MXPA06007376A - Fc-ERYTHROPOIETIN FUSION PROTEIN WITH IMPROVED PHARMACOKINETICS - Google Patents

Abstract

The present invention provides novel highly sialylated Fc-EPO fusion proteins preferably comprising a couple of modifications in the Fc- portion as well as in the EPO portion and having improved pharmacokinetics. Specifically, the Fc-EPO proteins have a prolonged serum half-life and increased in vivo potency. The Fc-EPO fusion proteins synthesized in BHK cells have dramatically prolonged serum half-lives and increased in vivo potency when compared to corresponding Fc-EPO fusion proteins produced in other cell lines, such as, for example, NS/O cells.

Description

FUSION PROTEIN OF Fc-ERITROPOYETIN WITH IMPROVED PHARMACOKINETICS FIELD OF THE INVENTION The present invention provides, as a rule, new highly sialylated Fc-EPO fusion proteins, with improved pharmacokinetics. Specifically, Fc-EPO proteins that have a prolonged serum half-life, and increased potency in vivo. The Fc-EPO proteins synthesized in BHK cells have dramatically prolonged serum half-lives, and have increased potency in vivo when compared to the corresponding Fc-EPO fusion proteins produced in other cell lines, such as, for example, NS / 0 cells. The present invention also relates to Fc-EPO wherein a pair of modifications in the Fe- portion, as well as in the EPO portion, have been carried out in order to obtain the respective molecules with further improved properties. BACKGROUND OF THE INVENTION Erythropoietin is a glycoprotein hormone necessary for the maturation of erythroid progenitor cells in erythrocytes. This is produced in the kidney and is essential in regulating the levels of red blood cells in the circulation. The conditions marked Ref.:172282 for low levels of tissue oxygen, increases the signal in the production of erythropoietin, which in turn stimulates erythropoiesis. The level of erythropoietin in the circulation is strictly regulated to ensure that red blood cells are made only in response to a long-term oxygen deficit. 70% of erythropoietin is cleared by receptor-mediated endocytosis. When erythropoietin binds to its receptor, the complex is endocytosed and degraded, thereby limiting the degree of signaling. The rest of the erythropoietin is cleared through renal filtration into the urine. As a result, erythropoietin has a relatively short serum half-life. The human erythropoietin of natural origin or the recombinant erythropoietin produced in mammalian cell contains three chains of N-linked oligosaccharides and one O-linked. N-linked glycosylation occurs in the asparagine residues located at positions 24, 38 and 83, while O-linked glycosylation occurs at a serine residue located at position 126 (Lai et al., (1986) J. Biol. Chem. 261: 3116; Broudy et al., (1988) Arch.

Biochem. Biophys, 265: 329). Oligosaccharide chains have been shown to be modified with terminal sialic acid residues. The N-linked chains typically have up to four sialic acids per chain, and the O-linked chains have up to two sialic acids. An erythropoietin polypeptide can therefore accommodate up to a total of 14 sialic acids. It has been shown that carbohydrate is required for the secretion of erythropoietin from cells, to increase the solubility of erythropoietin, and for the in vivo biological activity of erythropoietin (Dube et al., (1988) - J. Biol. Chem. 263: 17516; DeLo e et al., (1992) Biochemistry 31: 9871-9876). The administration of recombinant human erythropoietin has been effective in the treatment of hematopoietic disorders or deficiencies such as, for example, different forms of anemia, including those associated with renal insufficiency, HIV infection, blood loss and chronic disease. Erythropoietin is typically administered by intravenous injection. Since erythropoietin has a relatively short serum half-life, frequent intravenous injections are required to maintain a therapeutically effective level of erythropoietin in the circulation. Pharmaceutical compositions containing human erythropoietin of natural or recombinant origin are typically administered three times a week at a dose of about 25-100 units / kg. This form of erythropoietin therapy, although very effective, is very expensive and inconvenient because intravenous administration often requires a visit to a doctor or hospital. Currently, a hyperglycosylated analogue of recombinant human erythropoietin, the new erythropoiesis-stimulating protein (NESP), is available under the trade name Aranesp® (Amgen Inc., Thousand Oaks, California) for the treatment of anemia. Aranesp® can be administered less frequently than regular erythropoietin to obtain the same biological response. An alternative route of administration is subcutaneous injection. This form of administration can be performed by patients on site, and is more compatible with lens-release formulations that offer slower absorption from the site of administration, thereby causing a sustained release effect. However, significantly lower circulation levels are achieved by subcutaneous injection and, thus, frequent injections are required to achieve a desirable therapeutic effect. In addition, subcutaneous administration of protein drugs is generally more immunogenic than intravenous administration because the skin, as the major barrier to infection, is an immune organ that is rich in dendritic cells and has sensitive mechanisms to identify and respond to abrasions and foreign materials. Casadevall et al., Recently reported that patients receiving erythropoietin subcutaneously developed anti-erythropoietin antibodies (Casadevall et al (2002) N Engl. J. Med. 346 (7): 469-75). Accordingly, there is a need for more efficient therapy with erythropoietin requiring less frequent administrations. BRIEF DESCRIPTION OF THE INVENTION The present invention provides the fusion proteins of erythropoietin with improved pharmacokinetics compared, in various embodiments, to wild-type or naturally-occurring erythropoietin, to recombinant erythropoietin or to the hyperglycosylated erythropoietin analogue NESP (publication of PCT WO 00/24893). Accordingly, an object of the present invention is to simplify therapy with erythropoietin and reduce the costs associated with the treatment of humans or other mammals with hematopoietic disorders or deficiencies or other indications for the administration of erythropoietin. Specifically, the present invention provides a biologically active Fc-erythropoietin fusion protein (Fc-EPO) that has prolonged serum half life and increased potency in vivo. The "Fc-EPO fusion protein", as used herein, refers to a protein comprising a polypeptide having an Fe moiety and an erythropoietin moiety. The "Fe portion" as used herein, encompasses domains derived from the constant region of an immunoglobulin, preferably a human immunoglobulin, which includes a fragment, analogue, variant, mutant or derivative of the constant region. The "erythropoietin portion", as used herein, encompasses wild-type or naturally-occurring erythropoietin from human and other species, recombinant erythropoietin and erythropoietin-like molecules, including biologically active erythropoietin fragments, analogous variants , mutants, or derivatives of erythropoietin. In one aspect, the present invention provides the Fc-EPO proteins synthesized in BHK cells. The Fc-EPO fusion proteins of the invention, synthesized in BHK cells, have demonstrated dramatically prolonged serum half-lives and increased in vivo potency, when compared to the corresponding Fc-EPO fusion proteins produced in other cell lines, such as for example, NS / 0, PerC6, or 293 cells. The present invention also provides a population of highly sialylated Fc-EPO fusion proteins suitable for administration to a mammal. Highly sialylated Fc-EPO fusion proteins have longer serum half-lives, and increased potency in vivo compared, in various embodiments, to wild-type or naturally-occurring erythropoietin, to recombinant erythropoietin, to the hyperglycosylated erythropoietin analog NESP, or Fc-EPO fusion proteins of the same amino acid sequence, synthesized in NS / 0 cells , PerC6, or 293. According to the present invention, an Fc-EPO fusion protein may contain amino acid modifications in the Fe moiety that generally extend the serum half life of a Fe fusion protein. For example, such modifications of amino acids include mutations that decrease or substantially eliminate the binding to the Fe receptor or the activity of complement fixation. In addition, the Fc-EPO fusion protein may also contain modifications of amino acids in the erythropoietin portion that reduce endocytosis mediated by the EPO receptor or increase the biological activity of erythropoietin. In various embodiments, the present invention combines the benefits provided by an immunoglobulin fusion protein, amino acid modifications of Fe and portions of erythropoietin, and production in BHK cells (e.g., high levels of sialylation). The combined benefits have additive or synergistic effects that result in an Fc-EPO fusion protein with surprisingly long serum half-life and increased in vivo potency.

Accordingly, the present invention in one aspect relates to a BHK cell that contains a nucleic acid sequence encoding an Fc-EPO fusion protein. In one embodiment, the BHK cell of the present invention is adapted for development in a protein-free medium. In yet another embodiment, the BHK cell is adapted for growth in suspension. In still another embodiment, the BHK cell is adapted for growth in a protein-free medium, and in suspension. It has been found that the Fc-EPO fusion proteins produced from the BHK cells grown in a protein-free medium showed surprisingly increased and more homogeneous sialylation compared to the Fc-EPO fusion proteins produced from BHK cells developed in other media. In a preferred embodiment, the nucleic acid is stably maintained in the BHK cell. "Stably maintained nucleic acid" as used herein, refers to any nucleic acid whose rate of loss from a stem cell to a daughter cell is less than 3 percent in the absence of selective pressure, such as an antibiotic-based selection , to maintain the nucleic acid. Thus, when cells stably maintaining a nucleic acid are divided, at least 97 percent (and more preferably, more than 98, more than 99, or more than 99.5 percent) of the resulting cells contain the nucleic acid. When the resulting cells containing the nucleic acid are divided, at least 97% of the cells resulting from that (second) division will contain the nucleic acid. In addition, the number of copies per cell of the nucleic acid is not substantially reduced by repeated cell division. In a preferred embodiment, the stably maintained nucleic acid sequence is integrated from a chromosome of a BHK cell. The nucleic acid sequence that can encode the Fc-EPO fusion protein in any of the various configurations. In a preferred embodiment, the nucleic acid sequence encodes an Fc-EPO fusion protein that includes a Fe portion towards the N-terminus of the Fc-EPO fusion protein, and an erythropoietin portion towards the C-terminus of the fusion protein Fc-EPO. The Fe moiety generally encompasses regions derived from the constant region of an immunoglobulin, including a fragment, analogue, variant, mutant, or derivative of the constant region. In preferred embodiments, the Fe moiety is derived from a human immunoglobulin heavy chain, for example IgG1, IgG2, IgG3, IgG4, or other classes. In some embodiments, the Fc-EPO fusion protein does not include a variable region of an immunoglobulin. In one embodiment, the Fe protein includes a CH2 domain. In yet another embodiment, the Fe moiety includes the CH2 and CH3 domains.

In a preferred embodiment, the Fe moiety contains a mutation that reduces the affinity for an Fe receptor or reduces the effector function of Fe. For example, the Fe moiety may contain a mutation that removes the glycosylation site within the Fe moiety. a heavy chain of IgG. In some embodiments, the Fe moiety contains mutations, deletions or insertions of an amino acid position corresponding to Leu234, leu235, Gly236, Gly237, Asn297, or Pro331 of IgG1 (the amino acids are numbered according to the EU nomenclature). In a preferred embodiment, the Fe portion contains a mutation at an amino acid position corresponding to Asn297 of the IgG1. In alternative embodiments, the Fe moiety contains mutations, deletions or insertions at an amino acid position corresponding to Lew281, Leu282, Gly283, Gly284, Asn344, or Pro378 of IgGl. In some embodiments, the Fe portion contains a CH2 domain derived from a heavy chain of human IgG2 or IgG4. Preferably, the CH2 domain contains a mutation that removes the glycosylation site within the CH2 domain. In a modality, the mutation alters asparagine within the amino acid sequence Gln-Phe-Asn-Ser within the CH2 domain of the heavy chain of Ig2 or IgG4. Preferably, the mutation changes asparagine to a glutamine. Alternatively, the mutation alters phenylalanine and asparagine within the amino acid sequence Gln-Phe-Asn-Ser. In one embodiment, the amino acid sequence Gln-Phe-Asn-Ser is replaced with an amino acid sequence Gln-Ala-Gln-Ser. Asparagine within the amino acid sequence Gln-Phe-Asn-Ser corresponds to Asn297 of IgGl. It has been found that mutation of asparagine within the amino acid sequence Gln-Phe-Asn-Ser of IgG2 or IgG4 (for example, corresponding to Asn297 of IgGl) also surprisingly reduces the binding of the Fc-EPO fusion protein to the EPO receptor. Without wishing to be bound by any theory, the mutation of asparagine within the amino acid sequence Gln-Phe-Asn-Ser of IgG2 or IgG4 (eg, corresponding to Asn297 of IgGl) can induce a complete conformational change in the protein Fc-EPO fusion, which leads to dramatically improved pharmacokinetic properties. In yet another embodiment, the Fe moiety includes a CH2 domain and at least a portion of a hinge region. The hinge region can be derived from an immunoglobulin heavy chain, for example, IgGl, IgG2, IgG3, IgG4 or other classes. Preferably, the hinge region is derived from human IgGl, IgG2, IgG3, IgG4 or other suitable classes. More preferably, the hinge region derived from a human IgGl heavy chain. In one embodiment, the cysteine in the amino acid sequence Pro-Lys-Ser-Cys-Asp-Lys of the hinge region of IgG1 is altered. In a preferred embodiment, the amino acid sequence Pro-Lys-Ser-Cys-Asp-Lys is emplaced with an amino acid sequence Pro-Lys-Ser-Ser-Asp-Lys. In one embodiment, the Fe moiety includes a CH2 domain derived from a first antibody isotype, and a hinge region derived from a second antibody isotype. In a specific embodiment, the CH2 domain is derived from a heavy chain of human IgG1 or IgG4, while the hinge region is derived from an altered human IgG1 heavy chain. In a preferred embodiment, the Fe portion derived from an IgG sequence in which the amino acid sequence Leu-Ser-Leu-Ser near the C-terminus of the constant region is altered to eliminate the potential T-cell epitopes of binding. For example, in one embodiment, the amino acid sequence Leu-Ser-Leu-Ser is replaced with an amino acid sequence Ala-Thr-Ala-Thr. In yet another embodiment, the Fe moiety is derived from an IgG sequence in which the C-terminal lysine residue is replaced. Preferably, the C-terminal lysine, of an IgG1 sequence, is replaced with an amino acid other than lysine, such as alanine, to further increase the serum half-life of the Fe fusion protein.

In accordance with the present invention, the Fe moiety may contain one or more mutations described herein. Combinations of mutations in the Fe moiety generally have additive or synergistic effects on the extended serum half-life and the increased in vivo potency of the Fc-EPO fusion protein. Thus, in an exemplary embodiment, the Fe moiety can contain (i) a region derived from an IgG1 sequence, in which the amino acid sequence Lys-Ser-Lys-Ser is replaced with an amino acid sequence Ala-Thr -Ala-Thr; (ii) a C-terminal alanine residue instead of lysine; (iii) a CH2 domain of a hinge region that are derived from different antibody isotypes, eg, a CH2 domain of IgG2 and an altered hinge region of IgG1; (iv) a mutation that eliminates the glycosylation cycle within the CH2 domain derived from IgG2, for example, an amino acid sequence Gln-Ala-Gln-Ser instead of the amino acid sequence Gln-Phe-Asn-Ser within the domain CH2 derived from IgG2. The erythropoietin portion of the Fc-EPO fusion protein can be a full-length or wild-type wild-type erythropoietin, a recombinant erythropoietin, or an erythropoietin-like molecule, such as a biologically active erythropoietin fragment, analogous, variant , mutant or erythropoietin derivative.

Preferably, the erythropoietin portion is derived from a human erythropoietin. In some embodiments, the erythropoietin portion may contain amino acid modifications that reduce the binding affinity for the EPO receptor, or increase the biological activity of erythropoietin. In some embodiments, the erythropoietin portion contains at least one of the following mutations: Argl31 - > Glu and Argl39 - > Glu (the amino acid numbering based on the mature human erythropoietin sequence). In other embodiments, the erythropoietin portion contains at least one of the following mutations: His32 - »Gly, Ser3 - > Arg, and Pro90 - Ala. In yet another embodiment, the erythropoietin portion has a disulfide bond pattern distinct from human erythropoietin. For example, the erythropoietin portion may contain one or more of the following amino acid substitutions: a non-cysteine residue at position 29, a non-cysteine residue at position 33, a cysteine residue at position 88, and a residue of cysteine at position 139. In one embodiment, the erythropoietin portion contains cysteine residues at positions 7, 29, 88 and 161. In yet another embodiment, the erythropoietin portion further contains one or more of the following substitutions Hys32 - > Gly, Cys33 - Pro, and Pro90 - »Ala. In accordance with the present invention, the erythropoietin portion can contain any combination of the mutations described herein.

In some embodiments, the Fc-EPO fusion protein includes a linker between the Fe portion and the erythropoietin portion. If included, the linker generally contains between 1 and 25 amino acids and preferably does not have a protease cleavage site. The linker may contain an N-linked glycosylation site or an O-linked site to block proteolysis. For example, in one embodiment, the linker contains an amino acid sequence Asn-Ala-Thr. The present invention also relates to a method for producing an Fc-EPO fusion protein. The method includes the maintenance of BHK cells containing a nucleic acid sequence, which codes for the low Fc-EPO fusion protein or conditions suitable for the expression of the encoded Fc-EPO fusion protein, and recovering the fusion protein. Fc-EPO expressed. In one embodiment, BHK cells are cultured in a protein-free medium. In yet another embodiment, the BHK cells are cultured in suspension. In yet another embodiment, the BHK cells are cultured in a protein-free and suspension medium. In some embodiments, the nucleic acid is stably maintained in BHK cells. In general, the Fc-EPO fusion protein produced in BHK cells have a longer serum half-life than a corresponding Fc-EPO fusion protein produced in other cell lines, such as, for example, NS / 0 cells, PerC6, or 293.

The present invention provides a pharmaceutical composition containing the Fc-EPO fusion protein produced in BHK cells. In a preferred embodiment, the Fc-EPO fusion protein used in the pharmaceutical composition has not been treated to remove sialic acid residues. The pharmaceutical composition also includes a pharmaceutically acceptable carrier. The present invention also provides a method for treating a mammal when administering the pharmaceutical composition to the mammal. In some embodiments, the treated mammal has a hematopoietic disorder or deficiency. Because the Fc-EPO fusion proteins of the present invention have increased potency in vivo, and prolonged serum half-life, pharmaceutical compositions containing the Fc-EPO fusion proteins generally require less frequent administration compared to the compositions Pharmaceuticals containing the erythropoietin of natural or recombinant origin or the corresponding Fc-EPO fusion proteins in other cells. In a preferred embodiment, the pharmaceutical composition is administered less than three times per week (e.g., twice a week, weekly, or no more than once every ten days, such as once every two weeks, once a month or once every two months). In still another aspect, the present invention provides a method for selecting a BHK cell that stably maintains a nucleic acid encoding a fusion protein that includes a Fe portion and an erythropoietin portion. The method includes introducing into a BHK cell a nucleic acid sequence encoding hygromycin B and a nucleic acid sequence encoding the fusion protein; and culturing the BHK cell in the presence of hygromycin B. In one embodiment, the nucleic acid sequence encoding hygromycin B of the nucleic acid sequence encoding the fusion protein is present in a single nucleic acid. In yet another embodiment, the nucleic acid sequence encoding hygromycin B and the nucleic acid sequence encoding the fusion protein are present in two separate nucleic acids. In still another aspect, the present invention provides a population of purified Fc-EPO fusion proteins, suitable for administration to a mammal. In a preferred embodiment, the Fc-EPO fusion proteins include a Fe portion towards the N-terminus of the Fc-EPO fusion proteins and a portion of erythropoietin towards the C-terminus of the Fc-EPO fusion proteins. In a more preferred embodiment, the population of purified Fc-EPO fusion proteins is extensively sialylated, for example, having an average of 11-28 sialic acid residues per purified Fc-EPO fusion protein. Preferred highly sialylated populations of the Fc-EPO fusion proteins have an average of 13-28, 15-28, 17-28, 19-28, or 21-28 sialic acid residues per purified Fc-EPO fusion protein. . For example, a highly preferred sialylated population of the Fc-EPO fusion proteins averages from 20 to 22 sialic acid residues per purified Fc-EPO fusion protein. In a preferred embodiment, the purified Fc-EPO fusion proteins are synthesized in a BHK cell. In one embodiment, the BHK cell is adapted for growth in suspension. In yet another embodiment, the BHK cell is adapted for growth in a protein medium. In yet another embodiment, the BHK cell is adapted for growth in a protein-free and suspension medium. The highly sialylated population of purified Fc-EPO fusion proteins provided by the present invention have a longer serum half-life, as compared to a population of corresponding Fc-EPO fusion proteins produced in cells such as, for example, NS / 0, PerC6, or 293 cells. In accordance with the present invention, the Fe portion and the erythropoietin portion of the purified Fc-EPO merger portions may contain one or more mutations or modifications as described herein, providing a prolonged serum half-life and increased in vivo potency, with effects that are additive or synergistic with increased sialylation.

The present invention also provides a pharmaceutical composition containing the highly sialylated population of the purified Fc-EPO fusion proteins as described herein. A preferred pharmaceutical composition further includes a pharmaceutically acceptable carrier. The present invention further provides a method for treating a mammal that includes administering to the mammal a pharmaceutical composition containing the highly sialylated population of the purified Fc-EPO fusion proteins. In a preferred embodiment, the pharmaceutical composition is administered less than three times per week (for example, twice a week, weekly, or no more than once every ten days, such as once every two weeks, once a month or once every two months). • In summary, the invention relates to the following matters: A purified dimeric fusion protein, consisting essentially of a dimeric Fe portion of a human IgG molecule comprising a hinge region, a CH2 domain and a CH3 domain, and human erythropoietin (EPO), where each chain of the dimeric Fe moiety is linked via its C-terminus directly or via a peptide linker to the N-terminus of an EPO molecule, the fusion protein has the following properties: (i) the The molecule is highly sialylated by comprising 15-28 sialic acid residues; (ii) the CH2 domain is derived from human IgG2 and is modified by replacing the amino acid residues Phe and Asn, within the Gln-Phe-Asn-Ser sequence of the CH2 domain with Ala and Asn, thereby forming the Gln-Ala-Gln-Ser sequence within the CH2 domain, and (iii) the stretch of the amino acid sequence Leu-Ser-Leu-Ser near the C-terminus of the CH3 domain is replaced with Ala-Thr-Ala-Thr. • A respective dimeric Fc-EPO fusion protein, wherein, additionally, the C-terminal Lys residue of the CH3 domain is replaced with Ala. • A respective dimeric Fc-EPO fusion protein, wherein a hinge region is derived from human IgGl. • A respective dimeric Fc-EPO fusion protein, wherein the hinge region of IgGl is modified by replacing the amino acid residue Cys within the stretch of the Pro-Lys-Ser-Cys-Asp-Lys sequence of the region of hinge, with a residue of Being, thus forming the Pro-Lys-Ser-Ser-Asp-Lys sequence within the hinge region. • A respective dimeric Fc-EPO fusion protein, wherein the erythropoietin portion comprises at least one of the following amino acid substitutions: (i) a non-cysteine residue at position 29 of the EPO molecule, (ii) a non-cysteine residue at position 33 of the EPO molecule, (iii) a cysteine residue at position 88 of the EPO molecule, and (iv) a cysteine residue at position 139 of the EPO molecule. • A dimeric Fc-EPO fusion protein, respectively, wherein a non-Cys amino acid residue is at position 33 of the EPO molecule, instead of the original Cys residue, and a Cys residue is at position 88 of the molecule of EPO instead of the original Trp residue, thus making it possible for the EPO portion within the fusion protein to form a disulfide bond Cys2g-Cys88- • A respective dimeric Fc-EPO fusion protein, wherein the amino acid residue no Cys at position 33 is Pro. • A respective dimeric Fc-EPO fusion protein, wherein the EPO portion comprises one or more mutations selected from the group: (ii) Arg139 - > Glu139 (iii) His32 - > Gly32 (iv) Ser34 - Arg34 (v) Pro90 - > Ala90 • A respective dimeric Fc-EPO fusion protein, wherein the linker peptide comprises a glycosylation site. • A respective dimeric Fc-EPO fusion protein, wherein the glycosylation site comprises an amino acid sequence Asn-Ala-Thr. • A respective dimeric Fc-EPO fusion protein, which additionally comprises a CH1 domain.

• An Fc-EPO fusion protein, respectively, wherein the complete IgG molecule, including CH2, CH3 and optionally CH1, is derived from IgG2 and the hinge region is derived from IgG1. • A respective Fc-EPO fusion protein, wherein the complete IgG molecule, including CH2, CH3 and the hinge region, and optionally CH1, is derived from the IgG1. • A respective dimeric Fc-EPO fusion protein, wherein the fusion protein has 18-24, preferably 20-22 sialic acid residues. • A dimeric Fc-EPO fusion protein comprising the sequence: EPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQAQSTFRWSVLTWHQDWLNGKEYKCKVS NKGLPAPIEKTISKTK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPMLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSATATPGAA PPRLICDSRVLERYLL EAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEV WQGLALLSEAVLRGQA LLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAA SAAPLRTITADTFRKL FRVYSNFLRGKLKLYTGEACRTGDR (SEQ ID NO: 14) • A dimeric Fc-EPO fusion protein comprising the sequence: EPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQAQSTFRWSVLTVHQDWLNGKEYKCKVSN KGLPAPIEKTISKTK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKRTPPMLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSATATPGAA PPRLICDSRVLERYLL ? EAKEAENITTGCAEGPSi LNENITVPDTKVNFYAWKRMEVGQQAV EWQGLALLSEAVLRGQA LLVNSSQPCEALQLHDKAVSGLRSLTLLRALGAQKEAISPPDAASA APRQLRTITADTFRKL FRVYSNFLRGKLKLYTGRACRTGDR (SEQ ID NO: 15) • A DNA molecule encoding a fusion protein as specified above • A pharmaceutical composition suitable for the treatment of hematopoietic disorders of deficiencies in a mammal comprising an effective amount of an Fc-EPO fusion protein as specified above or in the claims, optionally, together with a pharmaceutically acceptable carrier, diluent or excipient. • A population of highly purified, sialylated Fc-EPO fusion proteins suitable for administration to a mammal, Fc-EPO fusion proteins comprising a Fe portion towards the N-terminus of the Fc-EPO fusion proteins and a portion of erythropoietin towards the C-terminus of the Fc-EPO fusion proteins, the population of the fusion proteins have an average of 15-28 sialic acid residues per purified Fc-EPO fusion protein and that is obtained by introducing a molecule of DNA encoding a respective Fc-EPO fusion protein within a BHK cell, and expressing, isolating and purifying the population of corresponding Fc-EPO fusion proteins, wherein the population has a longer whole half-life, compared to a population of corresponding Fc-EPO fusion proteins synthesized in NS / 0, PerC6 or 293 cells. • A corresponding population of purified Fc-EPO fusion proteins, wherein the population of fusion proteins has an average of 20-22 sialic acid residues per purified Fc-EPO fusion protein. • A corresponding population of purified Fc-EPO fusion proteins, wherein the BHK cell is adapted for human growth and free of proteins or in suspension. • A method for producing a population of recombinant, purified, highly sialylated Fc-EPO fusion proteins, comprising a Fe portion towards the N-terminus of the Fc-EPO fusion proteins and a portion of erythropoietin towards the C-terminus of the proteins of Fc-EPO fusion, the method comprises the steps of: (i) building a DNA molecule that encodes an Fc-EPO fusion protein, (ii) transforming a BHK cell with the DNA molecule into a protein-free medium or in suspension, (iii) expressing the population of the Fe fusion proteins encoded by the DNA molecule, (iv) harvesting, isolating and purifying the population of Fc-EPO fusion proteins. • A corresponding method, wherein the synthesized population of the fusion proteins have an average of 15-28, preferably 15-25, more preferably 20-22 sialic acid residues per purified Fc-EPO fusion proteins. • A method for selecting a BHK cell by stably maintaining a nucleic acid sequence encoding an Fc-EPO fusion protein comprising an Fe portion and an erythropoietin portion, the method comprising the steps of: (a) inserting into a BHK cell, a nucleic acid sequence encoding hygromycin B, and a DNA sequence encoding the Fc-EPO fusion protein; and (b) culturing the BHK cell in the presence. of hygromycin B. • A corresponding method, wherein the nucleic acid sequence encoding hygromycin B and the DNA sequence encoding the Fc-EPO fusion protein are present in a single DNA molecule. BRIEF DESCRIPTION OF THE FIGURES Figures 1A and IB describe an alignment of the amino acid sequences of the constant regions of the IgGl., Human IgG2 and IgG4. Figure 2 depicts a pharmacokinetic experiment with mice showing a correlation between the dose of Fc-EPO and the amount of decrease in serum concentrations of Fc-EPO during the alpha phase. In that experiment, a variant of susb-sialylated Fc-EPO synthesized in NS / 0 cells was used. Figure 3 describes the potential routes of elimination of the Fc-EPO fusion proteins and modifications to the fusion protein, which potentially modulate these routes. Figure 4 describes the responses of exemplary hematocrit in mice after administration of Fcg2h (FN >; AQ) -EPO. Figure 5 describes hematocrit responses in rat cues, after administration of the Fcg2h-EPO, Fcg2h-EPO (NDS), Fcg4h-EPO and Fcg4h (N> Q) -EPO proteins produced from BHK cells . Figure 6 describes hematocrit responses in cues in mice, after administration of Fcg2h-EPO (NDS) produced from BHK cells, Fcg2h-EPO (NDS) produced from NS / 0 cells, and NESP (by example, Aranesp®). Figure 7 depicts an exemplary nucleic acid sequence encoding a mature Fc-EPO protein. Figure 8 describes the pharmacokinetic profiles of Fcg2h (FN > AQ) -EPO produced from BHK and Fcg2h (FN> AQ) -EPO cells produced from NS / O cells in mice. Figure 9 describes the pharmacokinetic profiles of Fcg2h-EP0 (NDS) produced from BHK and Fcg2h EPO (NDS) cells produced from NS / O cells in mice.

Figure 10 describes the profiles of the pharmacokinetic proteins of Fcg2h-EPO (NDS) produced from BHK-21 cells, PERC6 cells and 293 cells in mice. Figure 11 describes the hematocrit responses in beagle dogs after treatment with the Fcg2h (FN - »AQ) -EPO proteins synthesized in BHK cells. DETAILED DESCRIPTION OF THE INVENTION The present invention provides an Fc-EPO fusion protein with improved pharmacokinetics. Specifically, the Fe protein provided by the present invention has a prolonged serum half life and increased potency in vivo. In one aspect, the present invention provides an Fc-EPO fusion protein synthesized in BHK cells. The Fc-EPO fusion proteins synthesized in BHK cells have demonstrated dramatically prolonged serum half-lives, and increased potency in vivo, when compared to the corresponding Fc-EPO fusion proteins produced in other cell lines, such as, for example, NS / O cells, perC6 or 293. In still another aspect, the present invention provides a population of highly sialylated Fc-EPO fusion proteins. The population of highly sialylated Fc-EPO fusion proteins has a longer serum half-life, compared to a corresponding population of Fc-EPO fusion proteins, with low levels of sialylation. According to the present invention, an Fc-EPO fusion protein can contain amino acid modifications in the Fe moiety extending in the half-life a serum of a Fe fusion protein, such as by decreasing or substantially eliminating the receptor binding activity of Fe, or modifications that reduce the activity of complement fixation. In addition, the Fc-EPO fusion protein may also contain amino acid modifications in the erythropoietin portion that produces endocytosis mediated by the EPO receptor, or increase the biological activity of erythropoietin. Fc-EPO Fusion Protein "Fc-EPO Fusion Protein" as used herein, refers to a protein comprising a polypeptide having at least two portions, namely, an Fe portion and an erythropoietin portion, which does not they are normally present in the same polypeptide. In preferred embodiments of the present invention, polypeptides having an Fe moiety and an erythropoietin moiety form homodimers; consequently, the Fc-EPO fusion protein is generally a dimeric protein "held together by one or more disulfide bonds, each polypeptide chain contains an Fe moiety and an erythropoietin moiety, however, the Fc-EPO fusion protein of the present invention can have any configuration that allows the erythropoietin moieties to be stably associated with Fe moieties, while maintaining erythropoietin activity, eg, such configurations include, but are not limited to, a simple polypeptide containing two Fe and Fe moieties. two portions erythropoietin, a simple polypeptide containing two Fe portions and one erythropoietin portion, a heterodimeric protein that includes a polypeptide containing one Fe moiety and one erythropoietin moiety, and one other polypeptide containing an Fe moiety, and other suitable configurations. Erythropoietin portion can be directly or indirectly linked to the po Fe rtion in various configurations. In one embodiment, the erythropoietin portion is directly linked to the Fe portion through a covalent bond. For example, the erythropoietin portion can be fused directly to the Fe portion either at its C-terminus or its N-terminus. In one embodiment, the C-terminus of the Fe moiety is fused to the N-terminus of the erythropoietin moiety, for example, Nterm-Fc-Cterm-Nterm-EPO-Cterm. In this configuration, the Fe portion is toward the N-terminus of the Fc-EPO fusion protein and the erythropoietin portion is toward the C-terminus. In yet another embodiment, the C-terminus of erythropoietin is fused to the N-terminus of the FC portion., for example, Nterm-EPO-Cterm-Nterm-Fc-Cerm. In this configuration, the erythropoietin portion is towards the N-terminus of the Fc-EPO fusion protein and the Fe portion is towards the C-terminus. In other embodiments, the erythropoietin portion is indirectly linked to the Fe moiety. For example, the Fc-EPO fusion protein may include a linker (L) between the Fe portion and the erythropoietin portion. Similar to direct fusion, the erythropoietin moiety is preferably fused to the C-terminus of the Fe moiety through a linker, for example, Nterm-Fc-Cterm-Nterm-EPO-Cterm. Thus, the Fe portion is towards the N-terminus of the Fc-EPO fusion protein and separated by a linker from the erythropoietin portion towards the C-terminus. Alternatively, the erythropoietin portion may be fused N from the Fe moiety. Through a linker, e.g., Nterm-EPO-Cterm-Nterm-FC-Cterm - Fe Sercation As used herein, "Fe moiety" encompasses the domains derived from the constant portion of an immunoglobulin, preferably a human immunoglobulin , including a fragment, analog, variant, mutant derived from the constant region. Suitable immunoglobulins include IgG1, IgG2, IgG3, IgG4 and other classes. The constant region of an immunoglobulin is defined as a naturally occurring or synthetically produced polypeptide, homologous to the C-terminal region of immunoglobulin, and may include a CH1 domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain , separately or in combination. A sequence alignment of the constant regions of human IgGl, IgG2, IgG3, IgG4 is shown in Figures 1A and IB. According to Paul, (1999) Fundamental Immunology 4a. Ed., Lippincott-Raven, the CH1 domain includes amino acids 118-215; the hinge region includes amino acids 216-230; the CH 2 domain includes amino acids 231-340; and the CH3 domain includes amino acids 341-447 (the amino acid positions are based on the IgG1 sequence). The hinge region binds to the CH1 domain to the CH2 and CH3 domains. In the present invention, the Fe portion typically includes at least one CH2 domain. For example, the Fe moiety may include hinge-CH2-CH3. Alternatively, the Fe portion may include all or a portion of the hinge region, the CH2 domain and / or the CH3 domain.

The constant region of an immunoglobulin is responsible for many important functions of the antibody, including binding to the Fe receptor (FcR) and complement fixation. There are five major classes of heavy chain constant reactions, classified as IgA, IgG, IgD, IgGE and IgM, each with characteristic effector functions designated by isotype. For example, IgG is separated into four subclasses?:? L,? 2,? 3 and? 4, also known as IgGl, IgG2, IgG3 and IgG4, respectively. IgG molecules interact with multiple cases of cellular receptors, including three classes of Fc? (Fc? R) specific for the IgG class of antibody, namely Fc? Rl, Fc? RII, and Fc? RIII. The sequences important for the binding of IgG to FcγR receptors have been reported as located in the CH2 and CH3 domains. The serum half-life of an antibody is influenced by the ability of that antibody to bind to an Fe receptor (FcR). Similarly, the serum half-life of immunoglobulin fusion proteins is also influenced by the ability to bind to such receptors (Gillies SD et al., (1999) Cancer Res. 59: 2159-66). Compared to those of IgGl, the CH2 and CH3 domains of IgG2 and IgG4 have biochemical undtable or reduced binding affinity to Fe receptors. It has been reported that immunoglobulin fusion proteins containing the CH2 and CH3 domains of the IgG2 and IgG4 had longer serum half-lives, compared to the corresponding fusion proteins containing the CH2 and CH3 domains of IgGl (U.S. Patent No. 5,541 / 087; Lo et al. (1998) Protein Engineering , 11: 495-500). Accordingly, the preferred CH2 and CH3 domains for the present invention are derivatives of an antibody isotype with binding affinity to the receptor and reduced effector functions, such as, for example, IgG2 or IgG4. The most preferred CH2 and CH3 domains are IgG2 derivatives. The hinge region is normally located C-terminal to the CH1 domain of the heavy chain constant region. In IgG isotypes, disulfide bonds typically appear within this hinge region, allowing the final tetrameric molecule to form. This region is dominated by prolines, serines and treonas. When included in the present invention, the hinge region is typically at least homologous to the immunoglobulin region of natural origin that includes the cysteine residues to form disulfide bonds that bind the two portions of Fe. The representative sequence of the hinge regions for human and mouse immunoglobulins can be found in Borrebaeck, C.A.K., ed., (1992) ANTIBODY ENGINEERING, A PRACTICAL GUIDE, G.H. Freeman and Co., The hinge regions suitable for the present invention can be derived from IgG1, IgG2, IgG3 and IgG4 and other classes of immunoglobulins. The hinge region of IgGl has three cysteines, two of which are involved in disulfide bond between the two heavy chains of the immunoglobulin. These same cysteines allow the formation of efficient and consistent disulfide bonds between the Fe portions. Therefore, a preferred hinge region of the present invention is derived from IgG1, more preferably from human IgG1. In some embodiments, the first cysteine within the hinge region of human IgGl is mutated to another amino acid, preferably serine. The hinge region of the IgG2 isotype has four disulfide bonds that tend to promote oligomerization and possibly incorrect disulfide bonding within the secretion in the recombinant systems. A suitable hinge region can be derived from an IgG2 hinge; the first two cysteines are each preferably mutated to another amino acid. The hinge region of IgG4 is known to form disulfide bonds that form interchain disulfide bonds efficiently. However, a hinge region suitable for the present invention can be derived from the hinge region of IgG4, preferably containing a mutation that improves the correct formation of the disulfide bonds between the portions derived from the heavy chain (Angal S., et al. (1993) Mol. Immunol., 30: 105-8). According to the present invention, the Fe moiety may contain the CH2 and / or CH3 domains and a hinge region, which are derived from different antibody isotypes, for example, a hybrid Fe moiety. For example, in one embodiment, the Fe portion contains the CH2 domains containing and / or CH3 derived from IgG2 or IgG4, and a mutant hinge region derived from IgG1. Alternatively, a mutant hinge region from another subclass of Fe is used in a hybrid IgG portion. For example, a mutant form of the IgG4 hinge that allows efficient disulfide bonding between the two heavy chains can be used, a mutant hinge can also be derived from a hinge of? GG2 in which the first two cysteines are each mutated to another amino acid. Such hybrid portions of Fe facilitate high level expression and improve the correct assembly of the Fc-EPO fusion proteins. The assembly of such hybrid Fe portions has been described in U.S. Patent Publication No. 20030044423 (e.g., U.S. Application No. 10 / 093,958), the description of which is incorporated by reference. at the moment .

In some embodiments, the Fe moiety contains amino acid modifications that generally extend the serum half-life of a Fe fusion protein. Such amino acid modifications include mutations that decrease or substantially eliminate Fe receptor binding or activity. fixation of the complement. For example, the glycosylation site within the Fe portion of an immunoglobulin heavy chain can be removed. In IgGl, the glycosylation site is Asn297. In other immunoglobulin isotypes, the glycosylation site corresponds to IgGl Asn297. For example, in IgG2 and IgG4, the glycosylation site is asparagine within the amino acid sequence Gln-Phe-Asn-Ser. Accordingly, an ASN297 mutation of IgG1 removes the glycosylation site in an Fe portion derived from IgGl. In one modality, Asn297 is replaced with Gln. Similarly, in IgG2 or IgG4, a mutation of asparagine within the amino acid sequence Gln-Phe-Asn-Ser removes the glycosylation site in a Fe portion derived from the heavy chain of IgG2 or IgG4. In one embodiment, asparagine is replaced with a glutamine. In other embodiments, the phenylalanine within the amino acid sequence Gln-Phe-Asn-Ser is further mutated to eliminate a potential non-own T cell epitope resulting from the mutation of asparagine. For example, the amino acid sequence Gln-Phe-Asn-Ser within a heavy chain of IgG2 or IgG4 can be replaced with an amino acid sequence Gln-ala-Gln-Ser. It has also been observed that the alteration of the amino acids near the binding of the Fe portion and the non-Fe portion can dramatically increase the serum life of the Fe fusion protein (PCT publication WO 01/58957, the description of the which is incorporated by reference herein). Accordingly, the binding region of the Fc-EPO fusion protein of the present invention may contain alterations which, relative to the naturally occurring sequences of an immunoglobulin heavy chain and erythropoietin, fall preferentially within about 10 amino acids within from the point of union. These amino acid changes may cause an increase in hydrophobicity by, for example, changing the C-terminal lysine of the Fe moiety to a hydrophobic amino acid such as alanine or leucine. In other embodiments, the Fe moiety contains amino acid alterations of the Leu-Ser-Leu-Ser segment near the C portion of the Fe portion of an immunoglobulin heavy chain. The amino acid substitutions of the Leu-Ser-Leu-Ser segment eliminate the epitopes of potential binding T cells. In one embodiment, the amino acid sequence Leu-Ser-Leu-Ser near the C-terminus of the Fe moiety is replaced with the amino acid sequence Ala-Thr-Ala-Thr. In other embodiments, the amino acids within the Leu-Ser-Leu-Ser segment are replaced with other amino acids such as glycine or proline. Detailed methods for generating amino acid substitutions of the Leu-Ser-Leu-Ser segment near the C-terminus of an IgG1, IgG2, IgG3, IgG4 or another class of immunoglobulin molecule have been described in the United States Patent Publication. No. 20030166877 (e.g., U.S. Patent Application No. 10 / 112,582), the description of which is incorporated by reference herein. Erythropoietin portion As used herein, "erythropoietin portion" encompasses wild type or naturally occurring erythropoietin from human and other species, recombinant erythropoietin and erythropoietin-like molecules, including biologically active erythropoietin fragments, analogues, variants, mutants or erythropoietin derivatives. Wild type or naturally occurring erythropoietin is a 34 KD glycoprotein hormone that stimulates the growth and development of red blood cells from erythropoietin precursor cells. Wild-type or naturally-occurring erythropoietin is produced in the kidney in response to hypoxia (e.g., the loss of red blood cells due to anemia) and regulates the growth of red blood cells and differentiation through interaction with their cognate cell receptor. Wild-type or naturally-occurring erythropoietin can be isolated and purified from blood (Miyake T., et al., (1997) J. Biol. Chem. 252: 5558-5564) or plasma (Goldwasser, E. , et al., (1997) Proc. Nati. Acad. Sci. USA, 68: 697-698) or urine. Recombinant or chemically synthesized erythropoietin can be produced using techniques well known to those skilled in the art. Two forms of recombinant human erythropoietin (rHuEPO) are commercially available: EPOGEN® from Amgen and PROCRIT® from Johnson & amp;; Johnson. As used herein, the biological activity of erythropoietin is defined as the ability to stimulate cell proliferation through interaction with the erythropoietin receptor. The erythropoietin functional assay can be conducted in vitro or in vivo. For example, the in vitro activity of erythropoietin can be tested in a cell-based assay. Specifically, the erythropoietin activity can be determined based on a TF-1 cell proliferation assay. TF-1 cells express EPO receptors. The proliferation of TF-1 cells that is determined by the incorporation of tritiated thymidine, is a function of the activity of erythropoietin (Hammerlling et al., (1996) J. Pharmaceutical and Biomedical Analysis, 14: 1455; Kitamura et al. , (1989) J. Cellular Physiol., 140: 323). The in vitro cell-based assay is described in more detail in Example 6. In vivo assays are typically conducted in animal models, such as, for example, mice and rats. Examples of in vivo assays include, but are not limited to, hematocrit (HCT) assays and reticulocyte assays. HCT assays measure the volume of red blood cells from a sample of blood taken from an animal treated with erythropoietin, and are performed by centrifugation of blood and capillary tubes and measuring the fraction of the total volume occupied by red blood cells sedimented. The HCT in vivo assay is described in more detail in Example 8. The reticulocyte assays measure the new red blood cells, also known as reticulocytes, which have recently differentiated from the precursor cells and still have nucleic acid remnants. characteristic of the precursor cells. Reticulocytes are measured by sorting red blood cells in a flow cytometer, after staining with a nucleic acid stain dye such as acridine orange or thiazole orange, and counting the positively stained reticulocyte fraction. A biologically active or functionally active erythropoietin-like molecule typically shares substantial amino acid sequence identity similarity (eg, at least about 55%, about 65%, about 75% identity, typically at least about 80%, and most typically about 90-95% identity) with the corresponding wild-type, or naturally occurring, sequences of erythropoietin, and possesses one or more of the wild-type erythropoietin functions. Thus, it is understood that the erythropoietin of the present invention specifically includes erythropoietin polypeptides having the amino acid sequences analogous to the wild-type erythropoietin sequences. Such proteins are defined herein as erythropoietin analogues. An "analogue" is defined herein to mean an amino acid sequence with sufficient similarity to the amino acid sequence of wild-type erythropoietin to possess the biological activity of the protein. For example, an erythropoietin analog can contain one or more amino acid changes in the amino acid sequence of the wild-type erythropoietin, it even possesses, for example, the ability to stimulate the production or maturation of red blood cells. Examples of such amino acid changes include additions, deletions or substitutions of amino acid residues. The erythropoietin of the present invention also encompasses the mutant proteins that exhibit higher or lower biological activity than the wild type erythropoietin, such as described in U.S. Patent No. 5,614,184. The erythropoietin of the present invention also encompasses the biologically active fragments of erythropoietin. Such elements may only include a part of the full-length amino acid sequence of erythropoietin, and still possess biological activity. As used herein, a "biologically active fragment" means a fragment that can exert a biological effect similar to the full-length protein. Such fragments can be produced by amino- and carboxyl-terminal deletions, as well as internal deletions. These can also include truncated and hybrid forms of erythropoietin. "Truncated" forms are shorter versions of erythropoietin, for example, with amino-terminal or carboxyl-terminal residues eliminated. Variations in the erythropoietin sequence The amino acid modifications can be introduced into the erythropoietin portion of the present invention to reduce the binding affinity to the EPO receptor.; to increase the stability of the protein; to increase the adoption of an active and correct conformation; to improve the pharmacokinetic properties; to improve the synthesis; or to provide other advantageous features. For example, endocytosis mediated by the EPO receptor is determined by the binding activity between the erythropoietin and the EPO receptor. The three-dimensional structure of a human erythropoietin complex and the EPO receptor demonstrate that the binding of erythropoietin to its receptor is dominated by positive charges on the surface of the erythropoietin and negative charges on the EPO receptor. Syed et al., (1998) Nature 395: 511. To reduce the rate of initiation of the link, mutations can be introduced to replace the positively charged amino acids that lie near the contact surface of the erythropoietin-EPO receptor. For example, in one embodiment, one or both of Argl31 and Argl39 of human erythropoietin can be replaced (the amino acid numbering of the EPO sequence which is based on mature human EPO). Preferably, Argl31 and Argl39 are replaced with glutamic acid, aspartic acid or other non-positively charged amino acids. Mutations in the erythropoietin of another species can be introduced to replace the amino acids corresponding to Argl31 and Arl39 of human erythropoietin. However, to preserve the biological activity of EPO, those residues that are at the center of the EPO EPO-receptor interaction must be edited when alterations are made in the EPO amino acid sequence. Alternatively, those regions or positions that could tolerate amino acid substitutions or alanine scanning mutagenesis can be determined empirically (Cunningham et al., (1989) Science, 244, 1081-1085). In this method, the selected amino acid residues are individually substituted with a neutral amino acid (eg, alanine) in order to determine the effects on biological activity. In one embodiment, the erythropoietin moiety contains at least one of the following mutations. His32 - »Gly and / or Ser34 - > Arg, and Pro90 - »Ala. In other embodiments, the cysteine substitutions are introduced into the erythropoietin to alter the cysteine-cysteine disulfide bond patterns, resulting in a new disulfide bond formation ("NDS mutations"). Human erythropoietin of natural origin, which appears to be unique among mammalian erythropoietins, has exactly four cysteines at positions 7, 29, 33 and 161 that form two disulfide bonds. One or more of these cistern residues, of the erythropoietin portion can be altered. To generate an altered disulfide bond, a cysteine residue is mutated to a structurally compatible amino acid such as alanine: or serine, and a second amino acid that is close in the three-dimensional structure is mutated to cysteine. For example, one of the amino acids Gln86, Pro8, Trp88, Glu89, and Leugi can be replaced by Cys. If Trp88 is replaced by Cys and Cys33 is replaced with another amino acid, the erythropoietin portion will form a disulfide bond Cys2g-Cys88 that is not found in human EPO. This linkage results in a fusion protein having higher activity than a fusion protein with a typical Cys2g-Cys33 disulfide bond. In addition, the Cys2g-Cys38 fusion protein shows a pronounced increase in activity, compared to the Cys29-Cys33 fusion protein, in the presence of other mutations in the erythropoietin portion of the fusion protein. Accordingly, in one embodiment of the present invention, the erythropoietin portion includes at least one of the following amino acid substitutions: a non-cysteine residue at position 29, a non-cysteine residue at position 33, a cysteine residue in the position 88, and a cysteine residue at position 139. In one embodiment, the erythropoietin portion contains cysteines at positions 7, 29, 88 and 161. In yet another embodiment, the erythropoietin portion also contains one or more of the following substitutions. . Hys32 - »Gly, Cys33 -» Pro and Pr? G0 - >; To. In an alternative embodiment, a completely new disulfide bond is added to the protein by mutation of two amino acids to cysteines. To compensate for possible stresses in the structure that Cys mutations can cause, in a preferred embodiment manipulated by Cys of this invention, the erythropoietin moiety also contains mutations designed to alleviate these potential stresses. Additional embodiments related to cysteine substitutions are described in PCT Publication WO 01/36489 (e.g., U.S. Application No. 09 / 708,506), the disclosure of which is incorporated by reference herein. Methods for introducing mutations in erythropoietin are well known in the art. For example, mutations can be introduced by site-directed mutagenesis techniques. It is important to note that a wide variety of site-directed mutagenesis techniques are available and can be used as alternatives to achieve similar results. Other techniques include, but are not limited to, random and semi-random mutagenesis. Linker The Fc-EPO fusion proteins according to this invention may include a linker molecule, preferably a peptide linker, between the Fe portion and the erythropoietin portion. A fusion protein with a linker can have improved properties, such as increased biological activity. A linker generally contains between 1 and 25 amino acids (for example, between 5 and 25 or between 10 and 20 amino acids). The linker can be designed not to include the protease cleavage site. In addition, the linker can contain an N-linked or an O-linked glycosylation site, to spherically inhibit proteolysis. Accordingly, in one embodiment, the linker contains an amino acid sequence Asn-Ala-Thr. Additional suitable linkers are described in Robinson et al. (1988), Proc. Nati Acad. Sci. USA: 95, 5929; and Application of the United States Serial No. 09 / 708,506. Glucosylation Human erythropoietin of natural origin and recombinant erythropoietin expressed in mammalian cells contain three N-linked and one O-linked oligosaccharide chains. N-linked glycosylation occurs in the asparagine residues located at positions 24, 38 and 83, while O-linked glycosylation occurs at a serine residue located at position 126 (lai et al., (1986) J. Biol. Chem., 261: 3116; Broudy et al., (1988) Arch. Biochem. Biophys., 265: 329). Oligosaccharide chains have been shown to be modified with terminal sialic acid residues. The N-linked chains typically have up to four sialic acids per chain and the 0-linked chains have up to two sialic acids. An erythropoietin polypeptide can therefore accommodate up to a total of 14 sialic acids. Sialic acid is the terminal sugar on the N-linked or 0-linked oligosaccharides. The degree of sialylation is variable from site to site from protein to protein, and may depend on the cell culture conditions, the types of cells and the particular cell clones that are used. It has been found that the Fe fusion protein of the present invention synthesized in BHK cells is highly sialylated. It has also been found that the degree of sialylation of the Fc-EPO fusion protein can also be increased by adapting the BHK cells for development in protein-free, suspension, or protein-free and suspension media. Other certain commonly used cell lines, such as NS / O, PerC6, or 293 cells fail to produce the highly sialylated Fc-EPO fusion protein, under standard culture conditions. The degree of sialylation of the Fc-EPO fusion protein produced from different cell lines can be determined by isoelectric focusing (IEF) gel electrophoresis by virtue of its highly negatively charged sialic acid residues; the details of the IEF gel electrophoresis are described in example 5B. The degree of sialylation of the Fc-EPO fusion protein produced in different cell lines can also be qualitatively confirmed by lectin binding studies using methods familiar to those skilled in the art. An example of a lectin binding assay is described in example 5B. Typically, a population of purified, highly sialylated Fc-EPO fusion proteins of the present invention averages from 11 to 28 sialic acid residues per outbreak. The highly sialylated, preferred populations of the Fc-EPO fusion proteins have an average of 13 to 28, 15-28, 17-28, 19-28 or 21-28 sialic acid residues per outbreak. For example, a highly sialylated and preferred population of the Fc-EPO fusion proteins have an average of 20 to 22 sialic acids per purified shoot. Another preferred population of Fc-EPO fusion proteins have an average of 23-28 sialic acid residues per purified Fc-EPO fusion protein. Pharmacokinetics of the sialylated Fc-EPO fusion protein One of the most important factors determining the in vivo biological activity of erythropoiesis stimulating agents is the length of time in which the serum concentration of the protein remains above the necessary threshold for erythropoiesis, which is determined by the pharmacokinetics of the agents that stimulate erythropoiesis. The pharmacokinetic profile of the highly sialylated Fc-EPO fusion protein is different from that of erythropoietin of natural or recombinant origin. The major difference is that the highly sialylated Fc-EPO fusion protein has a much longer serum half-life and a slower clearance, which leads to increased biological potency in vivo. Without wishing to be bound by any theory, it is believed that sialic acid residues increase negative charges on an erythropoietin molecule, resulting in decreased firing rate for negatively charged EPO receptor binding and decreased receptor-mediated endocytosis. EPO, lengthening the serum half-life. In addition, sialic acids also prevent erythropoietin proteins from being endocytosed by asialoglycoprotein receptors that bind to glycoproteins with exposed galactose residues. In general, most pharmacokinetic profiles of a therapeutic molecule such as erythropoietin show an initial drop in serum concentration (an alpha phase), followed by a more gradual decline (a beta phase) after administration.

Factors that influence the alpha phase According to the theory of small molecule pharmacokinetics, the alpha phase defines a volume of distribution that describes how a molecule is divided into compartments outside the blood. The drop observed in the alpha phase varies widely for different Fc-EPO fusion proteins synthesized in different cell lines. In theory, the difference could be due to the variation in the volume of distribution, or due to variations in inter-compartment traffic. However, it has been observed that there is a correlation between the degree of sialylation and the pharmacokinetic behavior of the Fc-EPO proteins in mice. For example, the Fc-EPO fusion proteins synthesized in BHK cells are highly sialylated and show the best pharmacokinetic profile. The Fc-EPO fusion proteins synthesized in NS / O cells are somewhat sialylated and have intermediate pharmacokinetic profile. The Fc-EPO fusion proteins synthesized in 293 and PerC6 cells have little or no sialylation and have a poor pharmacokinetic profile characterized by approximately a 100-fold drop in serum concentration in the first 30 minutes. Therefore, a key factor influencing the alpha phase of a particular Fc-EPO fusion protein is the distribution of the glycosylation species and the level of sialylation. The Fc-EPO fusion proteins that are subsylated disappear rapidly.

In addition, as shown in Figure 2, the degree of fall in serum concentrations of Fc-EPO, during the phase, alpha varies according to the dose, indicating that this behavior is saturable and more likely immediate by the recipient . It is possible that the receptor mediating the fall of the alpha phase is neither the EPO receptor nor the Fe receptor, but rather another receptor such as the asialoglycoprotein receptor. Aranesp® has reduced the binding affinity to EPO receptors compared to normal human erythropoietin because Aranesp® has increased negative charges as a result of additional N-linked glycosylation sites. However, Aranesp® and normal human erythropoietin show similar falls during the alpha phases. Furthermore, since in general the number of EPO receptors on the cell surface of an erythroid progenitor cell is only approximately 200, these results could be completely saturated at much lower doses of erythropoietin, than those used in Figure 2. perhaps it is unlikely that the Fe receptors are mediators of the dramatic fall in the alpha phase, because the Fc-EPO fusion proteins with a mutation eliminating the glycosylation site for example, a mutation of the corresponding amino acid of Asn297 of the IgGl, can still show a gradual fall in the alpha phase. In addition, although the CH2 regions of IgG2, when not aggregated, in general do not bind to the Fe receptors, the Fc-EPO proteins containing CH regions of IgG2 still show a significant fall during the alpha phase. Without wishing to be bound by any theory, the drop in serum concentration of an Fc-EPO fusion protein of the alpha phase can be mediated by the asialoglycoprotein receptors via the endocytosis mediated by the asialoglycoprotein receptor. The subsylated Fc-EPO fusion proteins contain exposed galactose residues that can be linked by the asialoglycoprotein receptor, resulting in endocytosis mediated by the asialoglycoprotein receptor. As a result, the subsylated Fc-EPO fusion proteins can disappear rapidly. Factors that include the beta phase The drop in serum concentrations of the Fc-EPO fusion proteins in the beta phase is less gradual compared to the fall in the alpha phase. For example, in mice, between 8 and 24 hours after administration, a 2 to 3 fold drop in serum concentrations of the Fc-EPO fusion proteins is observed. The difference in the fall during the alpha phase is also less drastic between the different Fc-EPO proteins synthesized in different cell lines. However, as in the alpha phase, the degree of sialylation correlates with the pharmacokinetic behavior in the beta phase. For example, Fc-EPO fusion proteins synthesized in BHK cells have a significantly improved beta phase compared to otherwise identical Fc-EPO fusion proteins synthesized in NS / O cells. Endocytosis mediated by the EPO receptor appears to be at least partially responsible for the fall in serum concentration of the Fc-EPO fusion proteins during the beta phase. Aranesp®, which has reduced binding affinity for EPO receptors compared to normal human erythropoietin, has a significantly improved beta phase compared to normal human erythropoietin, despite similar profiles in the alpha phase. The Fc-EPO fusion proteins of the invention generally show an improved beta phase compared to erythropoietin of natural or recombinant origin, indicating that the addition of the Fe moiety significantly raises the decline in serum concentration during the beta phase. It has also been observed that certain amino acid modifications in the Fe portion or in the erythropoietin portion can significantly improve the beta phase. For example, mutations that remove the glycosylation site in the Fe moiety improve the beta phase of the Fc-EPO fusion proteins. Mutagenesis that increase the stability of the erythropoietin moiety, for example, mutations genetically engineered disulfide bonds (e.g., NDS mutations) in the erythropoietin moiety, significantly improve the beta phase of the Fc-EPO fusion protein. In general, an improved beta phase extends the terminal serum half-life of an Fc-EPO fusion protein. Fc-EPO fusion protein elimination routes There are several possible routes of elimination of an erythropoietin protein molecule from the body. A wild-type or naturally-occurring erythropoietin protein molecule can be eliminated from the body by renal filtration and receptor-mediated endocytosis. Endocytosed erythropoietin is efficiently degraded. As described in Figure 3, the addition of a Fe portion to the erythropoietin portion is expected to essentially suppress the excretion of the Fc-EPO fusion protein through the kidney. As a result, endocytosis measured by receptor is the major route of elimination of an Fc-EPO fusion protein. In addition, the addition of an Fe moiety to the erythropoietin moiety is also expected to reduce degradation after internalization, because the Fc-Rn endosomal receptors are expected to recycle the fusion protein back out of the cell. In principle, at least three types of receptors can be mediators of the clearance of the Fc-EPO fusion protein, namely the Fe receptor, EPO reducer and the acialoglycoprotein receptor. The clearance of the Fc-EPO fusion protein through the Fe receptor should be significantly reduced by the use of a CH2 domain derived from IgG2 instead of a CH2 domain derived from IgG1 in the Fe moiety. The CH2 domains derived from IgG2 have approximately a 100 fold lower affinity for FcγRI, which has the highest affinity for IgGs, compared to the CH2 domains derived from IgGl. The interaction between the CH2 domain derived from IgG2 and Fc? RI is undetectable in most binding assays. However, the binding affinity of FcγR of the CH2 domain of IgG2 may still play a role in the clearance of the Fc-EPO fusion protein due to the mutation of asparagine by eliminating the glycosylation site in the CH2 domain which further reduces the binding to the Fe receptor and improves the pharmacokinetics of the Fc-EPO fusion protein. The NDS mutations have the effect of stabilizing the structure of erythropoietin, and as a result, it is expected to reduce a degradation of the Fc-EPO fusion protein after internalization. The Fc-EPO fusion proteins containing the NDS mutations have improved pharmacokinetic properties and increased serum half-life. Sialylation increases the negative charges of Fc-EPO fusion proteins, reducing the binding affinity of the Fe-EPO fusion protein to the EPO receptor. The sialylation also reduces the number of galactose residues exposed on the Fc-EPO fusion protein, reducing the binding affinity of the Fc-EPO fusion proteins for the asialoglucoprotein receptors. Accordingly, as described in Figure 3, sialylation reduces the endocytosis measured by the EPO receptor and the endocytosis measured by the sialoglycoprotein receptor, the highly sialylated Fc-EPO fusion proteins therefore have dramatically delayed clearance rates. , resulting in significantly increased serum half-lives. The addition of a Fe portion, the Fe alterations and the erythropoietin portions, and the sialylation, each reduce to the clearance of the Fc-EPO fusion proteins. The combined effects on clearance and serum half-life are additive or multiplicative. In vitro activity and in vivo potency of the Fc-EPO fusion protein The in vitro activity of the Fc-EPO fusion proteins can be tested in a cell-based assay. Specifically, the interaction between Fc-EPO and the EPO receptor can be determined based on the TF-1 cell proliferation assay. TF-1 cells express EPO receptors, therefore, the proliferation of TF-1 cells, which is determined by thymidine incorporation, tritiated is a function of erythropoietin activity (Hammerlling et al., (1996 ) J. Pharmaceutical and Biomedical Analysis, 14: 1455; Kitamura et al., (1989) J. Cellular Physiol. , 140: 323). In the present invention, the proliferation of TF-1 cells is a function of the interaction between the erythropoietin portion and the EPO receptors. Specifically, if a portion of erythropoietin of an Fc-EPO fusion protein has a reduced start rate for the EPO receptor, the Fe protein generally has a reduced activity in a cell-based assay (marked by an increased ED50 value). The data from the cell-based assays, which are relatively easy to obtain, correlate in general with the pharmacokinetics and the in vivo potency of the Fc-EPO protein. The reduced in vitro activity, which indicates a reduced ignition rate for the EPO receptor, correlates in general with the improved pharmacokinetic properties and the increased in vivo potency. In contrast, increased in vitro activity (marked with a decreased ED50 value), which indicates an increased firing rate for the EPO receptor, correlates in general with poor pharmacokinetic properties and potency is reduced in vivo. The in vivo biological activities of the Fc-EPO fusion proteins can be measured by assays conducted in animal models, such as for example, mice and rats. Examples of in vivo assays include, but are not limited to, hematocrit (HCT) assays and reticulocyte assays. HCT assays measure the volume of blood occupied with red blood cells (RBCs), and are performed simply by centrifuging the blood in capillary tubes, and measuring the fraction of the total volume occupied by the sedimented RBCs. Reticulocytes are new RBCs that have recently differentiated from precursor cells and characterized by containing remnants of nucleic acid from precursor cells. The reticulocytes are measured by sorting the red blood cells in a flow cytometer, then dyeing with a nucleic acid staining dye, such as, for example, acridine orange or thiazole orange, and counting the staining fraction. Typically, hematocrit and reticulocytes are measured twice a week. The reticulocyte data are, in a sense, a first derivative of hematocrit data. The reticulocyte counts are a measure of the production rate of red blood cells, while the hematocrits measure the total red blood cells. In a typical experiment, the hematocrit of the animals administered with the Fc-EPO fusion proteins will be increased and then returned to the baseline. When the hematocrit is high and the Fc-EPO proteins administered have disappeared from the circulatory system of the animal, the reticulocyte count goes below the baseline because erythropoiesis is suppressed. Reticulocytes normally emerge from the bone marrow 4 days after the precursors committed to the RBC destinations. However, in the presence of high levels of erythropoietin, reticulocytes will often leave the bone marrow after 1-3 days after administration. In response to an injection of the Fc-EPO fusion proteins, the hematocrit readings increase, remain stable, and then return to the baseline in an animal. Examples of such hematocrit responses are shown in Figures 4-6. The maximum rate of decline is approximately 7% of the blood volume per week in mice, which corresponds to the lifetime of the RBC cells of about 45 days in a mouse, and about 5% of the blood volume per week in rats, which corresponds to the RBC lifetime of approximately 65 days in a rat. The maximum rate of decrease presumably represents the destruction of the RBCs in the absence of new synthesis. If the biologically active Fc-EPO fusion proteins remain in the system at a concentration above the threshold for erythropoiesis, the level of the hematocrit will remain high and will not fall, even if the level of biologically active Fc-EPO is not detectable in pharmacokinetic experiments. It has been found that the pharmacokinetic properties of an Fc-EPO protein correlates with the in vivo potency of the protein. All features of the present invention that increase the pharmacokinetics of an Fc-EPO fusion protein, as discussed above, will improve in vivo potency in animal experiments. As shown in Table 1, such features include, for example, the addition of the Fe portion, the removal of the glycosylation site in the Fe moiety (eg, N-Q substitution at a position corresponding to IgGl Asn297), introduction of NDS mutations within the erythropoietin moiety, and high levels of sialylation by synthesis of Fc-EPO in BHK cells.

Table 1 Factors influencing the pharmacokinetics and biological activity of Fc-EPO proteins It has been found that, per portion of erythropoietin, Fcg2h (FN - »AQ) -Epo and Fcg2h-EPO (NDS) made from BHK cells, show the best pharmacokinetics and more potent in vivo biological activities. Fcg2h (FN? AQ) -Epo and Fcg2h-EPO (NDS) each have a longer serum half-life and more potent in vivo activity per erythropoietin portion than Aranesp®.

Synthesis of Fc-EPO fusion protein The Fc-EPO fusion protein of the present invention can be produced in suitable cells or in cell lines such as human or other mammalian cell lines. Suitable cell lines include, but are not limited to, baby hamster kidney cells (BHK), Chinese hamster ovary cells (CHO) (including cells deficient in dihydrofolate reductase (DHFR), and COS cells. In a preferred embodiment, BHK cells are used. To express the Fc-EPO fusion protein in suitable host cells (e.g., BHK cells), the nucleic acid sequences encoding the Fc-EPO fusion protein are first introduced into an expression vector using recombinant molecular techniques standards, familiar to those of ordinary experience in the art. The sequence encoding the erythropoietin portion is preferably optimized by codon for high level expression. Codon-optimized human erythropoietin was described in PCT publication WO 01/36489 (e.g., U.S. Application No. 09 / 708,506), the descriptions of which are incorporated by reference herein. An exemplary nucleic acid sequence encoding an erythropoietin portion is provided in SEQ ID No .: 1: GCCCCACCACGCCTCATCTGTGACAGCCGAGTGCTGGAGAGGTACCTCTTGGAGGC CAAGGAGGC CGAGAATATCACGACCGGCTGTGCTGAACACTGCAGCTTGAATGAGAACATCACCGTGCCT GACA CCAAAGTGAATTTCTATGCCTGGAAGAGGATGGAGGTTGGCCAGCAGGCCGTAGAAGTGTG GCAG GGCCTGGCCCTGCTGTCGGAAGCTGTCCTGCGGGGCCAGGCCCTGTTGGTCAACTCTTCCC AGCC GTGGGAGCCCCTGCAACTGCATGTGGATAAAGCCGTGAGTGGCCTTCGCAGCCTCACCACT CTGC TTCGGGCTCTGGGAGCCCAGAAGGAAGCCATCTCCCCTCCAGATGCGGCCTCAGCTGCTCC CCTC CGCACAATCACTGCTGACACTTTCCGCAAACTCTTCCGAGTCTACTCCAATTTCCTCCGGG GAAA GCTGAAGCTGTACACAGGGGAGGCCTGCCGGACAGGGGACAGATGA (SEQ ID NO: l) Exemplary nucleic acid sequences encoding a preferred Fe portion, eg, an Fe moiety that includes a CH2 domain derived from IgG2 and a hinge region derived from IgG1, was described in U.S. Patent Publication No. 20030044423 (for example, Request by the United States No. / 093,958), the description of which is incorporated by reference herein. In general, a nucleic acid sequence encoding an Fc-EPO fusion protein includes a nucleic acid sequence encoding a signal peptide (the leader sequence). The guiding sequence is cleaved during the secretion process. An exemplary nucleic acid sequence (SEQ ID No. 2) encoding a mature Fc-EPO protein, without a leader sequence, is shown in Figure 7. Suitable vectors include those appropriate for the expression of a host cell of a mammal The vectors can be, for example, plasmids or viruses. The vector will typically contain the following elements: the promoter and other regulatory elements "upstream" (5 ') the origin of replication, the ribosome binding site, the transcription termination site, the polylinker site, and the selectable marker which are compatible with the use of a mammalian host cell. The vectors may also contain elements that allow propagation and maintenance in prokaryotic host cells, as well. Vectors suitable for the present invention include, but are not limited to, PdCs-Fc-X and vectors derived from it, and phClO-Fc-X and vectors derived from it. The vectors encoding the Fc-EPO proteins are introduced into the host cells by standard techniques of cell biology, including transfection and viral techniques. Transfection means the transfer of genetic information to a cell using isolated DNA, RNA or synthetic nucleotide polymer. Suitable transfection methods include, but are not limited to, calcium phosphate mediated coprecipitation (Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press), lipofection (e.g. , Lipofectamine Plus from Life Technologies of Rockville, Maryland), transfection techniques mediated by DEAE-dextran, fusion with lysozyme or erythrocyte fusion, scraping, direct uptake, osmotic or sucrose shock, direct microinjection, indirect microinjection, such as technical routes mediated by erythrocytes, protoplast fusion, or by subjecting the host cells to electrical currents (eg, electroporation), to name but a few. The above list of transfection methods is not considered exhaustive, since other procedures to introduce genetic information into cells will undoubtedly be developed. To facilitate the selection of host cells containing the nucleic acid encoding the Fc-EPO fusion protein, the nucleic acid encoding the Fc-EPO fusion protein is typically introduced into a selection marker. The selection marker can be encoded by a nucleic acid sequence present on the same expression vector encoding the Fc-EPO fusion protein. Alternatively, the selection marker can be encoded with a nucleic acid sequence present on a different vector. In the latter case, the two vectors can be co-introduced into the host cells either by cotransfection or cotransduction. Suitable selection markers include, for example, hygromycin B (Hyg B) and dihydrofolate reductase (DHFR). Transient expression is useful for small-scale protein production and for rapid analysis of an Fc-EPO fusion protein. Host cells containing the DNA sequence encoding the Fc-EPO fusion protein are maintained under conditions suitable for the expression of the encoded Fc-EPO fusion protein. Cell culture methods, conditions and standard means can be used to maintain the host cells expressing the Fc-EPO fusion protein. Stably transfected cells are frequently preferred for large scale production, high level expression, and for other purposes. The stably maintained nucleic acid can be present in any of the various configurations in the host cell. For example, in one embodiment, the stably maintained nucleic acid sequence is integrated into a chromosome of a host cell. In other embodiments, the stably maintained nucleic acid sequence can be present as an extrachromosomal array, as an artificial chromosome, or in another suitable configuration. In one embodiment, BHK cells are used to synthesize the Fc-EPO fusion protein. In order to obtain a stably transfected BHK cell, a nucleic acid sequence encoding the fusion protein and a nucleic acid sequence encoding a selection marker are introduced into the BHK cells, preferably by electroporation, fusion of protoplasts or lipofection methods. The nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding a selection marker can be present on the same expression vector. Alternatively, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding a selection marker may be present or in separate vectors. The preferred selection marker for establishing a stable BHK cell is Hyg B. Other selection markers, such as DHFR, can also be used. Stably transfected clones are isolated and prepared by growing them in the presence of Hyg B at a suitable concentration (eg, 200, 250 or 300 micrograms / ml), in standard tissue culture medium, such as, for example, MEM medium. + FBS, DMEM / F-12 or VP-SFM available from Life Technologies, and other suitable means. The expression levels of the Fc-EPO fusion protein can be monitored by standard protein detection assays, such as, for example, ELISA, Western blot, dot blot, or other suitable assays, on samples from supernatants and culture media. High expression clones are selected and propagated on a large scale. Typically, the BHK cell is an adherent cell line and commonly developed in media containing sera, such as MEM + 10% heat-inactivated fetal bovine serum (FBS). However, BHK cells can be adapted for growth in suspension and in a serum-free medium, such as, for example, VP-SFM (Invitrogen Corp, cat # 11681-020) or Opti-Pro-SFM (Invitrogen Corp., cat # 12309). An exemplary adaptation process is described in Example 3. BHK cells adapted for growth in a serum-free medium can also be adapted for growth in a protein-free medium, such as, for example, DMEM / F-12 ( Invitrogen Corp., cat # 11039-021). An exemplary adaptation procedure is described in Example 3. Preferably, DMEM / F-12 is supplemented with suitable amino acids and other components such as, for example, Glutamine, protein hydrolysates such as HyPep 4601 (Quest International, cat # 5Z10419) e Hypep 1510 (International Quest, cat # 5X59053), Ethanolamine (Sigma, cat # E0135), and Tropolone (Sigma, cat # T7387). Suitable concentrations of each supplement can be determined empirically by those skilled in the art, with routine experimentation. The Fc-EPO fusion proteins synthesized in BHK cells grown in a protein-free medium, are sialylated to a greater degree, and show more homogeneous sialylation than the corresponding protein synthesized in cells grown in a medium containing serum (eg, MEM). + FBS) or a serum free medium but not free of protein (for example, VP-SFM). In addition, the Fc-EPO fusion protein obtained in this way is substantially non-aggregated, for example, approximately 98% of the total yield is not added. The protein yield from BHK cells grown in a protein-free medium is similar to that of BHK cells grown in a medium containing serum, for example, above 10 microgram / milliliter (mcg / ml). Thus, growth in suspension and / or in a protein-free medium offers a number of advantages, including 1) the improvement of the pharmacokinetics of the Fc-EPO fusion protein resulting from increased sialylation; and 2) the facilitation of downstream purification processes (3 ') because the proteins can be purified from cells grown in the suspension mode and in a protein-free medium. Purification The Fc-EPO purification is carried out following the procedures of good manufacturing practices (GMP), known to those skilled in the art. The protein is generally encoded to homogeneity or near homogeneity. Chromatographic purifications, such as those involving column chromatography, are generally preferred. In general, a purification scheme with an Fc-EPO fusion protein may include, but is not limited to, an initial protein capture step; a step of viral inactivation; one or more refining steps; a step of viral elimination; and a step of concentration and / or formulation of proteins. For example, chromatography resin materials that bind to the Fe portion of the fusion protein can be used to capture Fc-EPO proteins. Suitable resin materials, including, but not limited to, resins coupled to protein A. The refining steps may be included to remove contaminating components. For example, hydroxyapatite chromatography, Sepharose Q chromatography, size exclusion chromatography or hydrophobic interaction chromatography can be used to remove contaminants. A purification method using column chromatography based on protein A to bind the Fe moiety and purify the Fc-EPO fusion protein is described in example 12, since it is an optional method for virus inactivation and elimination. The purified proteins are generally concentrated to a desired concentration using ultrafiltration; diafiltered in a buffer of suitable formulation; sterilized by filtration; and assorted in jars. Administration Pharmaceutical compositions and routes of administration The present invention also provides the pharmaceutical compositions containing the Fc-EPO protein produced according to the present invention. These pharmaceutical compositions can be used to stimulate the production of red blood cells and to prevent and treat anemia. The conditions treatable by the present invention include anemia associated with a decline or loss of renal function (chronic renal failure), anemia associated with myelosuppressive therapy, such as chemotherapeutic or antiviral drugs (such as AZT), anemia associated with the progression of non-myeloid cancers, anemia associated with viral infections (such as HIV), and chronic disease anemia . Conditions that can lead to anemia in an otherwise healthy individual, such as an anticipated loss of blood during surgery, are also treatable. In general, any condition treatable with rHuEpo can also be treated with the Fc-EPO fusion protein of the invention. Formulations containing the Fc-EPO proteins In general, a formulation contains an Fc-EPO protein, a buffer and a surfactant in liquid form or in solid form. Solid formulations also include, but are not limited to, freeze-dried, spray-dried or spray-dried formulations. The liquid formulations are preferably water-based, but may contain other components, such as, for example, ethanol, propanol, propanediol or glycerol, to name but a few. The Fc-EPO proteins are formulated in aqueous solutions following the standard procedures of good manufacturing practices, known to those skilled in the art. In general, a formulation is generated by mixing defined volumes of aqueous solutions comprising suitable constituents at suitable concentrations. For example, a formulation typically contains the Fc-EPO protein at a concentration of 0.1 to 200 mg / ml, preferably 0.2 to 10 mg / ml, more preferably 0.5 to 6 mg / ml. The buffering components include any physiologically compatible substances which are capable of regulating the pH, such as, for example, citrate salts, acetate salts, histidine salts, succinate salts, maleate salts, phosphate salts, lactate salts, their acids or respective bases or mixtures thereof. The buffering components commonly used are citrate salts and / or their free acid. A formulation typically contains a buffering component at a concentration of 10 mmol / L, preferably 2 to 20 mmol / 1, more preferably 10 mmol / L. The surfactants for the Fc-EPO formulations can be any excipient used as a surfactant in the pharmaceutical compositions, preferably the polyethylene-sorbitan esters (Tweens®), such as polyoxyethylene (20) -sorbitanmonolaurate, polyoxyethylene (20) -sorbitan monopalmitate and polyoxyethylene. (20) -sorbitanmonostearate, and polyoxyethylene-polyoxypropylene copolymers. A formulation typically containing a surfactant at a concentration of 0.001 to 1.0% w / v, preferably 0.005 to 0.1% w / v, more preferably 0.01 to 0.5% w / v.

A formulation may also contain one or more amino acids. Suitable amino acids include, but are not limited to, arginine, histidine, orinithine, lysine, glycine, methionine, isoleucine, leucine, alanine, phenylalanine, tyrosine and tryptophan. In one embodiment, an Fc-EPO formulation contains glycine. Preferably, the amino acids are used in salt forms, for example, a hydrochloride salt. Applicable concentrations of amino acids are in the range of 2 to 200 mmol / liter, or 50 to 150 mmol / liter. Additionally, a formulation may contain sugars such as sucrose, trehalose, sorbitol; antioxidants such as ascorbic acid; preservatives such as phenol, m-cresol, methyl- or propyl-paraben; chlorobutanol; thiomersal; benzalkonium chloride; polyethylene glycols; Cyclodextrins and other suitable components. It is desirable that an Fc-EPO formulation is isotonic. For example, the osmolality of a formulation can be in the range of 150 to 450 mOsmol / kg. The pharmaceutical formulations have to be stable for the desired shelf life at the desired storage temperature, such as at 2-8 ° C, or at room temperature. In a useful formulation containing an Fc-EPO protein it is well tolerated physiologically, easy to produce, can be dosed accurately, and is stable during storage at 2 ° C-8 ° C or 25 ° C, during multiple cycles of freeze / thaw and mechanical stress; as well as other stresses such as storage for at least 3 months at 40 ° C. The stability of Fc-EPO formulations can be tested in a stress test. An exemplary stress test can be described in Example 13. Administration The pharmaceutical compositions containing the Fc-EPO fusion proteins produced according to the present invention can be administered to a mammalian host by any route. Thus, as is appropriate, administration can be oral or parenteral (e.g., i.v., i.a., s.c, i.m.), including routes of intravenous and intraperitoneal administration. In addition, administration may be by periodic injections of a bolus of the therapeutic agents, or may be performed more continuously by intravenous or intraperitoneal administration from an external reservoir (e.g., an intravenous bag). In certain embodiments, the therapeutic products of the present invention may be pharmaceutical grade. That is to say, in certain modalities they comply with standards of purity and quality control required for administration to humans. Veterinary applications are also within the intended meaning as used herein.

The formulations, for veterinary use and for human medical use, of the therapeutic agents according to the present invention typically include therapeutic agents in association with a pharmaceutically acceptable carrier and one or more other optional ingredients. The carrier (s) can be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not harmful to the container thereof. The pharmaceutically acceptable carriers, in this regard, are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. Except that any conventional medium or agent is incompatible with the active compound, the use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. The formulations can be conveniently presented in unit dosage form and can be prepared by any of the methods well known in the pharmacy / microbiology art. In general, some formulations are prepared by placing the therapeutic agent in association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include oral or parenteral, e.g., intravenous, intradermal, inhalation (e.g., after nebulization), transdermal (topical), transmucosal, nasal, buccal and rectal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and tonicity adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. A preferred method for administration of the Fc-EPO protein products of the invention is by parenteral routes (eg, IV, IM, SC or IP) and the compositions administered could ordinarily include therapeutically effective amounts of the product in combination with diluents, carriers and / or acceptable adjuvants. Effective doses are expected to vary substantially depending on the condition being treated, but it is currently expected that the therapeutic doses are in the range of 0.2 to 2 mcg / kg of body weight of the active material. Standard diluents such as human serum albumin are contemplated for the pharmaceutical compositions of the invention, as are standard carriers such as saline. Adjuvant materials suitable for use in the compositions of the invention include independently scored compounds for erythropoietic stimulatory effects, such as testosterones, progenitor cell stimulators, insulin-like growth factor, prostaglandins, serotonin, cyclic AMP, prolactin and triiodothyronine, as well as agents generally employed in the treatment of aplastic anemia, such as metholene, stanozolol and nandrolone. See, for example, Resegotti, et al. (1981), Panminerva Medies 23, 243-248; McGonigle, et al., (1984) Kidney Int., 25 (2), 437-444; Pavlovic-Kantera, et al., (1980) Expt. Hematol. , 8 (Sup. 8), 283-291; and Kurtz (1982) FEBS Letters, 14a (l), 105-108.

Also contemplated as adjuvants are the substance reported as enhancing the effects of, or synergizing with, Fc-EPO, such as adrenergic agonists, thyroid hormones, androgens and BPA, as well as the classes of compounds designated "hepatic erythropoietic factors". (see, Naughton et al., (1983) Acta. Hae at., 69, 171-179) and "erythrotropins" as described by Congote et al. in Excerpt 364, Proceedings 7th International Congress of Endocrinology, Quebec City, Quebec, July 1-7, 1984; Congote (1983), Biochem. Biophys. Res. Comm. , 115 (2), 447-483; and Congote (1984), Anal. Biochem., 140, 428-433, and "erythrogens" as described in Rothman, et al. (1982), J. Surg. Oncol., 20, 105-108. Solutions useful for oral or parenteral administration can be prepared by any of the methods well known in the pharmaceutical art, as described for example in Remington's Pharmaceutical Sciences, (Gennaro, A., ed.), Mack Pub., 1990. Formulations for parenteral administration may also include glycocholate for buccal administration, ethoxysalicylate for rectal administration, or citric acid for vaginal administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or in multi-dose vials made of glass or plastic. Suppositories for rectal administration can also be prepared by mixing the drug with a non-irritating excipient such as cocoa butter, other glycerides, or other compositions that are solid at room temperature and liquid at body temperatures. The formulations may also include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Formulations for direct administration may include glycerol and other high density compositions. Other potentially useful parenteral carriers for these therapeutic products include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for administration by inhalation may contain as excipients, for example, lactose, or they may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Retention enemas can also be used for rectal distribution. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions (where they are soluble in water) and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition can be sterile and can be fluid to the extent that easy syringability exists. This could be stable under the conditions of manufacture and storage, and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be caused by the inclusion in the composition of an agent delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by sterilization by filtration. In general, dispersions are prepared by incorporating the active compound in a sterile vehicle that contains a basic dispersion medium and the other ingredients required from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and lyophilization, which produces a powder of the active ingredient, plus any additional desired ingredient, from a solution previously sterilized by filtration, of the same. In one embodiment, the therapeutic agents are prepared with carriers that will protect against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biodegradable polymers can be used, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. Microsomes and microparticles can also be used. The oral and parenteral compositions can be formulated in unit dosage form for ease of administration and dose uniformity. The unit dose form refers to the physically discrete units suitable as unit doses for the subject to be treated; each unit containing a predetermined amount of the active compound is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the invention is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the technique of the composition, such as a compound active for the treatment of individuals.

Determination of the therapeutically effective amount of Fc-EPO and dosing frequency In general, therapeutic agents containing the Fc-EPO fusion proteins produced according to the present invention can be formulated for parenteral or oral administration to humans or other mammals , for example, in therapeutically effective amounts, for example, the amounts that provide appropriate concentrations of the drug to a target tissue, for a time sufficient to induce the desired effect. More specifically, as used herein, the term "therapeutically effective amount" refers to an amount of the Fc-EPO fusion proteins that gives an increase in the hematocrit to an objective hematocrit, or to a range of target hematocrit that provides benefit to a patient or, alternatively, keep a patient in an objective hematocrit, or within a range of target hematocrit. The amount will vary from one individual to another and will depend on a number of factors, including the total physical condition of the patient, the severity and underlying cause of the anemia, and ultimately the target hematocrit for the individual patient. An objective hematocrit is typically at least about 30%, or in a range of 30% to 38%, preferably above 38% and more preferably 40% to 45%. The general guidelines related to the target hematocrit intervals for rHuEpo are also found in the package insert. EPOGEN® dated 12/23/96 and are from 30% to 36%, or alternatively 32% to 38% as stated here. It is understood that such objectives will vary from one individual to another, such that the discretion of the physician may be appropriate in determining an effective target hematocrit for any given patient. However, the determination of an objective hematocrit is well within the level of experience in the art. A therapeutically effective amount of an Fc-EPO protein can be easily ascertained by a person skilled in the art. Example 15 describes a clinical protocol whose objective is to determine a therapeutically effective amount of an Fc-EPO protein once a week, once every two weeks, or once a month of dosing. For example, a dose range for administration once a week or once every two weeks is from about 0.075 to about 4.5 mcg of Fc-EPO per kg per dose. A dose range for administration once a month is 0.45 to 4.5 mcg of Fc-EPO per kg per dose. The effective concentration of the Fc-EPO fusion protein of the invention to be distributed in a therapeutic composition will vary depending on a number of factors, including the final desired dose of the drug to be administered and the route of administration. The effective dose to be administered is also likely to depend on variables such as the type and degree of disease or indication to be treated, the general health status of the particular patient, the relative biological efficacy (eg level of sialylation) of the distributed therapeutic products, the formulation of the therapeutic products, the presence and types of excipients in the formulation, and the route of administration. In some embodiments, the therapeutic products of this invention may be provided to an individual using typical unit doses deduced from studies in mammals using non-human primates and rodents. As described above, a dose unit refers to a unit dose that is capable of being administered to a patient, and which can be easily handled and packaged, remaining as a physical and biologically stable unit dose, comprising any of the therapeutic products as such or a mixture thereof with solid or liquid pharmaceutical carriers or diluents. The dosage frequency for a therapeutic product containing the Fc-EPO fusion protein will vary depending on the condition being treated and the target hematocrit, but in general it will be less than three times a week. The dosing frequency can be about once or twice a week. ~ The dosing frequency may also be less than about once a week, for example about once every two weeks (about once every 14 days), once a month or even once every two times. It is understood that the dosage frequencies actually used may vary to some extent from the frequencies described herein, due to variations in the responses by different individuals to erythropoietin and its analogs.; the term "approximately" is intended to reflect such variations. The invention also provides for the administration of a therapeutically effective amount of iron, in order to maintain increased erythropoiesis during therapy. The amount that is to be given can be easily determined by a person of experience in the art, based on therapy with rHuEpo. Additionally, the therapeutic products of the present invention may be administered alone or in combination with other molecules known to have a beneficial effect on the particular disease or indication of interest. By way of example only, useful cofactors include cofactors that alleviate symptoms, including antiseptics, antibiotics, antiviral and antifungal agents and analgesics and anesthetics.

Prodrug The therapeutic products or agents of the invention also include the "prodrug" derivatives. The term "prodrug" refers to a pharmacologically inactive (or partially inactive) derivative of a progenitor molecule that requires biotransformation, either spontaneous or enzymatic, within the body to release or activate the active component. Prodrugs are variations or derivatives of the therapeutic products of the invention having cleavable groups under metabolic conditions. The prodrugs become the therapeutic products of the invention that are pharmaceutically active in vivo, when they undergo solvolysis under physiological conditions or undergo enzymatic degradation. A prodrug of this invention can be called single, double, triple and so on, the number of biotransformation steps required to release or activate the active drug component within the organism, and indicating the number of functionalities present in a precursor type form. . Pro-drug forms often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard (1985) Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam; Silverman (1992 ) The Organic Chemistry of Drug Design and Drug Action, pp. 352-401, Academic Press, San Diego, California). In addition, the prodrug derivatives according to this invention can be combined with other characteristics to increase bioavailability. Ex vivo expression The Fc-EPO fusion protein of the present invention can be provided by expression methods in vivo. For example, a nucleic acid encoding an Fc-EPO fusion protein can be advantageously provided directly to a patient suffering from a hematopoietic disorder or deficiency, or can be provided to an ex vivo cell, followed by the administration of the living cell to the patient. In vivo gene therapy methods known in the art include the provision of purified DNA (for example as in a plasmid), providing the DNA in a viral vector, providing the DNA in a liposome or other vesicle (see, for example, U.S. Patent No. 5,827,703, which describes lipid carriers for use in gene therapy, and U.S. Patent No. 6,281,010, which provides adenoviral vectors useful in gene therapy). Methods for treating diseases by implantation of a cell that has been modified to express a recombinant protein are also known. See, for example, U.S. Patent No. 5,399,346, which discloses methods for introducing a nucleic acid into a primary human cell for introduction into a human. In vivo expression methods are particularly useful for distributing a protein directly to the target tissues or the cell compartment without purification. In the present invention, gene therapy using the sequence encoding Fc-EPO can find use in a variety of disease states, disorders and states of hematological irregularity, including anemia, in particular the correction of anemia of a type associated with chronic kidney failure and the like. A nucleic acid sequence encoding an Fc-EPO fusion protein can be inserted into an appropriate transcription or expression cassette and introduced into a host mammal as naked DNA or complexed with an appropriate carrier. Monitoring of the production of the active Fc-EPO protein can be performed by nucleic acid hybridization, ELISA, Western blotting, and other suitable methods known to the person of ordinary skill in the art. It has been found that a plurality of tissues can be transformed after the systemic administration of the transgenes. Exogenous DNA expression after intravenous injection of a cationic lipid carrier / exogenous DNA complex into a mammalian host, has been shown in multiple tissues, including T lymphocytes, in the reticuloendothelial system, cardiac endothelial cells, lung cells, and bone marrow cells, e.g., hematopoietic cells derived from the bone marrow. The in vivo gene therapy distribution technology as described in U.S. Patent No. 6,627,615, is non-toxic in animals, and transgene expression has been shown to last for at least 60 days after a single administration. The transgene does not appear to integrate into the host cell DNA at detectable levels in vivo, as measured by Southern analysis, suggesting that this technique for gene therapy will not cause problems for the host mammal by altering the expression of normal cell genes that activate oncogenes that cause cancer, or turn off tumor suppressor genes, which prevent cancer. EXAMPLES Example 1. Constructs coding for the Fc-EPO fusion proteins The plasmid phC10-Fcg2h (FN-AQ) -M1-EP0 coding for the Fc-EPO fusion protein containing a portion of normal erythropoietin and the plasmid phC10 -Fcg2h (FN-AQ) -Ml-EPO (NDS) which codes for an Fc-EPO fusion protein with the NDS mutations, were constructed as follows.

The nucleic acid sequence coding for human erythropoietin optimized by codons for high expression in mammalian cells. For example, SEQ ID NO: 3 shows an example of the coding sequences of mature human erythropoietin with codons modified to optimize translation. The 5 'end sequence was also modified to include a Sma I site to facilitate subcloning. SEQ ID NO: 3 CCCGGGtGCCCCACCACGCCTC ^ ^ AGGAGGCC SAATATCACGACcGGC CCTGACACO GTGAATTTCTATGCCTGGAAGAGGATG GTGGCAGGGCCTGGCCCIGCTGTCGGAAGC ^ ^ CCC ^ GCCGTGGGAGCCCCTGCAA ACTCTGCCTCGGGCTC GGGAGCCC- GAAGGA TCCCCT (G ACA T (CT ^ _ CTGCTGAC GGGGA GCTGTAC C ?? GCTGA GGGGAGG ^ (????????? The small letters indicate base differences of the wild-type human erythropoietin coding sequence.The changes are predicted to increase the level of expression in mammalian cells, but do not change the sequence of the expressed protein.) The NDS mutations were introduced into the erythropoietin moiety by site-directed mutagenesis as described in PCT publication WOOl / 36489, the descriptions of which are incorporated by reference herein, eg, a DNA fragment of Xmal-Xhol was used. containing a sequence form that encodes human erythropoietin with mutations that result in amino acid substitutions two His32Gly, Cys33Pro, Trp88Cys and Pro90Ala, as described in WO01 / 36489. The corresponding protein sequence is shown in SEQ ID NO: 4. APPRLICDSRVLERYLLEAKEAENITTCC ^ GI-AIiSEAVmGQA ^ WSSQPC ^ GI ^^ RTITADTFRKLFRVYSNFI-RGK] _IKLYTGFACRTGDR (SEQ ID NO: 4) A hybrid Fe portion, including a CH2 domain derived from IgG2 and a hinge region derived from IgGl, was constructed as described in U.S. Patent Publication No. 20020147311 and, for example, in WOOl / 058957. The Xmal-Xhol DNA fragment encoding an erythropoietin form was inserted into a plasmid vector, eg, pdCs -Fc-X, which codes for an altered hinge region of IgGl and a CH2 and CH3 region of IgG2, except that there were two groups of mutations (referred to herein as mutations of the MI group) that resulted in amino acid substitutions in the C-terminal region of CH3, such that the sequence at the C-terminal junction of CH3 and the N-terminus of EPO is as follows: .... TQKSATATPGA-APPRLI .... (SEQ ID NO: 5) The first group of mutations, which change the sequence KSLSLSPG (SEQ ID NO: 6) of the CH3 region of IgG2 to KSATATPG (SEQ ID NO: 7), as described in WO02 / 079232. The effect of the substitution of Leu-Ser-Leu-Ser (position 3 to position 6 of SEQ ID NO: 6) with Ala-Thr-Ala-Thr (position 3 to position 6 of SEQ ID NO: 7) ), is to eliminate the potential epitopes of non-human T cells, which may arise due to the binding between human Fe and human erythropoietin containing non-proprietary peptide sequences. The second group consisting of the substitution of simple amino acids K to A in the C-terminal amino acid of the CH3 region is described in WO01 / 58957. The expression vector pdCs-Fc-X for the expression of Fe fusion proteins was described by Lo et al. (1998) Protein Engineering 11: 495. The plasmid phClO-Fc-X was constructed from pdCs-Fc-X by replacing the coding region of the dihydrofolate-reductase (DHFR) gene that confers resistance to methotrexate with the gene which confers resistance to hygromycin B. A fragment of hygromycin B DNA Nhel / Nsil was obtained by PCR amplification of the gene of hygromycin B from the template plasmid pCEP4 (Invitrogen) using the primers 5 '-GCTAGCTTGGTGCCCTCATGAAAAAGCCTGAACTC-3' ( SEQ ID NO: 8) and 5'-ATGCATTCAGTTAGCCTCCCCCATC-3 '(SEQ ID NO: 9).

The PCR fragment was cloned into the TA cloning vector, pCR2.1 (Invitrogen), and its sequence confirmed. The plasmid phC10-Fcg2h-Ml-EPO (NDS) was generated by a triple ligation of the DNA fragments Nhel / AflI and AflII / Nsil from pdCs-Fcg2h-Ml-EP0 (NDS) and the fragment of hygromycin B Nhel / Nsel. In addition, a mutation leading to a double substitution of amino acids was introduced by PCR mutations, "FN >; AQ ", within the amino acid sequence Gln-Phe-Asn-Ser within the CH2 domain of the heavy chain of IgG2, which eliminates a potential epitope of T cells and N-linked glycosylation in the Fe moiety. The utagénicos primers 5 '-AGCAGGCCCAGAGCACGTTCCGTGTGGT-3' (SEQ ID NO: 10) and 5'-GAACGTGCTCTGGGCCTGCTCCTCCCGT-3 '(SEQ ID NO: 11) were paired respectively with a downstream primer (3') containing a SacII site 5 '~ CGCCGCGGGTCCCACCTTTGG -3 '(SEQ ID NO: 12) and an upstream primer (5') containing a PvuII site 5'-cccagctgggtgctgacacgt-3 '(SEQ ID NO: 13), and two overlapping DNA fragments were amplified from the DNA template pdC10-Fcg2h-Ml-EPO (NDS) In a second round of amplification, a fragment of Pvull / SacII containing the mutation (FN-> AQ) was amplified using the upstream primer (5 ') (SEQ ID NO: 13) and the current downstream primer (3 ') (SEQ ID NO: 12) from the PCR products from the PRI mere round of amplification. The Pvull / SacII fragment was cloned into a TA vector, pCR2. 1 (Invitrogen), and its sequence was verified. The construction pdC10-Fcg2h (FN> AQ) -Ml-EPO (NDS) was generated from a triple ligature of the PvuII / SacII fragment, an Xhol / SacII fragment from pdC10-Fcg2h-Ml-EPO and an XhoI fragment / PvuII from pdC10 -Fcg2h-Ml-EPO (NDS). To introduce the mutation FN > AQ within the plasmid phC10-Fcg2h-Ml-EPO, the appropriate DNA fragments from phC10-Fcg2h-Ml-EPO and from pdClO-Fcg2h (FN-AQ) -Ml-EPO were combined. The phC10 -Fcg2h-Ml-EPO and pdC10 -Fcg2h (FN- ^ AQ) -Ml-EPO constructs were digested with Xhol and Xbal, and the 5.7 kb Xhol / Xbal fragment, phC10-Fcg2h-Ml-EPO (NDS) was ligated with the fragment pdClO-Fcg2h (FN-AQ) -Ml-EPO of 1.9 kb, generating phC10-Fcg2h (FN-AQ) -Ml-EPO. To introduce the mutation EN- ^ AQ within the plasmid phC10-Fcg2h-Ml-EPO (NDS), the two appropriate fragments digested with Xhol / Smal from phC10-Fcg2h-Ml-EPO (NDS) and phC10 ~ Fcg2h (FN - > AQ) -Ml-EPO were ligated together, generating phC10 -Fcg2h (FN-AQ) -Ml-EPO (NDS). The amino acid sequence of Fc-EPO encoded by phC10-huFcg2h (FN > AQ) -ML-EPO is shown in SEQ ID NO: 14. EPKSSDKTHTCPPCPAPPVAGPSVITJPPKPKDTmiSRTPEVTCV VEVHNAKTKPREEQAQSTFRVVSVLTVVHQDWLNGKF ^ ^ ^ .KCKVSNKGLP QVYTLPPSREEMTKNQVSLTCLVKGFROSD VE ESNGQPE NYKTT ^ ^ VDKSRWQQG WSCSVMHEALHNHYTQKSATATPGAAPPRLICDSRVL AEHCSI ^ ENITVPGIEKVNFYAWKRMEVG ^ VDKAVSGLRSLTIl RALGAQKE? ISPPr ASAA ^ ACRTGDR (SEQ ID NO: 14) The amino acid sequence of Fc-EPO (NDS) encoded by pdC10-huFcg2h (FN-AQ) -Ml-EPO (NDS) is shown in the SEQ ID NO: 15 EPKSSDK ?? ICPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVV ^ VEVHNAK? ^ REEQAQSTFRWSVLTVVHQDWI ^^ QVYTLPPSREE? NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTO VDKSRWQQGN SCS \ MIEAI? NHYTQKSATATPGA ^ AEGPSIJSIENITVPDTKVNFMWKRMF ^ VDKAVSGIJ LTTIITEALGAQKFAISP ACRTGDR (SEQ ID NO: 15) The stretch of the underlined sequence represents the EPO portion, the doubly underlined sequence represents the hinge of IgGl and the unlined sequence represents the CH2 domain and CH3 of the modified IgG chain, where the sequence written in italics represents the CH3 domain. Example 2. Expression of Fc-EPO in various cell lines For rapid analysis of the fusion protein, a plasmid phC10-Fcg2h (FN-AQ) -Ml-EPO (NDS) or phClO-Fcg2h (FN-AQ) -Ml EPO was introduced into the appropriate tissue culture cells by standard methods of transient transfection, such as, for example, by co-precipitation of calcium phosphate-mediated DNA (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press), or by lipofection using Lipofectamine Plus (Life Technologies) according to the manufacturer's protocol. In order to obtain the stably transfected BHK-21 cells, a plasmid phC10 ~ Fcg2h (FN-> AQ) -Ml-EPO (NDS) or phC10-Fcg2h (FN-AQ) -Ml-EPO was introduced. within BHK-21 cells by electroporation. For high efficiency electroporation, BHK-21 cells grown in the MEM medium (supplemented with non-essential amino acand sodium pyruvate as recommended by the American Type Culture Collection (ATCC)), were washed once with PBS; and approximately 5xl06 cells were resuspended in 0.5 ml of PBS and incubated with 10 μg of linearized plasmid DNA in a Gene Pulser Bucket "11 with a 0.4 cm electrode gap (BioRad, Hercules, CA) on ice for 10 minutes. Electroporation was performed using a Gene Pulser "11 (BioRad, Hercules, CA) with settings at 0.25 V and 500 μF. The cells were allowed to recover for 10 minutes on ice, resuspended in growth medium, and plated on two 96-well plates. Hygromycin B (Hyg B) was added to the growth medium two days after transfection at a concentration of 300 micrograms / ml. Cells were fed every 3 days for two to three more times, and stable clones resistant to Hyg B appeared in 2 to 3 weeks. To identify stable clones that produce high levels of the Fc-EPO fusion protein, supernatants from the clones were evaluated by ELISA with anti-Fc antibodies. High production clones were isolated and propagated in the growth medium containing 300 micrograms / ml of Hyg B. For protein production purposes, BHK-21 cells were routinely grown in a supplemented DMEM / F-12 medium, or in another suitable medium such as VP-SFM (Life Technologies). The Fc-EPO fusion protein was harvested from the conditioned medium by standard normal flow filtration, and the clarified material was stored at 4 degrees Celsius until further purification. Typically, in a rotary bottle production mode, 6 to 12 mcg / ml of the Fc-EPO proteins were obtained from the BHK-21 cells. The Fc-EPO fusion proteins were also expressed in and recovered from the NS / 0 cells. The NS / 0 clones stably maintaining the plasmid pdClO-Fcg2h (FN-> AQ) -Ml-EPO or pdC10-Fcg2h (FN-AQ) -Ml-EPO (NDS) were established by methods previously described in the publication of the PCT WOOl / 36489, the full descriptions of which are incorporated by reference herein. Typically, yields of 50 to 100 mcg / ml of Fc-EPO protein were obtained from the NS / O cells. Example 3. Adaptation of BHK cells for growth in suspension and / or in protein-free media BHK is an adherent cell line commonly developed in media containing serum, such as, for example, MEM + 10% fetal bovine serum (FBS) inactivated by heat. To maintain and expand the BHK cells they are periodically (for example, at 4 day intervals) detached from the substrate, typically by the action of a trypsin-EDTA solution, diluted in fresh media and reseeded in appropriate containers. However, BHK cells can be adapted for growth in suspension and in serum free and / or protein free media by the following methods. In a typical adaptation process, BHK cells were first cultured in the 75:25 (v / v) mixture of MEM + FBS: target medium until the exponential stage, and subsequently cultured at an appropriate cell density at 50:50 (v. / v), 25:75 (v / v) and finally 0: 100 (v / v) of original medium: objective medium. During the adaptation process, the growth of BHK cells was monitored by visual inspection. The following serum free media were tested for adaptation: 293 SFM II (Invitrogen Corp., cat. # 11686-929), CHO-S-SFM II (Invitrogen Corp., cat. # 12052-098), VP-SFM (Invitrogen Corp., cat. # 11681-020), Opti-Pro SFM (Invitrogen Corp., cat. # 12309), Hybridoma CD (Invitrogen Corp., cat. # 11279-023), and H-SFM (Invitrogen Corp ., cat. # 12045-076). To change the BHK cells from an adherent cell line to a cell line in suspension during the adaptation process, the culture mixture was allowed to settle before each pass, and the top 25% of the cell suspension was removed and diluted in a medium cool. Because the cells that are added settle to the bottom of the culture vessels more rapidly than the single and double cells, the top 25% of the cell suspension generally consists of those cells that show the least amount of aggregation. In this way, each pass expands and enriches the BHK cells less prone to aggregation, and the cell lines in suspension of the BHK clones expressing Fc-EPO proteins were established by this method. It was found that BHK cells expressing the Fc-EPO proteins could be adapted for growth in serum-free media VP-SFM or Opti-PRO SFM, and suspension cultures were obtained. BHK cells expressing the Fc-EPO fusion proteins were not able to grow in the following serum-free media: 293 SMF II, CHO-S-SFM II, Hybridoma CD, and H-SFM. The BHK cells adapted for the serum-free medium, VP-SFM, were further adapted for growth in protein-free medium, for example, DMEM / F-12 (Invitrogen Corp., cat. # 11039-021) by sequentially culturing BHK cells, at an appropriate cell density, in a mixture 75:25 (v / v), 50:50 (v / v), 25:75 ( v / v), and finally 0: 100 (v / v) of VP-SFM: DEMEM / F-12. The DMEM / F-12 protein-free medium was supplemented with glutamine (6 mM final), 2 g / liter of HyPep 4601 (Quest International, Chicago, IL, cat. # 5Z10419), 2 g / liter of HyPep 1510 (Quest International, Chicago, IL, cat # 5X59053), 10 μl / liter (v / v) ethanolamine (Sigma, cat # E0135) and 5 μM Tropolone (Sigma, cat # T7387). A BHK cell line stably expressing the Fc-EPO fusion protein component to be grown in supplemented DMEM / F-12 was obtained by this method and maintained at high cell viability. Example 4. Purification and Characterization of Protein Aggregation Status For analysis, the Fc-EPO fusion proteins were purified from the cell culture supernatants via Protein A chromatography, based on the affinity of the Fe moiety. Protein A. The conditioned supernatant from the cells expressing the Fc-EPO proteins was loaded onto a column of Protein A Sepharose of Flow or Rapid pre-equilibrium (Fast-Flow Protein A Sepharose). The column was extensively washed with sodium phosphate buffer (150 mM sodium phosphate, 100 mM sodium chloride at neutral pH). The bound protein was eluted by a low pH sodium phosphate buffer (pH 2.5-3) (composition as described above) and the eluted fractions were immediately neutralized. To evaluate the aggregation state of the Fc-EPO fusion proteins produced by different cell lines, the purified samples of Protein A were analyzed by analytical size exclusion chromatography (SEC). The samples were fractionated by HPLC-SEC (for example, Super 3000 SW, TosoHaas, Montgomeryville, PA), in a run of fifteen minutes at a flow rate of 0.35 ml / minute. A substantial portion of the Fc-EPO proteins (eg, up to 90% to 100% of the total yield) produced from the BHK cells were non-aggregated. In addition, samples of the Fc-EPO fusion proteins analyzed by reducing SDS-PAGE (gel prevailing of 4% NuPAGE-12%, NuPAGE, Novex) revealed substantially a single band, indicating that the products were resistant to low degradation. or standard operating procedures.

The Fc-EPO fusion proteins purified from BHK developed in suspension, in serum-free medium, and / or in protein-free medium, were also characterized by SDS-PAGE and analytical SEC as described above. It was found that the proteins were sufficiently non-aggregated and not degraded, as the proteins synthesized in BHK cells developed in media containing serum. Example 5A Characterization of glycosylation patterns Serine 126 in human erythropoietin is in a sequence compatible with O-glycosylation, and is conserved in all mammalian erythropoietin proteins. However, serine 126 is in a "flexible loop" that is not packed tightly against the rest of the protein. In the absence of O-glycosylation, the erythropoietin region may be particularly sensitive to proteolysis. The O-glycosylation state in Serl26 in the Fc-EPO proteins produced in different cell lines was examined by reverse phase HPLC. The samples were denatured and produced, diluted in 0.1% trifluoroacetic acid (TFA), and injected into a reverse phase HPLC column (e.g., a Vydac C4 column, Grace Vydac). A gradient in 0.085% TFA in acetonitrile was applied and the retention times of the protein samples were recorded. It was found that Fc-EPO and Fc ~ g2h (FN-> AQ) -EPO synthesized in BHK-21 cells produced two major overlaps that partially overlap (Peak # 1 and Peak # 2). The peak fractions were further analyzed by tracing the peptide map. It was found that Peak # 1 corresponded to a form of Fc-EPO that was glycosylated in Serl26, as indicated by the absence of a signature peptide (Peptide # 36), while Peak # 2 corresponded to a form of Fc-EPO that was not glycosylated on Serl26, as indicated by the presence of the signature peptide (Peptide # 36). It was found that Ser126 is modified by O-glycosylation in approximately 60% of the Fc-EPO molecules produced from BHK cells, which is consistent with what has been reported for EPO of natural origin. In addition, the growth of the BHK cells in the protein-free DMEM / F-12 medium, supplemented, had a positive effect on the frequency of O-glycosylation. Example 5B. Characterization of sialylation patterns The degree of sialylation of the Fc-EPO fusion proteins synthesized in NS / O, BHK, 293 and PerC6 cells was compared by isoelectric focusing gel electrophoresis (IEF). In summary, the samples, concentrated at 2 mg / ml and desalted if necessary, were added to an equal volume of IEF sample buffer of pH 3-7, and run on a vertical pH Novex pH 3-7 prevalent IEF ( Novex, cat. # EC6655B / B2) for 2.5 hours, the first hour at 100V, the second hour at 200V and the last 30 minutes at 500V. The gel was then fixed, stained and destained. In a particular experiment, the following samples were compared (samples were derived from cells grown in medium containing serum): 1. Fcg2h-EP0 (NDS) of NS / O 2. Fcg2h-EPO (NDS) of BHK-21 3 Fcg2h-EPO of BHK-21 4. Fcg2h ("Delta Lys") - EPO of BHK-21 5. Fcg4h (FN-AQ "Delta Lys") - EP0 of BHK-21 6. Fcg4h ("Delta Lys") -EP0 of BHK-21 In this group, "Delta Lys" refers to a deletion of lysine at the C-terminus of the Fe domain (samples 4-6). Samples 1-3 have a mutation of this C-terminal lysine to an alanine. Therefore, this C-terminal lysine is absent in all samples, and there is no resulting charge difference between the samples. All cells were developed as adherent cells in medium containing serum. The samples were loaded on an IEF gel of pH 3-7 compared to the standards that were focused on pH 3.5, 4.2, 4.5, 5.2, 5.3, 6.0 and 6.9 (Serva Electroporesis, Germany). The first sample, Fcg2h-EPO (NDS) from NS / 0, migrated as a distribution of bands with isoelectric points between approximately pH 5.3 and 6.5; most of the intense bands were present at pH 6.0-6.1. The second sample, Fcg2h-EPO (NDS) from BHK-21, runs as a distribution of intense bands with isoelectric points at approximately pH 4.6 to pH 5.0, with fainter bands of pH 5.0 to approximately pH 6.0; the most intense bands were present at pH 4.8-4.9. The third and fourth samples, Fcg2h-EP0 from BHK-21 and Fcg2h ("Delta Lys") - EPO from BHK-21, respectively, both had a band distribution of approximately pH 4.7 to 6.0, with the most intense bands focused at approximately pH 5.3. The fifth and sixth samples, Fcg4h (FN- ^ AQ "Delta Lys") - EP0 of BHK-21 and Fcg4h ("Delta Lys") - EP0 of BHK-21, respectively, had an approach pattern similar to that of the second sample, for example, run as a distribution of intense bands with isoelectric points at about pH 4.6 to pH 5.0, with fainter bands of pH 5.0 at about pH 6.0. These results indicate that the synthesis of Fc-EPO fusion proteins in BHK cells generally resulted in a significantly more acidic product than identical or similar products synthesized in NS / 0 cells. In other experiments, Fcg2h-MI-EPO (NDS) samples from BHK cells were treated with neuraminidase, which removes sialic acid from oligosaccharides. The resulting samples treated with neuraminidase were run on an IEF gel and found to be focused as a few bands at pH 6. 9 and higher. When the bands formation patterns of the BHK cell samples with or without neuraminidase treatment and of the samples from NS / O cells were compared, approximately 27 different sialylated species were identified. The 27 species corresponded perfectly with the 28 different predicted species that could result from varying degrees of sialylation of an Fc-EPO fusion protein in homodimeric configuration. According to this analysis, Fcg2h-EPO with 4 to 5 sialic acid residues focused with the pH 6.9 marker, and Fcg2h-EP0 with 11 to 12 sialic acid residues focused with the pH 6.0 marker. It was found that a population of Fcg2h-EPO proteins synthesized in BHK cells appeared to have an average of 21 sialic acid residues per protein molecule. In contrast, a population of Fe (g2h) -EPO proteins synthesized in NS / O cells appeared to have an average of about 10 sialic acid residues per protein molecule. In subsequent experiments, BHK cells expressing the Fc-EPO proteins were adapted to serum-free growth conditions and conditions appropriate for large-scale production, eg, suspension conditions. The Fc-EPO proteins produced from the BHK cells grown in serum-free media, and in suspension, were analyzed by gel electrophoresis IEF as described above. These alterations in growth conditions resulted in displacements of at most, only 0.1 to 0.3 pH units at the isoelectric point of the most intense band. Samples of the Fc-EPO fusion proteins synthesized in free media of DMEM / F-12 supplemented proteins were similarly characterized by IEF gel electrophoresis. It was found that the protein product was sialylated to a greater degree and showed more homogeneous sialylation than the corresponding product from the cells grown in serum-free media such as VP-SFM. The degree of sialylation of the Fc-EPO proteins produced in different cell lines was also qualitatively confirmed by lectin binding studies. For example, the Fc-EPO fusion proteins were first separated by standard SDS gel electrophoresis and transferred by dots, then probed with modified lectins that recognize different portions of carbohydrate (eg., commercially available from Roche Applied Science, Indianapolis, IN), and linked lectins can be visualized. Suitable lectins include, but are not limited to, Sambucus nigra agglutinin (SNA) or Maackia amurensis agglutinin (MAA), which recognizes sialic acids with specific bonds, and Datura stramonium agglutinin (DAA), peanut agglutinin ( PNA) and jacaline, which recognize other regions of the carbohydrate moiety, such as the O-glycan core. Based on the lectin binding assays, the sialylation levels of the Fc-EPO fusion proteins produced in different cell lines could be determined. Example 6. In vitro biological activity of the Fc-EPO variants The in vitro activities of different Fc-EPO proteins were tested in a cell-based assay. The TF-1 cell line expresses the receptors of EPO, and consequently, under appropriate culture conditions, its incorporation of tritiated thymidine is a function of the activity of the EPO protein or similar to EPO (Hammerlling et al., (1996) J. Pharmaceutical and Biomedical Analysis, 14: 1455; Kitamura et al (1989) J. Cellular Physiol., 140: 323). Specifically, the TF-1 cells in active logarithmic phase were washed twice in a medium without EPO, and plated in approximately 10 4 cells / well in microtiter plates. The cells were then incubated in a medium with a serial dilution of the Fc-EPO variants for 48 hours. 0.3 μCi of 3H-thymidine were added to the wells ten hours before the cell proliferation assay. As controls, TF-1 cells were also incubated in the presence of recombinant human EPO, and the hyperglycosylated EPO analog Aranesp®.

The incorporation of radioactive thymidine was measured as total precipitable TCA counts. As shown in Table 2, the activities of the Fcg2h-Ml-EP0 molecules are comparable to that of recombinant human EPO. Some general conclusions can be obtained from this data. Consistent with previously reported results, EPO produced from CHO cells has an ED50 of approximately 0.7 ng / ml; this includes the EPO NIBSC standard, EPO from R &D Systems, and commercial Procrit, Aranesp is significantly less active in vi tro, presumably reflecting its reduced ignition speed due to its negative charges, similarly, Fc-EPO produced at from BHK cells is less active than Fc-EPO produced from NS / O cells, which is consistent with the observation that Fc-EPO proteins produced from BHK cells are highly sialylated, resulting in burdens Increased negatives on proteins Table 2 Example 7. Pharmacokinetic Analysis of the Fc-EPO Variants The pharmacokinetic profiles of different Fc-EPO proteins used in various cell lines were characterized based on the following experiments in vivo. In one experiment, as shown in Figure 8, approximately 14 mcg of the Fcg2h (N> Q) -EPO protein synthesized in NS / 0 cells and in BHK cells were intravenously administered into Swiss-Webster mice. At various time points after administration (eg, T = 0, 1/2, 1, 2, 4, 8 and 24 hours after administration), the blood samples were collected and the serum was prepared by centrifugation . The serum concentrations of Fc-EPO were determined by ELISA using anti-Fc antibodies. As shown in Figure 8, at 24 hours after the administration, more than 10% of the initial serum concentration of Fc-EPO derived from BHK remained in the serum, while less than 0.1% of the initial serum concentration. of Fc-EPO derived from NS / 0, remained in the serum. A similar experiment was performed with Fcg2h-EPO (NDS) proteins synthesized in NS / 0 cells and in BHK cells. Approximately 14 mcg of the Fcg2h-EPO protein (NDS) synthesized in NS / O cells and in BHK cells were administered intravenously in Swiss-Webster mice. Blood samples were collected at T = 0, 1/2, 1, 2, 4, 8, 24 and 36 hours after administration and serum Fcg2h-EP0 (NDS) concentrations were measured by anti-Fc ELISA. As shown in Figure 9, at 24 hours after the administration, more than 10% of the initial serum concentration of Fcg2h-EPO (NDS) derived from BHK remained in the serum, whereas less than 0.1% of the Initial serum concentration of Fcg2h-EP0 (NDS) derived from NS / O remained in the serum. The pharmacokinetic profiles of Fcg2h-EPO (NDS) produced in BHK-21 cells, PERC6 cells and 293 cells were also compared. Specifically, a plasmid expressing Fcg2h-Epo (NDS) was transiently transfected into BHK cells, 293 and PERC6. The expressed Fcg2h-Epo fusion proteins (NDS) were purified from different cell lines and injected intravenously into Swiss-Webster mice at a concentration of 1.7 micrograms per mouse. Blood samples were taken at T = 0, 1/2, 1, 2, 4, 8, 24, 48 and 72 hours, and the concentration of Fcg2h-Epo (NDS) in serum was measured by the anti-Fc ELISA. As shown in Figure 10, at 24 hours after the administration, more than 10% of the initial serum concentration of Fcg2h-EP0 (NDS) derived from BHK remained in the serum, while less than 1% of the concentration Initial serum Fcg2h-EP0 (NDS) derived from 293 cells remained in serum, and Fcg2h-EP0 (NDS) derived from PerC6 cells was almost undetectable in serum. Similar results were obtained with the Fcg2h (N-Q) -EPO proteins produced in BHK, PerC6 and 293 cells. Similar experiments were conducted in mice to compare the pharmacokinetic profiles of Fcg2h (N- >).; Q) -EPO, Fcg2h-EPO (NDS), Fcg2h-EP0 and Aranesp® (for example, NESP). The Fc-EPO variants used herein were synthesized from BHK cells. It was observed that, at 48 hours after administration, less than 10% of the initial serum concentration of Aranesp "remained in serum, whereas more than 10% of the initial serum concentrations of Fcg2h (N-> Q) - EPO and Fcg2h-EPO (NDS) remained in serum These results indicate that Fcg2h (N-Q) -EPO and Fcg2h-EPO (NDS) proteins produced from BHK-21 cells have much longer serum half-lives than that of Aranesp. " Example 8. In vivo potency of the Fc-EPO variants The biological activities in vivo of the different Fc-EPO variants were measured by the hematocrit assay (HCT) and the reticulocyte assays in rats and mice.

In an HCT experiment, the CD1 mice were injected intraperitoneally with the proteins Fcg2h (FN-AQ) -EPO synthesized in BHK cells at doses of 20 mcg / kg and 10 mcg / kg. Blood samples were taken from the mice on days 4, 7, 11 and 14, and centrifuged in capillary tubes. The amounts of red blood cells (RBCs) sedimented were measured as fractions of the total volume. As illustrated in Figure 4, in response to the injection of the Fcg2h (FN > AQ) -EPO proteins, the hematocrits first increased dramatically, then remained stable, and finally decreased. In yet another experiment, Sprague-Dawley rats were injected intraperitoneally with the following proteins synthesized in BHK cells. All animals were dosed at 42.5 mcg / kg. 1. Fcg2h-EPO 2. Fcg2h-EP0 (NDS) 4. Fcg4h-EP0 5. Fcg4h (N> Q) -EPO The HCT assays were performed with the blood samples taken from the mice injected as described above. As shown in Figure 5, in response to Fcg2h-EP0 (NDS) and Fcg2h-EP0, the amount of hematocrit in the injected rats remained stable for a prolonged period of time, indicating that the Fcg2h-EP0 (NDS) proteins and Fcg2h-EP0 have prolonged serum half-lives, and potent biological activity in vivo. It was also found that, as shown in Figure 5, that Fcg4h-EP0 and Fcg4h (N-> Q) -EPO showed a shorter stable period, and a more rapid decrease in serum concentration, compared to proteins Fcg2h-EP0 (NDS) and Fcg2h-EPO. In yet another experiment, the CDl mice were administered intraperitoneally with the following samples. 1. Fcg2h-EPO (NDS) from BHK cells at doses of 85 mcg / kg, 42.5 mcg / kg and 21.25 mcg / kg. 2. Fcg2h-EP0 (NDS) from NS / O cells at doses of 85 mcg / kg, 42.5 mcg / kg and 21.25 mcg / kg. 3. Aranesp® (for example, NESP) at doses of 50 mcg / kg, 25 mcg / kg and 12.5 mcg / kg. The amounts of protein were calculated based on the molecular weight of the protein, without carbohydrates. In this experiment, the molecular weight of the Fcg2h-EP0 (NDS) protein is based on a monomeric polypeptide. Accordingly, the ratio of molecular weights of Fcg2h-EPO (NDS) to NESP is about 1.71 to 1. Therefore, the dose ranges with each protein in this experiment were approximately equal.

As shown in Figure 6, the Fcg2h-EP0 (NDS) proteins synthesized in the BHK cells showed the best hematocrit profile in terms of potency and duration of effect, indicating that the Fcg2h-EPO (NDS) proteins from the cells BHK have longer serum half-lives, and more potent in vivo activities compared to Fcg2h-EP0 (NDS) from NS / O and NESP cells. The hematocrit profiles of Fcg2h-EP0 (NDS) from the NS '/ O and NESP cells are comparable. Example 9. Comparison of the Fc-EPO proteins with the CH2-CH3 domains derived from IgG2 and from IgG4 A comparison of the cell-based erythropoietin activities of various Fc-EPO proteins revealed that the fusion proteins with the CH2 domains and CH3 derived from IgG4 were in general less active than the corresponding proteins with CH2 and CH3 domains derived from IgG2. This conclusion is true for at least three types of Fc-EPO proteins, namely proteins with NDS mutations in the erythropoietin portion and synthesized in NS / O cells (Table 3), proteins with NDS mutations synthesized in BHK cells ( Table 4), and proteins with normal erythropoietin synthesized in BHK cells (Table 5). All the proteins compared in Tables 3 and 5 below have a modified hinge derived from IgGl and the MI group of mutations in the C-terminus of the Fe moiety. The activities of the proteins were determined by measuring the incorporation of tritiated thymidine within of the TF-1 cells stimulated by the proteins according to standard procedures described in Example 6. The activity is expressed as an ED50 in nanograms / ml of the erythropoietin portions. Table 3: Cell-based activities of the Fc-EPO fusion proteins with the NDS mutations and synthesized in NS / O cells Table 4: Cell-based activities of the Fc-EPO fusion proteins with the NDS mutations and synthesized in BHK cells Table 5: Cell-based activities of Fc-EPO fusion proteins with wild-type EPO and synthesized in BHK cells Activity data from in vitro cell-based assays can usually suggest pharmacokinetic profiles and in vivo potencies of proteins containing erythropoietin. In general, an activity decreased in vitro in a cell-based assay indicates a reduced ignition rate for the EPO receptor, which correlates with improved pharmacokinetic properties (eg, prolonged half-life) and enhanced in vivo activity. . However, the in vitro decreased activities of the Fc-EPO fusion proteins with the CH2 and CH3 domains derived from IgG4 do not correlate with the improved pharmacokinetics and increased biological activities in vivo. It was found that the pharmacokinetic profiles of the Fc-EPO fusion proteins with the CH2 and CH3 domains derived from IgG4 were in general indistinguishable from the corresponding proteins with the CH2 and CH3 domains derived from IgG2. It was also found that the Fc-EPO fusion proteins with CH2 and CH3 domains derived from IgG4 in general had less in vivo activity, compared to the corresponding proteins with the CH2 and CH3 domains derived from IgG2 (see Figure 5). Example 10 The effects of deletion of the glycosylation site on the Fe portion We conducted experiments to test the effects of glycosylation site removal on the Fe portion on in vitro activity, pharmacokinetics and potency in vivo. In particular, the Fc-EPO fusion proteins containing any of the CH2 and CH3 domains derived from IgG2 or the CH2 and CH3 domains derived from IgG4, were tested. Asparagine within the amino acid sequences Gln-Phe-Asn-Ser of IgG2 or IgG4, corresponding to Asn297 or IgGl, was replaced with a glutamine. In most experiments, phenylalanine with the amino acid sequence Gln-Phe-Asn-Ser was replaced with alanine to eliminate the possible epitopes of non-self T cells, which can result from the mutation of asparagine. As shown in Table 6, in the cell-based in vi tro assays, the ED50 values of the Fc-EPO proteins with the FN mutation > AQ eliminating the N-linked glycosylation site in the Fe moiety, are generally about 5 times smaller than those of the Fc-EPO proteins without the mutation, indicating removal of the N-linked glycosylation site, which resulted in an activity in vitro decreased in cell-based assays. Experiments were also conducted to test the effects of N-linked glycosylation elimination on pharmacokinetics and potency in vivo. CDl mice were treated with the Fcg2h-Ml-EPO, Fcg2h-Ml-EPO (NDS) and Fcg2h (N> Q) -Ml-EPO proteins synthesized in BHK cells at a dose of 42 mcg / kg each. It was observed that the Fcg2h (N-> Q) -Ml-EPO protein showed better pharmacokinetic profile than the corresponding protein without the N- ^ Q mutation. Therefore, the mutation N > Q, which removes N-linked glycosylation in the Fe portion derived from IgG2, resulted in improved pharmacokinetics (eg, prolonged serum half-life). The prolonged serum half-life can not be explained by an effect on the binding to the Fe receptors, because the CH2 and CH3 domains derived from IgG2 already have essentially no detectable link to the Fe receptor.

Table 6: Removal of the glycosylation site in the Fe portion reduces the cell-based in vitro activity of the Fc-EPO fusion proteins These effects are unexpected and surprising because the effects provoked by the elimination of the N-linked glycosylation in the Fe portions derived from IgG2 and IgG4 are the most consistent with the firing rate reduced by the erythropoietin receptor. Without wishing to be compromised by some theory, the removal of the N-linked glycosylation in the Fe portions derived from IgG2 and IgG4 can cause a complete conformational change on the Fc-EPO fusion protein. Example 11. Treatment of Beagle Dogs with Fc-EPO Fusion Proteins Synthesized in BHK Cells Fc-EPO fusion proteins were administered to beagle dogs to test effects on hematocrits, reticulocyte counts, and other blood parameters. Specifically, the Fcg2h (FN-AQ) -EPO proteins were purified from two stably transfected BHK cell lines independently, clone 65 and clone 187, and administered to beagle dogs intravenously. A male pachón dog and a female pachón dog were injected with each preparation according to the following scheme: Day O: 3 micrograms / kg Day 16. 10 micrograms / kg Day 23: 100 micrograms / kg At various time points after each administration, approximately 2 ml of blood was collected and blood parameters were measured, such as hematocrit, reticulocyte counts and other blood parameters. The hematocrit responses after the treatment are shown in Figure 11. After dosing with 3 mcg / kg of the Fc-EPO fusion proteins, the blood parameters did not increase from the normal range. Within a week after dosing with mcg / kg, the reticulocyte counts were increased to more than 3% of the total blood volume in three of the four animals, and the hematocrits were increased to 51 in one animal. Other blood parameters did not increase from the normal range. After dosing with 100 mcg / kg, the hematocrit counts rapidly increased, reaching peak levels of 57 to 62 and remaining above the normal range for five to six weeks. The reticulocyte counts remained elevated for two to three weeks. For each animal, the number of red blood cells per microliter of blood and hemoglobin, measured in grams per deciliter, were proportional to the number of hematocrits. These results indicated that the Fc-EPO proteins stimulate the production of red blood cells of normal size with normal hemoglobin content. Example 12. Purification of Fc-EPO proteins for clinical use Fc-EPO proteins are purified after standard GMP procedures known to those skilled in the art. The BHK-21 cells, from a production clone of a bank, are cultured in DM? M / F-12 medium (Invitrogen) supplemented with L-glutamine 2. 5 mM additional (Invitrogen), 2 g / liter each of HyPep 1501 and HyPep 4601 (Quest International, Chicago, IL), 10 μl / liter of ethanolamine (Sigma), and 5 μM Tropolone (Sigma) for 7 to 10 days in batch culture, while maintaining high cell viability (eg, above 80%). The conditioned medium is harvested and clarified by normal flux filtration, and is loaded onto a pre-equilibrated Rapid Flow of Protein A Shepharose (Pharmacia) column, which captures the fusion protein based on the affinity of Protein A for the Fe portion. The column is washed extensively with 15 column volumes of the sodium phosphate buffer containing 150 mM sodium phosphate and 100 mM sodium chloride at neutral pH. The bound protein is eluted at low pH with an additional 15 volumes of acid sodium phosphate buffer column pH 2. 5-3, but also containing 150 mM sodium phosphate and 100 mM sodium chloride. For viral inactivation, the pH of the combined peak fractions is adjusted to pH 3. 8 and incubated for an additional 30 minutes at room temperature. After 30 minutes of incubation, the combined fractions are neutralized and sterilized by filtration, then applied to a Fluj or Rapid Q-Sepharose ion exchange column (Pharmacia), which exploits the acidic pl of the Fc-EPO portions as a result of its extensive sialylation to effectively eliminate potential contaminants co-eluted with Fe proteins. Specifically, the neutralized fractions are loaded onto a Q-Sepharose Fast Flow anion exchange column (Pharmacia) at pH 5.0 and eluted with a gradient of sodium chloride solution. Fc-EPO fractions are then collected and combined for the subsequent analysis and for the additional purification process. For example, depuration with high blood concentration from the Q-sepharose column is applied to a reverse phase chromatography column to remove excess sodium chloride. The diluted eluent from the reverse phase column is further applied to a second column of Fluj o-Rapido Q-Sepharose (Pharmacia, 3 cm x 9 cm). Potential viral particles are then removed from the pool by nano-filtration (eg, Viresolve by Millipore). Optionally, additional purification steps, such as the hydroxyapatite column or a phenyl boronate column (cis-diols linkages), can be used. Finally, the purified proteins are concentrated to a desired concentration using ultrafiltration, and then diafiltered into a buffer of suitable formulation. The material is finally sterilized by filtration, and assortment in bottles at a pre-determined volume. Example 13. Stress test to determine the stability of the Fc-EPO protein formulations Vessels containing an Fc-EPO sample formulation, sample or a reference Fc-EPO formulation are stored at 40 ° C and at a humidity relative atmospheric of 75%, and by defined storage time (for example, 0 weeks, 4 weeks, 8 weeks, etc.). An aliquot sample is taken from each vial after a certain storage time and analyzed. Samples are evaluated visually under direct illumination with a cold light source for turbidity. The turbidity is additionally determined by the absorption measurement at 350 nm and 550 nm. In addition, the condition of the Fc-EPO protein in the samples and the presence of the protein degradation products are analyzed by analytical size exclusion chromatography (HPLC-SEC). It is found that a formulation containing 0.5 mg / ml Fc-EPO, 10 mM citrate pH 6.2, 100 mM glycine, 100 mM sodium chloride, 0.01% w / v polysorbate 20, had significantly increased stability compared to a reference solution. Example 14. A phase I study of the Fcg2h fusion protein (FN >; AQ) -Ml-EPO in humans A Phase I clinical trial of the fusion protein Fcg2h (FN> AQ) -Ml-EPO in humans is carried out as follows. The pharmacokinetic parameters are determined essentially as described for Aranesp® by MacDougall et al. (1999) J. Am. Soc. Nephrol. 10: 2392-2395, the teachings of which are incorporated by reference herein. The serum terminal half life of the fusion protein Fcg2h (FN> AQ) -Ml-EPO intravenously injected (dosed at 1 mcg / kg) in humans is found to be between approximately 20 and 30 hours. Thus, a dose of 1 mcg / kg, or approximately 70 mcg in an adult anemic patient, results in an initial serum concentration of approximately 10 ng / ml. Since the normal concentration of human erythropoietin is from about 0.04 to 0.25 ng / ml (Cazzola et al., (1998) Blood 91: 2139-2145), pharmacologically active levels of the Fc-EPO protein remain in the patient's system for at least 5 to 10 days. Example 15 A phase II dose finding study and dose schedule of the Fcg2h fusion protein (FN > AQ) -Ml-EPO Sequential, randomized, multi-center dose scale-up studies are initiated to investigate optimal dose of the dosing scheme for the fusion protein Fcg2h (FN> AQ) -Ml-EPO when administered by subcutaneous or intravenous injection in patients with chronic renal failure (CRF) receiving dialysis. In clinical practice, it is generally convenient to design the administration of the fusion protein Fcg2h (FN > AQ) -Ml-EPO to an individual anemic patient according to the following guidelines. An initial dose is administered and blood parameters such as hematocrit, hemoglobin, reticulocyte counts and platelet counts are monitored. The initial dose is typically between about 0.3 and 3 mcg / kg. A convenient initial dose is 1 mcg / kg. If the increase in hematocrit is less than 5 to 6 percent of the blood volume after 8 weeks of therapy, the dose should be increased. If the increase in hematocrit is greater than 4 percent of the blood volume over a 2-week period, or if the hematocrit is approaching 36%, the dose should be reduced. An exemplary dose scheme is as follows. Dosage once a week: 0.075, 0. 225, 0.45, 0.75, 1.5 and 4.5 mcg / kg / dose. Dosage once every two weeks: 0.075, 0.225, 0.45, 0.75, 1.5 and 4.5 mcg / kg / dose. Dosage once a month: 0.45, 0.75, 1.5 and 4.5 mcg / kg / dose.

The studies are carried out in two parts. The first part is a dose scale-up study designed to evaluate the dose of the Fcg2h fusion protein (FN> AQ) -Ml-EPO administered either once a week, once every two weeks, or once at month, which increases hemoglobin at an optimal rate over four weeks (greater than or equal to 1 g / dl, but less than 3 g / dl). The second part of each study is. designed to determine the dose required (when administered once a week, once every two weeks, or once a month by any of the intravenous or subcutaneous routes of administration) to maintain the hematocrit in the therapeutic target. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (25)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A purified dimeric fusion protein, consisting essentially of a dimeric Fe portion of a human IgG molecule comprising a hinge region, a CH2 domain and a CH3 domain, and human erythropoietin (EPO), characterized in that each chain of the Dimeric Fe moiety is linked via its C terminus directly or via a linker peptide to the N terminus of an EPO molecule, the fusion protein has the following properties: (i) the molecule is highly sialylated by comprising 15 to 28 acid residues sialic; (ii) the CH2 domain is derived from human IgG2 and is modified by replacing the amino acid residues Phe and Asn within the Gln-Phe-Asn-Ser sequence stretch of the CH2 domain with Ala and Asn, thereby forming the Gln-Ala-Gln-Ser sequence within the CH2 domain; and (iii) the stretch of the amino acid sequence Leu-Ser-Leu-Ser close to the C-terminus of the CH3 domain is replaced with Ala-Thr-Ala-Thr.

2. A dimeric Fc-EPO fusion protein according to claim 1, characterized in that additionally, the C-terminal Lys residue of the CH3 domain is replaced with Ala.

3. The dimeric Fc-EPO fusion protein according to claim 1 or 2, characterized in that the hinge region is derived from human IgGl.

4. A dimeric Fc-EPO fusion protein according to claim 3, characterized in that the IgG1 hinge region is modified by the replacement of the Cys amino acid residue within the stretch of the sequence Pro-Lys-Ser-Cys-Asp- Lys of the hinge region, with a residue Ser, thereby forming the Pro-Lys-Ser-Ser-Asp-Lys sequence within the hinge region.

5. A dimeric Fe fusion protein according to any of claims 1 to 4, characterized in that the erythropoietin portion comprises at least one of the following amino acid substitutions: (i) a non-cysteine residue at position 29 of the EPO molecule, (ii) a non-cysteine residue at position 33 of the EPO molecule, (iii) a cysteine residue at position 88 of the EPO molecule, and (iv) a cysteine residue in the position 139 of the EPO molecule.

6. A dimeric Fc-EPO fusion protein according to claim 5, characterized in that a non-Cys amino acid residue is at position 33 of the EPO molecule, instead of the original Cys residue, and a residue of Cys is at position 88 of the EPO molecule instead of the original Trp residue, thus making it possible for the EPO portion within the fusion protein to form a Cys29-Cys88-7 disulfide bond. A dimeric Fc-EPO fusion protein according to claim 6, characterized in that the non-Cys amino acid residue at position 33 is pro. 8. A dimeric Fc-EPO fusion protein according to any of claims 1 to 7, characterized in that the EPO portion comprises one or more mutations selected from the group: (ii) Arg139 ^ Glu139 (iii) His32- Gly32 (iv ) Ser3-Arg3 (v) Proso- > Ala90 • 9. A dimeric Fc-EPO fusion protein according to any of claims 1 to 8, characterized in that the linker peptide comprises a glycosylation site. 10 A dimeric Fc-EPO fusion protein according to claim 9, characterized in that the glycosylation site comprises an amino acid sequence Asn-Ala-Thr. eleven . A dimeric Fc-EPO fusion protein according to any of claims 1 to 10, characterized in that the complete IgG molecule is derived from IgG2 and the hinge regions are derived from IgG1. 12 A dimeric Fc-EPO fusion protein according to any of claims 1 to 11, characterized in that it additionally comprises a CH1 domain. 13 A dimeric Fc-EPO fusion protein according to any one of claims 1 to 12, characterized in that the fusion protein has 20 to 22 sialic acid residues. 14 A protein Fc-EPO fusion dimeric Zada characterization comprising the sequence:? EPKSSDKTHTCPPCPAPP GPSVFLFPPKPKDTIM ^ ^ VDGVF -NAKTKPRHEQAQSTFRWSVL GQPREPQVYTLPPSREEMTKNQVSLTCLVKGF ^ ^ SFFLYSKLTVDKRSWQQGN SCSVMHEALH EAKEAENITTGC EHCSIjNENITVPDTKVNF AWK ^^ LLWSSQPWEPLQ] _- HVDKAVSG] _- RSLTTL ^^ FRVYSNF GKLKLYTGEACRTGDR (SEQ ID NO: 14). 15. A protein Fc-EPO dimeric fusion sequence characterized by comprising: EPKSSDKIHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS VEXWE ^ ^^ HNAKTKPREEQAQSTFRWSVLTV GQPREPQVYTLPPSF-EEMTKNQVSLT (WGFYPSD ^ H ^ SFFLYSKLTVDKSRWQQGNWSCSVMHEALH EAKEAEOTTIGCAEGPSLNENITVPDTKV ^ 3 .. VNSSQPCEALQLHVDKAVSG1.RSLTILLRALGAQKEMSP FRVYSNFLRGKI.K YTGEACRTGDR (SEQ ID NO.: 15) A DNA molecule characterized in that it encodes a fusion protein according to any one of claims 1 to 15. 17 A pharmaceutical composition suitable for the treatment of hematopoietic disorders of deficiencies in a mammal, characterized in that it comprises, in an effective amount, an Fc-EPO fusion protein as specified according to any of claims 1 to 15, optionally together with a pharmaceutically acceptable carrier, diluent or excipient 18. A population of Fc-EPO fusion proteins highly sialylated, puri The Fc-EPO fusion proteins comprise a Fe portion towards the N-terminus of the Fc-EPO fusion proteins, and an erythropoietin portion towards the C-terminus of the Fc-EPO fusion proteins, suitable for administration to a mammal. , the population of the fusion proteins has an average of 15 to 28 sialic acid residues per purified Fc-EPO fusion protein and is obtainable by the introduction of a DNA molecule that codes for a respective Fc-EPO fusion protein within of a BHK molecule and expressing, isolating and purifying the population of corresponding Fc-EPO fusion proteins, characterized the population because it has a longer serum half life compared to a population of corresponding Fc-EPO fusion proteins synthesized in NS cells / O, PerC6 or 293. 19. A population of purified Fc-EPO fusion proteins according to claim 18, characterized in that the Fusion protein population averages from 20 to 22 sialic acid residues per purified Fc-EPO fusion protein. 20. A population of purified Fc-EPO fusion proteins, according to claims 18 or 29, characterized in that the BHK cell is adapted for growth in a protein-free or suspension medium. 21. A method for producing a population of recombinant, purified, highly sialylated Fc-EPO fusion proteins comprising a Fe portion towards the N-terminus of the Fc-EPO fusion proteins, and an erythropoietin portion towards the C-terminus of the Fc-EPO fusion proteins, the method is characterized in that it comprises the steps of: (i) building a DNA molecule that codes for an Fc-EPO fusion protein; (ii) transforming a BHK cell with the DNA molecule into a protein-free or suspension medium, (iii) expressing the population of the Fe fusion proteins encoded by the DNA molecule, (iv) harvesting, isolating and purifying the population of the Fc-EPO fusion protein. 22. A method according to claim 21, characterized in that the synthesized population of fusion proteins has an average of 15 to 28 sialic acid residues per purified Fc-EPO fusion protein. 23. A method according to claim 22, characterized in that the synthesized population of fusion proteins has an average of 20-22 sialic acid residues per purified Fc-EPO fusion protein. 24. A method for selecting a BHK cell stably maintaining a nucleic acid sequence encoding the Fc-EPO fusion protein, comprising an Fe portion and an erythropoietin portion, the method is characterized in that it comprises the steps of: ) introducing into a BHK cell a nucleic acid sequence encoding hygromycin B and a nucleic acid sequence encoding the Fc-EPO fusion protein; and (b) culturing the BHK cell in the presence of hygromycin B. 25. The method according to claim 24, characterized in that the nucleic acid sequence encoding hygromycin B and the nucleic acid sequence coding for the protein of Fc-EPO fusion are present in a simple DNA molecule.