RU2232163C2 - Conjugates of erythropoietin and polyethylene glycol, pharmaceutical compositions (variants), method for prophylactic and/or therapeutic treatment of disturbances and method for preparing conjugate or composition - Google Patents

Abstract

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to conjugate of glycoprotein erythropoietin that has at least one free amino-group and elicits biological activity in vivo providing elevating production of reticulocytes and erythrocytes by bone marrow cells. Conjugate is taken among the group involving human erythropoietin and its analogues that have sequence of human erythropoietin modified by addition of 1 to 6 sites of glycosylation or rearrangement of at least one site of glycosylation. This glycoprotein is bound covalently with 1-3 (lower)-alkoxypoly-(ethylene glycol) groups and each poly-(ethylene glycol) group is bound covalently with glycoprotein through linker of the formula -C(O)-X-S-Y. The C(O)-group of linker forms amide bond with one of these amino-groups wherein X means -(CH₂)_k- or -CH₂(O-CH₂-CH₂)_k- wherein k means 1-10; Y means

An average molecular mass of each poly-(ethylene glycol) fragment is from about 20 to about 40 kDa and molecular mass of conjugate is from about 51 to about 175 kDa. Also, invention relates to pharmaceutical compositions, composition, method for prophylaxis and/or therapeutic treatment and method for preparing conjugate. Invention provides preparing conjugates exhibiting the longer half-time life value in blood stream and plasma retention time, reduced clearance and higher activity in vivo.

EFFECT: improved preparing and treatment methods, valuable properties of conjugates.

39 cl, 3 dwg, 1 tbl, 4 ex

Description

BACKGROUND OF THE INVENTION

Erythropoiesis is a red blood cell formation process that serves to replenish cell destruction. Erythropoiesis is a controlled physiological mechanism, resulting in the formation of the number of red blood cells necessary for adequate oxygenation of the tissue. Naturally occurring human erythropoietin (hEPO) is a 165-amino acid glycoprotein that is produced by the kidney and is a humoral plasma factor that stimulates the formation of red blood cells (Carnot P. and Deflandre C., CRAcad. Sci. 143: 432 (1906); Erslev AJ, Blood 8: 349 (1953); Reissmann KR, Blood 5: 372 (1950); Jacobson LO, Goldwasser E., Freid W. and Pizak LF, Nature 179: 6331-6334 (1957)). Human EPO stimulates the division and differentiation of committed erythroid progenitors in the bone marrow. Human EPO exerts its biological activity by binding to receptors on erythoid precursors (Ktantz B.S. (1991) Blood 77: 419). Naturally occurring human erythropoietin is an acid glycoprotein present in plasma at low concentrations, which stimulates the replenishment of red blood cells that die during aging.

Erythropoietin was obtained by biological synthesis using recombinant DNA techniques (Egrie JC, Strickland TW, Lane J. et al., Immunobiol. 72: 213-224 (1986)) and is a product of the cloned human EPO gene inserted and expressed in tissue cells Chinese hamster ovary (CHO cells). Naturally occurring human erythropoietin is first translated into a 166 amino acid acid (ak) polypeptide chain containing arginine at position 166. As a result of post-translational modification, the arginine at position 166 is cleaved by carboxypeptidases. The primary structure of human EPO (165 ak) is shown in figure 1. The primary structure of human EPO (166 ak) is shown in figure 2. Two disulfide bridges are present between Cys ⁷ Cys ¹⁶¹ and Cys ²⁹ -Cys ^{33. The} molecular weight of the human EPO polypeptide chain without sugar fragments is 18,236 Da. In the intact EPO molecule, approximately 40% of the molecular weight is in the carbohydrate groups (Sasaki P., Bothner B. Dell A. and Fukuda M., J. Biol. Chem. 262: 12059 (1987)).

Since human erythropoietin plays a key role in the formation of red blood cells, this hormone can be used in the treatment of blood diseases, which are characterized by low production or production of abnormal red blood cells. In clinical settings, EPO is used, for example, to treat anemia in patients with chronic renal failure (CRF) (Eschbach JW, Egri JC, Downing MR et al., NEJM 316: 73-78 (1987); Eschbach JW, Abdulhadi MH, Browne JK et al., Ann. Intern. Med. 111: 992 (1988); Egrie JC, Eschbach JW, McGuire T., Adamson JW, Kidney Intl. 33: 262 (1988); Lim VS, Degowin RL, Zavala D et al., Ann. Intern. Med. 110: 108-114 (1989)) and AIDS sufferers and cancer patients undergoing chemotherapy (Danna RP, Rudnick SA, Abels RI in: M.V. Garnick (ed.) Erythropoietin in Clinical Application An International Perspective, New York, NY: Marcel Dekker; 1990: pp. 301-324). However, the bioavailability of commercially available protein-based therapeutic agents, such as EPO, is limited by their short plasma half-lives and sensitivity to protease degradation. These shortcomings prevent the manifestation of their maximum potential activity in a clinical setting.

Summary of the invention

Thus, the present invention relates to a new class of pegylated derivatives of erythropoietin. Physiologically active PEG-EPO conjugates of the invention include an erythropoietin glycoprotein having at least one free amino group and having in vivo biological activity causing an increase in the production of reticulocytes and red blood cells by bone marrow cells and selected from the group. including human erythropoietin and its analogues, which have a primary sequence of human erythropoietin, modified by adding from 1 to 6 glycosylation sites; this glycoprotein is covalently linked to 1-3 (lower) alkoxypoly (ethylene glycol) groups, each poly (ethylene glycol) group is covalently linked to the glycoprotein via a linker of the formula —C (O) —XSY, while the C (O) group of the linker forms an amide a bond with one of these amino groups, X is - (CH ₂ ) _k or —CH ₂ (O — CH ₂ —CH ₂ ) _k -, where k is 1-10, Y is

the average molecular weight of each poly (ethylene glycol) fragment is from about 20 to about 40 kDa and the molecular weight of the conjugate is from about 51 to about 175 kDa. The invention also relates to compositions containing conjugates described herein, wherein the percentage of conjugates in the composition in which n is 1 is at least 90%.

Compared to unmodified EPO (i.e., EPO without attached PEG) and conventional PEG-EPO conjugates, the conjugates of the present invention have a longer half-life in the bloodstream and a retention time in plasma, reduced clearance and higher in vivo activity in clinical conditions. The conjugates of the invention can be used for the same purposes as EPO. In particular, the conjugates of the invention can be used to treat patients by stimulating the division and differentiation of committed erythroid progenitors in the bone marrow, similar to that used to treat EPO patients.

The present invention also relates to a method for treating anemia in humans. The invention also relates to a method for producing products comprising erythropoietin glycoprotein, which involves the covalent interaction of the ε-amino group of the erythropoietin protein lysine with a bifunctional reagent to produce an amide-coupled intermediate. The bifunctional reagent contains a reactive group and a protected thiol group. The amide-coupled intermediate is then covalently reacted with an activated polyethylene glycol derivative to produce products of the present invention including erythropoietin glycoprotein.

Brief Description of the Drawings

Figure 1 - the primary structure of human EPO (165 amino acids).

Figure 2 - the primary structure of human EPO (166 amino acids).

Figure 3 - in vivo activity of pegylated EPO, determined using analysis using normocytic mice (mice with normocytes (mature red blood cells)).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "erythropoietin protein", "erythropoietin", "EPO" or "erythropoietin glycoprotein" refers to a glycoprotein having the sequence shown in figure 1 (SEQ ID NO: 1) or figure 2 (SEQ ID NO: 2), or to a protein or polypeptide that is practically homologous to them, whose biological properties are associated with stimulation of red blood cell production and stimulation of division and differentiation of committed erythroid precursors in the bone marrow. In the context of the present description, the concept of EPO protein includes those proteins that are modified intentionally, for example, using site-directed mutagenesis, or as a result of random mutations. These concepts also include analogs having from 1 to 6 additional glycosylation sites, analogues having at least one additional amino acid at the carboxyl end of the protein, wherein the additional amino acid (s) includes (are) at least one glycosylation site, and analogues having an amino acid sequence that includes rearrangement of at least one glycosylation site, for example, analogues that are described in EP-A 640619. These concepts include both naturally occurring and gender recombinantly studied human erythropoietin.

The term “practically homologous” refers to the particular sequence under consideration, for example, a mutant sequence that differs from the sequence being compared with the presence of one or more substitutions, deletions or additions, the “pure” effect of which does not lead to an unfavorable functional difference between the considered sequence and the sequence with which the comparison is made. For the purposes of the present invention, sequences that are more than 95% homologous, having equivalent biological properties and equivalent expression characteristics, are considered to be substantially homologous. In order to evaluate homology, shortening of the mutant sequence should be neglected. Sequences having lower degrees of homology, comparable in biological activity and having equivalent expression characteristics, are considered almost equivalent,

The term "fragment" of an EPO protein refers to any protein or polypeptide whose amino acid sequence corresponds to the sequence of a portion or fragment of an EPO protein, and which has the biological activity of EPO. Fragments include proteins or polypeptides obtained by proteolytic cleavage of the EPO protein or using conventional chemical synthesis methods. An EPO protein or fragment thereof is “biologically active” if administration of a protein or fragment to a human leads to stimulation of red blood cell production and stimulation of division and differentiation of committed erythroid precursors in the bone marrow. The determination of the biological activity of the EPO protein is carried out using generally accepted tests well known in the art, using for this purpose one or different types of mammals. The following is an acceptable test that can be used to demonstrate biological activity.

The term “therapeutically effective amount” refers to that amount of a product comprising the erythropoietin glycoprotein that is necessary for the in vivo biological activity to increase the production of bone marrow cells by reticulocytes and red blood cells. The exact amount of the product, including the erythropoietin glycoprotein, is preferably determined by factors such as the particular type of disease to be treated, the condition of the patient being treated, and the other ingredients in the composition. Pharmaceutical compositions that contain a product comprising the erythropoietin glycoprotein can be of a form that is highly effective when administered in various ways to a sick person suffering from blood diseases, which are characterized by low production or production of abnormal red blood cells. Average therapeutically effective amounts of a product comprising erythropoietin glycoprotein may vary and, in particular, should be based on the recommendations and prescriptions of a qualified healthcare provider.

The present invention relates to products including the erythropoietin glycoprotein, which have an in vivo biological activity that causes an increase in the production of bone marrow cells of reticulocytes and red blood cells, which are represented by formula 1:

where X and Y are as defined above, m is from about 450 to about 900; n is 1-3; R is lower alkyl and P is erythropoietin glycoprotein without an amino group or amino groups which (s) form an amide bond with X. As will be described below, the preparation and purification of EPO are well known in the art. By EPO is meant a naturally-occurring or recombinant protein, preferably human, obtained from any conventional source, such as tissue, cultures of naturally-occurring or recombinant cells, as well as from protein synthesis. This term includes any proteins with EPO activity, such as muteins or other modified proteins. Recombinant EPO can be obtained by expression in CHO, BHK, or HeLa cells using the recombinant DNA method or by endogenous gene activation, i.e. when the expression of erythropoietin glycoprotein occurs as a result of endogenous gene activation. Preferred EPO species for the preparation of products comprising the erythropoietin glycoprotein are human EPO species. More preferably, the EPO species are human EPO having the amino acid sequence shown in FIG. 1 (SEQ ID NO: 1) or in FIG. 2 (SEQ ID NO: 2), most preferably the amino acid sequence of human EPO shown in FIG. 1 (SEQ ID NO: 1).

The human erythropoietin protein can also be modified by introducing at least one additional glycosylation site, for example, 1-6 additional glycosylation sites, as a result, they have the amino acid sequences shown below (but not limited to). The following conventions indicate that the sequence shown in FIG. 1 is modified by replacing the native amino acid in the position indicated by the upper index with the amino acid indicated to the left of the upper index.

Asn ³⁰ Thr ^{32 of} FIG. 1;

Asn ⁵¹ Thr ^{53 of} FIG. 1;

Asn ⁵⁷ Thr ^{59 of} FIG. 1;

Asn ^{69 of} FIG. 1;

Asn ⁶⁹ Thr ^{71 of} Figure 1;

Ser ⁶⁸ Asn ⁶⁹ Thr ⁷¹ Figure 1;

Val ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ Figure 1;

Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ Figure 1;

Ser ⁸⁷ Asn ⁸⁸ Gly ⁸⁹ Thr ⁹⁰ Figure 1;

Ser ⁸⁷ Asn ⁸⁸ Gly ⁸⁹ Thr ⁹⁰ Figure 1;

Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ Thr ^{92 of} FIG. 1;

Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ Ala ^{162 of} FIG. 1;

Asn ⁶⁹ Thr ⁷¹ Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ Figure 1;

Asn ³⁰ Thr ³² Val ⁸⁷ Asr ⁸⁸ Thr ⁹⁰ Figure 1;

Asn ⁸⁹ Ile ⁹⁰ Thr ^{91 of} Figure 1;

Ser ⁸⁷ Asn ⁸⁹ Ile ⁹⁰ Thr ^{91 of} Figure 1;

Asn ¹³⁶ Thr ^{138 of} FIG. 1;

Asn ¹³⁸ Tbr ^{140 of} FIG. 1;

Thr ^{125 of} FIG. 1 and

Pro ¹²⁴ Thr ^{125 of} Fig. 1.

The human erythropoietin protein may also be an analogue having at least one additional amino acid at the carboxyl end of the glycoprotein, the additional amino acid comprising at least one glycosylation site, i.e. the glycoprotein has a sequence comprising the sequence of human erythropoietin and a second sequence at the carboxyl end of the sequence of human erythropoietin, the second sequence containing at least one glycosylation site.

An additional amino acid sequence may include a peptide fragment derived from the carboxyl end of the human chorionic gonadotropin. Preferably, the glycoprotein is an analog selected from the group consisting of (a) human erythropoietin having the amino acid sequence SerSerSerSerLysAlaProProProSerLeuProSerProSerArgLeuProGlyProSerAspThrProIle LeuProGln (SEQ ID NO: 3) from the end of the boxbox; (b) an analogue indicated in section (a), further comprising Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ EPO; and (c) the analogue indicated in section (a), further comprising Asn ³⁰ Thr ³² Val ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ EPO.

The human erythropoietin protein may also be an analogue having an amino acid sequence that includes rearrangement of at least one glycosylation site. The rearrangement can be a deletion of any of their N-linked carbohydrate sites in human erythropoietin and the addition of an N-linked carbohydrate site at position 88 of the amino acid sequence of human erythropoietin. Preferably, the glycoprotein is an analog selected from the group consisting of Gln ²⁴ Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ EPO; Gln ³⁸ Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ EPO and Gln ⁸³ Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ EPO.

Erythropoietin analogs with additional glycosylation sites are described in EP-A 640619 to Elliot, published March 1, 1995, the contents of which are incorporated herein by reference.

In the formula 1, R may be any lower alkyl which has a linear or branched alkyl group containing from 1 to 6 carbon atoms, such as methyl, ethyl, isopropyl etc. Preferred alkyl is methyl.

In the formula 1, X is - (CH ₂ ) _k or —CH ₂ (O — CH ₂ —CH ₂ ) _k -, where k is from 1 to about 10. Preferably k is from 1 to about 4, more preferably k is 1 or 2. Most preferably, X is (CH ₂ ).

In the formula 1, Y denotes

Preferably Y is

Most preferably, Y is

In formula 1, the value of m is chosen so that the resulting conjugate of formula 1 has physiological activity comparable to the activity of unmodified EPO, this activity can be the same, higher, or only make up part of the activity of unmodified EPO. “M” refers to the number of ethylene oxide units in the PEG moiety. One PEG subunit, i.e. - (OCH ₂ CH ₂ ) -, has a molecular weight of about 44 Da. Thus, the molecular weight of the conjugate (including the molecular weight of EPO) depends on the value of m. The term “about” with respect to molecular weight means specific values that are within an acceptable range of values of this value when determined using conventional analytical methods, “m” is an integer from about 450 to about 900 (which corresponds to a molecular weight of from 20 to 40 kDa), preferably from about 550 to about 800 (which corresponds to a molecular weight of from about 24 to 35 kDa) and most preferably "m" has a value of from about 650 to about 700 (which corresponds to a molecular weight of sce from about 29 to 31 kDa).

In formula 1, “n” denotes the number of ε-amino groups of the lysine amino acid in the erythropoietin protein covalently linked to PEG via an amide bond. The conjugate of the invention may have one, two or three PEG groups per EPO molecule. “n” is an integer ranging from 1 to 3, preferably “n” is 1 or 2, and more preferably “n” is 1.

Preferred erythropoietin glycoprotein products are represented by the formulas:

where P, R, X, m and n are as defined above.

Most preferred erythropoietin-containing glycoprotein products are represented by the formula:

where P, R, X, m and n are as defined above.

Other preferred erythropoietin-containing glycoprotein products are represented by the formulas:

where P and n have the above meanings.

Most preferred erythropoietin-containing glycoprotein products are represented by the formula:

where P and n have the above meanings.

Preferred are compounds in which X is - (CH ₂ ) _k -, preferably compounds in which k is from 1 to 4, most preferably compounds in which X is - (CH ₂ ) -.

The invention also relates to the conjugates described above, in which m is an integer from 550 to 800, preferably m is an integer from 650 to 700.

Preferred compounds of the invention are those wherein n is 1 and / or R is methyl.

The invention further relates to compounds in which the average molecular weight of each poly (ethylene glycol) moiety is from about 24 to about 35 kDa, more preferably up to about 30 kDa.

In addition, the invention relates to compounds in which the glycoprotein is covalently linked to one or two blocked (lower) alkoxy groups of the poly (ethylene glycol) moieties, preferably with one blocked (lower) alkoxy group of the poly (ethylene glycol) moiety.

In a preferred embodiment, the poly (ethylene glycol) moieties are blocked by a methoxy group.

According to a most preferred embodiment, the invention relates to the above compounds in which X is - (CH ₂ ) -, m is an integer from 650 to 700, n is 1, R is methyl and the average molecular weight of each poly (ethylene glycol) moiety is approximately 30 kDa.

Another object of the invention is a method of treating anemia in humans, comprising administering to the person a therapeutically effective amount of a product of formula 1 comprising the erythropoietin glycoprotein.

The next object of the invention is a method for producing an erythropoietin-containing glycoprotein product that has an in vivo biological activity that causes an increase in the production of reticulocytes and red blood cells by bone marrow cells, which comprises the following steps:

(a) covalent interaction of the ε-amino group of the erythropoietin protein lysine represented by the formula P- [NH ₂ ] _n with a bifunctional reagent represented by the formula Z-CO-XSQ to obtain an amide-coupled intermediate represented by the formula:

P- [NH-CO-XSQ] _n ,

where P denotes an erythropoietin protein that does not contain an amino group forming an amide bond; n is an integer from 1 to 3; Z represents a reactive group, for example, NHS esters of a carboxylic acid; X is - (CH ₂ ) _k , or —CH ₂ (O — CH ₂ —CH ₂ ) _k -, where k is from 1 to about 10; and Q represents an alkanoyl type protecting group, for example acetyl;

(b) covalently reacting an amide bond intermediate obtained in step (a) with an activated polyethylene glycol derivative represented by the formula W- [OCH ₂ CH ₂ ] _m -OR to obtain an erythropoietin-containing glycoprotein product represented by the formula

where W is a sulfhydryl reactive form Y; m is an integer from about 450 to about 900; R is lower alkyl; and Y is

In this embodiment, the bifunctional reagent is preferably N-succinimidyl-S-acetylthiopropionate or N-succinimidyl-S-acetylthioacetate, Z is preferably N-hydroxysuccinimide, and the activated polyethylene glycol derivative W- [OCH ₂ CH ₂ ] _m -OR is preferably selected from the group including iodoacetylmethoxy-PEG, methoxy-PEG-vinyl sulfone and methoxy-PEG-maleimide.

Another object of the present invention is a composition containing conjugates, each of the conjugates comprising a erythropoietin glycoprotein, which has at least one free amino group and has an in vivo biological activity that causes an increase in the production of reticulocytes and red blood cells by bone marrow cells, and is selected from the group including human erythropoietin and its analogues, which have the primary structure of human erythropoietin, modified by adding from 1 to 6 glycosyliro sites or by rearrangement of at least one glycosylation site; and the glycoprotein is covalently linked to 1-3 (lower) alkoxypoly (ethylene glycol) groups, each poly (ethylene glycol) group is covalently linked to the glycoprotein via a linker of the formula -C (O) -XSY, while the C (O) -group of the linker forms an amide bond with one of these amino groups,

X is - (CH ₂ ) _k or —CH ₂ (O — CH ₂ —CH ₂ ) _k -,

k is 1-10,

Y stands for

the average molecular weight of each poly (ethylene glycol) fragment is from about 20 to about 40 kDa, and the molecular weight of the conjugate is from about 51 to about 175 kDa; and the percentage of conjugates in which n is 1 is at least 90%. Preferred the above conjugate-containing composition is a composition in which the percentage of conjugates for which n is 1 is at least 90%, more preferably in which the percentage of conjugates in which n is 1 is at least 92% and still more preferably in which the percentage of conjugates for which n is 1 is at least 96%, and most preferably in which the percentage of conjugates for which n is I is from 90 to 96%.

In addition, the present invention relates to a pharmaceutical composition comprising a conjugate or composition, as described above, and a pharmaceutically acceptable excipient, and to the use of a conjugate or composition, as defined above, for the preparation of drugs for the treatment or prevention of diseases related with anemia in patients with chronic renal failure (CRF), AIDS patients, and for the treatment of cancer patients undergoing chemotherapy. The invention further relates to a method for the prophylactic and / or therapeutic treatment of disorders including anemia in patients with chronic renal failure (CRF), AIDS patients and cancer patients undergoing chemotherapy, comprising the step of administering to the patient a composition as described above.

The invention also relates to a method for producing the conjugates or composition described above, which comprises covalently linking thiol groups with erythropoietin glycoprotein and combining the resulting activated erythropoietin glycoprotein with a poly (ethylene glycol) derivative (PEG). In addition, the invention relates to conjugates and compositions, as defined above, obtained using any of the methods described above, and to conjugates and compositions, as defined above, for the treatment of diseases associated with anemia in patients with chronic renal failure ( CRF), AIDS patients and for the treatment of cancer patients undergoing chemotherapy.

EPO protein expression methods

Erythropoietin (EPO) is a human glycoprotein that stimulates the formation of red blood cells. Its preparation and therapeutic use are described in detail, for example, in US patents 5547933 and 5621080, EP-B 0148605, Huang S.L., Proc. Natl. Acad. Sci. USA 2708-2712 (1984), EP-B 0205564, EP-B 0209539 and EP-B 0411678, as well as Lai R.N. et al., J. Biol. Chem. 261 3116-3121 (1986), Sasaki, N. et al., J. Biol. Chem. 262 12059-12076 (1987). Erythropoietin for therapeutic purposes can be obtained by recombination methods (EP-B 0148605, EP-B 0209539 and Egrie J.C., Strickland T.W., Lane J. et al., Immunobiol. 72: 213-224 (1986)). The expression of proteins, including EPO, by endogenous gene activation is well known in the art and is described, for example, in US Pat. Nos. 5,733,761, 5,641,670 and 5,733,746 and published international patent applications WO 93/09222, WO 94/12650, WO 95/31560, WO 90/11354, WO 91/06667 and WO 91/09955, the contents of each document are incorporated herein by reference.

Methods for the expression and production of erythropoietin in serum-free medium are described, for example, in WO 96/35718 in the name of Burg published on November 14, 1996 and in EP-A 513738 in the name of Koch published on June 12, 1992.

Purification Methods of Human EPO Protein

In addition to the methods described in the above publications, it is also known that in serum-free medium, fermentation of recombinant CHO cells that contain the EPO gene can be carried out. Such methods are described, for example, in EP-A 0513738, EP-A 0267678 and in general form by T. Kawamoto et al., Analytical Biochem. 130 (1983) 445-453, EP-A 0248656, by Kowar J. and Franek F. Methods in Enzymology 421 (1986) 277-292, Bavister B., Expcology 271 (1981) 45-51, EP-A 0481791, EP-A 0307247, EP-A 0343635, WO 88/00967.

EP-A 0267678 describes the use of ion exchange chromatography on S-Sepharose, preparative reverse phase HPLC on a C ₈ column, and gel filtration to purify EPO obtained in serum-free culture after dialysis. In this context, the gel filtration step can be replaced by high-speed ion exchange chromatography on S-Sepharose. It is also suggested that prior to ion exchange chromatography, dye chromatography is performed on a Blue Trisacryl column.

The purification method of recombinant EPO is described by Nobuo I. et al., J. Biochem. 107 352-359 (1990). According to this method, before the purification steps, EPO is treated with a solution of Tween®20, phenylmethylsulfonyl fluoride, ethyl maleimide, pepstatin A, copper sulfate and oxamic acid.

Several publications, including WO 96/35718 in the name Burg, published November 14, 1996, describe a method for producing erythropoietin by serum-free fermentation (EPOsf). Below, as an example, one of the methods for producing EPO is described, which is the starting product for PEGylation.

Biological analysis to determine the specific activity of EPO and EPO conjugates

The specific activity of EPO or EPO conjugates according to the present invention can be determined by various methods known in the art. The biological activity of the purified EPO proteins of the present invention is manifested in the fact that the administration of EPO protein by injection to sick people in bone marrow cells increases the production of reticulocytes and red blood cells compared with unexposed injections or control groups of people. The biological activity of EPO proteins or fragments thereof, obtained and purified according to the present invention, can be assessed using the methods described in Pharm. Europa Spec. Issue Erythropoietin BRP Bio, 1997 (2).

Another biological method for determining the activity of EPO protein, based on the use of normocytic mice, is described in example 4.

Methods for producing pegylated EPO

Methods for preparing the erythropoietin glycoprotein products represented by Formula 1 are covalently linking EPO thiol groups (“activation”) and combining the resulting activated EPO with a poly (ethylene glycol) derivative (PEG). The first step in the preparation of the PEGylated EPO of the present invention is the covalent bonding of thiol groups through the NH ₂ groups of EPO. This activation of EPO is carried out using bifunctional reagents that carry a protected thiol group and an additional reactive group, such as activated esters (e.g. succinimidyl esters), anhydrides, sulfonic acid esters, carboxylic acid and sulfonic acid halides, respectively. The thiol group is protected by groups known in the art, for example acetyl groups. These bifunctional reagents have the ability to interact with the ε-amino groups of lysine-containing amino acids by forming an amide bond. The first stage of the reaction is shown below:

where EPO, n and X have the above meanings and Z is a reactive group known in the art, for example, an N-hydroxysuccimide (NHS) substituent of the formula

In a preferred embodiment, the activation of the ε-amino groups of lysine is carried out by interaction with bifunctional reagents having a succinimidyl fragment. Bifunctional reagents can carry different forks of the spacers, for example, - (CH ₂ ) _k or -CH ₂ - (O-CH ₂ -CH ₂ -) _k fragments, where k has values from 1 to about 10, preferably from 1 to about 4 and more preferably 1 or 2 and most preferably 1. Examples of these reagents are N-succinimidyl-S-acetylthiopropionate (SATP) and N-succinimidyl-S-acetylthioacetate (SATA)

Acetylthioalkylcarbonic-NHS-ester type

2- (Acetylthio) (ethoxy) _k- acetic acid NHS ester, where k is as defined above.

The preparation of bifunctional reagents is known in the art. The precursors of 2- (acetylthio) (ethoxy) _k- acetic acid NHS esters are described in DE-3924705, and the derivatization of the acetylthio compound is described in March J. in Advanced Organic Chemistry, McGraw-Hill, 1977, 375-376. SATA is commercially available (Molecular Probes, Eugen, Oregon, USA; and Pierce, Rockford, Illinois).

The number of thiol groups that must be added to the EPO molecule can be selected by adjusting the reaction parameters, i.e. protein concentration (EPO) and protein / bifunctional reagent ratio. Preferably EPO is activated by covalent binding using from 1 to 5 thiol groups per EPO molecule, preferably from 1.5 to 3 thiol groups per EPO molecule. These ranges correspond to the statistical distribution of thiol groups in the EPO protein population.

The reaction is carried out, for example, in an aqueous buffer solution, pH 6.5-8.0, for example, in 10 mm potassium phosphate, 50 mm NaCl, pH 7.3. A bifunctional reagent can be added to DMSO. After completion of the reaction, preferably after 30 minutes, the reaction is stopped by the addition of lysine. Excess bifunctional reagent can be removed using methods well known in the art, for example, dialysis or column filtration. The average amount of thiol groups added to EPO can be determined by the photometric methods described, for example, in Grasetti D.R. and Murray J.E., J. Appl. Biotechnol., 119, 41-49 (1967).

After the above reaction, a covalent combination of an activated polyethylene glycol (PEG) derivative is carried out. Acceptable PEG derivatives are activated PEG molecules with an average molecular weight of from about 20 to about 40 kDa, more preferably from about 24 to about 35 kDa, most preferably about 30 kDa.

Activated PEG derivatives are known in the art and, for example for PEG vinylsulfone, are described by Morpurgo M. et al., J. Biocohj. Chem., P. 363 ff. (1996). Branched chain and PEG species can be used to prepare compounds of formula 1. Examples of reactive PEG reagents are iodoacetylmethoxy-PEG and methoxy-PEG-vinyl sulfone:

The use of these iodine-activated compounds is known in the art and is described, for example, by Hermanson G.T. in: Bioconjugate Techniques, Academic Press, San Diego (1996), pp. 147-148.

Most preferably, various types of PEG are activated using maleimide (alkoxy-PEG-maleimide) such as methoxy-PEG-maleimide (MM 30000; Shearwater Polymers Inc.)

The structure of alkoxy-PEG-maleimide is given below:

where R and m have the above meanings.

The most preferred derivative is

where R and m have the above meanings.

The coupling reaction with alkoxy-PEG maleimide proceeds after in situ cleavage of the thiol protecting group in an aqueous buffer solution, for example, 10 mM potassium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.2. Cleavage of the protective group can be carried out, for example, with hydroxylamine in DMSO at 25 ° C, pH 6.2 for about 90 minutes. For the PEG modification, the molar ratio of activated EPO / alkoxy-PEG maleimide should be from about 1: 3 to about 1: 6, preferably 1: 4. The reaction can be stopped by the addition of cysteine and the interaction of the remaining thiol (-SH) groups with N-methylmaleimide or other suitable compounds that can form disulfide bridges. As a result of the interaction of the remaining active thiol groups with a protecting group, such as N-methylmaleimide, or another suitable protecting group, EPO glycoproteins in the conjugates of the invention may include such protecting groups. In general, using the described process, it is possible to obtain a mixture of molecules having a different number of thiol groups protected by a different number of protective groups, depending on the number of activated thiol groups on a glycoprotein that are not conjugated to PEG-maleimide.

While N-methylmaleimide forms a similar type of covalent bond when it is used to block the remaining thiol groups on the PEGylated protein, the presence of disulfide compounds results in an intermolecular sulfide / disulfide exchange reaction to form a blocking reagent linked to the formation of the disulfide bridge. Preferred blocking agents for this type of blocking reaction are oxidized glutathione (GSSG), cysteine and cystamine. While cysteine does not introduce a pure charge into the PEGylated protein, the use of blocking reagents such as GSSG or cystamine leads to the introduction of an additional negative or positive charge.

Further purification of the compounds of formula 1, including the separation of mono-, di and tri-PEGylated EPO species, can be carried out by methods well known in the art, for example column chromatography.

Pharmaceutical Compositions

On the basis of the products of the invention comprising the erythropoietin glycoprotein according to methods known in the art, pharmaceutical compositions suitable for injection can be prepared in combination with a pharmaceutically acceptable carrier or excipient. For example, suitable compositions are described in WO 97/09996, WO 97/40850, WO 98/58660 and WO 99/07401. Among the preferred pharmaceutically acceptable carriers suitable for preparing the products of the invention are human serum albumin, human plasma proteins, etc. The compounds of the present invention can be prepared in 10 mM sodium / potassium phosphate buffer, pH 7, which contains a tonicity-enhancing agent, for example 132 mM sodium chloride. Optionally, the pharmaceutical composition may contain a preservative. The pharmaceutical composition may contain various amounts of erythropoietin, for example 10-1000 μg / ml, for example 50 or 400 μg.

Treatment of blood diseases characterized by low production or production of abnormal red blood cells

Administration of the erythropoietin glycoprotein-containing products of the present invention leads to the formation of red blood cells in humans. Thus, the administration of erythropoietin-containing glycoprotein products replenishes this EPO protein, which is important for the formation of red blood cells. Pharmaceutical compositions containing erythropoietin glycoprotein products can be of a form that is highly effective when administered to a sick person suffering from blood diseases in various ways, which is characterized by low production or production of abnormal red blood cells, which is either an independent disease or one of the symptoms of the condition or diseases. Pharmaceutical compositions may be administered by injection, such as subcutaneous or intravenous injection. The average amount of the product, including the erythropoietin glycoprotein, can vary and, in particular, should be based on the recommendations and prescriptions of a qualified healthcare provider. The exact amount of the conjugate is preferably determined by factors such as the particular type of disease to be treated, the condition of the patient being treated, and other ingredients of the composition. For example, you can enter from 0.01 to 10 μg / kg of body weight, preferably from 0.1 to 1 μg / kg of body weight, for example, once a week.

This description provides links to various publications. The contents of these publications are incorporated herein by reference in order to more fully characterize the prior art.

Below the invention is explained in more detail by examples, which serve only to illustrate the preparation of compounds and compositions according to the invention, but do not limit its scope.

EXAMPLES

EXAMPLE 1. Fermentation and purification of human EPO

a) Inoculum production and fermentation

From the Working Cell Bank created from an EPO-producing CHO cell line (ATCC CRL8695 strain described in EP 41167 (Genetic Institute) can be used), one vial is taken from the liquid storage tank nitrogen frozen in the gaseous phase. Cells are transferred to glass rotating flasks and cultured in bicarbonate-buffered medium in an incubator in a humid atmosphere of CO ₂ . A typical serum-free medium used for inoculum and fermentation is described in EP-A 513738 in the name of Koch published on June 12, 1992 or in WO 96/35718 in the name of Burg published on November 14, 1996, for example, includes DMEM / F12 (e.g., manufactured by JRH Biosciences / Hazleton Biologies, Denver, USA, serial number 57-736) and optionally sodium bicarbonate, L + glutamine, D + glucose, recombinant insulin, sodium selenite, diaminobutane, hydrocortisone, ferrous sulfate (P) , asparagine, aspartic acid, serine and a stabilizer for mammalian cells, such as, for example, polyvinyl alcohol, methyl cellulose, polydextran, polyethylene glycol, Pluronic F68, a plasma-increasing polygeline (HEMACCEL®), or polyvinylpyrrolidone (WO 96/35718).

Cultures are evaluated using a microscope with respect to the absence of contaminating microorganisms and cell density is determined. These tests are carried out at each stage of cleavage.

After the initial growth period, the cell culture is diluted with fresh medium until the initial cell density is obtained and subjected to a second growth cycle. This procedure is repeated until a culture volume of approximately 2 liters is obtained in a rotating flask. After receiving approximately 12 doubled volumes of 1-5 l of this culture, they are used as inoculum for a 10-liter fermenter designed to produce an inoculum.

After 3-5 days, the culture in a 10 liter fermenter can be used as an inoculum for a 100 liter fermenter designed to produce an inoculum.

After culturing for another 3-5 days, the culture in a 100-liter fermenter can be used as an inoculum for 1000 l of fermenter designed to produce the product.

b) Collection and separation of cells

Apply a process with periodic recharge, i.e. after reaching the desired cell density, approximately 80% of the culture is harvested. The remaining culture is replenished with fresh culture medium and cultivated until the next harvest. One production cycle consists of a maximum of 10 consecutive cell harvests: 9 partial harvests and 1 complete harvest at the end of the fermentation. Charges are made every 3-4 days.

A certain amount of collection is transferred to a chilled vessel. Cells are removed by centrifugation or filtration and discarded. The EPO-containing supernatant obtained in the centrifugation step is separately filtered and collected in a second chilled vessel. In the cleaning process, each collection is processed separately.

A typical purification method for EPO protein is described in WO 96/35718 in the name of Burg, published November 14, 1996. This purification method is described below as an example.

a) Chromatography using blue Sepharose (Blue Sepharose)

Blue Sepharose (Pharmacia) consists of Sepharose granules, to the surface of which Cibacron blue dye is covalently bonded. Since EPO is more strongly associated with Blue Sepharose than most non-protein contaminants, some protein contaminants and PVA (polyvinyl acetate), EPO levels may be increased at this stage. Elution of the filled Blue Sepharose column is carried out by increasing the concentration of salt, as well as the pH value.

The column is filled with 80-100 L of Blue Sepharose, regenerated with NaOH and equilibrated with equilibration buffer (sodium / calcium chloride and sodium acetate). The acidified and filtered supernatant obtained from the fermenter is charged. After loading is completed, the column is washed first with a buffer similar to a balancing buffer that contains a higher concentration of sodium chloride, and then with Tris buffer. The product is eluted with Tris buffer and collected as a single fraction in accordance with the main elution profile.

b) Chromatography using Butyl Toyopearl

Butyl Toyopearl 650 C (Toso Haas) is a matrix based on polystyrene with which aliphatic butyl moieties are covalently bonded. Since EPO binds more strongly to this gel than most contaminants and PVA, it can be eluted using an isopropanol-containing buffer.

The column is filled with 30-40 L Butyl Toyopearl 650 C, regenerated with NaOH, washed with Tris buffer and equilibrated with Tris buffer containing isopropanol.

The concentration of isopropanol in the eluate obtained by Blue Sepharose chromatography was adjusted to the concentration of isopropanol in equilibration buffer and loaded onto a column. Then the column is washed with equilibration buffer with a high concentration of isopropanol. The product is eluted with elution buffer (Tris buffer with a high concentration of isopropanol) and collected as a single fraction in accordance with the main elution profile.

c) Chromatography using hydroxyapatite ultragel (Hydroxyapatite Ultragel)

Hydroxyapatite Ultragel (Biosepra) consists of hydroxyapatite incorporated into an agarose matrix to improve mechanical properties. EPO has a low affinity for hydroxyapatite and, as a result, can be eluted with lower phosphate concentrations than protein contaminants.

The column was filled with 30-40 L of Hydroxyapatite Ultragel and regenerated with a buffer containing potassium phosphate / calcium chloride and NaOH, and then with Tris buffer. Then it is balanced with Tris buffer low in isopropanol and sodium chloride.

The eluate obtained by Butyl Toyopearl chromatography is loaded onto the column. The column is washed sequentially with equilibration buffer and Tris buffer without isopropanol and sodium chloride. The product is eluted with Tris buffer low in isopropanol and sodium chloride and collected as a single fraction according to the main elution profile.

d) reverse phase HPLC on Vydac C4 material

The material for Vydac C4 HPLC-V (Vydac company) consists of silica gel particles with C4 alkyl chains on their surface. The separation of EPO from protein contaminants is based on differences in the strength of hydrophobic interactions. Elution is carried out in a gradient of acetonitrile in diluted trifluoroacetic acid.

Preparative HPLC was performed using a stainless steel column (filled with 2.8-3.2 L of Vydac C4 silica gel). Hydroxyapatite Ultragel chromatography eluate was acidified by the addition of trifluoroacetic acid and loaded onto a column filled with Vydac C4. A gradient of acetonitrile in diluted trifluoroacetic acid is used for washing and elution. Fractions are collected and immediately neutralized with phosphate buffer. Collect fractions containing EPO that comply with IPC requirements.

e) Chromatography using DEAE-Sepharose

DEAE-Sepharose (Pharmacia) consists of diethylamino-ethyl (DEAE) groups covalently bonded to the surface of Sepharose granules. The binding of EPO to DEAE groups is mediated by ionic interactions. Acetonitrile and trifluoroacetic acid were passed through the column without delay. After these substances are washed, trace amounts of contaminants are removed by washing the column with acetate buffer with low pH. The column is then washed with neutral phosphate buffer and EPO is eluted with a buffer with increased ionic strength.

The column is filled with DEAE-Sepharose designed for high flow chromatography. The column volume is adjusted in such a way as to guarantee loading of EPO at the rate of 3-10 mg EPO / ml gel. The column is washed with water and equilibrated with buffer (sodium / potassium phosphate). The pooled fractions obtained after elution using HPLC were loaded and the column was washed with equilibration buffer. Then, the column is washed with washing buffer (sodium acetate buffer), and then washed with equilibration buffer. After that, EPO is eluted from the column using an elution buffer (sodium chloride, sodium / potassium phosphate) and collected as a single fraction in accordance with the main elution profile.

The eluate obtained from the column filled with DEAE-Sepharose, adjusted to a certain conductivity. The resulting substance for the preparation of a medicinal product is filtered under sterile conditions into Teflon vessels (Teflon) and stored at -70 ° C.

EXAMPLE 2. Covalent binding of thiol groups to EPO

This example describes the results of determining the reaction conditions for covalent binding of thiol groups to EPO. To determine the conditions, various amounts of a reagent containing a blocked thiol group, in this case SATA or SATP (dissolved in DMSO up to 10 mg / ml), are added to the EPO solution, in this case to 1 ml of EPO solution with a concentration of 5 mg / ml in 10 mM potassium phosphate, 50 mM NaCl, pH 7.3. The reaction mixture was stirred for about 30 minutes (25 ° C) and the reaction was stopped by adding a 1 M lysine solution in an amount of 1 mM. Excessive amounts of SATA and SATP are removed by dialysis in countercurrent flow with 10 mM potassium phosphate, 50 mM NaCl and 2 mM EDTA, pH 6.2. After removal of the protective acetyl group using hydroxylamine, the amount of thiol groups covalently linked to EPO is determined photometrically using dithiodipyridine according to the method described by Grasetti D.R. and Murray J.E. In: Appl. Biochem. Biotechnol. 119, pp. 41-49 (1967).

The table presents data on the number of thiol groups covalently linked to the EPO molecule

EXAMPLE 3. Modification of activated EPO using methoxy-PEG-maleimide

A) Activation of EPO

100 mg of EPO obtained according to example 1 (190,000 international units (IU) / mg, determined by analysis using normocytic mice), activate SATA (molar ratio EPO: SATA = 1/5) according to the method described in example 2. Obtained as a result, EPO ("activated EPO") carrying covalently bound blocked thiol groups is separated from by-products such as N-hydroxysuccinimide or unreacted SATA by dialysis according to the method described in example 1. A solution containing 4.5 mg / ml of activated EPO in 10 mM potassium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.2.

B) PEGylation of activated EPO

380 mg of methoxy-PEG maleimide, having, as illustrated above, the “most preferred” structure (MM 30,000; Shearwater Polymers Inc., Hunstville (Alabama, USA)), is dissolved in the above solution containing 95 mg of activated EPO (4 5 mg / ml in 10 mM potassium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.2). As a result, the molar ratio of activated EPO and methoxy-PEG maleimide in solution is 1: 4. By adding a 1 M aqueous solution of hydroxylamine to 30 mM, a pH of 6.2 of the solution described above, the covalently bound blocked thiol groups of activated EPO are released. As a result, in the solution of activated EPO in the reaction mixture are free thiol (-SH) groups. After the thiol groups are unblocked, a coupling reaction is immediately carried out between activated EPO, now already containing free thiol (-SH) groups, and methoxy-PEG-maleimide for 90 minutes (stirring, 25 ° C.). The coupling reaction is terminated by adding a 0.2 M aqueous cysteine solution to a 2 mM reaction mixture. After 30 minutes, the excess of free thiol groups of activated EPO that did not react with methoxy-PEG maleimide was blocked by adding a 0.5 M solution of N-methylmaleimide in DMSO until a concentration of 5 mM was reached. After 30 minutes, the resulting reaction mixture, already containing PEGylated EPO species, was dialyzed in countercurrent with 10 mM potassium phosphate, pH 7.5 for ≥15 hours.

C) Purification of PEGylated EPO species

The following purification process is carried out to isolate PEGylated EPO species from the reaction mixture: 50 ml of a Q-Sepharose ff column is equilibrated with 10 mM potassium phosphate, pH 7.5. The reaction mixture obtained in step B) is loaded onto a column (flow rate: 3 column volumes (OK) / h). To separate unreacted methoxy-PEG-maleimide, the column was washed with 5 OK with 10 mM potassium phosphate, pH 7.5. Pegylated EPO species are separated by elution with an increasing salt gradient in 5 OK buffers (10 mM potassium phosphate, pH 7.5) and 5 OK buffers B (10 mM potassium phosphate, 500 mM NaCl, pH 7.5) at a flow rate of 3 OK / h . Due to the presence of a NaCl gradient, the PEGylated EPO species (tri-, di- and mono-PEGylated EPO species) are first eluted, and then the non-PEGylated EPO species. The eluate fraction containing PEGylated EPO species (tri-, di- and mono-PEGylated EPO species) is combined and filtered (filtration under sterile conditions using a 0.2 μm filter).

The content and purity of tri-, di- and mono-PEGylated EPO species are evaluated using SDS-PAGE with Coomassie brilliant blue staining (Laemmli, Nature, 227, 680-685 (1970)), and the protein concentration is determined at 280 nm according to Beer's law -Lamberta. The apparent molecular weight of the EPO species, determined by SDS-PAGE electrophoresis, is approximately 68 kDa (mono-PEGylated EPO species), approximately 98 kDa (di-PEGylated EPO species) and approximately 128 kDa (tri-PEGylated EPO species).

Additional separation of tri-, di- and mono-PEGylated EPO species can be carried out by chromatography, for example gel filtration (Superdex, pg 200; Pharmacia).

The determination of the biological activity of an in vivo eluate containing tri-, di- and mono-PEGylated species is carried out according to the method described in example 4.

EXAMPLE 4. In vivo activity of pegylated EPO, determined using analysis using normocytic mice

Biological analysis using normocytic mice is known in the art (Pharm. Europa Spec. Issue Erythropoietin BRP Bio, 1997 (2)), this method is described in the monograph on erythropoietin Ph. Eur. BRP Samples diluted with BSA-PBS. Normal healthy mice aged 7-15 weeks are injected subcutaneously with 0.2 ml of EPO fraction containing unPEGylated EPO or tri-, di- or mono-PEGylated EPO obtained according to the method described in example 2. Within 4 days, starting after 72 h after administration, take blood by puncture of the tail vein and dilute so that in 1 ml of a solution stained with 0.15 μm acridine orange was 1 μl of blood. The staining time is 3-10 minutes. The number of reticulocytes is determined microfluorometrically using a flow cytometer, analyzing a histogram of red fluorescence. The number of reticulocytes is given in the form of absolute values (for 30,000 red blood cells analyzed). Data are presented for a group of 5 mice every day, with mice taking blood only once.

EPO bound to methoxy-PEG maleimide described in Example 3, unmodified EPO and buffer solution are administered to mice. The results are shown in figure 3. The results indicate a higher activity and prolonged half-life of PEGylated EPO species, which is characterized by a significant increase in the number of reticulocytes and a shift in the maximum number of reticulocytes when using the same dose per mouse.

Claims (39)

1. The conjugate, which includes the erythropoietin glycoprotein, which has at least one free amino group and has an in vivo biological activity that causes an increase in the production of reticulocytes and red blood cells by bone marrow cells, and which is selected from the group consisting of human erythropoietin and its analogues, which have the sequence human erythropoietin, modified by adding from 1 to 6 glycosylation sites or rearrangement of at least one glycosylation site, this glycopr oein is covalently linked to 1-3 (lower) alkoxypoly (ethylene glycol) groups, each poly (ethylene glycol) group is covalently linked to a glycoprotein via a linker of the formula -C (O) -XSY, while the C (O) -group of the linker forms an amide bond with one of these amino groups, X is - (CH ₂ ) _k - or -CH ₂ (O-CH ₂ -CH ₂ ) _k -, k is 1-10, Y stands for

the average molecular weight of each poly (ethylene glycol) fragment is about 20-40 kDa and the molecular weight of the conjugate is about 51-175 kDa. 2. The conjugate according to claim 1 of the formula

where X and Y have the meanings indicated in claim 1, m is 450-900; n is 1-3; R is lower alkyl and P is erythropoietin glycoprotein without an amino group or amino groups which (s) form an amide bond with X. 3. The conjugate according to any one of the preceding claims

where P, R, X, m and n have the meanings specified in paragraph 2. 4. The conjugate according to claim 1 or 2 of the formula

where P, R, X, m and n have the meanings specified in paragraph 2. 5. The conjugate according to any one of the preceding paragraphs, where X denotes - (CH ₂ ) _k -. 6. The conjugate according to claim 5, where k is 1-4. 7. The conjugate according to any one of the preceding paragraphs, where X is —CH ₂ -. 8. The conjugate according to any one of the preceding paragraphs, where m denotes an integer from 550 to 800. 9. The conjugate according to any one of the preceding paragraphs, where m denotes an integer from 650 to 700. 10. The conjugate according to any one of the preceding paragraphs, where n is 1. 11. The conjugate according to any one of the preceding paragraphs, where R is methyl. 12. The conjugate according to any one of the preceding paragraphs, where the average molecular weight of each poly (ethylene glycol) fragment is approximately 24-35 kDa. 13. The conjugate according to any one of the preceding paragraphs, where the average molecular weight of each poly (ethylene glycol) fragment is approximately 30 kDa. 14. The conjugate according to any one of the preceding paragraphs, where the glycoprotein is covalently linked to one or two blocked (lower) alkoxy groups of poly (ethylene glycol) fragments. 15. The conjugate according to any one of the preceding paragraphs, where the poly (ethylene glycol) fragments are blocked by a methoxy group. 16. The conjugate according to any one of the preceding paragraphs, where X is —CH ₂ -, m is an integer from 650 to 700, n is 1, R is methyl, and the average molecular weight of each poly (ethylene glycol) moiety is about 30 kDa. 17. The conjugate according to any one of the preceding paragraphs, where the erythropoietin glycoprotein is human erythropoietin. 18. The conjugate according to any one of the preceding paragraphs, where the erythropoietin glycoprotein is expressed by endogenous gene activation. 19. The conjugate according to any one of the preceding paragraphs, where the erythropoietin glycoprotein has the sequence shown in figure 1 (SEQ ID NO: 1) or in figure 2 (SEQ ID NO: 2). 20. The conjugate according to claim 19, where the erythropoietin glycoprotein has the sequence shown in figure 1 (SEQ ID NO: 1). 21. The conjugate according to any one of claims 1 to 16, where the glycoprotein has a human erythropoietin sequence modified by adding from 1 to 6 glycosylation sites. 22. The conjugate according to any one of claims 1 to 6, 8 and 10, where the glycoprotein has a human erythropoietin sequence modified by a modification selected from the group including Asn ³⁰ Thr ³² ; Asn ⁵¹ Thr ⁵³ ; Asn ⁵⁷ Thr ⁵⁹ ; Asn ⁶⁹ ; Asn ⁶⁹ Thr ⁷¹ ; Ser ⁶⁸ Asn ⁶⁹ Thr ⁷¹ ; Val ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ ; Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ ; Ser ⁸⁷ Asn ⁸⁸ Gly ⁸⁹ Thr ⁹⁰ ; Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ Thr ⁹² ; Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ Ala ¹⁶² ; Asn ⁶⁹ Thr ⁷¹ Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ ; Asn ³⁰ Thr ³² Val ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ ; Asn ⁸⁹ Ile ⁹⁰ Thr ⁹¹ ; Ser ⁸⁷ Asn ⁸⁹ Ile ⁹⁰ Thr ⁹¹ ; Asn ¹³⁶ Thr ¹³⁸ ; Asn ¹³⁸ Thr ¹⁴⁰ ; Thr ¹²⁵ and Pro ¹²⁴ Thr ¹²⁵ . 23. The conjugate according to any one of the preceding paragraphs, where the glycoprotein has a sequence comprising a sequence of human erythropoietin and a second sequence at the carboxyl end of the sequence of human erythropoietin, and the second sequence contains at least one glycosylation site. 24. The conjugate according to item 23, where the second sequence includes a sequence derived from the sequence of the carboxyl end of the human chorionic gonadotropin. 25. The conjugate according to paragraph 24, where the glycoprotein has a sequence selected from the group including (a) human erythropoietin having the amino acid sequence SerSerSerSerLysAlaProProProSerLeuProSerProSerArgLeuProGlyProSerAspThrProIleLeuProGln (SEQ ID NO: 3) extending from the carboxyl end; (b) the sequence indicated in (a) modified with Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ ; and (c) the sequence indicated in (a) modified with Asn ³⁰ Thr ³² Val ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ . 26. The conjugate according to any one of claims 1 to 16, where the glycoprotein has a human erythropoietin sequence modified by rearrangement of at least one glycosylation site. 27. The conjugate of claim 25, wherein the rearrangement comprises a deletion of any of the N-linked carbohydrate sites in human erythropoietin and the addition of an N-linked carbohydrate site at position 88 of the human erythropoietin sequence. 28. The conjugate of claim 14, wherein the glycoprotein has a human erythropoietin sequence modified by a modification selected from the group consisting of Gln ²⁴ Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ EPO; Gln38Ser ⁸⁷ Asn88Thr90EPO; and Gln ⁸³ Ser ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ EPO. 29. The conjugate according to any one of claims 1 to 28, intended for the treatment of diseases associated with anemia in patients with chronic renal failure (CRF), AIDS patients and for the treatment of cancer patients undergoing chemotherapy. 30. A pharmaceutical composition for treating diseases related to anemia in patients with chronic renal failure (CRF), AIDS patients and for treating cancer patients undergoing chemotherapy, comprising conjugates, where each of the conjugates includes erythropoietin glycoprotein having at least one a free amino group and possessing in vivo biological activity, causing an increase in the production of reticulocytes and red blood cells by bone marrow cells, and selected from the group comprising human erythropoietin and its analogues, which have the sequence of human erythropoietin modified by adding from 1 to 6 glycosylation sites or by rearrangement of at least one glycosylation site, this glycoprotein is covalently linked to 1-3 (lower) alkoxypoly (ethylene glycol) groups, each poly ( ethylene glycol) group is covalently linked to the glycoprotein via a linker of the formula -C (O) -XSY, while the C (O) -group of the linker forms an amide bond with one of these amino groups, X denotes - (CH ₂ ) _k - or -CH ₂ ( O-CH ₂ -CH ₂ ) _k -, k about means 1-10, Y means

the average molecular weight of each poly (ethylene glycol) fragment is about 20-40 kDa and the molecular weight of the conjugate is about 51-175 kDa, in which at least 90% of the conjugates are conjugates in which n is 1, and pharmaceutically acceptable excipients. 31. The composition of claim 30, wherein the percentage of conjugates in which n is 1 is at least 92%. 32. The composition of claim 30, wherein the percentage of conjugates in which n is 1 is 90-96%. 33. The composition comprising the conjugate according to any one of claims 1 to 27, where the percentage of conjugates in which n is 1 is at least 90%. 34. The composition according to p, where the percentage of conjugates in which n is 1 is at least 92%. 35. The composition of claim 33, wherein the percentage of conjugates in which n is 1 is at least 96%. 36. The composition according to p, where the percentage of conjugates in which n is 1, is 90-96%. 37. A pharmaceutical composition having in vivo biological activity, causing an increase in the production of reticulocytes and red blood cells by bone marrow cells, comprising the composition according to any one of claims 30-36 or the conjugate according to any one of claims 1 to 29 and a pharmaceutically acceptable excipient. 38. A method for the prophylactic and / or therapeutic treatment of disorders including anemia in patients with chronic renal failure (CRF), AIDS patients and cancer patients undergoing chemotherapy, comprising the step of administering to the patient a conjugate according to any one of claims 1 to 29 or a composition according to any one pp. 30-37. 39. A method of producing a conjugate according to any one of claims 1 to 29 or a composition according to any one of claims 30 to 37, which provides (a) covalent interaction of the e-amino group of a lysine of an erythropoietin protein represented by the formula P- [NH ₂ ] _n with a bifunctional reagent represented by the formula ZOXSQ to obtain an amide-coupled intermediate represented by the formula

where P denotes an erythropoietin protein containing no amino group forming an amide bond; n is an integer from 1 to 3; Z is a reactive group; X is (CH ₂ ) _k or —CH ₂ (O — CH ₂ —CH ₂ ) _k -, where k is from 1 to about 10; Q is a protecting group; (b) covalently reacting an amide-bonded intermediate obtained in step (a) with an activated polyethylene glycol derivative represented by the formula W- [OCH ₂ CH ₂ ] _m -OR to give an erythropoietin-containing glycoprotein product represented by the formula

where W is a sulfhydryl reactive form Y; m is an integer from about 450 to about 900; R is lower alkyl and Y is

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