UA73468C2 - Conjugate of erythropoietin, compositions containing it, and methods for prevention and/or treatment of diseases - Google Patents

Abstract

The present invention refers to conjugates of erythropoietin with poly(ethylene glycol) comprising an erythropoietin glycoprotein having at least one free amino group and having the in vivo biological activity of causing bone marrow cells to increaseproduction of reticulocytes and red blood cells and selected from the group consisting of human erythropoietin and analogs thereof which have sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site; said glycoprotein being covalently linked to "n" poly(ethylene glycol) groups of the formula -CO-(CH2)x-(OCH2CH2)m-OR with the carbonyl of each poly(ethylene glycol) group forming an amide bond with one of saidamino groups; wherein R is lower alkyl; x is 2 or 3; m is about 450 to about 900; n is from 1 to 3; and n and m are chosen so that the molecular weight of the conjugate minus the erythropoietin glycoprotein is from 20 kilodaltons to 100 kilodaltons.

Description

Description of the invention

Erythropoiesis is the process of producing erythrocytes, which occurs in order to compensate for 2 destruction of cells. Erythropoiesis is a regulated physiological mechanism that ensures the level of availability of erythrocytes sufficient for the appropriate saturation of tissues with oxygen. Natural human erythropoietin (PERO) is produced by the kidneys and is a humoral plasma factor that stimulates the production of erythrocytes (Carnot P. (Sagpoi R.) and Deflander K. (OyePapage S.) (1906), S. MK. Asai. 5si., 143:432; Erslev

A.J. (EgviemAu.) (1953), Viosd, 8:349; Reissmann K.R. (Keiizztapp KK) (1950), Viosa, 5:372; Jacobson L.O. 70 (dasorzop I.0.), Goldwasser E. (SoIdulazzeg E.), Freud V. (Rgeii MU.) and Plzak L.F. (Rigak I.B.) (1957), Maishge 179:6331-4). Natural ERO stimulates the division and differentiation of committed erythrocyte progenitor cells in the bone marrow and exerts its biological activity by binding receptors on erythrocyte progenitor cells (Krantz BS (Kgapig 8.5.) (1991), Viosa 77:419).

Erythropoietin was obtained biosynthetically using recombinant DNA technology (Egri 12 J.K. (Edgie 9.S.), Strickland T.V. (ZiisKiapa T.M/), Lane J. (Iape .) and others, (1986), Ittiporbio|. 72:213-224); said erythropoietin is a product of a cloned human EPO gene that was inserted and expressed in Chinese hamster ovary tissue cells (CHO cells). The primary structure of the predominant fully processed form of PERO is illustrated by Sequence Mo 1. There are two disulfide bridges between Suz"-Suv'b! and Suv?9-Suv3Z. The molecular weight of the EPO polypeptide chain without the sugar component is 18,236 daltons. In an intact EPO molecule, approximately 4,095 molecular mass is accounted for by hydrocarbon groups that glycosylate the protein at the glycosylation sites of the mentioned protein (Sasaki G. (Zazaki N.), Bothner B. (Voijpeg

V.), Dell A. (Oeiyi A.) and Fukuda M. (RGiKiada M.) (1987), 9. VioY. Spet., 262:12059).

Since human erythropoietin is an integral part of the process of formation of erythrocytes, the mentioned hormone is suitable for the treatment of blood diseases characterized by a low level or impaired production of ch 29 erythrocytes. Under clinical conditions, ERO is used in the treatment of anemia in patients with chronic renal failure (CKD) (EshbachJ.V. (Evzsprasi U.MU.), Egri J.K. (Edgi 9U.S.), Downing M.R. (YUOzhm/Pipd M.K.) and others, (1987) MEM 316:73-78; Eshbach J.V. (Ezsprasii U.MU.), Abdulgadi M.H. (Avrashnadi M.N.), Brown J. K. (Vhom/pe .).K.) and others, (1989) App. Ipiegp. Med. 111:992; EgriJ.K. (Edgie U. S.), Eshbach J. V. (Ezsprasy ..MU. ), MakPr T. (MesOtsige T.), Adamson JW (Adatwop MU.) (1988) Kiapeu Ipi. 33:262; Lim VS (it M.5.), Degovin. RL. co (Oegsdomip K.I), Zavala D. (lamaia 0.) and others, (1989) App. Ipiegp. May. 110:108-114), AIDS and cancer patients undergoing chemotherapy (Danna R .P. (YuOappa K.R.), Rudnik S.A. (Kiapisk 5.A.), Abels

Р.

Ipiegpayopa! Reggeresiime, New York, NY: Magse! YuekKeg; 1990: shor. 301-324). However, the bioavailability of commercially available protein therapeutics, such as EPO, is limited by their short plasma half-life and susceptibility to protease cleavage. These shortcomings prevent their maximum use in clinical practice.

The present invention provides an erythropoietin conjugate, and said conjugate includes an erythropoietin glycoprotein having at least one free amino group and a biological activity, i.e., that said glycoprotein induces bone marrow cells to increase the production of reticulocytes and erythrocytes, and -o it is selected from the group consisting of human erythropoietin and its analogues, which have the sequence c of human erythropoietin, modified by adding from 1 site to 6 sites of glycosylation or rearrangement: with" at least one site of glycosylation; said glycoprotein is covalently bound to "n" poly(ethylene glycol) groups of the formula -СО-(СНоХ-(ОСНоСНо)п-ОК, where -СО (ie, the carbonyl) of each poly(ethylene glycol) group forms an amide bond with one of said amino groups; where K is lower alkyl; X-1 15 is 2 or 3; t is from about 450 to about 900; H is 1-3; and n and t are selected such that the molecular weight of said conjugate minus glycoprotein erythropoietin is from 20

Ge) kilodaltons to 100 kilodaltons. This invention additionally provides compositions that include the conjugates described above, and the percentage of said conjugates in said composition, where n is 1, is at least ninety percent. (o) 50 Compared to unmodified EPO (i.e. EPO without attached polyethylene glycol (PES)) and traditional REOS-ERO co-conjugates, the conjugates of the present invention have an increased half-life in the circulatory system and plasma residence time, reduced elimination time from the body and increased clinical activity of ip mimo.

Conjugates according to this invention have the same application options as ERO. In particular, the mentioned conjugates of the present invention are suitable for treating patients by stimulating the division and differentiation of committed erythrocyte progenitor cells in the bone marrow in the same way that ERO is used for

HF) treatment of patients. 7 The present invention provides conjugates, said conjugates comprising an erythropoietin glycoprotein having at least one free amino group and a biological activity of ip mimo, wherein said glycoprotein induces bone marrow cells to increase the production of reticulocytes and erythrocytes, and it is selected from the group consisting of human erythropoietin and its analogs having a sequence of human erythropoietin modified by adding from 1 site to 6 glycosylation sites or rearranging at least one glycosylation site; said glycoprotein is covalently bound to "n" poly(ethylene glycol) groups of the formula -СО-(СН.)-(ОСНоСНо) OK, where -СО (i.e., carbonyl) of each poly(ethylene glycol) group forms an amide bond with one from the mentioned amino groups; where K is lower alkyl; X is equal to 2 or 3; t is from about 450 to about 900; n equals 1-3; and p and t are selected such that the molecular weight of said conjugate minus the erythropoietin glycoprotein is from 20 kilodaltons to 100 kilodaltons.

It was found that the conjugates of this invention can be used in the same way as unmodified EPO. However, the conjugates of the present invention have increased circulatory half-life and plasma residence time, reduced elimination time, and increased clinical activity. Due to the mentioned improved properties, the conjugates of the present invention can be administered once a week instead of three times a week in the case of unmodified EPO. It is believed that the reduced frequency of administration will improve the patient's compliance with the treatment with a consequent improvement in the final result of the treatment, as well as an improvement in the patient's quality of life. Compared to traditional EPO conjugates linked to 7/0 Ppoly(ethylene glycol), it was found that conjugates having the molecular weight and linker structure of the conjugates of this invention have increased activity, stability, plane under curve, increased half-life in the circulatory system and improved cost profile.

The conjugates of the present invention can be administered to patients in a therapeutically effective amount in the same manner as EPO. A therapeutically effective amount is the amount of conjugate necessary to ensure the biological activity of ip mimo, which forces bone marrow cells to increase the production of reticulocytes and erythrocytes. The exact amount of conjugate is a subjective point, which is subject to the influence of such factors as the exact determination of the condition to be treated, the condition of the patient to be treated, and also other ingredients of said composition. For example, from 0.01 μg/kg to 1 μg/kg of body weight, preferably from 0.1 μg/kg to 1 μg/kg of body weight can be administered, for example, once a week.

Pharmaceutical compositions comprising said conjugate may be formulated at a concentration effective for administration by various means to a human patient suffering from a blood disorder characterized by low or impaired production of red blood cells. Average therapeutically effective amounts of said conjugate may vary and, in particular, should be based on the recommendations and prescriptions of a qualified physician. high school

Said glycoprotein products of erythropoietin obtained according to the present invention can be incorporated into pharmaceutical compositions suitable for injection with a pharmaceutically acceptable carrier or diluent by methods known in the art. For example, the description of the relevant compositions was given in applications UMO 97/09996, UMO 97/40850, UUO 98/58660 and MO 99/07401. Preferred pharmaceutically acceptable carriers for the preparation of dosage forms of the products of this invention include human serum albumin, plasma proteins, etc. The compounds of the present invention can be incorporated into a dosage form together with 10 mM sodium/potassium phosphate buffer (pH 7) containing an isotonic agent, for example, 132 mM sodium chloride. Optionally, the composition of the mentioned pharmaceutical composition may include a preservative. Different amounts of erythropoietin can be included in g of the mentioned pharmaceutical composition, for example, from 1 Ωkg/ml to 100 Ωkg/ml, for example, 5 Ωkg or 40 Ωkg. and)

The term "erythropoietin" or "EPO" refers to a glycoprotein having an amino acid sequence represented by Sequence Mo 71 or Sequence Mo 2, or an amino acid sequence substantially homologous to those whose biological properties are associated with the stimulation of erythrocyte production. and stimulating the division and differentiation of committed erythrocyte progenitor cells in the bone marrow. As used in this description, the mentioned terms mean such proteins that have been modified either intentionally, for example, by site-directed mutagenesis, or accidentally, through mutations. These terms also mean analogs having from 1 additional site to 6 additional glycosylation sites, analogs having at least one additional amino acid at the carboxy-terminal region of said glycoprotein, wherein said additional amino acid includes at least one glycosylation site, and analogs that have an amino acid sequence that includes rearrangement of at least one glycosylation site. These

The terms mean both natural human erythropoietin and human erythropoietin obtained by -I recombinant DNA methods.

Said conjugates of erythropoietin according to the present invention can be represented by Formula 1: (95)

Ge) 50 where x, t, n and K correspond to the definition that was given before. In Formula I, P is a residue of erythropoietin glycoprotein, the description of which is given (for example, without an amino group or amino groups that form an amide bond with the mentioned carbonyl group, which is represented in Formula I), which has a biological activity that is in forcing bone marrow cells to increase the production of reticulocytes and erythrocytes. 59 According to the preferred embodiment of the present invention, K is methyl. According to the preferred option, t is equal to

GF) is about 650 to about 750, and n is preferably 1.

HF According to the most preferred version of the embodiment of the present invention, K is methyl, t is from approximately 650 to approximately 750, and n is 1, i.e., the mentioned conjugate, according to the definition given before, has the formula 69 Tonao(snosno)psNgsNoSNoSO- M P where t is from 650 to 750, n is 1 and P corresponds to the definition that was given before. According to the preferred option, t is approximately 680 on average.

According to a preferred option, the mentioned glycoprotein conjugate, the definition of which was given before, is human erythropoietin. Human erythropoietin and analog proteins, as defined previously, can be expressed by activation of an endogenous gene. Preferred human erythropoietin glycoproteins are glycoproteins having Sequence Mo 1 and Sequence Mo 2, most preferred being glycoproteins having Sequence Mo 1.

In addition to this, P can be selected from the group consisting of residues of human erythropoietin and its analogs having from 1 additional site to b additional glycosylation sites. As described in more detail below, the preparation and purification of EPO is well known in the art. The mentioned term EPO means a natural or recombinant protein, preferably human, which has been obtained from any conventional source, for example, tissues, by protein synthesis, using cell culture with natural or recombinant cells. The scope of the mentioned term covers any protein that has 70 EPO activity, for example, mutins or proteins that have been modified in some other way. Recombinant EPO can be obtained by expression in CHO, BNK or Neg cell lines! and, using recombinant DNA methods or endogenous gene activation. The expression of proteins, including EPO, by activation of an endogenous gene is well known in the art and is disclosed, for example, in US Patent Nos. 5,733,761, 5,641,670 and 5,733,746 and international application publications MoMo UUO 93/09222, MO 94/12650, MO 95 /31560, UMO 90/11354, MO 91/06667 and

MO 91/09955; each of said documents is incorporated herein by reference. The preferred type of EPO for obtaining glycoprotein products of erythropoietin is human EPO. In a more preferred embodiment, said species of EPO is human EPO having an amino acid sequence represented by Sequence Mo 1 or Sequence Mo 2, in a more preferred embodiment having an amino acid Sequence Mo 1.

According to one of the variants of the embodiment of the present invention, P can be a residue of a glycoprotein analog that has from 1 additional site to 6 additional glycosylation sites. Protein glycosylation, with the help of one or more oligosaccharide groups, occurs at certain sites along the polypeptide backbone and significantly affects the physical properties of said protein, namely, protein stability, secretion, subcellular localization, and biological activity. As a rule, two types of glycosylation occur. O-linked oligosaccharides are attached to serine or threonine residues, while M-linked oligosaccharides are attached to asparagine residues. One type of oligosaccharide that is part of both M-linked and O-linked oligosaccharides is M-acetylneuraminic acid (sialic acid), which is i) a family of amino sugars that contain 9 or more carbon atoms. Sialic acid is usually the terminal residue of both M-linked and O-linked oligosaccharides and, since it carries a negative charge, it imparts acidic properties to said glycoprotein. The composition of human erythropoietin, which has 165 amino acids, includes three M-linked oligosaccharide chains and one O-linked oligosaccharide chain, which make up approximately 4095 of the total molecular weight of the mentioned glycoprotein. X,

M-linked glycosylation occurs on asparagine residues located in positions 24, 38 and "g 83; O-linked glycosylation occurs on the serine residue located at position 126. The mentioned oligosaccharide chains are modified by terminal sialic acid residues. As a result of enzymatic me)

35 removal of all sialic acid residues from the resulting glycosylated erythropoietin results in the loss of ip imimo activity, but not ip migo activity, since sialylation of erythropoietin prevents its binding (with subsequent removal) by the renal binding protein.

The glycoproteins of the present invention include analogs of human erythropoietin with one or more substitutions in the amino acid sequence of human erythropoietin, resulting in an increase in the number of sialic acid attachment sites. Said glycoprotein analogues can be obtained by site-directed mutagenesis with additions, deletions or changes of amino acid residues that increase or change the number of sites available for glycosylation. Glycoprotein analogs, the levels of sialic acid content of which;" exceed the corresponding levels of content in human erythropoietin, obtained, as a rule, by adding glycosylation sites that do not disturb the secondary or tertiary conformation necessary for biological activity.

The mentioned glycoproteins according to this invention also include analogs that have increased levels of addition of -I hydrocarbons at the glycosylation site, which, as a rule, involve the replacement of one or more amino acids in the immediate vicinity of the M-linked or O-linked site. The glycoproteins of the present invention also include analogs having one or more amino acids protruding from the carboxy-terminal region of their erythropoietin and providing at least one additional hydrocarbon site. Glycoproteins according to this invention 5p also include analogs that have an amino acid sequence that includes the rearrangement of at least one

Me. glycosylation site. Such rearrangement of the glycosylation site involves the deletion of one or more glycosylation sites of human erythropoietin and the addition of one or more natural glycosylation sites.

Increasing the number of hydrocarbon chains on erythropoietin, and thus the amount of sialic acid on erythropoietin molecules, can provide advantageous properties, for example, increased solubility, increased resistance to proteolysis, reduced immunogenicity, increased serum half-life, and increased biological activity. Erythropoietin analogs with additional glycosylation sites

F) are disclosed in more detail in the application for European patent 640619 in the name of Elliott (EiPoU), which was published on March 1, 1995.

According to a preferred embodiment of the present invention, the glycoproteins of the present invention include an amino acid sequence that includes at least one additional glycosylation site, for example (but not limited to) erythropoietins that include the sequence of human erythropoietin modified by a modification selected from given below: b5

Avp3otpg32,

AvpZITng53;

Avpi" ТЯ;

Avpbo;

AvpbUtygi;

BegbzAdvpo Ti,

Uaiv7 Ap Tito;

Vegat Ap To;

Bei87 Ap ASU TvgYao; begiAvpi Throne, beta" Ap Gid av:

AvpbTig? and Bet7AvpeeTnIoo: 70 Avp3iotn in av, AvpeTvgo:

Avp8U Petri; begi7Avpiya pet.

AvpizeTgi 38.

Avpiz8Tvg140-

Tigi""; and

Rgo!124Trg125,

The notation used herein for amino acid sequence modification means that the position(s) of the corresponding unmodified protein (e.g., PERO Sequence Mo 1 or Sequence Mo 2) indicated by the superscript(s) is replaced by the amino acid(s) , which immediately precedes the corresponding superscript(s).

Said glycoprotein may also be an analogue having at least one additional amino acid at the carboxy-terminal region of the glycoprotein, wherein said additional amino acid includes at least one glycosylation site, i.e. said conjugate, as defined above, also relates to to a compound where said glycoprotein has a sequence that includes a sequence of human erythropoietin and a second sequence at the carboxy-terminal region of said human erythropoietin sequence, wherein said second sequence includes at least one glycosylation site. Said additional amino acid may include a peptide fragment that was obtained from said carboxy-terminal region of human chorionic d) gonadotropin. In a preferred embodiment, said glycoprotein is an analog selected from the group consisting of (a) human erythropoietin having the amino acid sequence Zeg Zeg Zeg Zeg I uz Aia Rgo Rgo

Rgo Zeg Ge Rgo beg Rgo Zeg Agu Gey Rgo Su Rgo Zeg Avr Tyg Rgo Ne Gey Rgo bip (Sequence Mo Z), which departs from said carboxy-terminal region; (B) analog (a), which additionally includes Zeg"AzpUZTpgOERO; and so 3o (c) analog (a), which additionally includes AzpTpgMaIZ"AvpeZTNg OZERO, (Te)

Said glycoprotein may also be an analog having an amino acid sequence that includes a rearrangement of at least one glycosylation site. Said rearrangement may include deletion of any of said M-linked hydrocarbon sites of human erythropoietin and addition of M-linked Ge) hydrocarbon site at position 88 of said amino acid sequence of human erythropoietin. According to the preferred option, the mentioned glycoprotein is an analog selected from the group consisting of siP?beg"Avp88TygoErO; SiPZveg"Azpe8TNgoErO; and SiPZvVeg"Avpe8TygOErO,

As used herein, the term "lower alkyl" refers to a straight or branched alkyl group having from one to six carbon atoms. Examples of lower alkyl groups are methyl, ethyl, and isopropyl. In this invention, K is any lower alkyl. Preference is given to conjugates where K is methyl. w-in

The mentioned symbol "t" means the number of residues of ethylene oxide (OSH2CH3) in the mentioned poly(ethylene oxide) c group. One RES subunit of ethylene oxide has a molecular weight of approximately 44 daltons. Thus, the molecular weight of the mentioned conjugate (including the molecular weight of EPO) depends on the mentioned number "t". In said conjugates of the present invention, "t" is from about 450 to about 900 (corresponding to a molecular weight of about 20 kilodaltons to about 40 kilodaltons), preferably from about 650 to about 750 (corresponding to a molecular weight of about 30 kilodaltons). Said number t is chosen in such a way that the resulting conjugate according to the present invention has a physiological activity comparable to that of unmodified EPO, and the activity may be the same, greater than, or a fraction of the corresponding activity of unmodified EPO. A molecular weight that corresponds "approximately" to a certain number, 5p, means that said molecular weight is within reasonable limits of that number, as determined by conventional analytical methods. Said number "t" is chosen such that the molecular mass of each poly(ethylene glycol) group covalently bound to said glycoprotein (erythropoietin) is from about 20 kilodaltons to about 40 kilodaltons, and, in a preferred embodiment, is about 30 kilodaltons.

In the said conjugates according to the present invention, the mentioned number "n" is the number of polyethylene glycol groups covalently linked to free amino groups (including E-amino groups of the amino acid lysine and/or amino-horse (F) amino groups) of the erythropoietin protein through an amide connection (connections). The conjugate of this invention may have one,

GI has two or three RES groups per EPO molecule. "n" is an integer from 1 to 3, according to a preferred option, "n" is equal to 1 or 2, and according to a more preferred option, "n" is equal to 1. The mentioned compounds of Formula I can be obtained from a known polymeric material: posnnemonosvo y o a de K and t are as defined above by condensing said compound b of Formula II with erythropoietin glycoprotein. Compounds of Formula II, where X is 3, are alpha-lower alkoxysuccinimide esters of poly(ethylene glycol) butyric acid (lower alkoxy-RTES-5BA). Compounds of Formula II, where X is 2, are alpha-lower alkoxysuccinimide esters of poly(ethylene glycol) propionic acid (lower alkoxy-PEO-5RA).

Any conventional method of reacting an activated ether with an amine to give an amide can be used. In the aforementioned reaction described earlier, the succinimide ester exemplified is the leaving group that affords the amide. The use of succinimide esters, such as compounds of formula II, to form conjugates with proteins is disclosed in US Pat. No. 5,672,662, issued 3 September 1997 to Harris (Nuggets) et al.

The composition of human EPO includes nine free amino groups, the mentioned amino-terminal amino group plus the mentioned 7/0 E-amino group of 8 lysine residues. It was established that in the case of combining the polyethylene glycolization reagent with the ZVA compound of the formula I at pH 7.5, the protein-to-protein ratio of 1:3 and the reaction temperature of 20-25 2С, a mixture of mono-, di-, and trace amounts of tri-polyethylene glycolized species. In the case of using the ZPA compound of Formula II as a polyethylene glycolization reagent, under similar conditions, with the exception that the ratio of protein: RES was equal to 1:2, mainly mono-polyethylene glycolized species were obtained. 75 Polyethylene glycolized ERO can be administered in the form of a mixture or in the form of various polyethylene glycolized species, which were separated using cation exchange chromatography.

By changing the reaction conditions (for example, the ratio of reactants, pH, temperature, protein concentration, reaction time, etc.), it is possible to change the relative amount of the mentioned various polyethylene glycolized species.

Human erythropoietin (EPO) is a human glycoprotein that stimulates the formation of red blood cells. A detailed description of its preparation and therapeutic use is given, for example, in US patents MoMo 5,547,933 and 5,621,080, EP-B 0148605, in the work of Huang S.L. (Nyapo 5.1), Rgos. Mai). Asad All together. OA (1984) 2708-2712, EP-B 0205564, EP-B 0209539 and EP-B 0411678, as well as in the works of Lai PG (Gai RN) and others, 9. Vioi. Spent 261 (1986) 3116-3121 and Sasaki G. (ZazakKi N.) and others, 9. Vioi. Spent 262 (1987) 12059-12076. Erythropoietin for therapeutic use can be obtained by recombinant DNA methods (EP-B 0148605, EP-B 0209539,

AgriJ. K. (Edgpie 9. C), Strickland T. W. (5igiskiapa T. MU.), Lane J. (I ape 9.) and others, (1986) Ittypobio!. 72:213-224). at

A description of the methods of expression and production of erythropoietin in a serum-free medium is provided, for example, in the application MO 96/35718 in the name of Barga (Vygo), which was published on November 14, 1996, and in the European patent application Mo 513738 in the name of Koch (Kosi ), which was published on July 12, 1992. In addition to the aforementioned publications, it is known that serum-free fermentation of recombinant cells can be carried out

CHOs that include the EPO gene. The description of such methods is given, for example, in EP-A 0513738, EP-A 0267678 and, in x, general form, in the work of Kawamoto T. (Kazhatoyu T.) and others, Apaiuis! Viosyet. 130 (1983) 445-453, ЕР-А «хг 0248656, in the work of Kovar J. (Kozhmag).) and Franek F. (Egapek R.), Meijodz ip Epgutoiodu 421 (1986) 277-292, in the work of BavisterB. (Vamizieg V.), Ehrsoiodu 271 (1981) 45-51, in ER-A 0481791, ER-A 0307247, ER-A 0343635, MO and. z5 88/00967. what

EP-A 0267678 describes ion-exchange chromatography on 5-Sepharose (5-Zerpagose), preparative high-performance liquid chromatography with a reversed phase on a C5 column, and gel filtration for the purification of EPO, which was obtained in a serum-free culture after dialysis. In this regard, the gel filtration step can be replaced by ion-exchange fast flow chromatography on Z-Sepharose. Before ion-exchange chromatography, it is also suggested to carry out chromatography with ssh with staining on a Vice Ttgizasgui column. and Description of the method of purification of recombinant ERO is given in the work of Nobuo I. (Mobio 1.) and others, U. Viospet. 107 "" (1990) 352-359. In this method, however, the EPO is treated with a solution of Tmeepe 20, phenylmethylsulfonyl fluoride, ethylmaleimide, pepstatin A, copper sulfate, and oxamic acid before the purification steps. Publications, including UMO application 96/35718 in the name of Barga (Vygo), which was published on November 14, 1996, reveal the method of obtaining erythropoietin by serum-free fermentation (EROSION).

The specific activity of the EPO or EPO conjugates of the present invention can be determined using a variety of assays known in the art. The biological activity of the purified EPO proteins according to the present invention is such that it results from the introduction of the mentioned protein. ERO to a human patient by injection is an increase in the production of reticulocytes and erythrocytes by bone marrow cells compared to subjects who were not injected, or with

Fo subjects of the control group. The biological activity of EPO proteins or their fragments, which have been obtained and co-purified in accordance with the present invention, can be tested by the methods given in the work of Annable (Appabier) and others, You. Ma. NIV Oh (1972) 47:99-112 and in Rpagt. Eygora Res. Izvtse Eguipgoroiivyp VKR Vio 1997 (2). A description of another biological test for determining the activity of the EPO protein, a normocytemic test on mice, is given in Example 4.

The present invention provides a composition comprising conjugates as described in (F, above). A composition comprising at least ninety percent mono-RES conjugates, i.e., a composition with t equal to 1 can be prepared as shown in Example 5. In general, mono-PES conjugates of erythropoietin glycoproteins are preferred because they have higher activity than di-PES 60 conjugates. The percentage of mono -RES of the conjugates, as well as the ratio of mono- and di-RES species can be adjusted by collecting wider fractions around the elution peak to reduce the percentage of mono-RES or by collecting narrower fractions to increase the percentage of mono-RES in said composition. yield and activity are approximately ninety percent mono-RES conjugates Sometimes it may be desirable to have compositions in which, for example, at least ninety-two percent or at least 65 ninety-six percent of said conjugates are mono- RES you house (n equals 1). According to an embodiment of the present invention, the percentage of conjugates where H is equal to 1 is from ninety to ninety-six percent.

The present invention also relates to suitable pharmaceutical compositions comprising a conjugate or composition as described above and a pharmaceutically acceptable excipient.

The conjugates and compositions of the present invention are particularly suitable for the preparation of medicaments for the treatment or prevention of diseases associated with anemia in patients with chronic renal failure (CKD).

AIDS and for the treatment of cancer patients undergoing chemotherapy.

An additional embodiment of the present invention is related to a method of prevention and/or therapeutic treatment of diseases involving anemia in patients with chronic renal failure (CKD), AIDS and cancer patients undergoing chemotherapy, which includes the step of administering to the patient the composition according to the description, 70 which was given before that.

In addition, the present invention relates to a process for the preparation of compounds of the preceding description, which comprises the condensation of a compound of Formula III (c) possniaenasooo (c) with erythropoietin glycoprotein, where K, m and x are as defined in was given before that.

The present invention also relates to compounds as defined above for the treatment of diseases associated with anemia in patients with chronic renal failure (CKD), AIDS and cancer patients undergoing chemotherapy.

This invention will be better understood by reference to the following examples, which illustrate, but are not limited to, the invention described.

EXAMPLES

EXAMPLE 1: Fermentation and purification of human EPO a) Preparation and fermentation of inoculum

One tube with a working cell line from the Cell Line Bank (Uuogkipda Seili WapKk), which is derived from the CHO cell line that produces ERO (ATSS (American Type Culture Collection) SC. 8695, which is disclosed in EP 411678 (Sep. )), are removed from the gas phase of the tank for storage in liquid nitrogen. The mentioned cells are transferred to glass roller flasks and cultivated in a bicarbonate-buffered environment in a CO incubator. so 3o Typical serum-free media used for inoculum preparation and fermentation are disclosed in (Se) European Patent Application No. 513738 in the name of Koch (Kosi), which was published on July 12, 1992, or in MO Application 96/35718 in the name of i Barga (Vygo), which was published on November 14, 1996, include, for example, as C medium, UMEM/E12 (Dulbecco's modified Eagle's medium) (eg, "9 KN medium of Wiozsiepsez/Naliep Wioiodiez, Denver, USA , order Mo 57-736) and, in addition, sodium bicarbonate, L-glutamine, L-glucose, recombinant insulin, sodium selenite, diaminobutane, - hydrocortisone, iron sulfate (II), asparagine, aspartic acid, serine and a stabilizer for cells mammals, for example, polyvinyl alcohol, methylcellulose, polydextran, polyethylene glycol, Rishgopis 68, plasma substitute polygelin (NEMASSEI 9) or polyvinylpyrrolidone (UUO 96/35718). " 20 The mentioned cultures are subjected to a microscopic check for the absence of contaminating microorganisms and the cell density is determined. These checks are carried out at each stage of cleavage. c After the initial growth period, the mentioned cell culture is diluted with fresh nutrient medium to :c» the original cell density and maintained for one more growth cycle. This procedure is repeated until approximately 2 liters of culture volume is obtained in each glass roller flask. After approximately 12 repetitions, from 1 liter to 5 liters of the mentioned culture is obtained, which is subsequently used as an inoculum - for a 10-liter inoculum fermenter.

After 3-5 days, the mentioned culture in a 10-liter fermenter can be used as an inoculum for a 100-liter inoculum fermenter. After another 3-5 days of cultivation, the mentioned culture in a 100-liter fermenter can be used as an inoculum for a 1000-liter production fermenter. b) r) Collection of cells grown in culture and their separation "c. The process of periodic feeding is used, that is, after reaching the required cell density, approximately 8095 cultures are collected. The residual culture is fed with fresh culture medium and cultured until the next collection of cells grown in culture. One production run includes at most 10 consecutive cell collections: 9 partial collections and 1 total full collection at the end of fermentation. Cells grown in culture are harvested every 3-4 days. (F) A certain volume of collected cells is transferred to a cooling vessel. Cells are removed by centrifugation

GI or filtering and get rid of them. The supernatant from EPO, which is obtained after the centrifugation step, is filtered and collected in the second cooling vessel. Each collection of cells grown in culture is treated separately during purification.

A typical process for the purification of EPO protein is disclosed in UUO 96/35718 in the name of Barga (Vygo), which was published on November 14, 1996. Below is an explanation of the said cleaning process. a) Chromatography on a Vise Zerpagose column

Vine Zerpagose (RINAgtasia company) is sepharose beads, the surface of which is covered with the covalently bound blue dye Syrasgop. Because EPO binds to Vice Zerpagose much more strongly than most non-protein-like impurities, some protein-like impurities, polyvinyl acetate, and EPO may be enriched at this stage. The elution of the mentioned Vice Zerpagoze column is carried out by increasing the salt concentration, as well as the pH.

The aforementioned column is filled with 80-100 l of Vice Zerpagose, regenerated with the help of Mason and balanced according to

With the help of an equilibration buffer (sodium chloride/calcium and sodium acetate). The column is loaded with acidified and filtered fermentation supernatant. After loading is complete, the mentioned column is washed first with a buffer that resembles an equilibration buffer, which includes a higher concentration of sodium chloride, then with a buffer based on TRIS buffer. The product is eluted with TRIS-buffer-based buffer and collected in one fraction according to the main 7/0 elution profile.

C) Chromatography on a Vshchui column! Toworeagi.

Higher! Touoreagi 650 C (Tozo Naaz company) is a matrix based on polystyrene, with which aliphatic butyl residues are covalently bound. Since EPO binds to this gel more strongly than most impurities and polyvinyl acetate, it has to be eluted with a buffer containing 7/5 isopropanol.

The aforementioned column is filled with 30-40 liters of Vshu! Tooreagi! 650 C, regenerated with Mahon, washed with buffer based on TRIS-buffer and equilibrated with buffer based on TRIS-buffer, which includes isopropanol.

The Vise Zerpagose eluate is adjusted to the concentration of isopropanol in the column equilibration buffer and loaded onto the column. After that, the column is washed with an equilibration buffer with an increased concentration of isopropanol. The product is eluted with an elution buffer (buffer based on

TRIS buffer with a high content of isopropanol) and collected in one fraction according to the main elution profile. c) Chromatography on a Nuadgohuarache OPygode column! Nuagohuaraitse SHOPGODE! (Viozerga company) is hydroxyapatite, which is included in the composition of the agarose matrix to improve mechanical properties. EPO has a low affinity for hydroxyapatite and due to this it can be eluted at i) lower phosphate concentrations than protein impurities.

The column is loaded with 30-40 L of Nuagohuaraiie Shgodei, regenerated first with a buffer containing potassium phosphate/calcium chloride and Mahon, then with a buffer based on TRIS buffer. co

After that, the column is equilibrated with a buffer based on TRIObuffer, which includes small amounts of isopropanol and sodium chloride. ise)

The eluate, which contains EPO, which was obtained after chromatography on a Vshu column! Toworeagig!, again «g loaded into the mentioned column. The column is washed again with equilibration buffer and buffer based on TRIS buffer without isopropanol and sodium chloride. The product is eluted with a buffer based on TRIS buffer, me), which includes a small concentration of potassium phosphate, and collected in one fraction according to the main elution profile. a) High-performance reversed-phase liquid chromatography on a Mudas C4 column

The Mudas C4 packing (Mudas company) for high-performance reversed-phase liquid chromatography is silica gel particles on the surface of which there are Ca4-alkyl chains. "

The separation of EPO from impurities resembling protein is based on the difference in the strength of hydrophobic interactions. z c Elution is carried out with a gradient solution of acetonitrile in diluted trifluoroacetic acid.

Preparative high-performance liquid chromatography is performed on a stainless steel column (which is filled with 2.8-3.2 liters of Mudas C4 silica gel). The eluate obtained after column chromatography

Nudgohuarashe SHOPKgode!i, acidified by adding trifluoroacetic acid and loaded into the column

Mudas C4. For washing and elution, a gradient solution of acetonitrile in diluted -I trifluoroacetic acid is used. Fractions are collected and immediately neutralized with phosphate buffer. Fractions

EROs that meet the requirements for the level of impurities that resemble protein are combined. o f) Chromatography on a OEAE Zerpagose column. The mentioned material OEBEAE (diethylaminoethyl) Zerpagose (RNagtasia company) consists of 5-diethylaminoethyl (OEAE) groups, which are covalently bound to the surface of sepharose beads. Connection of ERO with

Me, mentioned OEAE groups are mediated by interionic interactions. Acetonitrile and trifluoroacetic acid are passed through the column without delay. After washing out the mentioned substances, trace impurities are removed by washing the column with an acetate buffer with a low pH. After that, the column is washed with a neutral phosphate buffer and EPO is eluted with a buffer with an increased ionic strength.

The mentioned column is filled with fast-flowing OEAE Zerpagoze. The volume of the mentioned column is regulated in such a way that the load of EPO is 3-100 mg of EPO/ml of gel. The column is washed with water and

F) balancing buffer (sodium/potassium phosphate). The combined fractions of the eluate, which was obtained as a result of high-performance liquid chromatography, are loaded, and the column is washed with equilibration buffer. After that, the mentioned column is washed first with washing buffer (sodium acetate buffer), then with equilibration buffer. After that, EPO is eluted from the column using the elution buffer (sodium chloride, sodium/potassium phosphate), which is collected in one fraction according to the main elution profile.

The eluate, which was obtained after chromatography on an OEAEE Zerpagoze column, is brought to a certain level of electrical conductivity. The obtained medicinal substance is filtered under sterile conditions into Teflon 65 vials and stored at a temperature of -7026.

EXAMPLE 2: Polyethylene glycolization of EPO using ТРЕОС-5ВА

EPO purified by the serum-free procedure of Example 71 (EPO) had a homogeneous structure as determined by analytical methods and showed a typical isoform pattern, which included 8 isoforms. Its specific biological activity, based on the results of determination using a normocytemic test on mice, was equal to 190,000 international units/mg. Methoxy-PEsO-ZBA was used as a polyethylene glycolizing agent, which is a compound of Formula II, where K is methyl; X -- 3; and t is from 650 to 750 (on average, about 680, corresponding to an average molecular weight of about 30 kilodaltons).

Polyethylene glycolization reaction 70 To one hundred milligrams of EPOzvia (9.71 ml of a 10.3 mg/ml concentrated solution of EPOz5vi, 5.48 μmol) was added 1 Oml of 0.1 M potassium phosphate buffer, pH 7.5, which included 506 mg of methoxy-RES-ZVA ( 30 kilodaltons, 16.5 μmol) (which was obtained from Speagmaeg Research Co., Ips., Huntsville, Ala.) and stirred for 2 hours at room temperature (20-232C). The final protein concentration was equal to 5 mg/ml, the protein:reactive RES ratio was 1:3. After two hours, the reaction was stopped by adjusting pH 75 to 4.5 using glacial acetic acid and the resulting reaction mixture was stored at -2090 until ready for purification.

Purification 1. Conjugation mixture: Approximately 28 ml of 5P-BZERNACOSE RE (sulfopropyl cation exchange resin) was added to an AMISOM glass column (2.2 x 7.5 cm) and the column was equilibrated with 20 mM acetate buffer, pH А4.5, at a flow rate of 150 ml/hour . Six milliliters of the mentioned reaction mixture, which included 30 mg of protein, was diluted five times with the help of equilibration buffer and applied to the mentioned column.

The unadsorbed material was washed with buffer, the adsorbed RES conjugate mixture was eluted from the column with 0.175M Mass in the equilibration buffer. Unmodified EROS, which still remained in the column, was eluted with 750 mM Massi. The column was re-equilibrated with the original buffer. with

The samples were analyzed by electrophoresis in a polyacrylamide gel in the presence of sodium dodecyl sulfate (505-RACE) and the degree of their polyethylene glycolization was determined. It was found that the 0.175 M Mass eluate i) contained mono-, di-, as well as trace amounts of tri-polyethylene glycolized species, while the 750 mM Mass eluate contained unmodified EPOZ35i. 2. Di-RES and mono-GEOS-EROS solutions: The purified conjugate mixture, which was eluted from the mentioned column in its previous step, was diluted 4 times with buffer, re-introduced to the mentioned column and washed as described. Di-REO-EROSION and mono-RES-EROSION were separately eluted from the column with 0.1M Masi and OD75M Masi, respectively. Elution was also carried out with 750 mM Masi in order to elute "And any residual unmodified EPO from.

Alternatively, said reaction mixture was diluted 5-fold with acetate buffer and applied to a 5P-Zerpagozve column (approximately 0.5 mg protein/ml gel). The column was washed, and the adsorbed mono-RES-EROS, di-RES-EROS and unmodified EROS were eluted according to the description given in the previous section.

Results «

RES-EROzvi was synthesized by chemical conjugation of an unbranched molecule of RES with a number average molecular weight of 30 kilodaltons. RES-EROSION was obtained as a result of the reaction between - with the primary amino groups of EPO and a derivative of the succinimide ester of REOS-butyric acid (30 kilodaltons) with the formation of an amide bond. "» The summarized results are presented in Table 1. The composition of the purified conjugate mixture included mono- and di-REOS-EROZvii. according to the results of analysis by means of electrophoresis in a polyacrylamide gel in the presence of sodium dodecyl sulfate (5ZO5-RACE), the mentioned mixture did not have unmodified RES -ERO zion. The conjugate mixture -I was 23.4 mg or 7895 of the starting material. According to the results of the separation of mono- and di-PEO-ERO3 by means of cation exchange chromatography, it was established that the ratio of mono- and di-RES in

Mn of the mentioned conjugate mixture was almost equal to 1:1. After completion of the mentioned reaction, the ratio of individual t» mono:di:unmodified components was 40:38:20 (95). The total yield was almost quantitative.

FO so z dolav o Nemozhrkivnya 1505 o o EXAMPLE 3: Polyethylene glycolization of ERO with the help of tRES-ZRA Different aliquots of EROz5ii, which was used in Example 2, were introduced into the reaction with methoxy-REO-5RA (30 kilodaltons) (ZPpeagmayeg company

Roiutegues, Ips., Huntsville, Ala.). The reaction was carried out at a protein reagent ratio of 1:2 and the purification was carried out in accordance with Example 2. Mainly mono polyethylene glycolized form was obtained.

EXAMPLE 4: The activity of polyethylene glycolized EPO ip mimo, determined using a normocytemic 65 test on mice

Normocytemic bioassay on mice is known in this field (Rnagt. Eigora Zres. Izzce Eguipgoroivyip VKR Vio

1997 (2)); the method is given in the monograph devoted to erythropoietin (RA. Eig. VKR.). Samples were diluted

B5A-RV5. Normal healthy mice aged 7-15 weeks were injected subcutaneously with 0.2 ml of the EPO fraction, which included non-polyethylene glycolized EPO, or tri-, di-, or mono-polyethylene glycolized EPO from Example 2 or 3. After 6 days, blood samples were taken by puncturing the tail vein, which was diluted in such a way that 1 ml of blood corresponded to 1 ml of 0.15 μmol of acridine orange dye solution. The duration of staining was from 3 minutes to 10 minutes. The number of reticulocytes was determined microfluorometrically using a flow cytometer by analyzing the fluorescence histogram induced by red light. The number of reticulocytes was given in absolute numbers (per 30,000 blood cells that were analyzed). For the 7/0 data presented, each group included 5 mice per day, the blood sample from said mice was collected only once.

In separate experiments, mice were administered a single dose of unmodified EPO (25ng EPO), a mixture

RES(ZBA)-EPO from Example 2 (1 ng conjugate), mono- and di-polyethyleneglycolized erythropoietins from Example 2 (1 ng conjugate), RES(ZPA)-EPO from Example C (1 ng conjugate) and buffer solution. The results obtained /5 are shown in Table 2. The mentioned results show an extremely high activity and a prolonged half-life of polyethylene glycolized EPO species, as indicated by a significantly increased number of reticulocytes and a shift in the maximum number of reticulocytes when using the same dose per mouse (1Ong) compared to with a dose of 25 ng in the case of unmodified EPO. nn

NI nni Ah tn kilo-daltons) kilo-daltons) |kilo-daltons) IREO-ERO 5VA buffer cm 5 o radyudyny! 10601 in 16071666 in co

EXAMPLE 5: Obtaining mainly topo-RES-ERO

Polyethylene glycolization reaction

Starting with 100 mg (5.448 μmol) of EROS in 100 mM potassium phosphate buffer (pH 7.5), which was obtained according to Example 1, 329 mg (10.9 μmol) of the RES-ZBA reagent (30 kilodaltons) was added, which was dissolved in 3 ml of 1 mM HCl. The necessary amount of 100 mM potassium phosphate buffer (pH 7.5) was added to bring the volume of the mentioned reaction mixture to 2000 ml. The final protein concentration was equal to 5 mg/ml, so the ratio of protein:RES reagent was 1:2. The mentioned reaction mixture was stirred for 2 hours at an ambient temperature of 3° (20-22). After 2 hours, the mentioned reaction was stopped by adjusting the pH to 4.5 with glacial acetic acid and the resulting reaction mixture was stored in a frozen state at a temperature of -202C until readiness for cleaning.

Purification « du The reaction mixture from the previous step was diluted 1:5 with 10 mM sodium acetate, pH 4.5, and added to ZOOml with

ZR-Zerpagose EE (sulfopropyl cation exchange resin), which was loaded into a 4.2 x 19 cm column. The aforementioned column was previously equilibrated with the same buffer. The flow that came out of the above-mentioned column was monitored at 280 nm using an ultraviolet monitor from the company BiYop; the received data were recorded using the self-record of the Kirr apa 7opep company. The column was washed with 30 Oml or 1 layer volume of equilibration buffer to remove excess reagents, by-products of the reaction and oligomeric - 15 REO-ERO. After this, washing was carried out with 2 volumes of a layer of 100 mM Mass in order to remove di-RES-ERO.

After that, mono-RES-ERO was eluted with 200 mM Masi. During the elution of mono-RES-ERO, the first 5 Oml of the protein (95) peak was removed, and mono-TES-ERO was collected as a 150 ml fraction. The unmodified EROZZi, which remained in the column, was eluted with 750 mM Masi. All buffers before elution were dissolved in equilibration buffer. All eluted samples were analyzed using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (o) 50 (5ZO5-RACE) and high performance gel chromatography (5ES). After that, the collective mixture of mono-PEO-ERO, which was obtained from the mentioned 1500 ml fraction, and which did not include unmodified EROZZI in an amount that would be amenable to determination, was concentrated to approximately 4.5-7.5 mg/ml and diafiltered to storage buffer (10 mM potassium phosphate, 100 mM Mass, pH 7.5). Concentration/diafiltration was performed using a TEBE Zuviet Microsystems equipped with a MiPSroghe Reisop Hi Viotach 50 band-limited membrane (50 kilodaltons) at ambient temperature (F). Concentrated mono-RES-EPO was filtered under sterile conditions and stored

Ge frozen at a temperature of -2026.

Approximately 7,595 EROs were polyethylene glycolated. After purification, the total yield was approximately 3096 mono-RES-EPO with no detectable amount of unmodified EPOS-EPO and approximately 2595 di-RES-EPO. The remaining part of the protein consisted of oligomers and EPO, which was not subjected to polyethylene glycolization. The composite mixture of mono-RES-ERO, which was obtained from a 150 ml fraction, included approximately 9095 mono-REO-ERO and approximately 1095 di-REO-ERO/

SEQUENCE LISTING (1) GENERAL INFORMATION: 65 (Y) Applicant: (А) Name: R. Noitanp-i a Kospe DAO

(B) Street: 124 OSgepgaspegvigazze (C) City: Basel (E) Country: Switzerland (R) Postal code (index): СН-4070 (b) Phone: (61)688 11 11 (Н) Telefax: (61) 688 13 95 (I) Telex: 962 292 Pig sp (ii) Name of the invention: Erythropoietin conjugates (ii) Number of sequences: Z (m) Computer-readable form: (A) Type of carrier: Floppy disk 70 ( C) Computer: IVM RS compatible (C) Operating system: UUOKO (O) Program: Raiepiip Keyeaze 2.0 "170" RaibepiChp Meg. 2.0 21051 "211" 165 "2122 RT "213" Noto zariepz "00" 1 ma Rko Rko Agd Geo T1e Suv Avr bek Aho Uai pe bip Ak Tug Bez 5 10 15 Tech b15 Aza uz biz dia bij Avp IZe Tvk Tv biu Suv Aa Sich Niz 20 25 30

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Claims (1)

The formula of the invention "from . . Sh. g. 1. Conjugate, wherein said conjugate comprises an erythropoietin glycoprotein having at least one free amino group and a biological activity of i mimo, which is that said glycoprotein induces bone marrow cells to increase the production of reticulocytes and erythrocytes, and its is selected from the group -I, which includes human erythropoietin and its analogues, which have the sequence of human erythropoietin C, modified by adding from 1 site to 6 glycosylation sites or rearranging at least one glycosylation site, while the said glycoprotein is covalently linked to "n" CHK» poly(ethylene glycol) groups of the formula bu 0000 bO(СНОКОСНоСН»)t ОВ, where -CO of each poly(ethylene glycol) group forms an amide bond with one of the mentioned amino groups; where (Che K is lower alkyl, x is 2 or 3, t is from about 450 to about 900, n is 1-3 and n and t are chosen in such a way that the molecular weight of said conjugate excluding glycoprotein F, erythropoietin is from 20 kilodaltons to 100 kilodaltons. co 2. Conjugate according to claim 1 of the formula: РА-ИМНСО-(СНоЖ(ОСНо СН) то ОВ, (I) 60 where x, t, n and K correspond to the definition given in claim 1, and P is a glycoprotein residue without p amino group (amino groups), which forms an amide bond (bonds) with the mentioned poly(ethylene glycol) group (groups). 3. Conjugate according to any of the previous items, where K is methyl. 4. The conjugate of any of the preceding items, wherein t is from about 650 to about 750. 65 5. The conjugate of any of the preceding items, wherein n is 1. 6. Conjugate according to any of the previous items, where K is methyl; t is from about 650 to about 750; tap - 1. 7. The conjugate according to any of the previous clauses, which has the formula IsSNZO(СНШНОЮ)ТСНоСНоСНоСО-МНИ-Р, where t is from 650 to 750, n is 1 and Р corresponds to the definition given in clause 2. 8. Conjugate according to any of the preceding clauses, wherein said glycoprotein is human erythropoietin. 9. Conjugate according to any of the previous claims 1-7, where the said glycoprotein of human erythropoietin is expressed by activation of an endogenous gene. 10. Conjugate according to any of the previous claims 1-9, where said glycoprotein has the sequence represented by /y/0 in ZEAYU MO. 11. Conjugate according to any of the previous claims 1-8, where said glycoprotein has the sequence of human erythropoietin modified by adding from 1 site to 6 glycosylation sites. 12. Conjugate according to any of the previous claims 1-11, where said glycoprotein has a sequence of human erythropoietin modified by a modification selected from the group consisting of: Avpotigzg, Avp?TAg, Avpo/Tpgo", AvpbZ. AvpZTyg, Zeg8dapeTng I, maiI8"Azpe8Trgzo, Zeg"Avpe8Trgto, Zeg"Avp8eiuaTngzo, with Zeg"AvpeaTngotTrg2, o Zeg"Avp88Tng Oda 162, AvpUZTpg Zeg"Azp88TNgzo, AvpZotpgZ2uai|8'Azp88Tngzo, so ZegTzotAvpeZ, segTzotAvpeZ1) Avp/ZbTrg 138, "Avp'ZVaTng, so Tyg!25 and Rgo'yaTrng 125, M. 13. Conjugate according to any of the preceding claims 1-12, wherein said glycoprotein has a sequence that includes a human erythropoietin sequence, and a second sequence at the carboxy-terminal region of said human erythropoietin sequence, wherein said second sequence includes at least one site « glycosylation. 14. Conjugate according to claim 13, wherein said second sequence includes a sequence that was obtained from the carboxy-terminal sequence of human chorionic gonadotropin. h" 15. Conjugate according to claim 13, where said glycoprotein has a sequence selected from the group consisting of: (a) the sequence of human erythropoietin and the sequence presented in 5BO and MOZ at the carboxy-terminal region of said sequence of human erythropoietin ; Ш- (B) sequence (a) modified by Zeg"AzpUZTngO and oo (c) sequence (a) modified by AzpUTng2uaI8"Avp8Tngo, their 16. Conjugate according to any of claims 1-7, where said glycoprotein has a sequence of human erythropoietin modified by rearrangement of at least one glycosylation site. Ge») 20 17. The conjugate of claim 16, wherein said rearrangement comprises deletion of any M-linked glycosylation sites of human erythropoietin and addition of an M-linked site glycosylation at position 88 of the sequence of human erythropoietin. 18. Conjugate according to claim 17, wherein said glycoprotein has the sequence of human erythropoietin, modified by a modification selected from the group consisting of: sipZegZ"DAvpe8TrNgO, F) spe" AvpeaTngo and g sipZvego"AvpUtnggo, 19. The composition, which includes conjugates, and each of said conjugates includes an erythropoietin glycoprotein having at least one free amino group and a biological activity i mimo, which consists in the fact that said glycoprotein forces bone marrow cells to increasing the production of reticulocytes and erythrocytes, and is selected from the group consisting of human erythropoietin and its analogs, which have a sequence of human erythropoietin modified by adding from 1 site to 6 glycosylation sites or rearranging at least one glycosylation site, while said glycoprotein in 65 Each mentioned conjugate has poly(ethylene glycol) groups covalently bound to "n" of the formula -б0-СНоХ-(ОСНоСНо)т- OK, where -СО of each poly(ethylene glycol) group forms an amide bond with one of said amino groups, where in each of said conjugates K is lower alkyl, x is 2 or 3, t is from about 450 to about 900, n is 1-3, and n and t is chosen in such a way that the molecular weight of each conjugate, excluding glycoprotein erythropoietin, is from 20 kilodaltons to 100 kilodaltons; the percentage of conjugates where n is 1 is at least ninety percent. 20. The composition, which includes conjugates according to any of claims 1-18, where the percentage of conjugates in which n is 1 is at least ninety percent. 21. The composition according to claims 19 or 20, where the percentage of conjugates in which n is 1 is at least ninety-two percent. 70 22. The composition according to claim 21, where the percentage of conjugates in which n is 1 is at least ninety-six percent. 23. The composition according to claims 19 or 20, where the percentage of conjugates in which n is 1 is from ninety percent to ninety-six percent. 24. Pharmaceutical composition, which includes a conjugate or composition according to any of claims 1-23 and a pharmaceutically acceptable carrier. 25. Use of the conjugate or composition according to any one of claims 1-23 for the preparation of medicaments for the treatment or prevention of diseases associated with anemia in patients with chronic renal failure (CKD), AIDS and for the treatment of cancer patients who subjected to chemotherapeutic measures. 26. A method of prevention and/or therapeutic treatment of diseases, including anemia in patients with chronic renal failure (CKD), AIDS and cancer patients undergoing chemotherapy, which includes the step of administering to the patient the composition according to any of claims 1-23. 27. The method of obtaining compounds according to any of claims 1-23, which includes the condensation of the compound of the formula ІІ with Во(СН.сН,О) (СН.), СооМ о 27/t 2/x (ee) (Се) ( 1) with erythropoietin glycoprotein, where K, t and x correspond to the definition according to any of claims 1-6. "AND 28. Compounds according to any of claims 1-3, obtained using the method according to claim 27. 29. Compounds according to any one of claims 1-23 for the treatment of diseases associated with anemia in patients with chronic renal failure (CKD), AIDS and cancer patients undergoing chemotherapy. - Official bulletin "Industrial Property". Book 1 "Inventions, useful models, topographies of integrated microcircuits", 2005, M 8, 15.08.2005. State Department of Intellectual Property of the Ministry of Education and Science of Ukraine. - and? -i (95) sh» (o) ІЧ e) ime) bo b5