Method for producing long-acting EPO-fc fusion protein

Abstract

The present invention relates to a method for producing an Fc fusion protein comprising an EPO-hybrid Fc fusion protein (i.e., epoetin alpha) having a C-terminal lysine content of about 1–10% and a sialic acid content of about 17–27 mol / mol. The method comprises culturing host cells transformed with DNA encoding the Fc fusion protein and DNA encoding human α-2,3-sialyltransferase under optimized culture conditions, and purifying the Fc fusion protein using an optimized purification process including anion exchange chromatography.

Description

Method for producing a sustained-release EPO-FC fusion protein

[0001] This patent is a continuation-in-part of U.S. patent application Ser. No. 13 / 493,508, filed June 11, 2012, which now incorporates the contents of U.S. patents US 8,586,531 B2 and US 7,867,491 B2 (issued January 11, 2011) and AU2016334954 B2 (filed October 6, 2016). The entire contents of the above patents are incorporated herein by reference.

[0002] The present invention relates to a method for producing a sustained-release erythropoietin (EPO)-Fc fusion protein, and more particularly, to an EPO-Fc producing cell line that produces a sustained-release EPO-Fc fusion protein in a highly sialylated form, and a method for producing a sustained-release erythropoietin (EPO)-Fc fusion protein using the cell line.

[0003]

[0004] Endogenous erythropoietin (EPO) is a glycoprotein hormone naturally produced by renal tubular cells. It stimulates red blood cell production, a process called erythropoiesis. In humans, most EPO is produced by renal cortical peritubular cells, but in fetuses, the liver is the primary site of production (Jelkmann, 2016). Small amounts of EPO are also produced in the spleen, liver, bone marrow, lungs, and brain. Oxygen pressure (pO2) directly regulates EPO production: lower pO2 levels lead to greater EPO production. Low hemoglobin levels indirectly stimulate EPO production.

[0005] Erythropoiesis-stimulating agents (ESAs) are recombinant versions of EPO produced pharmacologically in cell culture using recombinant DNA technology. Examples of ESAs include epoetin alfa, darbepoetin alfa, and methoxy polyethylene glycol-epoetin beta (Jelkmann, 2016). The latter two are long-lived EPOs, each with an additional carbohydrate side chain and additional pegylation.

[0006] ESAs are generally indicated in conditions where red blood cell production is impaired. The two main FDA-approved indications for ESAs are anemia due to chronic kidney disease (CKD) and chemotherapy-induced anemia in cancer patients (Noxon et al, 2017). Other approved indications include anemia due to zidovudine treatment in HIV infection, support for patients receiving autologous blood transfusions, anemic patients undergoing elective surgery (both preoperatively and postoperatively), and anemia in premature infants.

[0007] In CKD, where kidney damage limits EPO production in the tubular cells surrounding the kidney, ESA treatment alleviates anemia (Jelkmann, 2013). ESAs are effective in both dialysis and non-dialysis patients in CKD.

[0008] Endogenous EPO and ESAs act by binding to the EPO receptor (EPOR), a cell surface receptor highly expressed on erythroid progenitor cells. EPO binding triggers EPOR activation, including receptor dimerization and / or reorientation, which then induces the JAK / STAT and PI3K-AKT signaling cascades (Watowich, 2011; Cokic et al., 2012). In erythroid progenitor cells, EPOR activation promotes survival, cell proliferation, and differentiation into reticulocytes.

[0009] Polypeptides, such as EPO, generally have low stability and are easily denatured. Furthermore, these polypeptides have a short half-life in the body, as they are degraded by proteases in the blood and are easily eliminated through the kidneys and liver. Therefore, to maintain blood concentrations and efficacy of protein therapeutics containing polypeptides as pharmacological agents, frequent administration is necessary. However, most protein therapeutics are administered by injection, which causes pain in patients.

[0010] In addition to additional glycan and PEG attachments, fusing protein therapeutics with Fc fragments derived from immunoglobulin molecules has been shown to increase stability and serum half-life. Immunoglobulins are a major component of blood. Human immunoglobulins include various classes, including IgG, IgM, IgA, IgD, and IgE.

[0011] Immunoglobulins are composed of four polypeptide chains, two heavy chains and two light chains, which are linked by disulfide bonds to form a tetramer. Each chain consists of a variable region and a constant region. The heavy chain constant region is further divided into three or four domains (CH1, CH2, CH3, and CH4) depending on the isotype. The Fc region of the heavy chain constant region contains hinge, CH2, CH3, and / or CH4 domains, depending on the Ig isotype.

[0012] Since the discovery that sialic acid-degrading enzymes (sialidase) reduce the in vivo biological activity of EPO, the relationship between sialic acid and EPO's biological activity has been actively studied. Subsequent studies have shown that attaching sialic acid to the surface of recombinant EPO protein prevents proteolysis, extends its in vivo half-life, and ultimately enhances its efficacy. Sialic acid affects EPO's in vivo activity by preventing galactose residues on the EPO surface from binding to the asialoglycoprotein receptor, which is highly expressed on the surface of hepatocytes and removes the target glycoprotein from the circulation.

[0013] Numerous published studies have demonstrated that sugar residues play an essential role in maintaining the intrinsic potency of glycoproteins, and this role is particularly crucial in human EPO. In other words, EPO activity is lost without sugar residues, and EPO's in vivo activity is significantly influenced by the composition of sugars, particularly the content of sialic acid.

[0014] Meanwhile, the sialic acid content of the EPO-Fc fusion protein can be increased by adding N-acetylmannosamine to the culture medium. The EPO-Fc protein obtained through this process is typically a mixture of EPO-Fc fusion proteins with a high sialic acid content and EPO-Fc fusion proteins with a low sialic acid content. Since EPO-Fc fusion proteins with a high sialic acid content have a longer in vivo half-life than EPO-Fc fusion proteins with a low sialic acid content, there is a need to develop a technology that can selectively produce EPO-Fc fusion proteins with a high sialic acid content.

[0015] The production of EPO-Fc fusion proteins requires culturing transformed host cells to express large quantities of EPO-Fc fusion proteins, followed by purification. Therefore, optimization of cell culture and purification techniques is crucial for mass production of high-quality EPO-Fc fusion proteins.

[0016] Efepoetin alfa, an EPO-hybrid Fc fusion protein, is a homodimer in which each subunit is composed of EPO and a hybrid Fc derived from human IgD and human IgG4 (Figure 1). The complete amino acid sequence of efepoetin alfa is shown in Figure 2 (SEQ ID NO: 2; Patent No. US 8,586,531 B2). The amino acid sequence of EPO in efepoetin alfa is identical to that of native EPO, which has a half-life of approximately 5 hours in the blood.

[0017] The function of the EPO fragment in the ephepoetin alpha structure was confirmed by: (1) EPOR binding as determined by surface plasmon resonance (SPR) assays (Figures 3A-3D), and (2) its ability to induce proliferation of UT-7 cells in a dose-dependent manner (Figure 4). UT-7 is a human leukemia cell line with megakaryocyte characteristics established from the bone marrow of patients with acute megakaryoblastic leukemia. UT-7 cells show strict dependence on IL-3, GM-CSF, or EPO (Komatsu et al., 1991).

[0018] The fusion of EPO fragments to hybrid Fc was designed to (1) increase the molecular weight of the molecule, thereby evading renal clearance, and (2) extend the half-life of EPO by binding to FcRn (Figure 5) and thereby avoiding cellular degradation. By combining the CH2 and CH3 domains of IgG4 with the CH2 domain and hinge region of IgD, the hybrid Fc structure lacks immune effector activities such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) (Figure 6), and complement-dependent cytotoxicity (CDC) (Figure 7). It also provides maximum hinge flexibility for EPO to bind to its receptor and eliminates Fab-arm exchange in blood, a unique property of human IgG4 (Rispens et al. 2011).

[0019] An in vivo proof-of-concept study of epepoetin alfa was performed in a rat model of renal failure to mimic human CKD (Fig. 8). Briefly, Sprague-Dawley rats were surgically removed for 5 / 6 of the kidney to induce renal failure. Eight rats per group were randomly assigned to receive a single subcutaneous injection of the test article, including GX-E2 (epepoetin alfa produced in the GX-E2 cell line), Aranesp (darbepoetin alfa), Mircera (methoxy polyethylene glycol-epoetin beta), Epokine (epoetin alfa), or GX-E2 formulation buffer (vehicle control). Blood samples were collected, and blood parameters (e.g., reticulocytes, red blood cells, hematocrit, and hemoglobin) were measured twice a week for 60 days after treatment using an automated hematology analyzer.

[0020] There were no treatment-related deaths during the experimental period. There was no difference in body weight gain between treated and untreated animals, indicating that the treatment was well tolerated.

[0021] The reticulocyte percentage is the percentage of immature red blood cells in whole blood. It is used as an indicator of bone marrow function and the body's ability to produce red blood cells. Conversely, the hematocrit percentage indicates the percentage of red blood cells in whole blood, and the hemoglobin level indicates the blood's oxygen-carrying capacity. Therefore, hematocrit and hemoglobin levels correlate well with the red blood cell count. As shown in Figure 8, the red blood cell count rapidly increased after ESA injection, peaking on day 5, indicating ESA activity in red blood cell progenitor cells. As reticulocytes mature into red blood cells, the reticulocyte count decreases, while the red blood cell count, hemoglobin, and hematocrit levels increase. Since the half-life of RBCs in the circulation is approximately 120 days, the RBC count reaches a plateau as the ESA effect wears off. Compared with untreated controls, high and intermediate doses of GX-E2, Aranesp, and Mircera showed statistically significant therapeutic effects based on these parameters. High-dose GX-E2 administration showed longer-lasting effects compared to other treatments.

[0022] The invention presented herein is a robust commercial manufacturing process capable of producing EPO Fc fusion proteins, such as epepoetin alfa, with consistent quality attributes demonstrated to be safe and effective for the treatment of anemia in CKD patients, with acceptable yields. Three key indicators of consistent product quality are C-terminal lysine cleavage, sialic acid content, and glycosylation profile.

[0023] C-terminal lysine (CTL) truncation is a common phenomenon during the biological production of monoclonal antibodies. Because lysine carries a single net positive charge, this can result in a wide range of charge modifications. The impact of C-terminal lysine truncation has been studied, and it has been demonstrated that human IgG1 with zero, one, or two C-terminal lysines exhibit no difference in binding to FcRn and FcγRIIIa, thus not affecting the in vivo half-life or immune effector activity of human IgG (Faid et al. 2021). Furthermore, Cai et al. demonstrated that CTL is rapidly cleared from infused antibodies in vivo by endogenous circulating carboxypeptidases in the bloodstream (Cai et al. 2011). While CTL has little impact on product safety and efficacy, the CTL content of recombinant antibodies or Fc fusion proteins can be used as a measure of manufacturing consistency and product homogeneity.

[0024] Unlike CTLs, negatively charged sialic acid, present as N-acetylneuraminic acid attached to the glycan chain terminally as previously mentioned, has been shown to affect the half-life of many glycoproteins, including recombinant EPO, epoetin alfa (Chen et al. 2022). One likely mechanism is by inhibiting glycoprotein binding to mannose and asialoglycoprotein receptors, which are known to remove glycoproteins from the circulation. Our study also showed that despite the presence of the Fc fragment, the high sialic acid content of the EPO-hybrid Fc fusion protein unexpectedly significantly contributed to the prolonged in vivo half-life, resulting in a sustained pharmacodynamic effect (Figure 12). Given the importance of sialic acid in product efficacy, sialic acid content should be maintained within a range that reflects clinical trial data tested in the target indication.

[0025] Protein glycosylation is a hallmark of post-translational modification in mammalian cells. Glycosylation profiles vary primarily depending on the production cell line and cell culture conditions. Biosynthetic production processes generate numerous molecular variants, but the levels of these variants are tightly controlled both in upstream manufacturing and downstream processes that can enrich for specific molecular variants. Like CTL and sialic acid content, glycosylation profiles can also be used as a measure of manufacturing consistency.

[0026]

[0027] Cai B., et al. (2011). “C-terminal lysine processing of human immunoglobulin G2 heavy chain in vivo”.Biotechnol Bioeng., 108, 404-412.

[0028] Chen B., et al. (2022). “Impact of N-linked glycosylation on therapeutic proteins”.Molecules, 27, 8859.

[0029] Cokic VP., et al. (2012). “JAK-STAT and AKT pathway-coupled genes in erythroid progenitor cells through ontogeny”.J Transl Med.,10, 116.

[0030] Faid V., et al. (2021). “C-terminal lysine clipping of IgG1: impact on binding to FcgRIIIa and neonatal Fc receptors”.Eur J Pharm Sci., 159, 105730.

[0031] Jelkmann W. (2016). “Erythropoietin”. Front Horm Res. 47, 115-127.

[0032] Jelkmann W. (2013). "Physiology and pharmacology of erythropoietin". Transfus Med Hemother. 40, 302-309.

[0033] Komatsu N., et al. (1991). "Establishment and characterization of a human leukemic cell line with megakaryocytic features: dependency on granulocyte-macrophage colony-stimulating factor, interleukin 3, or erythropoietin for growth and survival".Cancer Res.,51, 341-348.

[0034] Lewis AM., et al. (2016). "Understanding and controlling sialylation in a CHO Fc-fusion process".PLoS ONE.11(6), e0157111. doi:10.1371 / journal.pone.0157111.

[0035] Miyazaki Y., et al. (1997). "Establishment and characterization of a new erythropoietin-dependent acute myeloid leukemia cell line, AS-E2".Leukemia,11, 1941-1949.

[0036] Noxon V., et al. (2017). "Tale of two erythropoiesis-stimulating agents: utilization, dosing, litigation, and costs of Darbopoetin and Epoetin among South Carolina medicaid-covered patients with cancer and chemotherapy-induced anemia".J Oncol Pract.13, e562-e573.

[0037] Pyzik M., et al. (2015). "FcRn: the architect behind the immune and non-immune functions of IgG and albumin".J Immunol.194(10), 4595-4603.

[0038] Rispens T., et al. (2011). "Mechanism of immunoglobulin G4 Fab-arm exchange".J Am Chem Soc.133, 26, 10302-10311.

[0039] Shields RL., et al. (2001). "High resolution mapping of the binding site on human IgG1 for FcRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcR".J Bio Chem. 276, 6591-6604.

[0040] Torres M., et al. (2018). "Process and metabolic engineering perpectives of lactate production in mammalian cell cultures".Chem Eng. 22, 184-190.

[0041] Watowich SS. (2011). “The erythropoietin receptor: molecular structure and hematopietic signaling pathways”.J Investig Med.,59(7), 1067-1072.

[0042] Xu X. (2017). “Safety, pharmacokinetics and sialic acid production after oral administration of N-acetylmannoseamine (ManNAc) to subjects with GNE myopathy”.Mol Genet Metab.122, 126-134.

[0043]

[0044]

[0045] An object of the present invention is to provide an EPO-Fc producing cell line and a method for producing the same for producing an EPO-Fc fusion protein having a desired CTL content, sialic acid content, isoelectric point (pI) range and specific glycosylation profile required for sustained activity in vivo.

[0046] Another object of the present invention is to provide a method for producing an EPO-Fc fusion protein.

[0047] The purpose achieved by the present invention is not limited to the above-mentioned purpose, and other purposes not mentioned above will be clearly understood by those skilled in the art to which the present invention pertains from the following description.

[0048]

[0049] To achieve the above object, one embodiment of the present invention provides an EPO-Fc fusion protein producing cell line transformed with an α-2,3 sialyltransferase gene.

[0050] In one embodiment of the present invention, the α-2,3 sialyltransferase may comprise the base sequence of SEQ ID NO: 1. In another embodiment of the present invention, the α-2,3 sialyltransferase may be composed of the base sequence of SEQ ID NO: 1.

[0051] In one embodiment of the present invention, the EPO-Fc fusion protein may bind to and activate endogenous EPO receptor at a level similar to or better than EPO BRP.

[0052] In one embodiment of the present invention, the EPO-Fc fusion protein may induce proliferation of erythroid progenitor cells and differentiation into reticulocytes at a level similar to or better than EPO BRP.

[0053] In one embodiment of the present invention, the EPO-Fc fusion protein may induce proliferation of an EPO-dependent cell line, such as UT-7, at a level similar to or better than EPO BRP.

[0054] In one embodiment of the present invention, the Fc may lack immune effector activities, including but not limited to ADCC (antibody-dependent cytotoxicity), ADCP (antibody-dependent cellular phagocytosis), and CDC (complement-dependent cytotoxicity) activities that are detrimental to cells expressing the EPO receptor.

[0055] In one embodiment of the present invention, the Fc may bind to FcRn only in a pH range representative of the endosomal compartment of mammalian cells and dissociate from FcRn at neutral pH, thereby avoiding cellular degradation, thereby extending the half-life of the Fc fusion protein.

[0056] In one embodiment of the present invention, the Fc may be increased in molecular weight to avoid renal clearance, thereby extending the half-life of the Fc fusion protein.

[0057] In one embodiment of the present invention, the EPO-Fc may comprise the amino acid sequence of SEQ ID NO: 2.

[0058] In one embodiment of the present invention, the EPO-Fc fusion protein may include an EPO sequence and a hybrid Fc sequence corresponding to amino acid residues 1-193 and amino acid residues 194-438, respectively, in the amino acid sequence of SEQ ID NO: 2.

[0059] In one embodiment of the present invention, the Fc may comprise 30 amino acid residues of the hinge region of human IgD and 8 amino acid residues of the N-terminus of the CH2 domain (amino acid residues 194-231 of SEQ ID NO: 2), and 100 amino acid residues of the C-terminus of the CH2 domain of human IgG4 and 107 amino acid residues of the CH3 domain (amino acid residues 232-438 of SEQ ID NO: 2).

[0060] The properties and methods for preparing EPO Fc fusion proteins are described in detail, for example, in “Erythropoietin immunoglobulin fusion proteins”, Sehwan Yang, Young Chul Sung, Patent No.: US 8,586,531 B2 (filed June 11, 2012, issued November 19, 2013) and “Immunoglobulin fusion proteins”, Sehwan Yang, Young Chul Sung, Patent No.: US 7,867,491 B2 (filed May 30, 2008, issued January 11, 2011), all of which are incorporated herein by reference.

[0061] In some embodiments of the present invention, the host cell expressing the EPO-Fc fusion is a cell line derived from Chinese hamster ovary (CHO), including DHFR-deficient CHO-DG44 cells and CHO DG44 α-2,3 ST cell line (Accession No.: KCLRF-BP-00525). Some embodiments of the present invention also include GX-E4 cell line clone numbers 8P-4 and 10P-1. GX-E4 cell line clone number 8P-4 is stored at Biovian Oy, Genexine, Kalbio Global Medika. GX-E4 cell line clone number 10P-1 is stored at Genexine.

[0062] To achieve the above object, another embodiment of the present invention provides a method for producing an EPO-Fc producing cell line, the method comprising the steps of introducing an EPO-Fc gene into a host cell, selecting an EPO-Fc producing host cell expressing an EPO-Fc fusion protein, then transforming the host cell again with a hygromycin resistance vector into which an α-2,3 sialyltransferase gene has been introduced, and selecting the hygromycin resistant host cell as an EPO-Fc producing cell line. The cell line overexpresses α-2,3 sialyltransferase.

[0063] To achieve the above object, another embodiment of the present invention provides a method for producing an EPO-Fc fusion protein having the following quality characteristics: a CTL content of about 1% to about 10%, a sialic acid content of about 17 mol / mol to about 27 mol / mol, and a pI of about 4.0 to about 6.0. In another embodiment, a method for producing an Fc fusion protein is included, which comprises feeding-batch culturing a host cell transformed with DNA encoding an Fc fusion protein and human α-2,3 sialyltransferase, and isolating and purifying the Fc fusion protein produced in the cell line.

[0064] In one embodiment of the present invention, the method for producing the EPO-Fc fusion protein comprises the following steps: (a) a step of culturing the EPO-Fc fusion protein producing cell line in a fed-batch manner; and (b) a step of isolating and purifying the EPO-Fc fusion protein produced in the cell line.

[0065] In one embodiment of the present invention, the EPO in the EPO-Fc fusion protein may further comprise a modification for additional glycosylation.

[0066] In one embodiment of the present invention, the EPO-Fc fusion protein may further comprise PEG.

[0067] In one embodiment of the present invention, the EPO-Fc fusion protein may have specific quality characteristics including one or more of the following: C-terminal lysine content, N- and O-glycan profile, sialic acid content, and pI range.

[0068] In some embodiments of the present invention, the CTL content of the EPO-Fc fusion protein is about 1% to about 10%, specifically about 2% to about 9%, for example about 3% to about 7%, or about 5%. In some embodiments, the sialic acid content of the EPO-Fc fusion protein is about 17 mol / mol to about 27 mol / mol, specifically about 20 mol / mol to about 25 mol / mol. In some embodiments, the pI of the EPO-Fc fusion protein is about 4.0 to about 6.0, specifically about 4.5 to about 5.3.

[0069] In one embodiment of the present invention, the N- and O-glycan profile may comprise a non-glycosylation content of about 0-20% at position N24, about 0-5% at position N38, about 10-17% at position N83, about 0-10% at position N261, and about 50-70% at position S126.

[0070] In one embodiment of the present invention, the N- and O-glycan profile may comprise glycans containing sialic acid at about 50-90% at position N24, about 55-95% at position N38, about 40-80% at position N83, about 0-30% at position N261, and about 15-40% at position S126.

[0071] In one embodiment of the present invention, the fed-batch culture can be performed under conditions including N-acetyl-D-mannosamine (ManNAc) and galactose.

[0072] In one embodiment of the present invention, the N-acetyl-D-mannosamine may be at a concentration of between about 1 mM and about 20 mM, and the galactose may be at a concentration of between about 1 mM and about 40 mM.

[0073] In one embodiment of the present invention, the fed-batch culture can be performed under temperature conditions of about 34°C to about 38°C and pH conditions of about pH 6.7 to about pH 7.5.

[0074] In one embodiment of the present invention, the fed-batch culture may be a cell line culture for 9 to 11 days.

[0075] In one embodiment of the present invention, the fed-batch culture may be performed by lowering the temperature by 2-5°C 3 or 4 days after cell line inoculation.

[0076] In one embodiment of the present invention, the fed-batch culture can be performed by changing the temperature from about 29°C to about 36°C on the 3rd to 4th day of culture.

[0077] In one embodiment of the present invention, the fed-batch culture can be performed until the viability of cells in the culture medium reaches 70% or less.

[0078] In one embodiment of the present invention, the separation and purification of the EPO-Fc fusion protein can be performed at room temperature.

[0079] In another embodiment of the present invention, the isolation and purification of the EPO-Fc fusion protein, excluding viral inactivation, may be performed at room temperature.

[0080] In one embodiment of the present invention, the separation and purification of the EPO-Fc fusion protein may be performed at a temperature of 17°C to 27°C.

[0081] In one embodiment of the present invention, separation and purification may be performed using affinity chromatography or ion exchange chromatography. In another embodiment, the ion exchange chromatography is anion exchange chromatography.

[0082] In one embodiment of the present invention, the ion exchange chromatography may comprise an anion exchange chromatography process that facilitates binding of acidic species of the EPO-Fc fusion protein to the resin using an equilibration buffer having a pH of about 6.00 to about 6.50. In another embodiment, the ion exchange chromatography may comprise an anion exchange chromatography process that facilitates binding of acidic species of the EPO-Fc fusion protein to the resin using an equilibration buffer having a pH of about 6.10 to about 6.30.

[0083] In one embodiment of the present invention, the ion exchange chromatography may include an anion exchange chromatography process using an equilibration buffer having a conductivity of about 6.00 to about 10.50 mS / cm and an elution buffer having a conductivity of about 11.50 to about 16.00 mS / cm to facilitate separation of the EPO-Fc fusion protein having the above quality characteristics. In another embodiment, the ion exchange chromatography may include an anion exchange chromatography process using an equilibration buffer having a conductivity of about 8.00 to about 10.00 mS / cm and an elution buffer having a conductivity of about 12.00 to about 15.00 mS / cm to facilitate separation of the EPO-Fc fusion protein having the above quality characteristics.

[0084] In another embodiment of the present invention, the pH of the equilibration buffer in the anion exchange step is from about 6.00 to about 6.50, and the conductivity is from about 6.00 to about 10.50 mS / cm, and the pH of the elution buffer is from about 6.00 to about 6.50, and the conductivity is from about 11.50 to about 16.00 mS / cm.

[0085] In some embodiments of the present invention, the pH of the equilibration and elution buffers in the anion exchange step may be from about 6.10 to about 6.30, the conductivity of the equilibration buffer may be from about 8.00 to about 10.00 mS / cm, and the conductivity of the elution buffer may be from about 12.00 to about 15.00 mS / cm.

[0086] In some embodiments of the present invention, the Fc fusion protein is an EPO-Fc fusion protein, wherein the Fc fragment (1) lacks immune effector activities such as ADCC (antibody-dependent cellular cytotoxicity), ADCP (antibody-dependent cellular phagocytosis), and CDC (complement-dependent cytotoxicity) so as not to attack cells expressing EPO receptors, and (2) prolongs the half-life of the Fc fusion molecule, which is achieved by (2a) increasing the molecular weight of the Fc fusion protein to avoid renal clearance, and (2b) binding to FcRn only in the pH range indicative of the endosomal compartment of mammalian cells, and dissociating from FcRn at neutral pH to avoid cellular degradation.

[0087] Its Fc fragment comprises an immunoglobulin constant region and hinge region derived from an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, IgM or a combination thereof (e.g., IgG4 and IgD) sequence and a modified sequence of the immunoglobulin subtype.

[0088] In some embodiments of the present invention, the EPO-Fc fusion protein (1) binds to and activates endogenous EPOR at levels equal to or greater than the EPO BRP, and (2) induces proliferation and differentiation of erythroid progenitor cells into reticulocytes that mature into red blood cells. The EPO fragment can be derived from native EPO or an EPO-related structure that comprises a modified EPO sequence and / or additional glycan structures or additional PEG.

[0089] The method of the present invention can be used in a process for manufacturing Efepoetin alfa, an EPO-hybrid Fc fusion protein, in a commercial manufacturing process as described above, and can particularly contribute to enhancing the efficacy of EPO-hybrid Fc fusion proteins. Efepoetin alfa is a protein used, for example, to treat anemia in humans.

[0090] The methods disclosed herein can be used, for example, in commercial manufacturing processes, for example, to improve sialic acid content associated with longer in vivo half-life and batch homogeneity, in manufacturing processes used to produce epepoetin alfa, an EPO hybrid Fc fusion protein used to treat anemia in humans.

[0091]

[0092] The present invention relates to a method for producing a long-acting EPO fusion protein. According to the method of the present invention, highly sialylated forms of long-acting erythropoietin fusion proteins with increased in vivo half-lives can be efficiently produced through optimization of cell lines, culture methods, culture conditions, purification conditions, etc.

[0093] The effects of the present invention are not limited to the effects mentioned above, and it should be understood that the effects of the present invention include all effects that can be inferred from the composition of the present invention described in the description of the invention or the attached claims.

[0094]

[0095] Figure 1 shows the structure of the efepoetin alfa protein. Efepoetin alfa is a homodimer. Each subunit contains erythropoietin (EPO), the hinge and partial CH2 domains of IgD, and partial CH2 and CH3 domains of IgG4.

[0096] Figure 2 shows the full-length amino acid sequence of epepoetin alfa (SEQ ID NO: 2). Black: EPO sequence. Underlined black: signal peptide. Bold: human IgD heavy chain partial sequence (30 amino acids of the hinge region + 8 amino acids from the N-terminus of the CH2 domain). Italic: human IgG4 heavy chain partial sequence (100 amino acids from the C-terminus of the CH2 domain + 107 amino acids from the CH3 domain).

[0097] Figures 3a to 3d show binding to the EPO receptor (EPOR) measured by surface plasmon resonance (SPR) assay. BSA and Rituxan (anti-CD20 antibody) are negative controls. EPO-BRP is an EPO reference standard and a positive control. GX-E2 is epepoetin alpha produced in the GX-E2 cell line.

[0098] Figure 4 shows the ability of GX-E2 (epepoetin alfa) to induce proliferation of the EPO-dependent UT-7 cell line in a dose-dependent manner. EPO-BRP is used as a positive control.

[0099] Figure 5 shows the binding of ephepoetin alfa to human FcRn as measured by SPR assay.

[0100] Figure 6 shows the results of the FcγRI ELISA. Rituxan and Enbrel (human TNFR2 and human IgG1 Fc fusion protein) are positive controls. GX-E2 is epepoetin alpha produced in the GX-E2 cell line.

[0101] Figure 7 shows the C1q ELISA results. Rituxan and Enbrel are positive controls.

[0102] Figure 8 shows the erythropoiesis-stimulating activity of GX-E2 (epepoetin alfa) in a rat model of chronic kidney disease (CKD). RBC, red blood cells; HGB, hemoglobin; HCT, hematocrit; RET, reticulocytes. A single dose of the test substance was injected into surgically induced CKD rats at the indicated doses. Blood parameters were measured twice a week for 60 days.

[0103] Figure 9 shows representative IEF gel electrophoresis profiles of epepoetin alfa. RS, reference standard. S1-S3, epepoetin alfa samples.

[0104] Figure 10 shows representative peptide mapping results for epepoetin alfa. Red chromatogram, formulation buffer. Green chromatogram, epepoetin alfa reference substance.

[0105] Figure 11 shows representative in vivo bioassay results.

[0106] Figure 12 shows the ability of GX-E2 (epepoetin alfa) with the indicated sialic acid contents (numbers in parentheses) to induce changes in hemoglobin levels in normal mice. Sialic acid content unexpectedly influences the efficacy of epepoetin alfa.

[0107] Figure 13 shows the amino acid sequence (SEQ ID NO: 3) of α-2,3 sialyltransferase having an HA tag.

[0108] Figure 14 shows the detailed construct of the pCI-hygro-α-2,3 st-IRES-pERP plasmid expressing α-2,3 sialyltransferase.

[0109] Figure 15 shows a schematic diagram of the generation of the GX-E4 cell line.

[0110] Figure 16 shows Western blot analysis of cell pools transfected with α-2,3 sialyltransferase using anti-HA antibodies. C is the control. Lanes 6-10 correspond to cell pools transfected with the indicated α-2,3 sialyltransferase.

[0111] Figure 17 shows an IEF gel electrophoresis of a GX-E2 cell pool transformed with α-2,3 sialyltransferase. Lane 2 is purified GX-E2 (epepoetin alfa). Lanes 3-7 are cell cultures of GX-E2 cell pools transformed with the indicated α-2,3 sialyltransferase. Lane 8 is a GX-E2 cell culture.

[0112] Figure 18 shows IEF gel electrophoresis of a single subclone of GX-E4. The GX-E4 cell line is a GX-E2 cell line transformed with α-2,3 sialyltransferase.

[0113] Figure 19 shows the growth curve of cells cultured in the presence of the indicated additives (feed).

[0114] Figure 20 shows the IEF gel electrophoresis results of cells cultured in the presence of the indicated additives.

[0115] Figure 21 shows the IEF gel electrophoresis results of a DOE (design of experiments) study on ManNAc and galactose concentrations.

[0116] Figure 22 shows the additive condition-prediction profiler plot.

[0117] Figure 23 shows a schematic diagram of the upstream manufacturing process for epepoetin alfa. IPC, process control (critical control parameters that must meet acceptance criteria before the process can proceed). IPM, process monitoring (control parameters for informational purposes). VCD, viable cell density.

[0118] Figure 24 shows IEF gel electrophoresis of cell cultures in a fed-batch bioreactor at designated culture times. Compared to a purified GX-E2 reference standard, cell cultures harvested on day 10 contained more acidic species than cell cultures harvested on days 14, 15, or 16.

[0119] Figure 25 shows a schematic diagram of the downstream manufacturing process of epepoetin alfa.

[0120] Figure 26 shows a predictive profiler plot obtained from a DOE study for the production bioreactor stage.

[0121] Figure 27 shows the viable cell density and lactic acid concentration curves of six batches of GX-E4 during 10 days of production bioreactor cultivation. Batches 1-3: pH 7.0 and temperature change on day 3. Batches 4-6: pH 6.80 and temperature change on day 4.

[0122] Figure 28 shows the N-glycan profile at position N24.

[0123] Figure 29 shows the N-glycan profile at position N38.

[0124] Figure 30 shows the N-glycan profile at position N83.

[0125] Figure 31 shows the N-glycan profile at position N261.

[0126] Figure 32 shows the O-glycan profile at position S126.

[0127]

[0128] The following describes exemplary implementations of the present invention and definitions of terms used in this document.

[0129] The present invention relates to an EPO-Fc producing cell line.

[0130] The EPO-Fc producing cell line of the present invention is a cell line transformed with the α-2,3 sialyltransferase gene.

[0131] The above EPO-Fc producing cell line can have its function improved by additionally introducing and overexpressing α-2,3 sialyltransferase.

[0132] The above α-2,3 sialyltransferase gene may be composed of, for example, sequence number 1, but is not limited thereto.

[0133] Transfection can be performed using methods, conditions, and means known in the art. For example, a cell line derived from an existing EPO-Fc master cell bank (MCB) can be retransfected with α-2,3-sialyltransferase using electroporation.

[0134] The present invention also relates to a method for producing an EPO-Fc producing cell line.

[0135] The method of the present invention comprises the following steps: introducing an EPO-Fc gene into a host cell, selecting an EPO-Fc-producing host cell expressing an EPO-Fc fusion protein, retransforming the selected EPO-Fc-producing host cell with a hygromycin-resistant vector into which an α-2,3-sialyltransferase gene has been introduced, and selecting a host cell having hygromycin resistance among the retransformed EPO-Fc-producing host cells as an EPO-Fc-producing cell line.

[0136] Here, EPO-Fc, EPO-Fc producing cell line, α-2,3 sialyltransferase gene and re-transfection are as described above.

[0137] The host cell may be, for example, a CHO cell, more specifically a CHO dhfr(-) cell or a CHO DG44 cell.

[0138] The EPO-Fc producing cell line produced by the method of the present invention has enhanced function by overexpressing α-2,3-sialyltransferase and can produce a highly sialylated EPO-Fc fusion protein.

[0139] An EPO-Fc-producing cell line is a cell line used to express an EPO-Fc fusion protein. For example, an EPO-Fc-producing cell line is a cell line obtained by retransforming a CHO host cell transformed to express EPO-Fc with the α-2,3 sialyltransferase gene. As a more specific example, the EPO-Fc-producing cell line may be the CHO DG44 α-2,3 ST cell line (Accession Number: KCLRF-BP-00525).

[0140] The present invention also provides a method for producing an Fc fusion protein comprising an EPO-Fc fusion protein having a C-terminal lysine (CTL) content of about 1% to about 10%, a sialic acid content of about 17 mol / mol to about 27 mol / mol, and a pI of about 4.0 to about 6.0, the method comprising an upstream process comprising feeding-batch culturing a host cell transformed with a DNA encoding the Fc fusion protein and a DNA encoding human α-2,3 sialyltransferase in a culture medium containing N-acetyl-D-mannosamine and galactose, and using an equilibration buffer having a pH of about 6.00 to about 6.50 and a conductivity of about 6.00 to about 10.50 mS / cm and an elution buffer having a pH of about 6.00 to about 6.50 and a conductivity of about 11.50 to about 16.00 mS / cm. Downstream processes are included, including controlling the anion exchange chromatography step.

[0141] In another embodiment, the medium may contain N-acetyl-D-mannosamine at a concentration of about 1 mM to about 20 mM, preferably about 5 mM to about 15 mM, more preferably about 10 mM, and galactose at a concentration of about 1 mM to about 40 mM, preferably about 20 mM to about 35 mM, more preferably about 32 mM.

[0142] Additionally, the badge may additionally contain glutamine, methotrexate, and hygromycin.

[0143] For example, fed-batch culture can be performed at a temperature of 34°C to 38°C at the start of culture. Additionally, fed-batch culture can be performed at a pH of 6.7 to 7.5 at the start of culture.

[0144] Additionally, the fed-batch culture can be performed at a temperature changed from 29°C to 36°C by lowering the temperature by 2 to 5°C 3 to 4 days after cell inoculation.

[0145] By varying the temperature as described above, i.e., maintaining the temperature at 34 to 38°C during the cell growth phase and lowering the temperature by 2 to 5°C to 29 to 36°C during the protein production phase, protein production can be increased. Furthermore, as the temperature is lowered, the enzymatic activity of sialidase in CHO cells decreases, allowing the sialic acid in the protein to remain stable without being degraded.

[0146] The above culturing may be performed, for example, until the viability of the cells in the culture medium reaches 80% or less or 70% or less, but is not limited thereto. More specifically, the culturing may be performed for as short a time as possible until the viability of the cells in the culture medium reaches 70% or less.

[0147] The term “about” as used herein may mean a range of ±20%, ±15%, ±10%, ±5%, or ±1% of the stated value.

[0148] As used herein, the term "Fc fusion protein" or "EPO-Fc fusion protein" refers to a protein resulting from the fusion or linkage between an Fc fragment and another protein comprising a biologically active molecule such as EPO. In some embodiments of the invention, the Fc fragment of the EPO-Fc fusion protein is a hybrid of a human IgD sequence and a human IgG4 sequence, as illustrated in FIGS. 1 , 2 , and SEQ ID NO: 2. This Fc fragment is also referred to as a hybrid Fc fragment.

[0149] The term "Fc fragment" or "Fc" as used herein refers to a protein fragment comprising the heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3) of an immunoglobulin molecule, but excluding the variable regions of the heavy and light chains of the immunoglobulin and the light chain constant region 1 (CL1). The heavy chain constant region may further comprise a hinge region.

[0150] In some embodiments, the hybrid Fc fragment of the invention binds to neonatal Fc receptor (FcRn) but lacks binding ability to Fc gamma receptor (FcγR) and C1q (complement component 1q).

[0151] FcRn, which preferentially binds IgG under acidic conditions, was first discovered for its role in the transfer of maternal IgG to offspring via the placenta. It was later discovered that FcRn protects IgG from cellular degradation. Proteins, including IgG, are continuously internalized by vascular endothelial cells and immune cells through a nonspecific cellular process called pinocytosis. Once inside the cell, IgG binds to FcRn as the pH of the endosome becomes acidic. FcRn-bound IgG is then recycled back to the cell surface, where it is dissociated from FcRn at a higher pH. Proteins that do not bind to FcRn are transported to lysosomes for degradation. This mechanism extends the half-life of IgG (reviewed in Pyzik et al., 2015).

[0152] Human IgG, particularly IgG1, can mediate potent immune effector activities, including ADCC, ADCP, and CDC, which can injure or kill cells upon binding. ADCC is mediated primarily by the interaction of the Fc fragment of IgG with FcγRIIIa, which is expressed primarily on NK cells. ADCP is mediated primarily by FcγRIIa, which is expressed primarily on macrophages. In contrast, CDC is mediated by the interaction of the Fc fragment with C1q, which triggers the classical complement pathway. In some embodiments, the immune effector activity of the EPO-Fc fusion protein is assessed using an ELISA using FcγRI and C1q. In this assay, FcγRI was chosen to replace FcγRIIIa and FcγRIIa because FcγRI binds much more avidly to similar epitopes on the Fc fragment than FcγRIIIa and FcγRIIa (Shields et al., 2001). Other cell-based assays known to the skilled artisan using primary human NK cells or NK cell lines, human macrophages or macrophage cell lines, or appropriate target cell lines can be used to assess the immune effector activity of Fc fusion proteins, such as EPO-Fc fusion proteins.

[0153] In another embodiment, the Fc fragment of the EPO-Fc fusion protein lacks Fab-arm exchange. Human IgG4 is an unusually dynamic antibody with the ability to undergo half-molecule exchange or Fab-arm exchange, resulting in an asymmetric, bispecific antibody with two different antigen-binding sites (Rispens et al., 2011). In some embodiments, combining the hinge region of IgD and eight amino acid residues of the CH2 domain of human IgD with partial CH2 and CH3 domains of IgG4 eliminates the Fab-arm exchange ability of the human IgG4 Fc fragment.

[0154] The term "Efepoetin alfa" refers to an EPO-Fc fusion protein, the structure and amino acid sequence of which are shown in FIGS. 1 and 2 , respectively. In some embodiments, the GX-E2 protein is efepoetin alfa produced by a GX-E2 cell line. In other embodiments, the GX-E4 protein is efepoetin alfa produced by a GX-E4 cell line. In yet another embodiment, the GX-E4 cell line is a GX-E2 cell line co-transformed with DNA encoding human α-2,3 sialyltransferase.

[0155] CTL content is expressed herein as the percentage of C-terminal peptides possessing CTL and can be calculated using the following equation: CTL content (%) = (number of C-terminal peptides possessing CTL) / (total number of C-terminal peptides) x 100. The number of C-terminal peptides corresponds to the number of Fc fusion subunits since each C-terminal peptide is derived from a separate Fc fusion subunit. In some embodiments of the methods described herein, the CTL content of the Fc fusion protein or EPO-Fc fusion protein is from about 1% to about 10%, specifically from about 2% to about 9%, more specifically from about 3% to about 7%.

[0156] The sialic acid content is expressed as the molar amount of sialic acid per molar amount of Fc fusion protein, wherein the molar sialic acid content is determined using resorcinol and UV spectroscopy as described in Example 2. Other methods for measuring the molar sialic acid content, such as high performance liquid chromatography (HPLC), may be used instead of the resorcinol method. In some embodiments of the methods described herein, the sialic acid content of the Fc fusion protein or EPO-Fc fusion protein is from about 17 mol / mol to about 27 mol / mol, more specifically from about 20 mol / mol to about 25 mol / mol.

[0157] The isoelectric point (pI) range used herein refers to the pI range of a purified Fc fusion protein or EPO-Fc fusion protein as determined by IEF gel electrophoresis as described in Example 1. Other methods for separating glycoproteins by charge, such as ion exchange chromatography, may be used instead of IEF gel electrophoresis. In some embodiments of the methods described herein, the pI range of the Fc fusion protein or EPO-Fc fusion protein is from about 4.0 to about 6.0, more specifically from about 4.5 to about 5.3. In other embodiments, the pI of the glycoprotein represents the sum of all charges associated with CTLs, sialic acids, glycans, amino acid residues, and post-translational modifications, including deamidation.

[0158] The term "upstream process" as used herein refers to the initial steps of a biomanufacturing process, including all steps associated with growing host cells, producing the desired protein by the host cells in a cell culture system, harvesting or collecting the desired protein from the cell culture, and removing cells and cell debris from the cell culture (also called a clarification step).

[0159] The term "downstream process" as used herein refers to the later stages of a biomanufacturing process, including all steps involved in purifying the desired protein, removing product-related and process-related impurities, inactivating and removing potential anomalous substances, and formulating the desired protein (also known as the active pharmaceutical ingredient (API)).

[0160] In the context of biomanufacturing processes, the term "drug substance" or "DS" is used to describe an API, which is part of a "drug product" or "DP", which represents the finished dosage form.

[0161] The term "product-related impurities" refers to impurities derived from the product / active pharmaceutical ingredient (e.g., degradation and aggregation products, structural heterogeneity). These types of impurities are common in biological products due to their inherent heterogeneity and physicochemical properties. These impurities should be removed or reduced because they can reduce efficacy, safety, and / or PK.

[0162] The term “process-related impurities” refers to impurities that originate in the manufacturing process (e.g., protein A, host cell DNA, and host cell proteins that leach from the resin of an affinity chromatography column). These impurities are inherent in the manufacturing process of biological products. These impurities have a high potential to cause serious, unwanted side effects in patients and therefore must be sufficiently reduced during the purification process.

[0163] Methods for producing an Fc fusion protein having one or more of the defined quality characteristics include using any host cell known in the art to produce glycoproteins, including mammalian cells, such as Chinese hamster ovary (CHO) cell lines, such as DHFR-deficient CHO-DG44 cells, CHO-K1SV cells (Lonza Biologics, Cambridge, MA, USA), and CHO-K1 cells (American Tissue Culture Collection (ATCC), Manassas, VA, USA). Other representative host cells are SP2 / 0-Ag14 cells (ATCC) and NS0 (European Collection of Authenticated Cell Cultures (ECACC, Salisbury, Wilshire, UK)). Cells can be cultured in single cell suspension or anchorage-dependent manner using culture media and feeds suitable for the host cells. Selection of suitable culture media is within the knowledge of those skilled in the art. In some embodiments, the culture medium is a serum-free medium or an animal component-free medium.

[0164] In some embodiments of the present invention, the host cell is a CHO-DG44 cell line with a mutation in the DHFR (dihydrofolate reductase) gene. This cell line was developed in the laboratory of Dr. Lawrence Chasin (Columbia University, NY, USA). Due to the defect in the DHFR gene, the CHO-DG44 cell line requires hypoxanthine and thymidine for growth. Clonal selection strategies using the DHFR gene are well known to those skilled in the art.

[0165] In some embodiments, the gene encoding the protein of interest is amplified by increasing the concentration of methotrexate (MTX), which inhibits DHFR. This strategy for increasing the productivity of transfected cell lines is also common knowledge to those skilled in the art.

[0166] Various culture systems are known in the art, including shake flasks, roller bottles, and bioreactors (e.g., stirred tank bioreactors). Culture medium can be added in a single batch process or in a fed-batch process, where small amounts of culture medium are added periodically. Host cells can also be cultured in a perfusion culture, which involves continuously removing a fixed amount of culture medium from the culture and replacing it with a similar amount of fresh medium. Perfusion cultures can generally achieve higher cell densities and can be maintained for longer periods of time than batch cultures. Perfusion cultures also allow for repeated harvests. However, the present invention also provides evidence that there is an inverse correlation between the total amount of protein produced in a cell culture and the level of sialylation of the protein product. Therefore, the primary goal of upstream processes is not high protein yield, but rather high levels of acidic proteins, which represent proteins with a high sialic acid content.

[0167] The present invention also provides a method for increasing the sialylation level of a biomolecule, comprising using a host cell transformed with DNA encoding a sialyltransferase enzyme, including human α-2,3-sialyltransferase and 19 other sialyltransferases of the same class involved in the biosynthesis of sialoglycans or sialic acid-containing glycans. The method further comprises culturing the host cell in the presence of N-acetyl-D-mannosamine, a precursor of sialic acid.

[0168] The term "N-acetyl-D-mannosamine" is an uncharged monosaccharide and the first identified precursor in the sialic acid biosynthetic pathway (Xu et al., 2017). In some embodiments of the present invention, N-acetyl-D-mannosamine is added to cell culture to promote sialic acid biosynthesis.

[0169] A method for producing an Fc fusion protein having one or more defined quality characteristics comprises controlling the pH of a cell culture to a range of about 6.70 to about 7.50, more specifically, about 6.80 to about 7.00. The pH in the culture can be controlled using several approaches known in the art. These include using buffers in the culture medium, sparging the culture medium with CO2 to lower the pH, adding a base to the culture to raise the pH, adding an acid to the culture to lower the pH, removing accumulated CO2 through sparging with N2 and agitation and increased headspace aeration, and combinations thereof. Commonly used base solutions to raise the culture pH include sodium bicarbonate, sodium hydroxide, potassium hydroxide, and ammonium hydroxide. Acid solutions used to lower the culture pH include phosphoric acid and sulfuric acid.

[0170] Methods for producing Fc fusion proteins having one or more of the defined quality characteristics may also include controlling the harvest time of the Fc fusion protein. In some embodiments, the Fc fusion protein may be harvested within 14 days after cell inoculation, specifically within 13 days after inoculation, and preferably within 10 days after inoculation. In one embodiment of the present invention, controlling the harvest time maximizes the production of acidic species of the glycoprotein, which exhibits a high sialic acid content, as shown in Example 9.

[0171] The term "harvest" as used herein refers specifically to collecting a desired protein from a cell culture. The term "controlling the harvest time" refers to adjusting the period during which the host cell produces the desired protein to collect the desired protein for purification in a subsequent step.

[0172] A method for producing an Fc fusion protein having one or more defined quality characteristics includes downstream process control. Downstream processing primarily aims to remove components other than the EPO-Fc fusion protein contained in the culture medium and can be performed using methods, conditions, and means known in the art. For example, host cell-derived impurities can be removed by subjecting the culture medium to multiple filtration and purification processes. Host cell-derived impurities can include protein impurities, including abnormal peptides, such as various aggregates and fragments, DNA impurities, endogenous viruses, exogenous viruses, and other particles, which can originate from the host cells and supplement materials.

[0173] Additionally, in the purification process, the EPO-Fc fusion protein can be purified by, for example, protein A purification, hydroxyapatite (HAP) purification, etc.

[0174] In the present invention, separation and purification are performed at room temperature, except for the low-pH virus inactivation step described below.

[0175] Because EPO is a hormone and is unstable at room temperature, purification is generally performed at low temperature (5±3°C). However, in the method of the present invention, purification can be performed at room temperature (22±5°C) because the EPO-Fc fusion protein has high stability.

[0176] In addition, if purification is performed at low temperatures, it is expensive and poses problems to the safety of workers, but in the present invention, these problems can be overcome because separation and purification are performed at room temperature.

[0177] Purification may additionally include a low-pH virus inactivation step after protein A purification, and the low-pH virus inactivation step may be performed under conditions of 10°C to 15°C, pH 3.5 to pH 3.8.

[0178] The separation and purification can be performed, for example, using affinity chromatography or ion exchange chromatography. Ion exchange chromatography may include anion exchange chromatography. The anion exchange chromatography process described herein relies on the reversible adsorption of charged solute molecules (in this case, negatively charged) to immobilized, oppositely charged groups. This process is divided into four steps: equilibration, sample loading and wash, elution, and regeneration. This process is highly dependent on the pH and conductivity (or ionic strength) of the buffer used in the process. The buffer pH determines the electrical charge state of the protein and, therefore, the binding properties of the molecule to the immobilized resin. For example, at pH 6.0, a glycoprotein with a pI of 4.0 is negatively charged, and a glycoprotein with a pI of 8.0 is positively charged. Under these conditions, only glycoproteins with a pI of 4.0 bind to the positively charged anion exchange resin, while glycoproteins with a pI of 8.0 pass through the column. The buffer conductivity determines the exchange capacity during the binding and elution process steps. For example, low conductivity allows binding of the target molecule, whereas high conductivity elutes the glycoprotein from the column. In some embodiments, the pH of the equilibration, wash, and elution buffers is about 6.00 to about 6.50, more specifically about 6.10 to about 6.30. In other embodiments, the equilibration / wash buffer conductivity is about 6.00 to about 10.50 mS / cm, more specifically about 8.00 to about 10.0 mS / cm. In another embodiment, the elution buffer conductivity is from about 11.50 to about 16.00 mS / cm, more specifically from about 12.00 to about 15.00 mS / cm.

[0179] The term "quality attribute" as used herein refers to a physical or chemical property related to product quality. The pI range determined by IEF gel electrophoresis and the sialic acid content confirmed by the resorcinol assay are directly correlated to the PK, efficacy, and safety of a product and are therefore classified as "critical quality attributes" (CQAs), which are physical or chemical properties that must be within the appropriate limits, ranges, or distributions to ensure the desired product quality. CTLs, on the other hand, are not known to affect the PK, immunogenicity, efficacy, and safety of a product and are therefore not part of the CQAs. Nevertheless, CTLs can be used as a measure of manufacturing consistency and product homogeneity.

[0180] As used herein, the terms "controlling" or "control" refer to monitoring and adjusting process parameters or conditions to produce a desired output. Controlling involves directly or indirectly measuring the substance or parameter being controlled and using these measurements to correct for the desired result.

[0181] In some embodiments, to reduce the risk of adventitious agent contamination, the raw materials used in manufacturing the Fc fusion protein are preferably free of animal components, GMP grade, and suitable for biological manufacturing.

[0182] The term "erythropoietin" or "EPO" refers to a glycoprotein hormone naturally produced by peritubular cells in the kidney that stimulates red blood cell production, also known as erythropoiesis. Erythropoiesis is initiated when EPO binds to the EPO receptor (EPOR) on erythroid progenitor cells, which triggers receptor dimerization and / or receptor reorientation, followed by the JAK / STAT and PI3K / AKT signaling cascades (Watowich, 2011; Cokic et al., 2012). The JAK / STAT pathway refers to the Janus kinase and signal transducer and activator of transcription pathway, and the PI3K / AKT pathway refers to the phosphatidylinositol 3-kinase and Ak strain transforming pathway. The JAK / STAT and PI3K / AKT pathways are both well-known membrane-to-core signaling modules. In the case of EPO and erythroid progenitor cells, they induce the expression of various genes that promote the survival, proliferation, and differentiation of erythroid progenitor cells into reticulocytes. Reticulocytes are immature red blood cells that mature into erythrocytes within 1-2 days after release into the peripheral blood. Although EPO is highly expressed in erythroid progenitor cells, it is also expressed at lower levels in several tissues, such as the brain, heart, and bone. Therefore, in addition to erythropoiesis, EPO exerts other biological effects in various tissues.

[0183] The term "chronic kidney disease" or "CKD" refers to a type of kidney disease in which kidney function is gradually lost over months to years. Because the kidneys' primary function is to remove waste products from the blood and return this purified blood to the body through a mechanism called "renal clearance" or "renal / kidney filtration," patients with CKD eventually require dialysis to remove waste products from the blood once their kidneys stop functioning. Because the renal clearance system has a molecular weight cutoff of approximately 45 kDa, molecules with a molecular weight greater than this cutoff have a longer half-life in the peripheral blood, and vice versa. Therefore, one way to increase the half-life of a therapeutic molecule is to increase its molecular weight by fusing it with another molecule, such as the EPO-Fc fusion protein described herein, such as an Fc fragment. In the case of an Fc fusion protein, the Fc fragment can bind to FcRn, preventing cellular degradation and further increasing the lifespan of the molecule in the circulation.

[0184] In addition to waste removal, another function of kidney cells, particularly peritubular cells of the renal cortex, is the production of EPO (Jelkmann, 2016). In fact, the kidneys produce most of the EPO in the body. Because EPO levels are reduced, patients with renal failure due to other causes, such as chronic kidney disease or the use of toxic small-molecule drugs (e.g., chemotherapy) to treat unrelated conditions, often suffer from anemia.

[0185] The term "rat model of renal failure" used herein refers to an animal model of renal failure using rats. Animal disease models have been developed to investigate the biology of disease and evaluate the effectiveness of potential therapeutics. Rat models of renal failure can be induced by surgically removing the kidney or by chemotherapy.

[0186] The term "erythropoiesis-stimulating agent" or "ESA," as used herein, refers to a recombinant version of EPO produced pharmacologically using recombinant DNA technology and biological processes. ESAs are used to treat anemia associated with renal failure. Examples of ESAs include epoetin alfa (brand name Epokine), darbepoetin alfa (brand name Aranesp or NESP), and methoxy polyethylene glycol-epoetin beta (brand name Mircera). Aranesp and Mircera are long-lived EPOs that contain an additional carbohydrate side chain or additional pegylation, respectively.

[0187] The term "EPO BRP" refers to the European Pharmacopoeia EPO Biological Reference Preparation, an EPO reference substance or reference standard. EPO BRP is used as a positive control in several experiments presented herein. The term "reference standard" or "reference material" refers to a highly purified and well-characterized substance suitable for testing the identity, strength, quality, and purity of a pharmaceutical product.

[0188] The term "in vivo bioassay," as used herein, refers to an assay that uses animals to measure the efficacy of an API. In some embodiments, the experiment is conducted using normocytic mice, meaning mice with normal red blood cell counts. The efficacy of the test item is directly compared to the efficacy of a reference substance or reference standard.

[0189] In some embodiments of the present invention, the ability of an EPO-Fc fusion protein to stimulate erythroid progenitor cells can be assessed using a simple binding assay, such as surface plasmon resonance (SPR) technology using recombinant EPOR. In other embodiments, the same assessment can be performed using cell proliferation assays using EPO-dependent cell lines, such as the UT-7 cell line (Komatsu et al., 1991) and the AS-E2 cell line (Miyazaki et al., 1997; Riken BioResource Research Center, Tsukuba, Ibaraki, Japan).

[0190] The term "SPR" refers to an optical technique for detecting the interaction of two different molecules, one mobile and the other immobilized on a thin gold film. The interaction between the two molecules causes a small change in refractive index at the gold surface, which can be quantified using an instrument such as the Biacore 3000 (Biacore, Uppsala, Sweden).

[0191] Protein glycosylation is a characteristic post-translational modification of mammalian cells. Glycosylation profiles largely depend on the production cell line and cell culture conditions. Biosynthetic production processes generate multiple molecular variants, but the level of molecular variation is tightly controlled in both upstream manufacturing processes and downstream processes that can enrich for specific molecular variants. Similar to CTL and sialic acid content, glycosylation profiles can also be used as a measure of manufacturing consistency. In some embodiments, the glycosylation profile of epepoetin alfa is evaluated using the method described in Example 13, and the results of N- and O-glycan profiles demonstrate a high degree of similarity between the EPO-Fc fusion proteins produced by the GX-E2 and GX-E4 cell lines, indicating that the manufacturing process for EPO-Fc fusion proteins can produce drug substances with robust and consistent quality characteristics.

[0192] The term "process control" as used herein refers to a series of operations or process parameters and detection parameters that regulate and monitor a manufacturing process to provide a product that meets predefined acceptance criteria for each quality attribute to ensure product quality. In some embodiments of the present invention, the acceptance criteria for each quality attribute, the criticality of each quality attribute, the ranges of process parameters, the criticality of process parameters, the control strategies including in-process control (IPC) and in-process monitoring or testing (IPM or IPT), and the detection parameters are defined based on process and analytical development results, process characterization results, and manufacturing experience. The term "IPC" as used herein refers to a critical control parameter that must be met according to acceptance criteria before the process can proceed. The terms "IPM" or "IPT" as used herein refer to parameters collected during the manufacturing process for informational purposes only.

[0193] The term "Design of Experiments" or "DOE" refers to a systematic and efficient method that allows scientists and engineers to study the relationships between multiple input variables (aka factors) and a key output variable (aka response). It is a structured statistical approach to data collection and discovery. DOE is a common approach in process characterization, with one of its goals being to establish a functional relationship between input process parameters and output quality characteristics. The outcome of a process characterization experiment is to identify process parameters that affect a product's critical quality attributes (CQAs) and their acceptable ranges or limits. Process parameters that affect CQAs are called critical process parameters, and their acceptable ranges or limits must be met to ensure product quality.

[0194] One embodiment of the present invention is a peptide mapping method that ensures product characterization. In this method, a sample is denatured to remove N-glycans, the main glycans of epepoetin alfa. The sample is then digested with trypsin, and the resulting peptides are separated using UPLC. The unique chromatographic profile of the test sample is then compared to that of a reference substance. Because this method encompasses all tryptic peptides of epepoetin alfa, including the N- and C-terminal peptides, the CTL content can be assessed using the same method.

[0195] As used herein, the term "cell bank" refers to a cell line preserved and stored under controlled conditions for future use in various biotechnological applications, including the creation of other cell banks and the production of biological drug substances. The term "Research Cell Bank" or "RCB" refers to a cell bank used for research and development purposes. An RCB is the end result of cell line development, the process of developing a cell line for producing biological products. The RCB is often used to create a "Master Cell Bank (MCB)," which is an aliquot of a single pool of cells prepared from a selected cell clone under defined conditions, distributed into multiple containers, and stored under defined conditions to serve as the primary source of cells for the production of biological drugs. The term "Working Cell Bank (WCB)" refers to a cell bank prepared from the MCB under defined culture conditions and used directly to manufacture biological products. Both the MCB and WCB undergo quality control testing, genetic stability assessment, and cell recovery assessment.

[0196]

[0197] Example

[0198]

[0199] Example 1. Isoelectric focusing (IEF) gel electrophoresis

[0200] The C-terminal lysine residue carries a single net positive charge, whereas sialic acid carries a single net negative charge. Since sialic acid contributes to the glycosylation profile, it also influences the charge of proteins. The charge characteristics of epepoetin alfa are measured using inductively coupled plasma (IEF) gel electrophoresis, where the electrophoretic profile of a test sample is compared to that of an epepoetin alfa reference substance. IEF gel electrophoresis is a method for separating proteins based on their isoelectric point (pI), the pH at which the molecule carries no net electrical charge. The experiment is performed using an IEF gel with a pH gradient from 3.0 to 7.0. The sample is mixed 1:1 with sample buffer and loaded onto the IEF gel. Electrophoresis running conditions: 100 V for 1 hour, 200 V for 1 hour, and 400 V for 45 minutes. The gel was then stained with Coomassie blue and destained using a destaining solution. A representative example of the IEF gel electrophoresis profile of epepoetin alfa is shown in Figure 9. The results show heterogeneous bands between pI 4.5 and 5.3, indicating that epepoetin alfa is an acidic protein with a negative charge at neutral pH. IEF gel electrophoresis results can reflect CTL and sialic acid content.

[0201]

[0202] Example 2: Sialic acid content

[0203] Sialic acid content is part of the glycosylation profile and is directly measured by a calorimetric method using resorcinol and UV spectroscopy. The sample is first treated with a weak acid to liberate sialic acid, which is then reacted with resorcinol. Boiling the mixture at 100–105°C for 30 minutes produces a reactive intermediate, which reacts with resorcinol to produce a chromogen, which can be extracted with butanol and butyl acetate to produce a chromophore. The resulting chromophore is measured at 580 nm using UV spectroscopy. The amount of sialic acid is determined using a standard curve, and the molar content of sialic acid per mole of epepoetin alpha is calculated. Representative examples of sialic acid content measurements are shown in Table 1. GX-E2 and GX-E4 are epepoetin alpha produced from the GX-E2 and GX-E4 cell lines, respectively. For descriptions of the two cell lines, see Examples 6 to 8.

[0204]

[0205] [Table 1]

[0206] Sialic acid content of epepoetin alfa

[0207]

[0208]

[0209] Example 3: Peptide mapping method

[0210] CTL content can be detected using a peptide mapping method. Briefly, the sample is first concentrated. The sample is then mixed with denaturing buffer and heated at 95°C for 10 minutes. The sample is then treated with PNGase F to remove N-linked glycans at 37°C for 3 hours. The protein is precipitated with acetone at -20°C. Residual free N-glycans are removed with 60% acetonitrile at -20°C for 1 hour. The sample is then denatured with 1 M dithiothreitol at 37°C for 30 minutes. 1 M iodoacetic acid is added to the sample and incubated at 37°C for 30 minutes for carboxymethylation. A buffer exchange is performed, and the sample is digested with trypsin at 37°C for 4 hours. Trypsin digestion is stopped by the addition of trifluoroacetic acid. The degraded peptides are separated using UPLC and detected with a UV detector at 214 nm. The chromatogram profile is compared with that of a reference substance. A representative example of the peptide mapping results is shown in Figure 10.

[0211]

[0212] Example 4: In vivo bioassay

[0213] The efficacy of epepoetin alfa was measured using an in vivo bioassay using normal red blood cell mice. Briefly, ~8-week-old B6D2F1 mice (F1 crossbreed of C57BL6 female and DBA2 male mice) were injected subcutaneously with 0.5 mL of 800, 400, or 200 ng / mL samples or a reference standard diluted in 0.1% BSA solution. Blood samples were collected 4 days after injection and analyzed by flow cytometry. The relative efficacy of the test item was compared with that of the reference standard. A representative example is shown in Figure 11.

[0214]

[0215] Example 5: Effect of sialic acid content on the efficacy of epepoetin alfa

[0216] Sialic acid content is known to determine the half-life of epepoetin alfa in vivo. To determine the effect of sialic acid content on the efficacy of epepoetin alfa in vivo, epepoetin alfa with various sialic acid contents was produced and purified using the GX-E2 cell line. Five batches of epepoetin alfa with sialic acid contents ranging from 19.1 to 22.6 mol / mol were injected into five groups of rats. The PK profiles and changes in hemoglobin levels were monitored for each group, and the results are presented in Table 2 and Figure 12, respectively.

[0217]

[0218] [Table 2]

[0219] PK profiles of epepoetin alfa with various sialic acid contents

[0220]

[0221]

[0222] As shown in Table 2, GX-E2 reference standard, GX-E2 #3, and GX-E2 #4 with sialic acid contents ranging from 21.9 to 22.6 mol / mol showed very similar exposures. They also induced similar hemoglobin responses (Fig. 12). In contrast, GX-E2 #1 with sialic acid content of 19.1 mol / mol showed significantly lower exposure compared to the GX-E2 reference standard (Table 2), which correlated with a significantly lower hemoglobin response (Fig. 12). GX-E2 #2 with sialic acid content of 20.0 mol / mol also showed approximately 30% lower exposure compared to the reference standard, and although the hemoglobin response was slightly reduced, the change was not significant. The vehicle control showed no change in hemoglobin levels. Overall, these results suggest that the in vivo efficacy of epepoetin alfa may unexpectedly vary depending on sialic acid content, despite the presence of the Fc fragment.

[0223]

[0224] Example 6: Transfection of α-2,3 sialyltransferase into GX-E2 cell line

[0225] Given that a high sialic acid content is required for the prolonged half-life and long-acting activity of epepoetin alfa, it is important to have an epepoetin alfa-producing cell line capable of producing a highly sialylated protein.

[0226] In CHO cells, epepoetin alpha undergoes N-glycosylation through post-translational modification (PTM). Since the N-glycosylation site of the protein is only created at aspartic acid, additional N-glycosylation does not occur unless a mutation occurs. In addition, when the target protein is overexpressed in CHO cells, the process of sufficient N-glycosylation through PTM does not proceed sufficiently, and the desired level of sialic acid is not produced. Therefore, it is very important to maintain the existing N-glycosylation so that it is produced well. For this purpose, α-2,3 sialyltransferase was added. The basic principle is that CMP-Neu5Ac is biosynthesized from Neu5Ac (N-acetylneuraminic acid) and CTP by enzymes present in the nucleus, and in the cytoplasm, CMP-Neu5Ac is hydroxylated to CMP-Neu5Gc, which is transported to the lumen of the Golgi membrane by the sialic acid transporter present in the Golgi membrane, and is specifically converted into a glycoprotein by sialin transferase in the Golgi apparatus. At this time, α-2,3 sialyltransferase forms a sialic acid by adding a galactose to the sugar. Therefore, α-2,3 sialyltransferase is important because it helps in the production of sialic acid of epepoetin alpha (Patrick et al, 2009, Optimal and consistent protein glycosylation in mammalian cell culture Figure 2).

[0227] Specifically, to increase the level of protein sialylation, the ST3GAL4 gene encoding α-2,3 sialyltransferase, an enzyme that catalyzes the transfer of sialic acid to the carbohydrate group of glycoproteins or glycolipids, was transfected into the GX-E2 cell line producing epepoetin alfa. The amino acid sequence of α-2,3 sialyltransferase with an HA tag is shown in Figure 13 (SEQ ID NO: 3). To generate a stable cell line expressing α-2,3 sialyltransferase, GX-E2 master cell bank (MCB) cells were transfected with the α-2,3 sialyltransferase-HA expression plasmid, pCI-hygro-α-2,3 st-IRES-pERP (Figure 14), via electroporation. A schematic diagram of the generation of the GX-E4 cell line is shown in Figure 15. Briefly, GX-E2 MCB cells were thawed and passaged several times in Ex-Cell CHO DHFR medium containing 4 mM glutamine, 1 g / L poloxamer 188, and 200 nM methotrexate (MTX) until stable cell growth. Cells with a viability of ≥95% were mixed with the pCI-hygro-α-2,3 st-IRES-pERP plasmid, and the mixture was subjected to electroporation at voltages of 1400 V, 1600 V, and 1700 V. Transformed cells were cultured in 6-well plates and selected with hygromycin.

[0228]

[0229] Example 7: Hygromycin Selection and Characterization of Transformed Cell Pools

[0230] Forty-eight hours after transfection, the transformed cells were seeded at 4.5 x 10 in 4-5 mL EX-CELL CHO DHFR- medium containing 4 mM L-glutamine, 200 nM MTX, 1 g / L poloxamer 188, and 100 μg hygromycin. 4Cells were seeded at a viable cell density of 10 cells / mL. Cells were cultured at 37°C in 5% CO2 for 16 days. Hygromycin selection was completed when cell viability exceeded 90%. Five transformed cell subpools (i.e., #6P, 7P, 8P, 9P, and 10P) were analyzed by Western blot using anti-HA antibody to confirm the presence of α-2,3 sialyltransferase (Fig. 16) and by IEF gel electrophoresis to confirm increased sialylation (Fig. 17). All five transformed pools expressed α-2,3 sialyltransferase and produced more sialylated protein (lanes 3–7, Fig. 17) compared to untransformed GX-E2 cells (lane 8, Fig. 17), as indicated by increased intensity of the low pI band.

[0231]

[0232] Example 8: Single-cell subcloning and characterization of GX-E4 subclones

[0233] Stable clones of the GX-E2 cell line transformed with α-2,3 sialyltransferase are referred to as the GX-E4 cell line. Single-cell subcloning of the transformed subpools #8P, 9P, and 10P was performed using limiting dilution (Fig. 15). Cells were seeded at 0.5–1 cell per well in 96-well plates (10 plates for each subpool). The clonality of the subclones was documented using a Clone Select Imager 4 hours (day 0), 7, and 14 days after seeding. After 14 days of culture, the culture medium was replaced with fresh medium, and the specific productivity was assessed the following day by Fc ELISA. Based on the results, a total of nine highly productive clones were selected and expanded. Five subclones were successfully expanded and tested for mycoplasma, specific productivity, and sialylation levels. All samples were negative for mycoplasma. Compared with the GX-E2 cell line, the GX-E4 subclones did not show a significant difference in specific productivity. However, GX-E4 subclones #8P-4 and #10P-1 produced more acidic proteins than the GX-E2 WCB, as shown in Figure 18. Therefore, these two subclones were selected as lead subclones and used to prepare a research cell bank (RCB). This subclone showed excellent stability in terms of cell growth and epepoetin alpha production up to 34 passages in shake flask culture and 7, 14, 21, 28, and 34 passages in fed-batch bioreactor culture. The long-term stability results are shown in Table 3.

[0234]

[0235] [Table 3]

[0236] Long-term stability of two-lead RCBs

[0237]

[0238] Candidate 3: GX-E4 Clone #8P-4, Candidate 5: GX-E4 Clone #10P-1.

[0239] T d : Ship time (hours).

[0240] Productivity Decrease: Comparison of specific productivity between 1 and 34 subcultures.

[0241] Badge condition W: Contains methotrexate and hygromycin;

[0242] Medium conditions W / O: Without methotrexate and hygromycin.

[0243]

[0244] As shown in Table 3, Candidate 3 (Clone #8P-4) exhibited a slightly better doubling time and less decline in productivity over time. Based on these results, the GX-E4 #8P-4 RCB was selected to generate the GX-E4 master and working cell banks (MCB and WCB). All cell banks were thoroughly evaluated, and the results met the acceptance criteria according to regulatory guidelines.

[0245]

[0246] Example 9: Upstream manufacturing process of epepoetin alfa

[0247] The goal of the upstream process is to express epepoetin alpha protein in cell culture and harvest it in sufficient quantities for subsequent purification and formulation. The upstream process begins with thawing the WCB and expanding the cells multiple times in shake flasks. The shake flask contents are then collected and inoculated into a fed-batch bioreactor. The bioreactor culture is harvested and clarified using depth filtration or continuous centrifugation before downstream processing. A schematic of the upstream process is shown in Figure 23. The upstream process is performed in a controlled environment using disposable or sterile product-contact equipment. Each process step is carefully controlled by defined process parameters and input and output process control parameters. This control strategy is derived from rigorous process development and characterization experiments, as well as from experience in laboratory, pilot, and commercial-scale manufacturing.

[0248] The present invention encompasses the upstream process illustrated in Figure 23. This novel cell culture mode has been established and used to produce clinical trial materials for Phase 3 clinical studies. Previously used perfusion culture had several drawbacks, including difficulties in finding optimal protein production conditions, a high risk of contamination due to long culture periods, and difficulties in scale-up. Therefore, the present invention changed the culture mode to a fed-batch mode, which minimizes contamination risk by using a bioreactor for a short period of 10-15 days.

[0249] The production bioreactor steps are described as follows. The bioreactor is filled with culture medium (EX-CELL CHO DHFR(-), 4 mM glutamine, 1.0 g / L poloxamer 188) and preheated before cell inoculation. Seed cells from passage 8 are inoculated into the production bioreactor to an initial inoculum density of 400,000–700,000 cells / mL. The post-inoculation survival rate should be ≥ 90%.

[0250] The culture in the bioreactor is maintained at 34-38°C, pH 6.7-7.5, and dissolved oxygen (DO) of 50%. pH, cell viability, cell density, and concentrations of metabolites such as glucose, lactate, glutamine, glutamate, and ammonia are regularly measured. Other parameters such as temperature, agitation speed, DO concentration, and pH are controlled throughout the culture period. On the third or fourth day after inoculation, the temperature is lowered by 2-5°C, reaching 29.0-36.0°C. Various feeds are added at predetermined concentrations and times throughout the culture period.

[0251] When cell viability falls below 70%, the contents of the production bioreactor are harvested. The cell culture harvest is purified by removing cells and cell debris using deep filtration or continuous centrifugation. The titer and purity of the target protein are monitored. The novel process, the subject of the present invention, increases titer by 7.4-fold compared to a conventional process using the GX-E2 cell line (Table 4).

[0252]

[0253] [Table 4]

[0254] Comparison of the GX-E2 and GX-E4 Upstream Processes

[0255]

[0256]

[0257] Various additives were screened to enhance glycosylation (including sialic acid content). Table 5 shows the culture conditions used in this experiment. Table 6 lists the various additives and concentrations tested in this experiment.

[0258]

[0259] [Table 5]

[0260] Culture conditions

[0261]

[0262]

[0263] [Table 6]

[0264] Feed additive testing

[0265]

[0266]

[0267] [Table 7]

[0268] Results of various additives (feed) to promote glycosylation

[0269]

[0270]

[0271] The results of the additive screening are shown in Table 7, Figures 19 and 20. The results in Table 7 show that the addition of ManNAc or ManNAc + cytidine increased the purity, whereas the addition of dexamethasone or galactose increased the titer. With regard to cell growth, the addition of dexamethasone, galactose, or ManNAc did not alter the growth curve (Figure 19). With regard to IEF gel electrophoresis, the addition of glucosamine, glucosamine + uridine, or ManNAc + cytidine increased basic species compared to the control, whereas the addition of galactose or ManNAc increased acidic species (Figure 20). Based on these results, galactose and ManNAc were selected as additives in the production bioreactor stage.

[0272] Next, the feeding conditions of galactose and ManNAc were optimized using a design of experiments (DOE) (see Example 11 for a detailed description of the DOE). Sixteen experiments were performed using ManNAc concentrations of 0–20 mM and galactose concentrations of 0–40 mM. The results are presented in Table 8, Figures 21 and 22. It was confirmed that the purity increased but the titer decreased as the ManNAc concentration increased (Table 8). In addition, lanes 6 and 8 of Figure 21 were confirmed to have lower pI values ​​compared to the other experimental results. A prediction profiler plot was generated based on the DOE results. Based on the profiler plot (Figure 22), productivity, and IEF gel profile, 10 mM ManNAc and 32 mM galactose were found to be the optimal feeding concentrations.

[0273]

[0274] [Table 8]

[0275] DOE results for galactose and ManNAc supply conditions

[0276]

[0277]

[0278] Another key finding in upstream process development was that the production bioreactor phase should be limited to 10 days. Many bioprocesses use a 14-day period to maximize cell growth and protein production. However, epepoetin alfa is a unique product that relies on high sialic acid content, so the process must maximize the production of acidic species. As shown in Table 9, extending the culture period to 14, 15, and 16 days significantly increased total protein titer and viable cell density.

[0279]

[0280] [Table 9]

[0281] Process Development Results: Bioprocess Incubation Period

[0282]

[0283]

[0284] However, as shown in Figure 24, cell cultures over a 14- to 16-day period contained less acidic species than cell cultures over a 10-day period. Due to these results, the production bioreactor culture period was set to 10 days with the feed schedule shown in Figure 23.

[0285]

[0286] Example 10: Downstream manufacturing process of epepoetin alfa

[0287] The goal of the downstream process is to purify epepoetin alfa by removing process-related and product-related impurities and potential adventitious substances, and to enrich for acidic species with high sialic acid content. Epepoetin alfa drug substance (DS) is purified from cell culture supernatant through the following unit operations (see Figure 25):

[0288] ·Protein A affinity chromatography to capture ephepoetin alfa using a protein A ligand that binds to the Fc fragment of the protein.

[0289] ·Virus inactivation using low pH buffer at 10 - 15°C.

[0290] ·Deep filtration to remove large impurities such as particles / sediments formed in previous process steps.

[0291] ·Ion exchange chromatography (IEX) 1 using ceramic hydroxyapatite type II resin to remove product-related impurities such as oligomers and process-related impurities such as host cell DNA.

[0292] ·Diafiltration for buffer exchange.

[0293] ·IEX2 chromatography using Q Sepharose Fast Flow (QFF) resin, a powerful anion exchanger. This chromatography step purifies the acidic species of epepoetin alfa while simultaneously removing basic species and other process-related impurities.

[0294] ·Virus filtration to remove any remaining viruses.

[0295] ·Ultrafiltration and diafiltration (UF / DF) 2 for concentrating and formulating epepoetin alfa DS.

[0296] ·Add polysorbate 20 to the formulated DS, adjust the final concentration, and store in the freezer.

[0297] Downstream processing is performed in a controlled environment using disposable or sterilized product-contact equipment. Each process step is carefully controlled by defined process parameters and process control parameters to ensure product quality throughout the manufacturing process. This control strategy is derived from rigorous process development and characterization experiments, as well as from laboratory, pilot, and commercial-scale manufacturing experience.

[0298] A distinctive feature of the downstream processing of epepoetin alfa is the separation and purification of acidic species by anion-exchange chromatography using a positively charged QFF resin in the IEX2 step. This step utilizes a buffer with a pH of 6.10–6.30 to facilitate the binding of protein species with a pI of ≤ 5.3. After extensive washing, the bound acidic species are eluted with a buffer with a conductivity of 12.10–14.9 mS / cm (Figure 25).

[0299] Compared to the GX-E2 process, the downstream purification process of the present invention includes the following changes.

[0300] ·All purification steps except the virus inactivation step are performed at controlled room temperature.

[0301] ·Larger columns are used because production scale has increased.

[0302] ·The order of the virus filtration process and the ultrafiltration / diafiltration process has been changed (ultrafiltration / diafiltration is performed after virus filtration).

[0303]

[0304] Details of the downstream purification process of epepoetin alfa are as follows.

[0305] (1) Protein A affinity chromatography

[0306] Affinity chromatography is the first step in a series of purification steps in the downstream process. The strategy employed in affinity chromatography is to bind a protein A ligand to the Fc region of the target protein. The target protein is selectively isolated from the protein pool present in the purified harvest. After an equilibration step, the purified harvest is loaded onto the column. Re-equilibration and washing steps are then performed to remove impurities weakly bound to the column. Finally, the Fc fusion protein is eluted from the column using a low pH buffer.

[0307] (2) Virus inactivation - low pH treatment

[0308] The eluate from the affinity chromatography was adjusted to 12.5 ± 2.5°C using a temperature-controlled mixer. The pH was adjusted to 3.65 ± 0.1 using pre-chilled Protein A elution buffer while stirring at 50–70 rpm. After reaching the target pH, the solution was incubated for 120 ± 10 minutes. The solution was then neutralized with 1 M Tris buffer until the pH reached 6.9 ± 0.1.

[0309] (3) Deep filtration

[0310] Deep filtration is performed to remove any remaining host cell proteins (HCPs) in the solution. This step is also performed to remove particles / precipitates formed during the pH adjustment step of virus inactivation. The depth filter is moistened with water for injection (WFI) and a buffer solution. After sufficient moistening, the solution is passed through the depth filter. A post-wash with a buffer solution is then performed to remove any remaining material from the depth filter. After deep filtration is complete, the solution is filtered through a 0.2 μm filter.

[0311] (4) Ion exchange chromatography 1 (IEX1)

[0312] Ion exchange chromatography is the second chromatography step. It operates in bind-elute mode to remove oligomeric impurities. The column is then washed to reduce impurity and endotoxin levels. After the equilibration step, the column is loaded with a depth filtration solution. A re-equilibration step is then performed to remove any remaining impurities in the column. This is followed by an elution step. The target protein is eluted first, and the impurities remain in the column. The remaining impurities bound to the column are washed with column wash buffer. Before washing, an equilibration step is performed to reduce phosphate levels to prevent crystal formation, which can interfere with resin performance.

[0313] (5) Ultrafiltration / Difiltration 1 (UF / DF 1)

[0314] The diafiltration step is performed in TFF mode using a polyethersulfone (PES) membrane with a molecular cutoff of 30 kDa. Before loading the sample, the system (skid and UF filter) is washed with 0.5 M NaOH and then with water in water (WFI) to remove the washing solution. After equilibrating the membrane with the equilibration buffer, each sample is loaded into the system, and the buffer is replaced with the loading buffer for the next chromatography step. The produced substances are captured by the filter and remain as the retentate, while the impurities flow through the filter and are discarded as the permeate. The diafiltration process is complete when the pH and conductivity of the solution in the permeate are equal to those of the exchange buffer.

[0315] (6) Ion exchange chromatography 2 (IEX2)

[0316] Ion exchange chromatography (anion exchange) is the final chromatographic step. It operates in bind-elute mode to remove basic protein impurities and fragmented proteins. After the equilibration step, the diafiltration residue is loaded onto the column. A re-equilibration step is then performed to remove any remaining impurities. Next, the elution step is performed. The target protein is eluted first, and the impurities remain in the column. The remaining impurities bound to the column are washed with a column wash buffer. Upon completion of the process, the column is washed again to reduce impurities and endotoxin levels.

[0317] (7) Virus filtration

[0318] Virus filtration is performed to remove virus particles present in a solution. It operates in steady-flow filtration mode at a constant pressure. A pressure vessel is used to store the liquid, and nitrogen gas is used to apply a constant pressure to facilitate virus filtration. In tandem mode, a prefilter with a pore size of 0.1 μm is used before the virus filter. After sufficiently moistening with water-in-oil (WFI) and buffer, the solution is passed through the virus filter. After virus filtration is complete, the solution is filtered through a 0.2 μm filter.

[0319] 8) Formulation exchange (UF / DF-2)

[0320] The formulation exchange (UF / DF-2) step was performed in TFF mode using a PES membrane with a molecular cut-off of 30 kDa. Before loading the sample, the system (skid and UF filter) was washed with 0.5 M NaOH and then with water in water (WFI) to remove the washing solution. After the equilibration step, each sample was loaded into the system. The resulting material was captured by the filter and remained as the retentate, while impurities passed through the filter and were discarded as the permeate. The buffer was replaced when the target concentration was reached. The buffer exchange process is complete when the pH and conductivity of the solution in the permeate are identical to those of the exchange buffer.

[0321] The purification and analysis results are shown in Tables 10 and 11 below. When the temperature conditions were changed from low temperature to room temperature and the buffer and pH of each purification step were optimized accordingly, the yield was confirmed to increase by 2.7 times (based on harvest after 10 days of culture).

[0322]

[0323] [Table 10]

[0324] Comparison of purification processes performed at low and room temperature

[0325]

[0326]

[0327] [Table 11]

[0328] Quality parameters of refined products at refrigerated and room temperature

[0329]

[0330]

[0331] Example 11: DOE Study for the Production Bioreactor Stage

[0332] One of the goals of process characterization experiments is to establish a functional relationship between input process parameters and output quality attributes. These experiments identify critical process parameters (CPPs), which are process parameters that influence the product's critical quality attributes (CQAs). CQAs are product critical quality attributes that influence patient safety, efficacy, PK / PD, and immunogenicity.

[0333] Process characterization began after the process was completed at the intended manufacturing scale. Process characterization experiments were performed on a scaled-down model that was qualified to represent the manufacturing scale. First, a Failure Model Effect Analysis (FMEA, as described in ICH Q8[R2]) tool was used to identify all potential CPPs (pCPPs). Using the pCPPs, process characterization experiments were performed using univariate or multivariate experimental approaches. Based on the results of these experiments, each pCPP was classified as a CPP or non-CPP. Acceptable ranges for these CPPs (input process parameters) were derived based on the acceptable ranges for the output characteristics. Acceptable ranges for non-CPPs were derived from process development / characterization and manufacturing batches.

[0334] In addition to product quality, process characterization experiments can also identify process parameters that impact process performance, such as yield. The results of a multivariate design of experiments (DOE) used to improve the yield of a manufacturing process are as follows.

[0335] To further characterize the production bioreactor process, a DOE using response surface methodology (RSM) was performed at a 1 L scale using a qualified downscaled model. In this experiment, we focused on four factors (i.e., pH, transfer temperature, feed timing, and galactose timing), while other process parameters were fixed as in the manufacturing process described in Figure 25. The final yield depended on a small proportion of GX-E4, which had a high sialic acid content and was difficult to distinguish during the purification step, so the product was purified up to the IEX2 stage. This experiment measured seven reactions as follows:

[0336] · Response to control strategy: Purity (%) at IEX2 stage

[0337] · Response to information: final viable cell count, final cell viability (%), yield in bioreactor stage (mg), purity in bioreactor stage (%), yield in IEX2 stage (%), total yield (%)

[0338] The lower screening range (LSR) and upper screening range (USR) for the three factors are shown in Table 12.

[0339]

[0340] [Table 12]

[0341] Production Bioreactors - Screening Scope for Four Elements

[0342]

[0343]

[0344] A total of 18 runs were conducted based on a central composite design with four factors and two central points. This experiment enabled response surface modeling of all major factors and two-factor interactions. The results are presented in Table 13.

[0345]

[0346] [Table 13]

[0347] Production Bioreactor - Response Data for Each DOE Run

[0348]

[0349]

[0350] Statistical analysis of the data generated the predictive profiler plot shown in Figure 26. Based on the predictive profiler plot, the pH 7.0 condition, which was the manufacturing process setting at the time, showed slightly lower viable cell count, % cell viability, and total yield compared to the pH 6.9 and pH 6.8 conditions.

[0351]

[0352] Example 12: Upstream Process Modification to Improve Final Yield

[0353] Based on the process characterization results, the pH in the production bioreactor was lowered from 7.0 to 6.8. Additionally, the temperature transition time was delayed from day 3 to day 4 to allow optimal cell growth. These minor changes yielded significant results.

[0354] First, final viable cell density (VCD) was improved and lactate concentration was significantly reduced (Figure 27). Under the new culture conditions, cells grew steadily, reaching a maximum VCD of approximately 5,000,000 cells / mL on day 10 (see batches #4-6 in Figure 27). In contrast, under the previous culture conditions, VCD peaked on day 7 and then declined (see batches #1-3 in Figure 27). Similarly, under the new culture conditions, lactate concentration remained low (< 2 g / L) throughout the 10-day culture period, whereas under the old culture conditions, it steadily increased, reaching > 4 g / L on day 10. Lactate concentrations exceeding 40 mM (or 3.6 g / L) in CHO cell systems severely impair cell growth (Torres et al., 2018).

[0355] Second, and most importantly, the new conditions increased the final yield by more than 20% at 50 L scale, from an average of 6.69 grams to 8.11 grams (Table 14). The yield percentage also improved significantly, from an average of 13.10% to 31.73%. The difference in yield percentage was primarily due to increased acidic species production rather than total protein production. Total protein produced under the new culture conditions was ~25.6 grams, while that produced under the original culture conditions was ~51 grams.

[0356]

[0357] [Table 14]

[0358] Comparison of yields between two production bioreactor conditions

[0359]

[0360]

[0361] The results presented in Tables 3 and 14 suggest an inverse correlation between total protein produced in the bioreactor and the sialylation level of epepoetin. This finding is supported by a study by Lewis et al. (Lewis et al., 2016), which found that sialic acid levels decreased as protein titer increased during a 14-day fed-batch culture of an Fc-fusion protein.

[0362]

[0363] Example 13: Consistent product quality of epepoetin alfa

[0364] The impact of a change in production cell line (from GX-E2 to GX-E4) and an upstream process change (from pH 7.0 to pH 6.8) on the quality attributes of epepoetin alfa was thoroughly evaluated. The study included the following characteristics:

[0365] ·Appearance, pH.

[0366] ·Contents: Protein content, excipient concentration, sialic acid content

[0367] ·Product purity / product-derived impurities: By SE-HPLC (size exclusion - high performance liquid chromatography), which separates molecules by size, and RP-HPLC (reverse phase - high performance liquid chromatography), which separates molecules by charge.

[0368] ·Functional properties: In vivo bioassay.

[0369] ·Host and process-derived impurities: Host cell DNA (HCD), host cell protein (HCP), residual protein A.

[0370] ·Safety: Sterility, endotoxin-free

[0371] ·Structural analysis:

[0372] o Primary structure: amino acid composition, peptide mapping, amino acid sequencing, N- and C-terminal integrity, post-transcriptional modifications (deamidation and oxidation).

[0373] o Higher order structure: disulfide bonds, free thiols and circular dichroism.

[0374] oGlycosylation: monosaccharide composition, sialic acid content, N-glycan profile, and O-glycan profile.

[0375] o Molecular weight: reduced / deglycosylated protein, non-reduced protein

[0376] Due to the unique biosynthetic production process and molecular characteristics of biological products, biologic drug substances may contain multiple molecular variants. The extent of molecular variation is controlled by a robust, product-specific manufacturing process. While most of the quality attributes mentioned above (e.g., process-derived impurities, purity, and general characteristics) are controlled by downstream purification processes, structural and functional properties largely depend on the production cell line and upstream processes. Three characteristic quality attributes of epepoetin alfa that demonstrate manufacturing consistency are sialic acid content, % CTL cleavage, and a unique glycosylation profile.

[0377] The N- and C-terminal integrity was assessed by RP HPLC with UV detection and mass spectrometry identification following endoprotease digestion. Quantification of N- and C-terminal variants was based on extracted ion chromatograms. The N-termini of epepoetin alfa produced by GX-E2 and GX-E4 cell lines were intact, whereas the C-terminal peptides contained two species: intact and CTL-free peptides. The ratios of mutated and unmutated peptides are shown in Table 15. Most epepoetin alfa proteins had a truncated C-terminus. The frequency of C-terminal truncation was largely similar among the four batches of epepoetin alfa.

[0378]

[0379] [Table 15]

[0380] C-terminal Lys cleavage frequency

[0381]

[0382]

[0383] To determine the glycosylation sites, epepoetin alfa was first digested with Glu-C or trypsin, and the resulting peptides were separated using RP-UPLC and analyzed using Q-TOF MS. The extracted ion chromatograms of the oxonium ions present in the N-acetylhexosamine of the glycopeptides were compared with the corresponding total ion chromatograms (TICs) to confirm the identity of the glycopeptides and identify the glycosylation sites. Epepoetin alfa has four N-glycosylation sites (N24, N38, N83, and N261) and one O-glycosylation site (S126). The N-linked glycosylation sites match the corresponding consensus sequence, but there is no consensus sequence for the O-linked glycosylation sites.

[0384] To determine the N- and O-linked glycan profiles, the nonlinear mass of each glycopeptide was measured and compared with the theoretical masses calculated from the amino acid sequence and various forms of glycans. The relative abundance of various forms of N-glycans was calculated as signal intensities, and the results of the analysis are summarized in Figures 28–32. The results indicate that epepoetin alfa produced in GX-E2 and GX-E4 cell lines, including the GX-E4 cell line produced by the modified upstream process (i.e., batch EF19001-T), are structurally similar. Most of the N-glycosylation sites were glycosylated, whereas approximately 60% of the O-glycosylation sites were glycan-free.

[0385] Overall, the results of the structural and quality evaluation of epepoetin alfa, including sialic acid content (Table 1), % CTL cleavage (Table 15) and glycosylation profile (Figures 28-32), showed that the sialic acid content and glycosylation profile can be determined by IEF gel electrophoresis, indicating that the manufacturing process described herein can produce epepoetin alfa drug substance with robust and consistent quality characteristics.

[0386]

[0387] 서열번호 1. α-2,3-sialyltransferase (nucleotide sequence)

[0388] ATGGTCAGCA AGTCCCGCTG GAAGCTCCTG GCCATGTTGG CTCTGGTCCT 50

[0389] GGTCGTCATG GTGTGGTATT CCATCTCCCG GGAAGACAGT TTTTATTTTC 100

[0390] CCATCCCAGA GAAGAAGGAG CCGTGCCTCC AGGGTGAGGC AGAGAGCAAG 150

[0391] GCCTCTAAGC TCTTTGGCAA CTACTCCCGG GATCAGCCCA TCTTCCTGCG 200

[0392] GCTTGAGGAT TATTTCTGGG TCAAGACGCC ATCTGCTTAC GAGCTGCCCT 250

[0393] ATGGGACCAA GGGGAGTGAG GATCTGCTCC TCCGGGTGCT AGCCATCACC 300

[0394] AGCTCCTCCA TCCCCAAGAA CATCCAGAGC CTCAGGTGCC GCCGCTGTGT 350

[0395] GGTCGTGGGG AACGGGCACC GGCTGCGGAA CAGCTCACTG GGAGATGCCA 400

[0396] TCAACAAGTA CGATGTGGTC ATCAGGTTGA ACAATGCCCC AGTGGCTGGC 450

[0397] TATGAGGGTG ACGTGGGCTC CAAGACCACC ATGCGTCTCT TCTACCCTGA 500

[0398] ATCTGCCCAC TTCGACCCCA AAGTAGAAAA CAACCCAGAC ACACTCCTCG 550

[0399] TCCTGGTAGC TTTCAAGGCA ATGGACTTCC ACTGGATTGA GACCATCCTG 600

[0400] AGTGATAAGA AGCGGGTGCG AAAGGGTTTC TGGAGACAGC CTCCCCTCAT 650

[0401] CTGGGATGTC AATCCTAAAC AGATTCGGAT TCTCAACCCC TTCTTCATGG 700

[0402] AGATTGCAGC TGACAAACTG CTGAGCCTGC CAATGCAACA GCCACGGAAG 750

[0403] ATTAAGCAGA AGCCCACCAC GGGCCTGTTG GCCATCACGC TGGCCCTCCA 800

[0404] CCTCTGTGAC TTGGTGCCACA TTGCCGGCTT TGGCTACCCA GACGCCTACA 850

[0405] ACAAGAAGCA GACCATTCAC TACTATGAGC AGATCACGCT CAAGTCCATG 900

[0406] GCGGGGTCAG GCCATAATGT CTCCCAAGAG GCCCTGGCCA TTAAGCGGAT 950

[0407] GCTGGAGATG GGAGCTATCA AGAACCTCAC GTCCTTC 987

[0408]

[0409] 서열번호 2. EPO-Fc (Efepoein alfa) (amino acid sequence)

[0410] MGVHECPAWL WLLLSLLSLP LGLPVLGAPP RLICDSRVLE RYLLEAKEAE 50

[0411] NITTGCAEHC SLNENITVPD TKVNFYAWKR MEVGQQAVEV WQGLALLSEA 100

[0412] VLRGQALLVN SSQPWEPLQL HVDKAVSGLR SLTTLLRALG AQKEAISPPD 150

[0413] AASAAPLRTI TADTFRKLFR VYSNFLRGKL KLYTGEACRT GDRRNTGRGG 200

[0414] EEKKKKEKE EQEERETKTP ECPSHTQPLG VFLFPKPKD TLMISRTPEV 250

[0415] TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRVVSVLTVL 300

[0416] HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT 350

[0417] KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR 400

[0418] LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK

[0419]

[0420] søkjølødhem 3. α-2,3-sialyltransferase - HA (amino acid sequence)

[0421] MVSKSRWKLL AMLALVLVVM VWYSISREDS FYFPIPEKKE PCLQGEAESK 50

[0422] ASKLFGNYSR DQPIFLRLED YFWVKTPSAY ELPYGTKGSE DLLLRVLAIT 100

[0423] SSSIPKNIQS LRCRCVVVG NGHRLRNSSL GDAINKYDVV IRLNNAPVAG 150

[0424] YEGDVGSKTT MRLFYPESAH FDPKVENNPD TLLVLVAFKA MDFHWIETIL 200

[0425] SDKKRVRKGF WRQPPLIWDV NPKQIRILNP FFMEIAADKL LSLPMQQPRK 250

[0426] IKQKPTTGLL AITLALHLCD LVHIAGFGYP DAYNKKQTIH YYEQITLKSM 300

[0427] AGSGHNVSQE ALAIKRMLEM GAIKNLTSFY PYDVPDYAGY PL

[0428]

[0429] Deposit information

[0430] Depositary: Korea Cell Line Research Foundation (KCLRF)

[0431] [Correction pursuant to Article 91 of the Rules 26.06.2025] Address: 101 Daehak-ro, Jongno-gu, Seoul 03080

[0432] Deposit Date: July 11, 2023

[0433] Accession number: KCLRF-BP-00525

[0434] [Correction pursuant to Rule 91 dated 26.06.2025]

Claims

1. EPO-Fc fusion protein producing cell line transformed with the α-2,3-sialyltransferase gene.

2. An EPO-Fc fusion protein producing cell line according to claim 1, wherein the α-2,3-sialyltransferase gene comprises the base sequence of sequence number 1.

3. An EPO-Fc fusion protein producing cell line according to claim 1, wherein the EPO-Fc fusion protein binds and activates the endogenous EPO receptor at a level similar to or better than EPO BRP.

4. An EPO-Fc fusion protein producing cell line according to claim 1, wherein the EPO-Fc fusion protein induces proliferation of erythroid progenitor cells and differentiation into reticulocytes at a level similar to or better than that of EPO BRP.

5. An EPO-Fc fusion protein producing cell line according to claim 1, wherein the EPO-Fc fusion protein induces proliferation of an EPO-dependent cell line, such as UT-7, at a level similar to or better than EPO BRP.

6. An EPO-Fc fusion protein producing cell line according to claim 1, wherein the Fc lacks immune effector activity, including but not limited to ADCC (antibody-dependent cytotoxicity), ADCP (antibody-dependent cellular phagocytosis), and CDC (complement-dependent cytotoxicity) activities that are detrimental to cells expressing the EPO receptor.

7. An EPO-Fc fusion protein producing cell line according to claim 1, wherein the Fc binds to FcRn only in a pH range representative of the endosomal compartment of mammalian cells and dissociates from FcRn at neutral pH, thereby avoiding cellular degradation and thereby extending the half-life of the Fc fusion protein.

8. An EPO-Fc fusion protein producing cell line according to claim 1, wherein the Fc increases the molecular weight to avoid renal clearance and thereby extends the half-life of the Fc fusion protein.

9. An EPO-Fc fusion protein producing cell line according to claim 1, wherein the EPO-Fc fusion protein comprises an EPO sequence and a hybrid Fc sequence corresponding to amino acid residues 1-193 and amino acid residues 194-438, respectively, in the amino acid sequence of SEQ ID NO:

2.

10. An EPO-Fc fusion protein producing cell line according to claim 1, wherein the Fc comprises 30 amino acid residues of the hinge region of human IgD and 8 amino acid residues of the N-terminus of the CH2 domain (amino acid residues 194-231 of SEQ ID NO: 2), and 100 amino acid residues of the C-terminus of the CH2 domain of human IgG4 and 107 amino acid residues of the CH3 domain (amino acid residues 232-438 of SEQ ID NO: 2).

11. A method for producing an EPO-Fc fusion protein, comprising the following steps: (a) a step of culturing a cell line producing an EPO-Fc fusion protein according to any one of claims 1 to 10 in a fed-batch manner; and (b) A step of isolating and purifying the EPO-Fc fusion protein produced in the above cell line.

12. A method according to claim 11, wherein EPO in the EPO-Fc fusion protein further comprises a modification for additional glycosylation.

13. A method according to claim 11, wherein the EPO-Fc fusion protein further comprises PEG.

14. A method according to claim 11, wherein the fed-batch culture is performed in the presence of N-acetyl-D-mannosamine (ManNAc) and galactose.

15. A method according to claim 14, wherein the N-acetyl-D-mannosamine (ManNAc) is present at a concentration of 1 mM to 20 mM, and the galactose is present at a concentration of 1 mM to 40 mM.

16. A method according to claim 11, wherein the fed culture is performed at a temperature of 34°C to 38°C and a pH of 6.7 to 7.

5.

17. A method according to claim 11, wherein the fed-batch culture is performed by culturing the cell line for 9 to 11 days.

18. A method according to claim 16, wherein the fed-batch culture is performed by lowering the temperature by 2 to 5 ℃ 3 or 4 days after cell line inoculation.

19. A method according to claim 11, wherein the separation and purification of the EPO-Fc fusion protein are performed at room temperature.

20. A method according to claim 19, wherein the separation and purification of the EPO-Fc fusion protein, excluding virus inactivation, is performed at room temperature.

21. A method according to claim 11, wherein the separation and purification of the EPO-Fc fusion protein is performed at 17°C to 27°C.

22. The method of claim 11, wherein the EPO-Fc fusion protein has specific quality characteristics including one or more of the following: C-terminal lysine content, N- and O-glycan profile, sialic acid content, and pI range.

23. A method according to claim 22, wherein the C-terminal lysine content of the EPO Fc fusion protein is about 1% to about 10%.

24. A method according to claim 22, wherein the C-terminal lysine content of the EPO Fc fusion protein is about 2% to about 9%.

25. A method according to claim 22, wherein the C-terminal lysine content of the EPO Fc fusion protein is about 3% to about 7%.

26. A method according to claim 22, wherein the sialic acid content of the EPO-Fc fusion protein is from about 17 mol / mol to about 27 mol / mol.

27. A method according to claim 22, wherein the sialic acid content of the EPO-Fc fusion protein is about 20 mol / mol to about 25 mol / mol.

28. A method according to claim 22, wherein the pI range of the EPO-Fc fusion protein is from about 4.0 to about 6.

0.

29. A method according to claim 22, wherein the pI range of the EPO-Fc fusion protein is about 4.5 to about 5.

3.

30. A method according to claim 22, wherein the N- and O-glycan profiles comprise a non-glycosylation content of about 0-20% at position N24, about 0-5% at position N38, about 10-17% at position N83, about 0-10% at position N261, and about 50-70% at position S126.

31. A method according to claim 22, wherein the N- and O-glycan profile comprises glycans containing sialic acid at about 50-90% at position N24, about 55-95% at position N38, about 40-80% at position N83, about 0-30% at position N261, and about 15-40% at position S126.

32. A method according to claim 11, wherein the separation and purification of the EPO-Fc fusion protein is performed using affinity chromatography or ion exchange chromatography.

33. A method according to claim 32, wherein the ion exchange chromatography comprises an anion exchange chromatography process that facilitates binding of acidic species of the EPO-Fc fusion protein to the resin using an equilibration buffer having a pH of about 6.00 to about 6.

50.

34. A method according to claim 32, wherein the ion exchange chromatography comprises an anion exchange chromatography process that facilitates binding of acidic species of the EPO-Fc fusion protein to the resin using an equilibration buffer having a pH of about 6.10 to about 6.

30.

35. A method according to claim 32, wherein the ion exchange chromatography comprises an anion exchange chromatography process using an equilibration buffer having a conductivity of about 6.00 to about 10.50 mS / cm and an elution buffer having a conductivity of about 11.50 to about 16.00 mS / cm to facilitate separation of the EPO-Fc fusion protein having the quality characteristics.

36. A method according to claim 32, wherein the ion exchange chromatography comprises an anion exchange chromatography process using an equilibration buffer having a conductivity of about 8.00 to about 10.00 mS / cm and an elution buffer having a conductivity of about 12.00 to about 15.00 mS / cm to facilitate separation of the EPO-Fc fusion protein having the quality characteristics.

Images