixture is titrated with 5 mM Na ₂ S ₂ 0 ₃ solution until the color disappears. The concentration of unreacted aldehyde groups can be calculated from the consumption of titrant.

An almost quantitative yield was achieved (> 98%). Hydroxyethyl starches with a higher molar mass (e.g. 130 kD, 250 kD, 400 kD), as well as hydroxyethyl starches with a lower molar mass (e.g. 10 kD, 25 kD, 40 kD), could be oxidized in similarly high yields according to this specification.

EXAMPLE 2

Selective oxidation of HES with Cu ²⁺ ions

A solution of 0.24 mmol of HES-130 kD in 10 ml of deionized water was prepared with heating. This solution was heated in a 100 ml round-bottom flask to a temperature of 70-80 ° C., and 1.17 mmol of stabilized Cu ²⁺ (eg Rochelle salt as stabilizer or other stabilizers) and aqueous dilute NaOH solution were added (final concentration 0.1 N NaOH). The temperature was then raised to 100 ° C. and the reaction was allowed to continue until a reddish color had developed. The reaction was stopped and the reaction mixture was cooled to 4 ° C. The reddish precipitate was removed by filtration. The filtrate was against T EP03 / 02083

19 deionized water dialyzed and then converted into the lactone as in Example 1 and lyophilized. The oxidation was quantitative (yield> 99%). Using this method, HES (e.g. HES-10 kD, HES-25 kD, HES-40 kD) and higher molecular HES species are also oxidized.

EXAMPLE 3

Coupling of selectively oxidized high molecular weight HES (ox-HES-130 kD) to human serum albumin (HSA)

4.3 g of ox-HES-130 kD and 200 mg of HSA (Sigma, Taufkirchen) were completely dissolved in water in a round-bottomed flask with a magnetic stirrer while heating gently. 30 mg of ethyldimethylaminopropylcarbodiimide (EDC), dissolved in water, were added to this solution. After 2 hours with very moderate stirring, a second portion of 30 mg EDC was added. After a further two hours with very moderate stirring, a third portion of 40 mg of the carbodiimide was added. The reaction mixture was left under these conditions overnight, dialyzed against distilled water for 15 hours and lyophilized. The success of the coupling was demonstrated by gel permeation chromatography, SDS-PAGE and carbohydrate-specific staining (Glyco-Dig-Kit from Roche-Boehringer, Basel) after blotting on a PVDF membrane. The yield of the coupling product was approximately 90%.

EXAMPLE 4

Coupling of selectively oxidized low molecular weight HES (ox-HES-10 kD) to human serum albumin (HSA)

7.4 g of ox-HES-10 kD and 50 mg of HSA were completely dissolved in water in a round-bottom flask with a magnetic stirrer. The reaction was carried out according to the procedure described for high molecular weight HES, with a total of 282 mg EDC being added in three aliquots. The reaction mixture was also dialyzed and lyophilized as described above. Analysis (as above) showed that the coupling product was obtained, but the yields were somewhat lower than when coupling with high molecular weight ox-HES. EXAMPLE 5

Coupling ox-HES-130 kD to Myoglobin (Mb)

4.3 g of ox-HES-130 kD were completely dissolved in water (6-7 ml) and then 100 mg Mb (Sigma, Taufkirchen), dissolved in 10 ml of 0.1 M phosphate buffer (pH 7.0), were added. The coupling reaction was started by adding 30 mg EDC. The addition of EDC was repeated every 2 hours until a total of 90 mg of the carbodiimide was consumed. The reaction mixture was then dialyzed against 50 mM phosphate buffer, pH 7.0 and lyophilized. The GPC showed a clear product peak, which was detected in the exclusion volume at 450 nm. From this a coupling yield of 88% could be calculated. The oxygen binding capacity of the hesylated myoglobin was approximately 76% of the binding capacity of the unmodified Mb.

EXAMPLE 6

Coupling of ox-HES-10 kD to superoxide dismutase (SOD)

A volume of an aqueous solution of ox-HES-10 kD (1.05 g / ml) was incubated with a volume of a 7 mg / ml SOD solution (Sigma, Taufkirchen) in 50 mM phosphate buffer, pH 7.6, at room temperature. The coupling reaction was initiated by adding 280 mg EDC in 5 portions over a period of 24 h. The course of the reaction was followed by GPC analysis in phosphate buffer and detection at 280 nm. After 24 h, 81% of the protein was found in the high molecular weight region of the separation column and the reaction was stopped after this time. The reaction mixture was subjected to diafiltration with a 30 kD membrane and then lyophilized. Mass spectrometric analysis of the product showed on average a molar ratio of HES to protein of approx. 3: 1.

EXAMPLE 7

Coupling ox-HES-130 kD to Streptokinase (SK)

3.8 g of ox-HES-130 kD were dissolved together with 35 mg of streptokinase (Sigma, Taufkirchen) in as little as 50 mM phosphate buffer, pH 7.2. At room temperature, 46.5 mg EDC and 20 mg 1-hydroxybenzotriazole hydrate (HOBt) added and the reaction held for a total of 24 h with gentle stirring. After dialysis and freeze-drying, approximately 78% of the protein was found as HES conjugate according to GPC analysis. In the SDS-PAGE, a marked increase in the molar mass of streptokinase was observed with silver staining. At the same time, carbohydrate structures in the high-molecular band were clearly demonstrated using the digoxigenin method.

EXAMPLE 8

Coupling ox-HES-130 kD to Human Interleukin-2 (IL-2)

45 mg ox-HES-130 kD were completely dissolved in 0.5 ml 50 mM Na phosphate buffer, pH 6.5, with gentle warming. After adding 0.25 mg of human IL-2 (Sigma, Taufkirchen), which made the solution opaque, the mixture was stirred at room temperature for 4-6 h. 5 mg EDC were then added in 4 portions, each with a time difference of 2 h, and stirring was continued overnight, resulting in a clear solution. Analysis with GPC showed approx. 65% coupling yield. ^l

EXAMPLE 9

Coupling ox-HES-25 kD to Human Tumor Necrosis Factor (TNFα)

0.3 mg of hTNFα (Sigma, Taufkirchen) was added to 86 mg of ox-HES-25 kD in approx. 0.4 ml of 0.1 M phosphate buffer (pH 7.0). The cloudy solution was stirred for about 2 h before 1 mg EDC and 0.5 mg HOBt were added. The stirring was continued for about 6 h, during which the solution became clear in the course of the reaction time. The coupling product was isolated by ultrafiltration and freeze drying and analyzed by GPC and detection at 280 nm. A coupling yield of approximately 74% was found.

EXAMPLE 10

Coupling ox-HES-130 kD to "Glucagon-like Peptide" (GLP-1)

7.4 g of ox-HES-130 kD were dissolved in the smallest possible volume of water with heating and gentle stirring. For this purpose, a solution of 10 mg GLP-1 in the amide form (Bachern, Switzerland) in 50 mM phosphate buffer, pH 7.4, was pipetted. The reaction was started by adding 35 mg EDC and carefully stirred for 2 h. This was repeated twice, since after this time no peptide peak could be recognized in the GPC analysis at 280 nm, ie an almost complete conversion to the coupling product had taken place. This coupling product was diafiltered with a 30 kD membrane and lyophilized from phosphate buffer solution. A 1: 1 stoichiometry between peptide and HES was concluded from the results of a MALDI mass spectroscopy.

Example 11

Coupling of high molecular weight HES (HES-130 kD) to human serum albumin (HSA)

9.75 g of HES-130 kD were completely dissolved in water (6-7 ml) and then 50 mg of HSA, dissolved in 1 ml of 0.1 M phosphate buffer (pH 7.4), were added. The reaction mixture was stirred with a magnetic stirrer. The solution was then mixed with NaBH ₃ CN (50-70 mg) and stirred gently for a few minutes. The solution was further stirred every two hours for 15 minutes. Another aliquot of NaBH ₃ CN (approximately 50 mg) was then added. At the end (after a reaction time of almost 36 h), a total of 285 mg NaBH ₃ CN had been used. The solution was then dialyzed and lyophilized. The analysis was carried out as described in Example 4. The coupling efficiency was about 65%.

Example 12

Coupling of low molecular weight HES (HES-10 kD) to human serum albumin (HSA)

4.5 g of HES were completely dissolved in water (4-5 ml) and then 50 mg of HSA, dissolved in 1 ml of 0.1 M phosphate buffer (pH 7.4), were added. When the solution was clear, if necessary by stirring with a magnetic stirrer, NaBH ₄ (50-70 mg) was added and mixed in with gentle stirring. The solution was left without stirring for two hours and then stirred every two hours for 15 minutes as in the reaction with high molecular weight HES. When the solution no longer showed bubbles (H ₂ evolution), another aliquot of NaBH ₄ (about 50 mg) was added. In the end there was a total of 180 mg NaBH ₄ been used. The solution was then dialyzed and lyophilized. The analysis was carried out by means of gel permeation chromatography (GPC), the yield was about 15%.

EXAMPLE 13

Coupling of HES-40 kD to asparaginase

3.0 g of HES-40 kD were completely dissolved in water (approx. 4 ml). A solution of 80 mg of asparaginase (Sigma, Taufkirchen) in 6 ml of 0.1 M borate buffer, pH 9.0, was added and the mixture was stirred until the reaction mixture was clear. The temperature was then raised to 37 ° C. and, after 2 h, about 50 mg of NaBH ₃ CN were added. This reaction cycle was repeated 3 times. To work up the product, the reaction mixture was dialyzed against 0.1 M phosphate buffer, pH 7.4. The yield of the coupling product was approx. 61%, with approx. 73% of the asparaginase activity being recovered.

EXAMPLE 14

Coupling of HES-130 kD to Human Interleukin-2 (IL-2)

50 mg HES-130 kD were completely dissolved in water (approx. 0.2 ml). A suspension of 0.25 mg human IL-2 (Sigma, Taufkirchen) in 0.2 ml 0.1 M borate buffer, pH 9.0, was added and the mixture was stirred until the reaction mixture was clear (4 h). 1 mg NaBH ₃ CN were added at intervals of 4 h and stirring was continued. After a further 24 h reaction time, dialysis was carried out against 0.1 M phosphate buffer, pH 7.4 and lyophilization. The yield of the coupling product was approximately 42% after analysis by GPC.

EXAMPLE 15

Coupling of HES-130 kD to insulin

4.0 g of HES-130 kD were completely dissolved in water (approx. 6 ml). 55 mg of insulin from bovine pancreas (Sigma, Taufkirchen) in 7.5 ml of 0.1 M borate buffer, pH 9.0, were added and the mixture was stirred at 37 ° C. for about 24 h. The reducing agent NaBH ₃ CN (60 mg in 30 ml) was slowly added dropwise over a period of 8 h. The mixture was then stirred for a further 24 h and the reaction mixture by ultrafiltration (30th kD) freed from salts and unreacted reagents. A stable coupling product was obtained by lyophilization. Approximately 55% of the insulin used was recovered as an HES conjugate.

EXAMPLE 16

Coupling of ox-HES-130 kD to superoxide dismutase (SOD)

130 mg ox-HES-130 kD were completely dissolved in 6 ml PBS pH 6 and then 10 mg SOD (Röche, Mannheim) dissolved in 1 ml PBS pH 6 were added. The coupling reaction was started by adding 10 mg EDC. The addition of EDC was repeated every 3 hours until 39 mg of the carbodiimide had been consumed. The reaction was checked by means of GPC at 258 nm. After 24 h, 50% of the protein was found in the high-molecular region of the separation column and the reaction was stopped. The reaction mixture was dialyzed against 25 mM phosphate buffer pH 7.2 and lyophilized. The SOD activity was 95% of the initial activity. The determination of the mass distribution of HES protein samples by means of GPC light scattering coupling resulted in a molar ratio of HES to protein of 1: 1.

EXAMPLE 17

Coupling of ox-HES 70 kD to glucagon

Glucagon (66 x 10 ^"9 mol, 0.23 mg), oxHES 70 kD (6.6 x 10 ^{" 6} mol, 123 mg) were dissolved in phosphate buffer (1 ml, pH 5) in a round bottom flask. 26 mg EDC were added in 10 portions at intervals of 1 h. After a reaction time of 24 h, the reaction was stopped by adding 10 ml of water. The coupling product was purified by after dialysis against water by GPC and ion exchange chromatography. After freeze-drying, 88 mg of white coupling product (73%) were obtained.

Claims

1. Hydroxyalkyl starch-protein conjugate, characterized in that the binding interaction between the hydroxyalkyl starch molecule and the protein is based on a covalent bond which is the result of a coupling reaction between (i) the terminal aldehyde group or a functional group resulting from this aldehyde group by chemical reaction of the hydroxyalkyl starch molecule and (ii) a functional group of the protein which is reactive with this aldehyde group or from the functional group of the hydroxyalkyl starch molecule which results therefrom, it being possible for the bond resulting directly from the coupling reaction to be modified, if appropriate, by a further reaction to form the covalent bond mentioned above. , Hydroxyalkyl starch-protein conjugate according to claim 1, characterized in that the functional group resulting from the terminal aldehyde group of the hydroxyalkyl starch molecule by chemical reaction is one of the functional groups of a bifunctional linker molecule with which the terminal aldehyde group has been reacted. , Hydroxyalkyl starch-protein conjugate according to claim 1 or 2, characterized in that the reactive functional group of the protein is one of the functional groups of a bifunctional linker molecule which has been coupled to the protein. , Hydroxyalkyl starch-protein conjugate according to claim 1 or 2, characterized in that the reactive functional group of the protein was introduced into the protein by recombinant modification of the original amino acid sequence. , Hydroxyalkyl starch-protein conjugate according to claim 1, 3 or 4, characterized in that the covalent bond is the result of a coupling reaction between one by selective oxidation of the terminal Aldehyde group formed carboxyl group or activated carboxyl group of the hydroxyalkyl starch molecule and a primary amino group or thiol group of the protein.

Conjugate according to claim 5, characterized in that the covalent bond is an amide bond which is the result of a coupling reaction between an activated carboxyl group formed by selective oxidation of the terminal aldehyde group of the hydroxyalkyl starch molecule and a primary amino group of the protein.

Conjugate according to claim 1, 3 or 4, characterized in that the covalent bond is an amine bond which is the result of a coupling reaction between the terminal aldehyde group of the hydroxyalkyl starch molecule and a primary amino group of the protein to form a Schiff base and reduce the Schiff ^'s Base to the amine.

Conjugate according to one of claims 1 to 7, characterized in that the hydroxyalkyl starch molecule has a molecular weight in the range from about 4 to about 1000 kD.

Conjugate according to claim 8, characterized in that the hydroxyalkyl starch molecule has a molecular weight of about 4 to about 50 kD.

Conjugate according to claim 8, characterized in that the hydroxyalkyl starch molecule has a molecular weight of about 70 to about 1000 kD.

Conjugate according to claim 10, characterized in that the hydroxyalkyl starch molecule has a molecular weight of about 130 kD.

Conjugate according to one of claims 1 to 11, characterized in that the hydroxyalkyl starch molecule has a degree of substitution of about 0.3 to about 0.7. Conjugate according to one of claims 1 to 12, characterized in that the hydroxyalkyl starch molecule has a C ₂ to C ₆ substitution ratio of 8 to 12.

Conjugate according to one of claims 1 to 13, characterized in that the hydroxyalkyl starch molecule is a hydroxyethyl starch molecule.

Conjugate according to one of claims 1 to 14, characterized in that the protein has a regulatory or catalytic function, a signaling or transport function or a function in the immune response or triggering an immune response.

Conjugate according to claim 15, characterized in that the protein from the group consisting of enzymes, antibodies, antigens, transport proteins, bioadhesion proteins, hormones and prohormones, growth factors and growth factor receptors, cytokines, receptors, suppressors, activators, inhibitors or a functional derivative or fragment of which is selected.

Conjugate according to claim 15 or 16, characterized in that the protein α-, ß- or γ-interferon, an interieukin, a serum protein, eg albumin or a coagulation factor, erythropoietin, myoglobin, hemoglobin, a plasminogen activator, BCGF, BDGF, EGF, FGF, NGF, PDGF, BDNF, CNTF, TGF-α, TGF-ß, a colony stimulating factor, a BMP, somatomedin, somatotropin, somatostatin, insulin, gonadotropin, α-MSH, triptorelin, prolactin, calcitonin, glucagon Glucagon-like peptide, for example GLP-1 or GLP-2, exendin, leptin, gastrin, secretin, an integrin, a hypothalamus hormone, for example an ADH, oxytocin, a liberin or statin, a thyroid hormone, for example thyroxine, thyrotropin, thyroliberine , a growth hormone, for example human growth hormone, LH, FSH, a pigment hormone, TNF-α or TNF-ß, hirudin, a lipoprotein or apo-lipoprotein, for example Apo-B, Apo-E, Apo-L _a , an oligolysin Protein, an RGD protein, a lectin or ricin, bee venom or a snake venom, an immunotoxin, Ragwe ed allergen Antigen E, an immunoglobulin, or a receptor for one of these proteins or a functional derivative or fragment of one of these proteins or receptors.

Conjugate according to claim 15 or 16, characterized in that the protein is an enzyme which consists of an asparaginase, arginase, arginine deaminase, adenosine deaminase, glutaminase, glutaminase aspartinase, phenylalanine ammonium lyase, tryptophanase, tyrosinase, superoxide dismutase, endotoxinase, catalase Trypsin, chymotrypsin, elastase, thermolysin, a lipase, uricase, adenosine diphosphatase, purine nucleoside phosphorylase, bilirubin oxidase, glucose oxidase, glucodase, gluconate oxidase, galactosidase, glucocerebrosidase, glucuronidodinase, hyaluronidokinase, hyaluronidase kinase, a -Kinase, DNAse, RNAse, lactoferrin, and functional derivatives or fragments thereof is selected.

A pharmaceutical composition comprising an effective amount of a conjugate according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier and optionally other excipients and active ingredients.

Use of a conjugate according to one of claims 1 to 18 or a composition according to claim 19 for the therapeutic or preventive treatment of humans or animals.

Process for the preparation of a hydroxyalkylstarch-protein conjugate according to one of claims 1 to 18, characterized in that in aqueous solution a coupling reaction between the terminal aldehyde group or a functional group of the hydroxyalkylstarch molecule resulting from this aldehyde group by chemical reaction and with or with this aldehyde group resultant functional group of the hydroxyalkyl starch molecule reactive functional group of the protein is carried out and that in the coupling reaction immediately resulting binding is optionally modified by a further reaction.

A method according to claim 21, characterized in that the reaction medium of the coupling reaction is water or a mixture of water and an organic solvent, the water content of the mixture being at least 80%.

Process according to claim 21 or 22, characterized in that the terminal aldehyde group of the hydroxyalkyl starch molecule is converted into the corresponding carboxyl functionality by selective oxidation and this is then reacted under activation conditions in aqueous solution with a free amino group of the protein, so that the hydroxyalkyl starch molecule is bound to the by an amide bond Protein is bound.

Process according to claim 23, characterized in that the selective oxidation of the aldehyde group with iodine or metal ions is carried out in aqueous basic solution.

A method according to claim 23 or 24, characterized in that the coupling reaction is carried out in the presence of a carbodiimide.

A method according to claim 25, characterized in that the carbodiimide is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC).

A method according to claim 21 or 22, characterized in that the terminal aldehyde group of the hydroxyalkyl starch molecule is coupled to a free amino group of the protein to form a Schiff base and the Schiff base formed is reduced to the amine, so that the hydroxyalkyl starch molecule is bonded to the amine Protein is bound. A method according to claim 27, characterized in that both coupling and reduction take place in aqueous solution.

A method according to claims 27 or 28, characterized in that the reducing agent is sodium borohydride, sodium cyanoborohydride or an organic boron complex.

Method according to one of claims 27 to 29, characterized in that the coupling and reduction reactions are carried out simultaneously.

Process for the preparation of hydroxyalkyl starch selectively oxidized at the terminal aldehyde group, characterized in that the hydroxyalkyl starch is reacted in a molar ratio of iodine to HAS of 2: 1 to 20: 1 in aqueous basic solution.

A method according to claim 31, characterized in that the molar ratio of iodine to HAS is about 5: 1 to 6: 1.

A method according to claim 31, characterized in that a) an amount of hydroxyalkyl starch is dissolved in warm distilled water and a little less than 1 mol equivalent of aqueous iodine solution is added, b) NaOH solution in a molar concentration which is about 5-15 times the iodine solution is slowly added dropwise to the reaction solution at intervals of several minutes until after the addition the solution begins to become clear again, c) a little less than 1 mol equivalent of aqueous iodine solution is again added to the reaction solution, d) the NaOH solution is added dropwise is resumed, e) steps b) to d) are repeated until about 5.5-6 mol equivalents of iodine solution and 11-12 mol equivalents of NaOH solution, based on the hydroxyalkyl starch, have been added, f) the reaction is then stopped and the reaction solution is desalted and subjected to cation exchange chromatography and the reaction product is obtained by lyophilization.

A method according to claim 33, characterized in that the aqueous iodine solution is an approximately 0.05-0.5 N iodine solution.

A method according to claim 33 or 34, characterized in that the molar concentration of the NaOH solution is about 10 times the iodine solution.

Process for the preparation of hydroxyalkyl starch selectively oxidized at the terminal aldehyde group, characterized in that the HAS is oxidized in aqueous alkaline solution with a molar excess of stabilized metal ions selected from Cu ²⁺ ions and Ag ⁺ ions.