DE29820466U1 - Recombinant glycoproteins and medicinal products containing them - Google Patents

Description

MUNICH RUSCHKE"HA^TMANJJI..B*ECril:eR Patent Attorneys

Pienzenauer-Str. 2 MMWrHPN Rppi im ** European Patent Attorneys

D-61679 Munich MUINUMtN-BtKLIN _{Dr|ng Hans Ruscnke 1832} . ₁₉₈₀

Tel.:+49- 89 9829 0272 Law firm . Dipl.-Ing. Hans E. Ruschke

Fax:+49- 89 9829 0273 Dipl.-Chem. Dr. G. Hartmann [+49-89 989 358]

PA DR. G. HARTMANN Pienzenauerslr. 2 D-81679 Munich Attorneys at Law

Horst Ch. Becker, DEA Anja Roxin *

Consultant

European Patent Attorney British Chartered Patent Agent Paul Madgwick B.Sc.

Office Berlin* Kurfürstendamm 125 A 298 20 466.5 D-10711 Berlin

H 1278a-Gbm 25.02.99

Recombinant glycoproteins and their containing drug

The invention relates to new recombinant glycoproteins, which act as messenger substances, signaling substances, promoters, stimulators and initiators in a variety of ways in the animal, especially human, circulation; as well as pharmaceutical agents containing them. The invention relates in particular to new recombinant human glycoproteins (rh glycoproteins), preferably new rh differentiation factors, in particular rh erythropoietin; new rh growth factors, in particular rh CSF (colony stimulating factor), rh GMCSF (granulocyte-monocyte stimulating factor); new rh thrombolytics, in particular rh tPA (tissue plasminogen activator) and rh urokinase; new rh thromboprophylactics, in particular rh antithrombin III; new rh coagulation factors, in particular rh factor 5 VIII and rh factor IX; new rh interferons, in particular

e-mail: Gunter.Hartmann@emails.de

KtoVAcc. PA Dr. G. Hartmann - Postbank Munich (bank code 700 100 80) Kto. Ko. 224162-808 - VAT No. DE 130 393 827

rh α-, ß- and γ-interferons; and new rh interleukins, in particular rh IL-2, IL-15, IL-16 and IL-17; processes for their preparation; pharmaceutical compositions containing them and their uses.
5

Glycoproteins are proteins that contain covalently bound carbohydrates (sugars). Glycoproteins, especially human glycoproteins, act as messengers, signaling substances, promoters, initiators or stimulators of metabolic processes in animals and humans, which directly or indirectly intervene in these metabolic processes. They are usually formed in the body in vivo or biosynthetically and, after they have fulfilled their specific function, are excreted or broken down by the body. The glycoproteins that are formed naturally in the body (so-called "native glycoproteins") are characterized by the fact that they are substituted by an acetyl group on the amino group of the aminoglycan residue. These native glycoproteins, which can be produced naturally in vivo or by biosynthetic means (cf. DE-PS 4 311 580), are effective agents in humans and animals for stimulating the growth and differentiation of human and animal cells of the immune system and for preventing the adhesion of leukocytes, thrombocytes and tumor cells to vascular endothelial cells; for stimulating the immune system, in particular T lymphocytes, for fighting infections, for treating immune deficiencies, tumor diseases including metastasis processes, infectious diseases and circulatory failure, in particular vascular occlusions and septicemia; for inhibiting the binding of a ligand to its sialylated cell surface receptor; for inhibiting the binding of a microbial phage or toxin to the host cell via a sialylated receptor by in vivo modulation of neuraminic acids; for the biosynthetic production of ligands or receptors with modified neuraminic acid and use of the same for competition

physiological or pathological ligand-receptor interactions; to influence the course of infection with human immunodeficiency viruses in vitro and to prevent infection with human immunodeficiency viruses in vivo ; and to treat parasitic diseases.

In animal organisms, glycoproteins occur as essential components of cell membranes and as soluble components of body fluids and the extracellular matrix. The carbohydrates are linked to form chains (oligosaccharides) and can be linked to the protein structure in different ways. They contain sialic acid (derivatives of 2-keto-3-deoxy-D-glycero-D-galacto-nonulopyranosidonic acid (KDN)) as an important component of the cell membrane, which plays an important role in biological processes.

Depending on their bond type, oligosaccharides are assigned to different groups. The oligosaccharides of acid thioglycoproteins are most often N-glycosidically bound to an asparagine residue of the polypeptide chain (N-glycans). This group includes secreted glycoproteins with different functions, e.g. soluble enzymes, immunoglobulins and hormones, and membrane glycoproteins, e.g. membrane enzymes, transport proteins and receptor proteins. Another group is made up of oligosaccharides that are O-glycosidically bound to a serine or threonine residue of the polypeptide chain via a galatose, N-acetylgalactosamine or xylose residue (O-0 glycans). Together with N-glycans, they also occur in immunoglobulins and other glycoproteins. The O-glycans also include the oligosaccharides of the proteoglycans, which are characterized by a particularly high carbohydrate content. In these glycoconjugates, which occur in the extracellular matrix, the oligosaccharides can be bound to the polypeptide structure via a galactose, N-acetylgalactosamine or xylose residue.

^S ..:X

The glycoproteins, consisting of monosaccharides and protein, are often grouped together with the glycolipids as glycoconjugates. The sugar components of the glycoproteins, which with a few exceptions make up less than 50% of the total glycoprotein, are linked to the peptide portion via O- or N-glycosidic bonds that can be cleaved by glycosidases. The carbohydrates found in the glycoproteins are hexoses (galactose, mannose, less frequently glucose), N-acetylhexosamine, N-acetylneuraminic acid, fucose and others. Affinity chromatography with plant lectins as ligands (e.g. concanavalin A or wheat germ agglutinin or others) is primarily suitable for identifying and determining the glycoproteins.

Glycoproteins include almost all membrane glycoproteins, serum proteins, plasma proteins, blood group substances, many enzymes and proteohormones, all antibodies, chalones, mucins, lectins, bindins, fibronectin, intrinsic factor, etc. As membrane or surface proteins, some glycoproteins play a role in the pathogenicity of viruses. Here, as in other receptor-specific cellular interactions, the carbohydrate components are responsible for recognition processes at the molecular level. Some bacteria and viruses attach to their target cells via certain sugar structures on receptors.

Oligosaccharide structures are particularly important with regard to cell-cell and cell-matrix interactions. The oligosaccharides of glycoproteins mediate the adhesion of neural cells and the binding of lymphocytes to specific endothelial cells. In addition, oligosaccharides can serve as antigenic determinants of glycoproteins. Carbohydrate-carbohydrate interactions are also important during embryogenesis and organogenesis.

&Iacgr;&iacgr;-V ! :

genes play a key role in specific cell recognition. The malignant transformation of cells is accompanied by characteristic changes in the oligosaccharide structures of glycoproteins. The extent to which altered oligosaccharide structures of tumor cells and cell glycoproteins are the cause or effect of tumor formation and metastasis has not yet been clearly clarified.

In cases of illness in which the immune system is involved, it is necessary to support the immune system by administering substances that stimulate cells of the immune system. The search for active substances to stimulate the immune system is therefore a preferred goal of pharmacological research. However, effective immune stimulants with few or no side effects are not yet known.

The aim of the invention was therefore to find new substances with the help of which it is possible to influence the metabolic processes controlled by glycoproteins as messengers, signaling substances, promoters, stimulators and initiators even more effectively, specifically and selectively than before. The aim of the invention was in particular to find new glycoproteins as messengers, signaling substances, stimulators, promoters and initiators for human and animal metabolism that can be used in lower doses and therefore have fewer undesirable side effects than the corresponding native substances.

It has now been found that this object can be achieved according to the invention by replacing the naturally occurring acetic acid substitution in the aminoglycan residue in the above-mentioned native glycoproteins by another acyl group, in particular by a (C ₃ -C ₇ )-acyl group, or a hydrocarbyl residue, in particular a (C ₃ -C ₇ )-

Alkyl, alkenyl or alkynyl residue. It has been shown that the new compounds obtained in this way have changed, in particular prolonged, pharmacokinetics compared to the biological half-life (half-life) of the corresponding known and commercially available native glycoproteins with an N-acetyl group in the aminoglycan residue. These new glycoproteins can be produced in the manner described below, in particular by genetic engineering, whereby recombinant glycoproteins, in particular recombinant human glycoproteins (rh glycoproteins), are obtained.

Due to their modified, particularly prolonged, pharmacokinetics, these substances can be used therapeutically in much lower doses, which can also significantly reduce the undesirable side effects previously observed when using the corresponding native substances. This is particularly true when the new glycoproteins are used in the form of their derivatives in which the OH groups of the monosaccharide portion are singly or multiply acylated, preferably acetylated.

According to a first aspect, the present invention relates to new recombinant glycoproteins, in particular recombinant human glycoproteins (rh glycoproteins) of the general formula (I)

0 '^_yN COOH

where:

R ¹ I is a linear or cyclic, unbranched or branched, optionally mono- or polyhydroxylated and/or ketylated (C3-C7) acyl radical, in particular (C3-C7) alkanoyl radical, or a linear or cyclic, unbranched or branched, optionally mono- or polyhydroxylated and/or ketylated (C3-C7) hydrocarbyl radical, in particular (C3-C7) alkyl, alkenyl or alkynyl radical,

R ₂ , R3/ R4 and R5, which may be the same or different, each represent hydrogen, a linear or branched alkyl radical having 1 to 20 carbon atoms (C _n H _2n+2 , η = 1 to 20); a linear or branched alkenyl radical having 3 to 20 carbon atoms (C _n H _2n * η = 3 to 20, position of the double bond on C _n η = 2 to 19); a linear or branched alkynyl radical having 3 to 20 carbon atoms (C _n H _2n _2, η = 3 to 20, position of the triple bond on C _n η = 2 to 19); an alkenyl or alkynyl radical having 2 or more double bonds or triple bonds with 4 to 20 carbon atoms; an aryl radical with 6 to 20 carbon atoms; a linear or branched, saturated or mono- or polyunsaturated acyl radical (-CO-Ri) ^m ^ a total of 1 to 20 carbon atoms including its mono- or polyhydroxylated analogues; an aroyl radical with 6 to 2 0 0 carbon atoms; a carbonylamide radical of the formula -CONH ₂ or -CONHRi; ^a linear ^or branched, saturated or mono- or polyunsaturated thioacyl radical (-CS-Ri) ^m ^ a total of 1 to 2 0 carbon atoms; or a thiocarbamide radical of the formula . CS-NH ₂ or'-CS-NHRi; where R ₁ is in each case hydrogen or a linear or branched, saturated or mono- or polyunsaturated alkyl radical

with 1 to 20 carbon atoms and each of the abovementioned radicals except H may optionally be mono- or polysubstituted by halogen, hydroxy, epoxy, amino, mercaptan, phenyl, phenol or benzyl groups, and

T is a mono-, di- or oligosaccharide residue with up to " 4 0 glycosidically linked, optionally branched sugar residues, which represent furanose and/or pyranose rings and contain 5 to 23 0 carbon atoms and are N- or O-glycosidically bound to polypeptides.

Preferably, in the above general formula (I):

R ₂ , R3, R4 and R5, which may be the same or different, each represent hydrogen, a linear or branched alkyl radical having 1 to 7 carbon atoms; a linear or branched alkenyl radical having 3 to 10 carbon atoms; a linear or branched alkynyl radical having 3 to 10 carbon atoms; an alkenyl or alkynyl radical having 2 or more double bonds or triple bonds having 7 to 12 carbon atoms; a phenyl radical; a linear or branched, saturated or mono- or polyunsaturated acyl radical (-CO-R1) having a total of 1 to 7 carbon atoms, in particular acetyl radical, including its mono- or polyhydroxylated analogues; an aroyl radical having 6 to 10 carbon atoms; a carbonylamide radical of the formula -CONH ₂ or CONHR^; a linear or branched, saturated or mono- or polyunsaturated thioacyl radical (-CS-Ri) having a total of 1 to 7 carbon atoms; or 5 a thiocarbamide radical of the formula -CS-NH ₂ or -CS-NHRi; where Ri is in each case hydrogen or a linear or branched, saturated or mono- or

polyunsaturated alkyl radical having 1 to 20 carbon atoms and each of the aforementioned radicals except H can optionally be mono- or polysubstituted by fluorine, chlorine, bromine or iodine, hydroxy, epoxy, amino, mercaptan, phenyl, phenol or benzyl groups.

According to a further preferred embodiment of the invention, in the general formula (I) T 10

for a saccharide residue with N-glycan structure of formula (Ia)

' / CN——Gal—»

Asn . ..,./71.4 ^-&kgr;,&Zgr;/&Igr;.&Iacgr; ..^l,-) ,~ _&ngr; , ,,.

Part II ^r ~ ^{<jN CN M} . ^CN &ldquor;. , ■ ( ^Ia )

a \ .6 e<i(v

■ Polypeptide chain

or Ser

where:

Gal = galactose, GN = N-acetyl-D-glucosamine, M = D-mannose, Fuc = fucose, Asn = asparagine, X = any amino acid except proline, Thr = threonine, Ser = serine, * = T-linkage site (1 to 6 molecular residues),

where both peripheral residues M can be substituted by 1 to 3 trisaccharides; or

for a saccharide residue with O-glycan structure of the general formula (Ib):

Part eirver J Poly?3ptide- for Thr chain

or - O

- GaI - polysaccharide or 1,'AcGa 1 or Xy 1

(Ib)

where: 10 GaI = Galactose Thr = Threonine Ser = Serine XyI = Xylose

NAcGaI = N-acetyl-galactosamine
* = T-junction,

where in the above formulas (Ia) and (Ib) the galactose (GaI) can be replaced by 2-deoxy-galactose or 2-deoxy-2-halide(F, Cl, Br, I)-galactose.

Preferably, especially when T represents a saccharide residue with N-glycan structure or a saccharide residue with O-glycan structure, GN represents a residue of the general formula (II):

R,Ö

(II)

in which R ¹ χ, R2 and R3 have the meanings given above, Rg has the same meanings as R2 and R3 and -NHR'i can assume the axial position in addition to the equatorial position, and in addition, in the case of equatorial

ler position of -NHR ¹ ! -OR2 can be in the axial position, and in which one or more or all of the groups -OR ₂ , -OR ₃ , -OR ₆ and/or the free OH group can be acylated by a linear or branched, saturated or mono- or polyunsaturated acyl radical (-CO-R ₁ ) with a total of 1 to 20, preferably 1 to 7 carbon atoms, preferably by the acetyl radical (-CO-CH ₃ ), including its mono- or polyhydroxylated analogues.
10

The above-mentioned radicals R^ to Rß preferably represent H or CH ₃ or (C ₁ -C ₇ J-ACyI, in particular acetyl.

In the NHR'^ group, R' ]_ preferably represents a (C ₃ -C ₇ )-acyl radical selected from the group consisting of propanoyl, isopropanoyl, pivaloyl or pivanoyl (tert-butyl-carbonyl), cyclopropanoyl, butanoyl, pentanoyl, hexanoyl, heptanoyl, crotonoyl and levulinyl, and/or a (C ₃ -C ₇ )-hydrocarbyl radical selected from the group consisting of propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof.

The above-mentioned new compounds according to the invention have altered, in particular prolonged, pharmacokinetics compared with the biological half-life of the corresponding known and commercially available natural (native) glycoproteins with R'i = acetyl.

The new compounds according to the invention are preferably

recombinant glycoproteins, especially recombinant human glycoproteins (rh glycoproteins), preferably rh differentiation factors, in particular rh erythropoietin;

rh growth factors, especially rh CSF (colony-stimulating factor) , rh GMCSF (granulocyte-monocyte-stimulating factor) ;

rh thrombolytics, especially rh tissue plasminogen activator (tPA) and rh urokinase;

to rh thromboprophylactics, especially rh antithrombin in;

rh coagulation factors, especially rh factor VIII and rh factor IX;

rh interferons, especially rh α-, ß- and γ-interferons; and

about rh interleukins, especially rh IL-2, IL-15, IL-16 and IL-17.

The above-defined new recombinant glycoproteins according to the invention, in particular rh glycoproteins, represent new classes of substances which can be used as potent active ingredients for the treatment of diseases in which cells of the specific and non-specific immune system, tumor cells, leukocytes, thrombocytes and vascular endothelial cells are involved. The new compounds according to the invention can act in a targeted manner via a modulation of membrane-bound receptors which are involved in the regulation of growth and differentiation as well as the adhesion of cells of the immune system, tumors and vessels. Immune stimulation is necessary in the healing processes of infectious and tumor-related diseases. In addition, the new compounds according to the invention prevent the adhesion of leukocytes or tumor cells to vascular endothelial cells in septic shock or metastasis. Due to their modified, preferably extended, biological half-life, these substances can be administered in a significantly reduced dosage. When using their derivatives acylated at the 5 OH groups of the monosaccharide residue, especially acetylated _, the dosage can be further reduced to about half. The inventive

The new recombinant glycoproteins can be used as stimulants for cells of the immune system. This makes it possible to significantly strengthen the immune system in immunocompromised organisms. They are characterized by high cell specificity and low to completely absent side effects, as well as high stability, especially biological stability.

The invention is explained in more detail below with reference to the specific glycoprotein rh erythropoietin, but is not limited thereto. Of course, as will be readily apparent to the person skilled in the art, the following statements also apply to the other new recombinant glycoproteins of the invention listed above.

Human erythropoietin (EPO) is a glycoprotein with 166 amino acids, 3 glycosylation sites at amino acid positions 34, 38 and 83 and a molecular weight of about 34,000 to 38,000. The proportion of glycosyl side chains in the molecular weight is about 40% (cf. Jacobs et al, "Nature" 313 . 806-809 (1985); and Dordal et al, "Endocrinology" 116 . 2293-2299 (1985)). EPO is formed in the kidney and transported to its physiological site of action, the bone marrow, via the bloodstream.

EPO can be isolated either from natural sources such as human urine (see e.g. Miyake et al, "J. Biol. Chem.", 252 . 5558-5564 (1977)), or it can be produced by genetic engineering (see e.g. EP-AO 148 605 and 0 267 678). Its physiological function is to stimulate erythropoiesis, ie it stimulates the growth and differentiation of erythrocyte precursor cells. It is widely used as a remedy for anemia patients, postoperative patients and patients who receive homodialysis after kidney excision. It is primarily used to

Therapy for renal anemia, i.e. anemia caused by the limitation or loss of kidney function. The loss of kidney function can have various causes, such as reduced blood flow, chronic inflammation, trauma, extirpation, hepatorhenal syndrome and dialysis measures caused by these diseases.

A method for isolating EPO from human urine containing EPO is already known (see, for example, US Patent 3,865,801). However, since the content of EPO in normal human urine is extremely low and is on the order of about 0.01 to 0.02% by weight of the total protein in the urine, it is difficult to produce EPO effectively in this way. A method is also already known from EP 0 116 446 by which highly pure erythropoietin can be produced and in which a polypeptide is used as an antibody. However, this document contains no information on the yields that can be achieved and this method is relatively difficult to carry out.

The gene for human EPO has been known for several years and was isolated and characterized from a fetal liver gene bank. It has been available for genetic engineering studies since 1985 (cf. Jacobs et al, "Nature" 313, 806-809 (1985)). EPO can be expressed in animal cells using genetic engineering methods.

The novel recombinant EPO according to the invention (like the other novel recombinant glycoproteins mentioned above) can be produced by genetic engineering by starting from pure erythropoietin from a material containing erythropoietin which has been obtained from cell culture supernatants. Eucaryotic cells, especially animal cells, in particular non-transformed cells, are used as culture cells.

and transformed CHO cells or COS7 cells or NaIm cells or fibroblasts are used, which are cultured in a conventional culture medium, preferably in an FCS, RPMI 164 0, HAT, Eagle MEM, Dulbecco MEM and/or Glasgow MEM medium, which contains an N-(C ₃ -C ₇ )-acylated and/or N-(C ₃ -C ₇ )-hydrocarbylated monosaccharide for biosynthesis as a glycan precursor, in a manner known per se and the rh EPO obtained in this way is optionally acylated or preferably acetylated on the OH groups of the monosaccharide residue, preferably by means of a suitable acid anhydride, in particular by means of acetic anhydride, once or several times or completely.

According to a further aspect, the present invention relates to a process for producing the novel recombinant glycoproteins of the invention defined above from a material containing one or more recombinant glycoproteins, in particular recombinant human glycoproteins, which was obtained from cell culture supernatants, which is characterized in that the culture cells used are eukaryotic, preferably animal cells, particularly preferably non-transformed or transformed CHO cells or C0S7 cells or Nalm cells or fibroblasts, in a conventional culture medium which contains, as glycan precursor, an N-(C ₃ -C ₇ )-acylated and/or N-(C ₃ -C ₇ )-hydrocarbylated monosaccharide for the biosynthesis of the amino sugars in a concentration of 0.001 to 50 mmol/l, preferably 0.5 to 20 mmol/l, at a temperature of 18 to 40 ⁰ C, preferably 30 ⁰ C, and at a pH of 6.0 to 8.2, preferably 7.2, in a manner known per se and the N-acyl derivative or N-hydrocarbyl derivative of the glycoprotein obtained thereby is separated and recovered in a manner known per se and optionally added to the OH or OR groups of the monosaccharide in a manner known per se, preferably by means of a suitable acid anhydride, in particular by means of acetic anhydride.

or multiply or fully acylated or preferably acetylated.

The preferred culture medium is FCS, RPMI 164 0, HAT, Eagle MEM, Dulbecco MEM and/or Glasgow MEM.

The glycan precursors used are preferably N-(C ₃ -C ₇ )-acyl and/or N-(C ₃ -C ₇ )-hydrocarbyl, in particular �N&I&Ogr; Propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl, N-levulinyl, and/or N-propyl, N-isopropyl, N-pivalyl or N-tert-butyl, N-cyclopropyl, N-butanyl, N-pentanyl, N-hexanyl, N-heptanyl, N-crotonyl and N-levulinyl-D-glucosamine, -D-galactosamine or -neuraminic acid, in particular -D-mannosamine, or a mixture thereof.

One of the essential features of the process according to the invention is that a cell culture medium is used which contains as glycan precursor an N-acylated monosaccharide for the biosynthesis of the amino sugars, for example N-propanoyl-D-glucosamine or N-propanoyl-D-galactosamine, which are suitable for replacing N-acetyl-D-glucosamine or N-acetyl-D-galactosamine or N-acetyl-neuraminic acid by N-propanoyl-D-glucosamine or N-propanoyl-D-galactosamine or N-propanoyl-neuraminic acid in the glycan chain of the EPO secreted from the culture cells. Direct precursors of neuraminic acid have proven to be particularly effective, namely N-propanoyl-D-mannosamine or N-butanoyl-D-mannosamine or N-pentanoyl-D-mannosamine or N-hexanoyl-D-mannosamine or N-cyclopropanoyl-D-mannosamine or a mixture of these sugars. The metabolic pathway of these amino sugar analogues has been elucidated (cf. Kayser et al, "J. Biol. Chem." 267 , 16934-16938 (1992)), and is reproduced below for N-propanoyl-D-mannosamine:

· Q · » · · ·· · · · · » · · ·
I · · · · · · · ·

/V-Propanoyl-D-mannosamine

ATP

/V-Acetylmannosamine kinase

/V-Propanoyl-D-mannosamine -6- (p)

PEP-".

&Idigr; /V-acetyl-neuraminic acid aldolase

/V-Propanoyl-neuraminic acid -9-(p)

A/-Acetyl-neuraminic acid .-9-phosphatase

/V-Propanoyl-neuraminic acid

CTP-^

1 CMP-W-acetyl-neuraminic acid synthetase

CMP-W-propanoyl-neuraminic acid

Acceptor ~\

&lgr; Sialyltransferase

/V-Propanoyl-neuraminic acid acceptor + CMP

where in the above reaction sequence:

ATP PEP-P

* CTP CMP

Adenosine triphosphate Phosphoenolpyruvate Phosphate

Cytidine triphosphate Cytidine monophosphate

Acceptor a protein that is in a state of glycosylation.

The following monosaccharides are required for the glycosylation of proteins: N-acetyl-D-glucosamine, N-acetyl-

D-galactosamine, D-mannose, D-galactose, L-fucose, N-acetyl-D-mannosamine and N-acetyl-neuraminic acid, which are typically linked together (cf. E. Buddekke, "Grundriß der Biochemie", 9th edition, p. 18 5 ff. (1994)).

The monosaccharides linked to oligosaccharides (glycans) are linked to the polypeptide either via the amino acid asparagine in the typical tripeptide sequence-asparagine-any amino acid (except proline)-serine or threonine or cysteine-N-glycosidically (N-glycans) or they are linked O-glycosidically via serine (O-glycans). In these glycans, N-acetyl-D-glycosamine can be replaced by N-propanoyl-D-glucosamine and/or N-acetyl-D-galactosamine can be replaced by N-propanoyl-D-galactosamine or by homologous compounds such as N-isopropanoyl, N-pivaloyl (i.e. N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl or N-levulinoyl monosaccharide or the corresponding (C3-C7) hydrocarbyl derivatives. In the case of the terminal sugar N-acetylneuraminic acid, replacement by N-propanoyl- or N-butanoyl- or pentanoyl- or N-hexanoyl- or N-cyclopropanoyl- or N-isopropanoyl-, or N-pivaloyl- or N-levulinoyl-neuraminic acid takes place.

The EPO synthesized and secreted by the cells contains a new structure in which the native monosaccharides are replaced by new, chemically modified monosaccharides through the introduction of one or more of these sugar analogues, which can be added to the cell culture medium individually or as a mixture. The resulting total compounds represent new compounds.

These metabolically modified new erythropoietins have new biological properties, in particular they have a higher biological stability. Explains

This is currently made possible by the fact that it is known that glycoproteins contain terminal N-acetyl-neuraminic acid residues that are linked to D-galactose (cf. A. Rosenberg, "Biology of Sialic Acids", Plenum Press, New York and London, pages 7-67 (1995)). Their function in this link is to protect this galactose from being recognized by cells and their galactose-specific receptors, which was shown by the studies of G. Ashwell in the above-mentioned Schauer reference. When these terminal galactoses have been recognized in glycoproteins, the recognized glycoproteins, which therefore no longer carry N-acetyl-neuraminic acid, are subjected to intracellular degradation. This N-acetyl-neuraminic acid, which is essential for biological stability, is subject to natural turnover. It is essentially determined by specific neuraminidases (or sialidases), which split off the N-acetyl-neuraminic acid. This causes these glycoproteins to age and they are subjected to natural degradation. These neuraminidases require the N-acetyl group in the N-acetyl-neuraminic acid for their full activity.

In the new EPO compounds according to the invention, the natural N-acetyl group is replaced by homologous N-(C3~C7) acyl groups and/or homologous N-(C3-C7) hydrocarbyl groups, for example by the N-propanoyl group. However, the other N-acyl and N-hydrocarbyl groups mentioned above are also effective replacement groups. The replacement of the N-acetyl neuraminic acid by the homologous N-acyl or N-hydrocarbyl compounds mentioned above therefore leads to a reduced affinity of the neuramminidases for these modified N-acyl or N-hydrocarbyl neuraminic acids. As a result, the modified neuraminic acid is split off at a much lower rate, it remains connected to the hydroprotein for longer and can perform the desired protective function for longer. This makes the glycoprotein more biologically stable. This means that parenteral

EPO administered in the manner described above can remain in the organism for longer. It is therefore active for longer. This means that the EPO dosage can be reduced, since the EPO modified according to the invention has the same biological activity with regard to stimulating new blood formation as native EPO. In addition, the EPO modified according to the invention is more stable.

The above description of the biological mechanism involved in the production and administration of rh erythropoietin naturally also applies to the other recombinant glycoproteins according to the invention, i.e. also to the above-mentioned coagulation factors, interferons, interleukins, growth factors, thrombolytics and thromboprophylactics.

According to a further aspect, the present invention relates in particular to the following compounds:

recombinant human erythropoietin (rh erythropoietin) for the treatment of anemias, in which the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C ₃ -C ₇ )-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl and/or an N-(C3-C7)-hydrocarbyl residue selected from the group N-propyl, N-isopropyl, N-pivalyl or N-tert-butyl, N-cyclopropyl, N-butanyl, N-pentanyl, N-hexanyl, N-heptanyl, N-crotonyl and N-levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof.

The new rh erythropoietins according to the invention preferably have a glycan residue which is selected from the following group:

21

NewAci»2-3Ga]/?I -

: -2.Mar.ut

Mar./?!-iGIcNAc,?!-JGIcNAc 3

-4GIcNAc/?!

-4g:cnac/?i .

NewAca2-3Ga!jJl -^GlcS'Ac/Jl -2

-i/il --iGlcN'Ac/Jl -4GIcNAc

NewAc<i2-3Ga]/?l -4GIeNAc^l -3GaI^Tl -4GIcNAe^l

-4G!cMAc/Jl

-2Mnnal /4

mm/fl 3

Fu'ol-6

l--4GlcNAc

2-3Gai^1-4GIcXAc/?!-3GiI/?!-4GItNAc/?!

l-3Ga!/?l-4GIcNAc^I-3GaJ/?l-4G:cNAc/?l-2M

Ne'xAco2-3Gal^l &mdash;lGlcNAc/?! -3GaI^l -JGk.VA·.·/?! -2

Fucsi-6

Min/71 -4GIcN 3

NeuAca2-3GuJ/Jl-4GIcNAc/?l-2M;inal ^ Fucel-6

Many?! -NiuAca2-3Ca)/?l-4GlcNAc/7l '

NcuAlu2-3GjI//1 -4GlcNAc ₁

-!Ck-NAc (&thetas;)

Man/Zl--! 3

cNA./;! -JGk-NAc

(O

22

NcuAcc2-3Ga!/!l-

Man/?1 &mdash;SCIcNAc/?! - JCIcNAc 3

NewAeai&mdash;3G;iJ/?l -iOlcNAc/fl -2M»iwl

Gal/ft-4GlcNAc,41 "^

NewAcci-3Ci]^I -4GIcNAc/?! -3Cal/?l -4CIcNAc^l ^

^6

_&khgr; Fucol-6 (YiS

Msn^n-4GlcNAc^I--!GlcNAc s 3 NcuAce2-3GiJ/?!-4GlcNAc^l-2Man<il

NcuAcu2-3GiJ/h-4GIcNACy?!-3GaJ^l -

Fucel-6 (\\

6 "^ V/

^ ^ -4GlcNAc

_x -3Gil/fl -4

-4GicNAc/!l

Ne^o£-3GaI/M-4G;cNAc:/n-2Mana1 _&khgr; Fucal-6

/3

Ncv>Ace2-3Gu2^1 -4GIcNAe/?! -2MI

Manjil-4GlcNAc^l &mdash;SGIeNAc (k)

-2Maaal _&khgr; · Fucffl-&oacgr;

Mün^l -4GIcNAc^l -4GIcNAc -

^ 3 (I)

N£uAecr2~3GaI/?! -4GIeNAc/?! -2Miiol

-4GIcSAc/?!

"6

NiüActx2-3Ga!/?i -4GIcNAr/?! -2MMCl

^{β x} (m)

Man/?!-4GIcNAe/?! -4GIcNAc s 3

CaJ/?! -JCIcNAc/!! -ZMancrl

Fucat6

6 ^* (n)

N«jA«f2-3Gj!/J!-4GIcNAcZn-2i\1anel ^ Fucat-6 Nhn/?1 -JGIcNAe/il -4CIcVAi.-

-2Nfan«l

' ^6 NiuAcu2-3Gai/fl-4G!cNAc//1-2Mjn'.·! _&khgr; Fuc.il-6 _&khgr; /-&Ngr;

Nbn/&iacgr;&Igr; -JOIcN ^- Ac//! -4GIeNAc

GjI//! -4GIcNACyJI-IMx-Iu! ^ 4

wherein Ac in the NeuAc residues and/or one or more or all Ac in the remaining residues is (are) replaced by a (C ₃ -C ₇ )-acyl residue and/or (C ₃ -C ₇ )-hydrocarbyl residue selected from the group consisting of propanoyl, isopropanoyl, pivaloyl or pivanoyl (tert-butylcarbonyl), cyclopropanoyl, butanoyl, pentanoyl, hexanoyl, heptanoyl, crotonoyl; levulinyl, propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl; including the mono- or polyhydroxylated and/or ketylated analogues thereof.

Further preferred rh glycoproteins according to the invention are the following:

rh growth factors, in particular rh GCSF and/or rh GMCSF, for the treatment of diseases of the blood cell forming system, in particular agranulocytosis or thrombocytopenia, in particular for stimulating the growth of white blood cells, in particular lymphocytes, granulocytes and monocytes, in which the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C ₃ -C ₇ )-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl, and/or an N-(C ₃ -C ₇ )-hydrocarbyl radical selected from the group N-propyl, N-isopropyl, N-pivalyl or tert-butyl, N-cyclopropyl, N-butanyl, N-pentanyl, N-hexanyl, N-heptanyl, N-crotonyl and " N-levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof;

rh Thrombolytics, in particular rh tPA or rh urokinase, 5 for the treatment of thrombi or for the prophylaxis of thrombi formation, in which the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneu-

ramic acid residues are replaced by an N-(C3-C7) acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinyl and/or an N-(C3-C7) hydrocarbyl residue selected from the group propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof;

rh Thromboprophylactics, in particular rh Antithrombin III, for the prophylaxis of thrombus formation, in which the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C3~ C7)-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl, and/or an N-(C3-C7)-hydrocarbyl residue selected from the group propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, Crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof, is replaced ;

rh coagulation factors, in particular rh factor VIII and/or rh factor IX, for the treatment of disorders of the coagulation system caused by a hereditary or 0 acquired deficiency of factor VIII and/or factor IX, in which the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C3-C7) acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl, and/or an N-

(C3-C7) hydrocarbyl radical selected from the group propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof;

rh interferons, in particular rh ot interferon compounds, for the treatment of malignant tumors, in particular melanomas, and of autoaggressive diseases, in which the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C ₃ -C ₇ )-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl, and/or an N-(C ₃ -C ₇ )-hydrocarbyl residue selected from the group propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof;

rh interferons, in particular rh ß-interferon compounds, for the treatment of all forms of multiple sclerosis and viral diseases, in particular viral hepatitis B, viral hepatitis C, viral hepatitis D and viral encephalitis, in which the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C ₃ -C ₇ )-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl and/or an N-(C ₃ -C ₇ )-hydrocarbyl residue selected from the group propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl,

F · &diams; · · I · · ·
I · · · ' · · · · ·

including the mono- or polyhydroxylated and/or ketylated analogues thereof;

rh interferons, in particular rh γ-interferon compounds, for the treatment of malignant tumors, viral hepatitis B ₇ , viral hepatitis C, viral hepatitis D and viral encephalitis, in which the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C3-C7)-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl, and/or an N-(C3-C7)-hydrocarbyl residue selected from the group propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof; and

rh interleukins, in particular rh IL-2, rh IL-15, rh IL-16 and rh IL-17, for the treatment of malignant metastatic tumors, in particular melanomas, in which the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C3-C7)-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl, and/or an N-(C ₃ -C ₇ )-hydrocarbyl residue selected from the group propyl, isopropyl, Pivalyl or " tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof.

The new compounds according to the invention are in particular those in which the natural N-

Acetyl group of N-acetyl-D-glucosamine has been completely or partially replaced by N-propanoyl or N-cyclopropanoyl or N-isopropanoyl or N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl) or N-butanoyl or N-pentanoyl or N-hexanoyl by biochemical modulation (biochemical engineering) by adding the corresponding N-acyl-D-glucosamine or N-acyl-D-galactosamine or N-acyl-neuraminic acid, in particular N-acyl-D-mannosamine derivative, to the culture medium in a final concentration in the medium of 0.001 to 50.0 mM, in particular 0.5 to 20 mM, especially 0.5 to 5 mM, wherein in said culture medium the D-glucose is completely or partially replaced by D-galactose may be replaced; and

specifically those in which the natural D-galactose has been completely or partially replaced by 2-deoxy-D-galactose by biochemical modulation (biochemical engineering) by adding 2-deoxy-D-galactose to the culture medium in a final concentration in the medium of 0.001 to 50 mM, in particular 0.5 to 20 mM, especially 0.5 to mM.

According to a further aspect, the present invention relates to pharmaceutical compositions which contain as active ingredient at least one rh glucosamine as defined above, optionally in combination with other active ingredients as well as conventional pharmaceutical carriers and/or excipients.

0 The pharmaceutical agents according to the invention preferably contain as active ingredient one or more recombinant glycoproteins, in particular recombinant human glycoproteins or a mixture of differently modified N-(C ₃ -C ₇ )-acyl and/or N-(C ₃ -C ₇ )-hydrocarbyl derivatives thereof.

The pharmaceutical agents according to the invention contain the rh glycoprotein active ingredient, which is preferably mono- or polyacylated, in particular acetylated, on the monosaccharide, preferably in an amount of 0.001 to 50% by weight, preferably 0.1 to 20% by weight, especially up to 10% by weight.

Particularly preferred pharmaceutical agents of the invention are those which contain as rh glycoprotein active ingredient: 10

recombinant human erythropoietin or derivatives thereof as defined above;

rh growth factors, in particular rh CGSF and/or rh GMCSF, as defined above;

rh thrombolytics, in particular rh tPA or rh urokinase, as defined above;

0 rh thromboprophylactics, in particular rh antithrombin III, as defined above;

rh coagulation factors, in particular rh factor VIII and/or rh factor IX, as defined above;

rh interferons, in particular rh α-, β- and γ-interferons, as defined above; or

rh interleukins, in particular rh IL-2, rh IL-15, rh IL-16 and rh IL-17, as defined above.

According to a further aspect, the present invention relates
35

the use of recombinant human erythropoietin and its derivatives as defined above,

for the treatment of anemia, particularly renal anemia, in a dosage of 0.001 to 200 mg/kg body weight;

the use of rh growth factors as defined above for the treatment of diseases of the blood cell-forming system, in particular agranulocytosis or thrombocytopenia, in particular for stimulating the growth of white blood cells, in particular lymphocytes, granulocytes and monocytes, in a dosage of 0.001 to 1.0 mg/kg body weight;

the use of rh thrombolytics as defined above for the treatment of thrombi or for the prophylaxis of thrombi formation in a dosage of 1 to 200 mg/kg body weight, preferably 50 to 100 mg/kg body weight;

the use of rh thromboprophylactics as defined above for the prophylaxis of thrombus formation in a dosage of 1 to 200 mg/kg body weight, preferably 50 to 100 mg/kg body weight;

the use of the rh coagulation factors as defined above for the treatment of disorders of the coagulation system caused by a hereditary or acquired deficiency of factor VIII and/or factor IX, in a dosage of 0.001 to 200, preferably 0.1 to 50 mg/kg body weight; 30

the use of rh α-interferons as defined above for the treatment of malignant tumors, in particular melanomas, and of autoaggressive diseases in a dosage of 0.001 to 10 μg/kg body weight, in particular 0.01 to 0.1 μg/kg body weight;

the use of rh ß-interferons as described above
for the treatment of all forms of multiple sclerosis and viral diseases, especially
of viral hepatitis B, viral hepatitis C, viral hepatitis D and viral encephalitis in a dosage of
0.001 to 10 μg/kg body weight, in particular from 0.01
up to 0.1 jug/kg body weight;

the use of rh γ-interferons as described above

defined for the treatment of malignant
Tumors, viral hepatitis B, viral hepatitis C,
Viral hepatitis D and viral encephalitis in a dosage of 0.001 to 0.1 mg/kg body weight, in particular 0.01 to 0.1 mg/kg body weight; and

the use of rh interleukins as defined above for the treatment of malignant metastatic tumors, in particular melanomas, in a
Dosage from 0.001 to 200 mg/kg body weight.

The rh glucoproteins according to the invention are preferably administered in a form in which the OH or OR groups
of its monosaccharide residue is acylated once or several times,
preferably acetylated, in which case the dosage for the correspondingly acylated or acetylated derivatives can be reduced to half the dosages indicated for the above-mentioned preferred uses.

Claims (45)

R2 a linear or cyclic, unbranched or branched, optionally mono- or polyhydroxylated and/or ketylated (C3-C7) acyl radical, in particular (C3-C7) alkanoyl radical, or a linear or cyclic, unbranched or branched, optionally mono- or polyhydroxylated and/or ketylated (C3-C7) hydrocarbyl radical, in particular (C ₃ -C ₇ ) alkyl, alkenyl or alkynyl radical, R3, R4 and R5, which may be the same or different, each represent hydrogen, a linear or branched alkyl radical having 1 to 20 carbon atoms (C _n H2n+2' η = 1 to 20); a linear or branched alkenyl radical having 3 to 20 carbon atoms (C _n H2 _n , η = 3 to 20, position of the double bond on C _n η = 2 to 19); a linear or branched alkynyl radical with 3 to 2 0 carbon atoms (C _n H _2n -2/ η = 3 to 20, position of the triple bond at C _n η = 2 to 19); an alkenyl or alkynyl radical with 2 or more double bonds or triple bonds with 4 to 20 carbon atoms; an aryl radical with 6 to 20 carbon atoms; a linear or branched, saturated or mono- or polyunsaturated acyl radical (-CO-R ₁ ) with a total of 1 to 20 carbon atoms including its mono- or polyhydroxylated analogues; an aroyl radical with 6 to 2 0 carbon atoms; a carbonylamide radical of the formula -CONH ₂ or -CONHR ₁ ; a linear or branched, saturated or mono- or polyunsaturated thioacyl radical (-CS-R ₁ ) with a total of 1 to 20 carbon atoms; or a thiocarbamide radical of the formula CS-NH ₂ or -CS-NHR ₁ ; where R ₁ is in each case hydrogen or a linear or branched, saturated or mono- or polyunsaturated alkyl radical 0 with 1 to 2 0 carbon atoms and each of the aforementioned radicals except H can optionally be mono- or polysubstituted by halogen, hydroxy, epoxy, amino, mercaptan, phenyl, phenol or benzyl groups, and
25 T is a mono-, di- or oligosaccharide residue with up to 40 glycosidically linked, optionally branched sugar residues, which represent furanose and/or pyranose rings and contain 5 to 230 carbon atoms and are N- or O-glycosidically bound to polypeptides. 2. Glycoproteins according to claim 1, characterized in that in the general formula (I) mean: 35 R ₂ , R3, R4 and R5, which may be the same or different, are each hydrogen, a linear or branched alkyl radical with 1 to 7 carbon atoms; a linear or branched alkenyl radical with 3 to 10 carbon atoms; a linear or branched alkynyl radical with 3 to 10 carbon atoms; an alkenyl or alkynyl radical with 2 or more double bonds or triple bonds with 7 to 12 carbon atoms; a phenyl radical; a linear or branched, saturated or mono- or polyunsaturated acyl radical (-CO-R ₁ ) with a total of 1 to 7 carbon atoms, in particular acetyl radical, including its mono- or polyhydroxylated analogues; an aroyl radical with 6 to 10 carbon atoms; a carbonylamide radical of the formula -CONH2 °or CONHR ₁ ; a linear or branched, saturated or mono- or polyunsaturated thioacyl radical (-CS-R ₁ ) with a total of 1 to 7 carbon atoms; or a thiocarbamide radical of the formula -CS-NH2 or -CS-NHR ₁ ; where R ₁ is in each case hydrogen or a linear or branched, saturated or mono- or polyunsaturated alkyl radical having 1 to 20 carbon atoms and each of the aforementioned radicals except H can optionally be mono- or polysubstituted by fluorine, chlorine, bromine or iodine, hydroxy, epoxy, amino, mercaptan, phenyl, phenol or benzyl groups, 3. Compounds according to claim 1 or 2, characterized in that in the general formula (I) T is for a saccharide residue with N-glycan structure of the formula oaer Ser where: Gal = galactose, GN = N-acetyl-D-glucosamine, M = D-mannose, Fuc = fucose, Asn = asparagine, X = any amino acid except proline, Thr = threonine, Ser = serine, * = T-linkage site (1 to 6 molecular residues), where both peripheral residues M can be substituted by 1 to 3 trisaccharides; or for a saccharide residue with O-glycan structure of the general formula (Ib): C Ser - O - GaI - polysaccharide Part of a J Polypeptide- ior Thr or ^kezte L I 1,'AcGa ' or Xyl where: Gal = Galactose 0 Thr = Threonine ■ Ser = Serine Xyl = xylose NAcGaI = N-acetyl-galactosamine * = T-junction, 35 where in the above formulas (Ia) and (Ib) the galactose (GaI) can be replaced by 2-deoxy-galactose or 2-deoxy-2-halide(F, Cl, Br, I)-galactose. 4. Compounds according to at least one of claims 1 to 3, characterized in that when T represents a saccharide residue with an N-glycan structure or a saccharide residue with an O-glycan structure, GN represents a residue of the general formula (II): 10 15 R-O' (II) in which R ¹ , R 2 and R 3 have the meanings given above, Re has the same meanings as R ₂ and R ₃ and -NHR 1 can also assume the axial position in addition to the equatorial position, and in which, in addition, when -NHR ¹ ! is in the equatorial position, -OR ₂ can be in the axial position, and in which one or more or all of the groups -OR ₂ , -OR _{3 /} -OR ₆ and/or the free OH group can be acylated by a linear or branched, saturated or mono- or polyunsaturated acyl radical (-CO-R ₁ ) with a total of 1 to 20, preferably 1 to 7 carbon atoms, preferably by the acetyl radical (-CO-CH 3), including its mono- or polyhydroxylated analogues. 5. Compounds according to at least one of claims 1 to 4, characterized in that R^ to Rg are H or CH ₃ or (C ₁ -C ₇ )-ACyI, in particular acetyl. 6. Compounds according to at least one of claims 1 to 5, characterized in that in the NHR'^ group R^ stands for a (C ₃ -C ₇ ) acyl radical selected from the group consisting of propanoyl, isopropanoyl, pivaloyl or pivanoyl (tert-butyl-carbonyl), cyclopropanoyl, butanoyl, pentanoyl, hexanoyl, heptanoyl, crotonoyl and levulinyl and/or a (C ₃ -C ₇ ) hydrocarbyl radical selected from the group consisting of propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof. 7. Compounds according to at least one of claims 1 to 6, characterized in that they have modified, in particular prolonged, pharmacokinetics compared with the biological half-life of the corresponding known and commercially available natural (native) glycoproteins with R ¹ 1 = acetyl. 8. Compounds according to at least one of claims 1 to 7, characterized in that they are a recombinant glycoprotein, especially a recombinant human glycoprotein (rh glycoprotein), preferably rh differentiation factors, especially rh erythropoietin; rh growth factors, especially rh CSF (colony-stimulating factor), rh GMCSF (granulocyte-monocyte-stimulating factor);
rh thrombolytics, especially rh tissue plasminogen-0 activator (tPA) and rh urokinase; ■ to rh thromboprophylactics, especially rh antithrombin III; rh coagulation factors, especially rh factor VIII and rh factor IX; rh interferons, especially rh α-, ß- and γ-interferons; and about rh interleukins, especially rh IL-2, rh IL-15, rh IL-16 and rh IL-17. 9. Compounds according to claim 8, characterized in that they are recombinant human erythropoietin (rh erythropoietin) for the treatment of anemias, in which the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C ₃ -C ₇ )-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl and/or an N-(C ₃ -C ₇ )-hydrocarbyl residue selected from the group N-propyl, N-isopropyl, N-pivalyl or N-tert-butyl, N-cyclopropyl, N-butanyl, N-pentanyl, N-hexanyl, N-heptanyl, N-crotonyl and N-levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof. 10. Compounds according to claim 9, characterized in that they have a glycan residue selected from the following group: NewAcff2-3G:d/?l - ci I -2.Mar.uI M3.-./J1 -JGIcNAc ₁ Sl -JCIcNAc 3 -4CIcNAc/?! zr.ßX&mdash; iGlcNAcyJl .-4GlcNAc N£uAcc2-3Gal^l -4GIcNAc^I -3GaI^l -4GIcN aI-3G1I/M -4GlcNAc/fl I -JGkNAc 4GkSAc ₁ Jl -3GMZfI -JGlcNAc/Jl -2
NeuAec2-3GaI^l - JGkNAcyJl Fucol-6 Man/?l-4GlcNAc^l-JGlcNAc 3 FuceI-6 Nei:A:o2-3Gal/?l -J -2.Manol Man/il -4
3 Fucul-6 k-SA ₁ -/;! -JCIcSAc NewAec2-3Ga!/?1 -4GIcSAc/?! ^ 6 KcuAc£i2-3Ga]/?l -4GIcNAc/?! -2Ma Ma.n/fl -4GIcNAc/?! -4GIoNAc Gal/ft -4GlcNAc;?! NewAceL2-3Ga2/)J &mdash;iGlcNAc/Ji-3GM^l -4GIcN New A cc2 ~3Gi)/?l- 4 GIcSAcJh -2M ana! ^i^l -4GIcNAe/?! Fucol-6 6 ¹ S Man/ft &mdash;ideSAcßl -4GIc ₁ NAc ^ 3 NsuAce2-3Gdl/n-4GIcNACyJl -3GaJ/n - l -3G«lyil -4CIeNAc^l -2Manel _v , , X ₆ rucei-o Man/il-4GIcN ^ 3 -2 Miob1 -4G!cNAc/3l Fuel-6 6 "*^ Min/?l-4G:cKAc/?I&mdash;!GIcNAc s -4GleNAc/?1 -2M Fu-el-6 6 I -4GlcNAc/i[ NeuAcu2 -3Cal/fi -4G!cNAc//l -3Gal//I -4GIoNac/(1 &mdash;lGlenAC NewAce2~3Gal/?l -4GlcNAc/7l -2.Ma.nal Ca!/n -4GlcS'Ac/?l -4G!cN'Ac/?l -4GlcNAi/Jl-2MMcl ^ ^6x Min^l -iClcNAe/Jl -4CIcNAc ^ 3 -4GkNAc/?! -ZMancr I -4GIeNAc/?! -iMancl ^ Fucol-6 6 ^F ^"- ⁶ - (O) Nhn/il -4GIcNAc//; -4GIcNAc ^v ' &iacgr;&ogr; wherein Ac in the NeuAc residues and/or one or more or all Ac in the remaining residues is (are) replaced by a (C ₃ -C ₇ )-acyl residue and/or (C ₃ -C ₇ )-hydrocarbyl residue selected from the group consisting of propanoyl, isopropanoyl, pivaloyl or pivanoyl (tert-butylcarbonyl), cyclopropanoyl, butanoyl, pentanoyl, hexanoyl, heptanoyl, crotonoyl; levulinyl, propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl; including the mono- or polyhydroxylated and/or ketylated analogues thereof. 11. Compounds according to claim 8, characterized in that they are rh growth factors, in particular rh GCSF and/or rh GMCSF, for the treatment of diseases of the blood cell-forming system, in particular agranulocytosis or thrombocytopenia, in particular for stimulating the growth of white blood cells, in particular lymphocytes, granulocytes and monocytes, in which 0 the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C ₃ -C ₇ )-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinyl, and/or an N-(C ₃ -C ₇ )-hydrocarbyl radical selected from the group N-propyl, N-isopropyl, N-pivalyl or tert-butyl, N-cyclopropyl, N-butanyl, N-pentanyl, N-hexanyl, N-heptanyl, N-crotonyl and N-levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof. 12. Compounds according to claim 8, characterized in that they are rh thrombolytics, in particular rh tPA 5 or rh urokinase, for the treatment of thrombi or for the prophylaxis of thrombi formation, in which the natural N-acetyl group of the glycan residues, in particular of the terminal N-acetylneuraminic acid residues, is replaced by an N- J I 11'· »··* (C ₃ -C ₇ )-acyl radical selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinyl and/or an N-(C ₃ -C ₇ )-hydrocarbyl radical selected from the group propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof. 10 13. Compounds according to claim 8, characterized in that they are rh thromboprophylactics, in particular rh antithrombin III, for the prophylaxis of thrombus formation, in which the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C ₃ -C ₇ )-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl, and/or an N-(C ₃ -C ₇ )-hydrocarbyl residue selected from the group propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof. 14. Compounds according to claim 8, characterized in that they are rh coagulation factors, in particular rh factor VIII and/or rh factor IX, for the treatment of disorders of the coagulation system caused by a hereditary or acquired deficiency of factor VIII and/or factor IX, in which the natural N-acetyl group of the glycan residues, in particular of the terminal N-acetylneuraminic acid residues, is replaced by an N-(C ₃ -C ₇ )-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl, 12 and/or an N-(C ₃ -C ₇ )-hydrocarbyl radical selected from the group consisting of propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof. 15. Compounds according to claim 8, characterized in that they are rh interferons, in particular rh a-interferon compounds, for the treatment of malignant tumors, in particular melanomas, and of autoaggressive diseases, in which the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C ₃ -C ₇ )-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl, and/or an N-(C ₃ -C ₇ )-hydrocarbyl residue selected from the group propyl, 0 isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof. 16. Compounds according to claim 8, characterized in that they are rh interferons, in particular rh ß-interferon compounds, for the treatment of all forms of multiple sclerosis and viral diseases, in particular viral hepatitis B, viral hepatitis C, viral hepatitis D and viral encephalitis, in which the '· natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C ₃ -C ₇ )-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl and/or an N-(C ₃ -C ₇ )-hydrocarbyl radical selected from the group propyl, isopropyl, pivalyl or tert-butyl, cy- clopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof.
5 17. Compounds according to claim 8, characterized in that they are rh interferons, in particular rh γ-interferon compounds, for the treatment of malignant tumors, viral hepatitis B, viral hepatitis C, viral hepatitis D and viral encephalitis, in which the natural N-acetyl group of the glycan residues, in particular of the terminal N-acetylneuraminic acid residues, is replaced by an N-(C3-C7)-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl, and/or a N-(C3-C7)-hydrocarbyl radical selected from the group propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, including the mono- or polyhydroxylated and/or ketylated analogues thereof. 18. Compounds according to claim 8, characterized in that they are rh interleukins, in particular rh IL-2, rh IL-15, rh IL-16 and rh IL-17, for the treatment of malignant metastasizing tumors, in particular melanomas, in which the natural N-acetyl group of the glycan residues, in particular the terminal N-acetylneuraminic acid residues, is replaced by an N-(C ₃ -C ₇ )-acyl residue selected from the group N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl and N-levulinoyl, and/or a N-(C3-C7)-hydrocarbyl radical selected from the group propyl, isopropyl, pivalyl or tert-butyl, cyclopropyl, butanyl, pentanyl, hexanyl, heptanyl, crotonyl and levulinyl, one- finally the mono- or polyhydroxylated and/or ketylated analogues thereof. 19. Compounds according to claims 8 to 18, in which the natural N-acetyl group of N-acetyl-D-glucosamine has been completely or partially replaced by N-propanoyl or N-cyclopropanoyl or N-isopropanoyl or N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl) or N-butanoyl or N-pentanoyl or N-hexanoyl by biochemical modulation (biochemical engineering) by adding the respective corresponding N-acyl-D-glucosamine or N-acyl-D-galactosamine or N-acyl-neuraminic acid, in particular N-acyl-D-mannosamine derivative to the culture medium in a final concentration in the medium of 0.001 to 50.0 mM, in particular 0.5 to 20 mM, especially 0.5 to 5 mM, where in the said culture medium, the D-glucose can be completely or partially replaced by D-galactose. 20. Compounds according to claims 8 to 19, characterized 0 characterized in that the natural D-galactose has been completely or partially replaced by 2-deoxy-D-galactose by biochemical modulation (biochemical engineering) by adding 2-deoxy-D-galactose to the culture medium in a final concentration in the medium of 0.001 to 50 mM, in particular 0.5 to 20 mM, especially 0.5 to 5 mM. 21. Recombinant glycoproteins according to at least one of claims 1 to 20, characterized in that they are obtainable from a material containing one or more recombinant GIy-0 coproteins, in particular recombinant human glycoproteins, which was obtained from cell culture supernatants by culturing eukaryotic, preferably animal cells, particularly preferably non-transformed or transformed CHO cells or COS7 cells or Nalm cells or fibroblasts, in a conventional culture medium which contains as glycan precursor an N-(C3- _C7 )-acylated and/or N- [C-^-j) -hydrocarbylated monosaccharide for the biosynthesis of the amino sugars in a concentration tration of 0.001 to 50 mmol/l, preferably 0.5 to 20 mmol/l, at a temperature of 18 to 40 ⁰ C, preferably 30 ⁰ C, and at a pH of 6.0 to 8.2, preferably 7.2, in a manner known per se and separating and isolating the resulting N-acyl derivative or N-hydrocarbyl derivative of the glycoprotein in a manner known per se and optionally single or multiple or complete acylation, preferably acetylation, of the OH or OR groups of the monosaccharide, preferably by means of a suitable acid anhydride, in particular by means of acetic anhydride, in a manner known per se. 22. Compounds according to claim 21, characterized in that an FCS, RPMI 1640, HAT, Eagle MEM, Dulbecco MEM and/or Glasgow MEM medium was used as the culture medium for their preparation. 23. Compounds according to claim 21 or 22, characterized in that for their preparation as glycan precursors N-(C ₃ -C ₇ )-acyl and/or N-(C ₃ -C ₇ )-hydrocarbyl, in particular N-propanoyl, N-isopropanoyl, N-pivaloyl or N-pivanoyl (N-tert-butylcarbonyl), N-cyclopropanoyl, N-butanoyl, N-pentanoyl, N-hexanoyl, N-heptanoyl, N-crotonoyl, N-levulinoyl, and/or N-propyl, N-isopropyl, N-pivalyl or N-tert-butyl, N-cyclopropyl, N-butanyl, N-pentanyl, N-hexanyl, N-heptanyl, N-crotonyl- and N-levulinyl-D-glucosamine, -D-galactosamine or -neuraminic acid, in particular -D-mannosamine, or a mixture thereof has been used. 24. Pharmaceutical agent which contains as active ingredient at least one compound according to claims 1 to 20, optionally in combination with other active ingredients and conventional pharmaceutical carriers and/or excipients. 25. Pharmaceutical agent according to claim 24, characterized in that it contains as active ingredient one or more recombinant binary glycoproteins, in particular recombinant human glycoproteins, or a mixture of differently modified N-(C ₃ -C ₇ ) acyl and/or N-(C ₃ -C ₇ ) hydrocarbyl derivatives thereof.
5 26. Pharmaceutical agent according to claim 24 or 25, characterized in that it contains the active ingredient(s), preferably mono- or polyacylated, preferably acetylated, on the monosaccharide, in an amount of 0.001 to 50% by weight, preferably 0.1 to 20% by weight, in particular 2 to 10% by weight. 27. Pharmaceutical agent according to claims 24 to 26, characterized in that it contains as active ingredient the compounds according to claims 9 to 10. 28. Pharmaceutical agent according to claims 24 to 26, characterized in that it contains the compounds according to claim 11 as active ingredient. 29. Pharmaceutical agent according to claims 24 to 26, characterized in that it contains as active ingredient the compounds according to claim 12. 5 30. Pharmaceutical agent according to claims 24 to 26, characterized in that it contains as active ingredient the compounds according to claim 13. 31. Pharmaceutical agent according to claims 24 to 0 26, characterized in that it contains as active ingredient the compounds according to claim 14. 32. Pharmaceutical agent according to claims 24 to 26, characterized in that it contains the compounds according to claim 15 as active ingredient. 33. Pharmaceutical agent according to claims 24 to
26, characterized in that it contains as active ingredient the compounds according to claim 16. 34. Pharmaceutical agent according to claims 24 to
26, characterized in that it contains as active ingredient the compounds according to claim 17. 35. Pharmaceutical agent according to claims 24 to
26, characterized in that it contains as active ingredient the compounds according to claim 18. 36. Compounds according to claims 9 and 10 for the treatment of anemias, in particular renal anemias, in a dosage of 0.001 to 200 mg/kg body weight
. 37. Compounds according to claim 11 for the treatment of diseases of the blood cell forming system, in particular agranulocytosis or thrombocytopenia, especially for Stimulating the growth of white blood cells, especially
Lymphocytes, granulocytes and monocytes, at a dosage of 0.001 to 1.0 mg/kg body weight. 38. Compounds according to claim 12 for the treatment of thrombi or for the prophylaxis of thrombi formation in a dosage of 1 to 200 mg/kg body weight, preferably from 50 to 100 mg/kg body weight. 0 39. Compounds according to claim 13 for the prophylaxis of Thrombus formation at a dosage of 1 to 200 mg/kg
body weight, preferably from 50 to 100 mg/kg, body weight. 5 40. Compounds according to claim 14 for the treatment of disorders of the coagulation system caused by a hereditary or acquired deficiency of factor VIII and/or factor IX in a dosage of 0.001 to 200, preferably 0.1 to 50 mg/kg body weight. 41. Compounds according to claim 15 for the treatment of malignant tumors, in particular melanomas, and of autoaggressive diseases in a dosage of 0.001 to 10 ^g/kg body weight, in particular from 0.01 to 0.1 body weight. 42'. Compounds according to claim 16 for the treatment of all forms of multiple sclerosis and viral diseases, especially viral hepatitis B, viral hepatitis C, viral hepatitis D and viral encephalitis in a dosage of 0.001 to 10 µg/kg body weight, in particular of 0.01 to 0.1 µg/kg body weight. 43. Compounds according to claim 17 for the treatment of malignant tumors, viral hepatitis B, viral hepatitis C, viral hepatitis D and viral encephalitis 0 in a dosage of 0.001 to 0.1 g/kg body weight, in particular 0.01 to 0.1 g/kg body weight. 44. Compounds according to claim 18 for the treatment of malignant metastatic tumors, in particular melanomas, in a dosage of 0.001 to 200 mg/kg body weight . 45. Compounds according to claims 1 to 20 in their form which is mono- or polyacylated, preferably acetylated, at the OH or OR groups of the monosaccharide residue for the purposes stated in claims 36 to 44 in a dosage which preferably corresponds to about half of the dosages stated in claims 36 to 44.