AU2016334954A1 - Composition comprising long-acting erythropoietin - Google Patents

Abstract

The present invention provides a composition containing long-acting erythropoietin, and a method for preparation thereof. More particularly, there is provided an EPO-Fc fusion protein composition with excellent bio-sustainability and high purity, wherein sialic acid content of EPO-Fc is 17 mol/mol or more, and host cell-derived protein (HCP) impurity is included in an amount of 100 ng/mg or less.

Description

The present invention provides a composition containing long-acting erythropoietin, and a method for preparation thereof. More particularly, there is provided an EPO-Fc fusion protein composition with excellent bio-sustainability and high purity, wherein sialic acid content of EPO-Fc is 17 mol/mol or more, and host cell-derived protein (HCP) impurity is included in an amount of 100 ng/mg or less.

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TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG).

Published:

— with international search report (Art. 21(3))

WO 2017/061780 PCT/KR2016/011169

Description

Title of Invention: COMPOSITION COMPRISING LONGACTING ERYTHROPOIETIN

Technical Field [1] The present invention relates to a pharmaceutical composition including long-acting erythropoietin and a method for preparation of the same.

Background Art [2] Erythropoietin (EPO) is a glycoprotein to promote differentiation of erythroblast in bone marrow and generate erythrocytes, which includes 165 amino acids. The EPO is mostly generated in kidneys and known to be also generated a little in a liver. As shown in chronic renal failure (CRF), for example, a loss in function of the kidney typically causes a decrease in EPO generation and a decrease of erythrocytes involved thereby. It has been found that EPO has excellent effects in treatment of anemia caused by CRF and other anemia due to diverse causes, and different hematopoietic functions such as surgical operation assistance.

[3] In general, polypeptide such as EPO has low stability and is easily degenerated and decomposed by a protein hydrolysis enzyme, that is, protease in the blood, thus being easily removed through the kidneys or liver. Therefore, the polypeptide has a short biological half-life period ('half-life'). Accordingly, in order to maintain a blood concentration and titer value of a protein medicine containing polypeptide as a pharmacological component, the protein medicine needs to be frequently administered to a patient. However, in a case of such a protein medicine to be administered in a type of injection formulation, often injecting to keep the blood concentration of active polypeptide may usually cause pain to the patient.

[4] In order to increase the half-life of EPO polypeptide, fusion protein prepared using immunoglobulin (Ig) has been currently developed. Ig is an important constitutional component of the blood. Human Ig (hlg) may include diverse classes such as IgG,

IgM, IgA, IgD and IgE.

[5] The immunoglobulin contains four polypeptide chains, and more particularly, two heavy chains and two light chains, wherein these chains are joined together through disulfide bonds in order to form a tetramer. Each chain consists of a variable region and a constant region. The constant region in the heavy chain ('heavy chain constant region') may be further divided into three or four sites such as CHI, CH2, CH3 and CH4 according to isotypes thereof. Fc region of the heavy chain constant region may include a hinge, CH2, CH3 and/or CH4 domains according to Ig isotypes.

[6] With regard to the half-life, chimeric proteins fused in the Fc region of the imWO 2017/061780 PCT/KR2016/011169 munoglobulin is known to show increased stability and a prolonged serum half-life.

[7] Further, it is known that adhering sialic acid to the surface of EPO protein may induce a prevention of protein degradation in the liver. Sialic acid protects a second galactose group in an asialoglycoprotein receptor to thus influence upon activity of EPO in a living body ('bio-activity'). It is also known that the sialic acid present in a glyco-chain of erythropoietin has a significant role in an extension of the half-life of erythropoietin, ultimately, influencing upon efficacy thereof. Further, since bio-activity of erythropoietin has been found to be inactivated by a reaction of sialidase, studies on a function of sialic acid to the bio-activity of erythropoietin have more actively conducted. In most cases, sugar used in a production of therapeutic agents has an essential and indispensable role in expressing inherent efficacy of glycoprotein. Especially, for human-derived erythropoietin, the role of sugar is absolutely required. In other words, if the sugar is not included, erythropoietin never shows activity and, according to a constitutional composition of sugar or a content of silaic acid at a terminal, bio-activity of EPO is significantly influenced.

[8] During the expression of host cells, the content of sialic acid in EPO-Fc may be increased by adding N-acetylmannosamine to a culture solution. The EPO-Fc obtained after the above process is generally a mixture of high sialic acid content EPO-Fc and low sialic acid content EPO-Fc. Herein, it is known that the high sialic acid content EPO-Fc usually has a longer half-life than the low sialic acid content EPO-Fc. Therefore, there is a need to develop a technique of selectively producing the EPO-Fc with an increased content of sialic acid.

[9] Production of the EPO-Fc needs culturing transformed host cells, expressing the EPO-Fc in great quantities, disintegrating the cells and purifying the EPO-Fc. Therefore, for mass-production of high quality EPO-Fc, it is very important to optimize culturing and purification techniques. There is still much room for improvement in such culturing and purification techniques currently developed, therefore, an urgent requirement is now to optimize an EPO-Fc manufacturing process through the development of the above techniques. Korean Patent Publication No. 897938 (registered on May 8, 2009) discloses immunoglobulin fusion proteins.

[ 10] [Prior Art Document] [11] Korean Patent Registration No. 0897938 (2009. 5. 8)

Disclosure of Invention Technical Problem [12] Accordingly, it is an object of the present invention to provide an EPO-Fc fusion protein composition with excellent sustainability in a living body ('bio-sustainability').

[13] In addition, another object of the present invention is to provide an EPO-Fc fusion

WO 2017/061780 PCT/KR2016/011169 protein composition with a high sialic acid content.

[14] Further, another object of the present invention is to provide an EPO-Fc fusion protein composition with a high purity.

[15] Furthermore, another object of the present invention is to provide an EPO-Fc fusion protein composition with reduced content of host cell-derived impurities.

Solution to Problem [16] The above object of the present invention will be achieved by the following characteristics:

[17] (1) An EPO-Fc composition, including EPO-Fc having a sialic acid content of 17 mol/mol or more, and host cell-derived protein impurity in an amount of 100 ng/mg or less.

[18] (2) The EPO-Fc composition according to the above (1), wherein the sialic acid content ranges from 20 to 28 mol/mol.

[19] (3) The EPO-Fc composition according to the above (1), wherein the EPO-Fc has pi < 6.0.

[20] (4) The EPO-Fc composition according to the above (1), wherein the EPO-Fc has 4.5 < pi < 5.3.

[21] (5) The EPO-Fc composition according to the above (1), wherein the host cellderived protein impurity is an aggregate or fragment of EPO-Fc.

[22] (6) The EPO-Fc composition according to the above (1), wherein the host cellderived protein impurity is contained in an amount of 60 ng/mg or less.

[23] (7) The EPO-Fc composition according to the above (1), further including host cellderived DNA impurity of 0.5 ng/mg or less.

[24] (8) A method for preparing an EPO-Fc composition, including: flowing an EPO-Fc cell culture solution at a flow rate of 4 vvd or less under conditions of a temperature ranging from 35°C to 39°C and 6.5 < pH < 7.5 to prepare a perfusion culture solution; obtaining an EPO-Fc pure solution from the perfusion culture solution; and adsorbing the EPO-Fc pure solution to an anion exchange resin to prepare a Q effluent that includes EPO-Fc having a sialic acid content of 17 mol/mol or more.

[25] (9) The method according to the above (8), wherein the preparation of Q effluent includes: adsorbing the EPO-Fc to the anion exchange resin; and eluting the adsorbed EPO-Fc using a buffer which contains 0.005 to 0.02 M sodium phosphate, 0.05 to 0.2 M L-arginine and 0.02 to 0.1 M sodium chloride, and has 6. 7< pH <7.1.

[26] (10) The method according to the above (8), wherein the step of obtaining of EPO-Fc pure solution includes passing the perfusion culture solution through a POD filter.

[27] (11) The method according to the above (8), wherein an amount of host cell-derived protein (HCP) impurity in the EPO-Fc composition is 100 ng/mg or less.

WO 2017/061780

PCT/KR2016/011169 [28] (12) The method according to the above (8), wherein the EPO-Fc cell culture solution is inoculated in an amount of 2.0xl0⁵ cells/mL or more onto an incubator.

[29] (13) The method according to the above (10), further including, after passing the perfusion culture solution through the POD filter, passing the above solution through a column filled with a protein A-bonded resin.

[30] (14) The method according to the above (8), wherein the EPO-Fc contained in the Q effluent has pi of 6.0 or less.

[31] (15) The method according to the above (8), further including filtering the Q effluent using a POD filter, ultrafiltration membrane and nano-filter, respectively.

Advantageous Effects of Invention [32] The EPO-Fc composition according to the present invention is characterized in that frequent parts of the terminal of sugar chain in the EPO-Fc are capped with sialic acid, thereby increasing a half-life and enhancing convenience of a patient.

[33] The EPO-Fc composition according to the present invention has reduced contents of host cell-derived protein and DNA impurities, thereby having a high EPO-Fc purity.

[34] The method for preparing an EPO-Fc composition according to the present invention may be suitable for selective purification of EPO-Fc having such characteristics as described above.

[35] The method for preparing an EPO-Fc composition according to the present invention may noticeably improve sialic acid adhesion to the EPO-Fc and enhance survival of host cells and process efficiency as well.

Brief Description of Drawings [36] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawing, in which:

[37] FIG. 1 is a diagram illustrating test results of an EPO-Fc isoelectric focusing examination.

Best Mode for Carrying out the Invention [38] The present inventors have completed the present invention on the basis of cell culture technique of host cells for improvement of productivity and sialic acid content, high sialic acid content EPO-Fc selection technique and purification technique for removal of host cell-derived impurities.

[39] The present invention discloses an EPO-Fc composition which contains EPO-Fc having 17 mol/mol or more of sialic acid, and 100 ng/mg or less of host cell-derived protein (host cell protein; HCP) impurity, as well as a method for preparation thereof.

[40] Hereinafter, the present invention will be described in detail.

[41] EPO-Fc fusion protein is formed by fusing a Fc region of human immunoglobulin

WO 2017/061780 PCT/KR2016/011169 heavy chain constant region into erythropoietin (EPO), and has a longer half-life than the EPO. Such an increased half-life refers to an increase in bio-sustainability of efficacy.

[42] Sialic acid present in a sugar chain of the EPO protects a second galatose group in an asialoglycoprotein receptor to thus greatly influence upon bio-activity of the EPO. Further, the sialic acid may induce a prevention of EPO degradation in the liver to thus increase the half-life.

[43] Accordingly, adhesion of sialic acid contained in the EPO-Fc fusion protein may provide effects of increasing the half-life in addition to half-life increase effect due to the Fc fusion.

[44] During the culture of EPO-Fc host cells, N-acetylmannosamine (NAM) may be added to a culture solution to increase the sialic acid content of the EPO-Fc. The EPOFc resulted after the above processes generally includes a mixture of high sialic acid content EPO-Fc and low sialic acid content EPO-Fc, wherein the high sialic acid content EPO-Fc is known to have a longer half-life than the low sialic acid content EPO-Fc.

[45] According to one embodiment of the present invention, with the number of sialic acid adhered to the EPO-Fc is increased, a pi value is decreased. Based on this aspect, using an anion exchange resin may selectively purify the high sialic acid content EPOFc. Preferably, the anion exchange resin containing cross-linked agarose is used. According to one embodiment, a Q-Sepharose resin manufactured by GE Healthcare Co. may be used.

[46] By a purification process using the anion exchange resin, EPO-Fc having pi < 6.0 may be selectively purified. In particular, EPO-Fc having a sialic acid content of 17 mol/mol or more may be obtained through this process. According to one embodiment, in a case of 4.5 < pi < 6.0, an EPO-Fc containing composition with a sialic acid content of 17 to 28 mol/mol may be obtained. According to another embodiment, in a case of 4.5 < pi < 5.3, an EPO-Fc containing composition with a sialic acid content of 20 to 28 mol/mol may be obtained.

[47] During the EPO-Fc production, host cell-derived protein (HCP) impurities may be mixed. The HCP impurity may include, for example, protein impurities containing abnormal peptides such as different aggregates and fragments possibly derived from host cells and other materials, DNA impurities, intrinsic or extrinsic viruses and other particles, or the like.

[48] Eliminating such the impurities may be a significant process directly influencing upon the quality of EPO-Fc fusion protein. When this composition is prepared through a series of processes according to the present invention, the HCP may be controlled to an amount of 100 ng/mg or less, and more preferably, 60 ng/mg or less. The host cellWO 2017/061780 PCT/KR2016/011169 derived DNA impurity may be possibly contained in an amount of 0.5 ng/mg or less.

[49] The method for preparing an EPO-Fc composition of the present invention may be completed as follows:

[50] The method for preparing an EPO-Fc composition of the present invention may include: (1) flowing an EPO-Fc cell culture solution at a flow rate of 4 vvd or less under conditions of a temperature ranging from 35°C to 39°C and 6.5 < PH < 7.5 to prepare a perfusion culture solution; (2) obtaining an EPO-Fc pure solution from the perfusion culture solution; and (3) adsorbing the EPO-Fc pure solution to an anion exchange resin to prepare an EPO-Fc containing Q effluent with a sialic acid content of 17 mol/mol or more.

[51] The perfusion culture solution in step (1) may be prepared as follows.

[52] First, EPO-Fc master cell bank (MCB) may be built from the cell bank for EPO-Fc studying (RCB), and EPO-Fc working cell-line bank (WCB) may be built from the EPO-Fc master cell bank (MCB).

[53] The culturing for building MCB and WCB may include sub-culturing cells in an EXcell CHO DHFR(-) liquid medium including L-glutamine and methotrexate under conditions of 3 to 7% CO2 and 37°C, then, dispensing the product in a cryo-vial for freezing and storage.

[54] After thawing the frozen and stored WCB cell-line, the thawed cell-line is provided and suspended in the medium to proliferate cells therein. For example, the subculture could be conducted using a shake flask in a CO2 incubator set up with 3 to 7% CO2 at 37°C in a ratio of 1:2 to 1:6 by an interval of 50 to 90 hours. Using several shake flasks in the culture may enable cultured cells to become a sufficient amount enough to be inoculated onto the cell incubator.

[55] In this case, an EPO-Fc culture medium may be used. The EPO-Fc culture medium may include, for example, EX-cell CHO DHFR(-) powder medium, L-glutamine, methotrexate and sodium bicarbonate.

[56] When a sufficient amount of host cells for inoculation is ensured through shake flask culture, the cells may be inoculated onto the main incubator to conduct perfusion culture. The main incubator used herein may be a 30L biological incubator.

[57] After inoculation onto the main incubator, a cell concentration of 2.0xl0⁵ cells/mL or more may be obtained.

[58] A culturing temperature of the main incubator may range from 35 to 39°C, or from 36 to 38°C, or the like.

[59] The acidity of the main incubator may be kept in a range of 6.5 < pH < 7.5 (after pH control), 6.8 < pH < 7.2 (after pH control), or the like.

[60] During perfusion culture, if necessary, the culture solution may be sampled and observed by a microscopy to monitor the condition of cells, and process examination

WO 2017/061780

PCT/KR2016/011169 may be performed by analyzing pH, the number of cells, cell activity, glucose concentration, glutamine concentration, ammonia concentration, osmotic pressure, or the like.

[61] A perfusion rate may be controlled to 4 vvvd or less. The perfusion rate may range from 0 to 4 vvd, 0 to 2 vvd, 1 to 3 vvd, 1 to 2 vvd, or the like.

[62] A perfusion culture medium used herein may include a medium prepared by adding N-acetylmannosamine to the EPO-Fc medium (that is, N-acetylmannosamine-added culture medium), a medium prepared by adding N-acetylmannosamine and glucose to the EPO-Fc culture medium (a production medium), or both of these media may be used in order.

[63] For example, according to the perfusion culture by 40L per day for 4 days, a total 140 to 180 kg of cell culture solution for EPO-Fc production may be obtained. Repeating the above collection of cell culture solution once to 10 times, or twice to five times, may provide more increased amount of cell culture solution for EPO-Fc production.

[64] The cell culture solution obtained as described above may become the perfusion culture solution in step (1) of the present invention.

[65] According to steps of preparing the perfusion culture solution of the present invention, 17 mol/mol or more, 17 to 28 mol/mol or 20 to 28 mol/mol of sialic acid may be adhered to the EPO-Fc. Further, survival of cells may be improved to thus enhance the productivity of EPO-Fc composition.

[66] The method for preparing EPO-Fc composition of the present invention may further include (2) gaining an EPO-Fc pure solution from the perfusion culture solution.

[67] The above step has a major purpose of removing other components except the EPOFc contained in the perfusion culture solution in step (1). This removal may include any conventional method used in the culture of cells. The removal method may be combined with at least one among filtration and purification processes as described below.

[68] After recovering the perfusion culture solution in step (1), this solution may be subjected to multiples steps for filtration and purification to thus eliminate the host cell-derived impurities. Such the host cell-derived impurities may include, for example, protein impurities containing abnormal peptides such as different aggregates and fragments, DNA impurities, intrinsic virus, extrinsic virus and other particles.

[69] The host cell refers to all cells used for EPO-Fc expression in step (1).

[70] According to one embodiment, in order to remove the host cell-derived protein (HCP) or DNA impurities contained in a host cell culture solution for EPO-Fc production, a POD depth filter may be used. The POD depth filter is usually used for removing cells, however, being used for removal of protein in the present invention.

WO 2017/061780 PCT/KR2016/011169 [71] Using the POD filter and the filtration filter as well, the host cell-derived protein (HCP) impurities such as protein aggregates, fragments and other particles may be removed.

[72] For example, as the POD filter, Millistak + (MA1HC10FS1) manufactured by Merck Milipore Co. may be used. Further, the filtration filter used herein may be Sartobran P sterile grade capsule manufactured by Sartorius Co.

[73] According to another embodiment, protein A purification, low pH virus inactivation and/or hydroxyapatite purification, etc., may be adopted for EPO-Fc purification.

[74] According to another embodiment, an ultrafiltration device, that is, a tangential flow filtration (TFF) membrane system may be used for concentration and buffer change. If the conductivity and acidity come within a reference range thereof through the buffer change, the concentration and buffer change processes are completed (Reference: conductivity 9.0+1.0 mS/cm, acidity pH 6.9+0.1).

[75] The EPO-Fc pure solution in step (2) may be prepared as described above.

[76] The method for preparing an EPO-Fc composition of the present invention may include (3) adsorbing the EPO-Fc pure solution to the anion exchange resin in order to prepare a Q effluent including EPO-Fc having a sialic acid content of 17 mol/mol or more.

[77] The above step has a major purpose of selecting specific one having a sialic acid content of 17 mol/mol or more of the EPO-Fc contained in the composition.

[78] This step may be conducted by passing the EPO-Fc pure solution in step (2) through a column filled with an anion exchange resin containing cross-linked agarose.

[79] According to one embodiment, the Q Sepharose resin of GE Healthcare Co. may be used, for example, a Q Sepharose Fast Flow resin shown in Table 1 below may be used.

[80] [Table 1]

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PCT/KR2016/011169

Q Characteristics of Sepharose Fast Flow
Matrix	6% highly cross-linked agarose
Average particle size	9 0 pm
Type of medium	Strong anion
Charged group	-N+(CH3)3
Total ionic capacity	0.18 to 0.25 mmol Cl“/mL medium
Dynamic binding capacity1	120 mg HSA/mL medium
Flow velocity2	400 to 700 cm/h
pH stability (CIP3)	1 to 14
pH stability (Working4)	2 to 12
Storage temperature	4°C to 30°C
Storage buffer	20% ethanol
Chemical stability	All commonly used buffers, 1 M NaOH, 8 M urea, 6 M guanidine hydrochloride, and 70% ethanol
Avoid	Oxidizing agents, anionic detergents, and buffers
1 Determination of dynamic binding capacity: DEAE Sepharose Fast Flow, Q Sepharose Fast Flow, SP Sepharose Fast Flow, and CM Sepharose Fast Flow: Samples were applied at 75 cm/h until 50% breakthrough. Columns: 0.5 * 5 cm. Buffers: 0.05 M Tris, 2 M NaCl (in the elution buffer), pH 7.5 (Q and DEAE) or 0.1 M acetate, 2 M NaCl (in the elution buffer), pH 5.0 (SP and CM). ANX Sepharose 4 Fast Flow (high sub): Sample was applied at 300 cm/h until 10% breakthrough. Column: 1.6 * 13 cm. Buffer: 0.05 M Tris, 1 M NaCl (in the elution buffer), pH 7.5. 2 1 bar (14.5 psi, 0.1 MPa), XK 50/30 column, bed height 15 cm (SP, CM, Q, and DEAE) or 1 bar (14.5 psi, 0.1 MPa), XK 50/60 column, bed height 25 cm (ANX). 3 Refers to the pH interval for regeneration and cleaning. 4 Refers to the pH interval where the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance.

[82] A process of preparing a Q effluent may include: equilibrating a Q-Sepharose resin by using an equilibration buffer containing 0.005 to 0.02 M sodium phosphate; passing the EPO-Fc pure solution through the Q-Sepharose resin to adsorb EPO-Fc; reequilibrating the Q-Sepharose resin by using the above equilibration buffer; and passing an effluent which contains 0.005 to 0.02 M sodium phosphate, 0.05 to 0.2 M F-arginine and 0.02 to 0.1 M sodium chloride and has an acidity of 6. 7 < pH <7.1, through the column to collect the Q effluent of EPO-Fc.

[83] Elution may be suitably repeated. For example, when the elution is repeatedly conducted 4 times, Q effluents Nos. 1 to 3 obtained in first to third purifications are stored in an ultra-low temperature freezer and, after completing fourth elution, all of Q

WO 2017/061780 PCT/KR2016/011169 effluents Nos. 1 to 4 may be subjected to pooling.

[84] Optionally, after the elution, filtration may be implemented using the POD filter. The POD filter used herein may include, for example, Millistak + (MA1HC10FS1) manufactured by Merck Milipore Co.

[85] Other than the above processes, optionally, a process of mounting a filtration filter on the column and a process of sterilizing and washing the Q-Sepharose resin by contacting the resin to CIP may be further included. According to one embodiment, the filtration filter used herein may include, for example, Sartobran P sterile grade capsule manufactured by Sartorius Co.

[86] EPO-Fc contained in the Q effluent obtained in step (3) may have pi < 6.0 and sialic acid content of 17 mol/mol or more. For example, EPO-Fc may be 4.5 < pi < 6.0 and the sialic acid content may range from 17 to 28 mol/mol, or EPO-Fc may be 4.5 < pi < 5.3 and the sialic acid content may range from 20 to 28 mol/mol.

[87] The method for preparing an EPO-Fc composition of the present invention may further include (4) filtering Q effluent through the POD filter, ultrafiltration membrane and/or nano-filter, respectively.

[88] According to the above step, the EPO-Fc may have an increased concentration, the buffer may be changed, and virus may be eliminated.

[89] The POD filter used herein may be Millistak + (MA1HC 10FS1) manufactured by Merck Milipore Co.

[90] The ultrafiltration membrane used herein may include, for example, the tangential flow filtration (TFF) membrane system. For example, after washing a membrane (Cut off: 30K) using an injection buffer, the membrane may become equilibrated using a formulation buffer. The formulation buffer may contain 0.01 M sodium citrate, 0.1 M glycine and 0.1 M sodium chloride, and have acidity of pH 6.2+0.2.

[91] After completing the equilibration, the purposed protein could be concentrated into about 1.5+0.3 mg/mL. If the recovered solution has a protein concentration of about 1.5+0.3 mg/mL, buffer exchange could be conducted by continuously adding a buffer for preparing a crude solution with the same volume. If conductivity and acidity of a finally recovered solution come within the reference range thereof, the concentration and buffer exchange processes could be completed (Reference: conductivity 12.0+2.0 mS/cm, acidity pH 6.2+0.2).

[92] When the concentration and buffer exchange processes of the finally recovered solution are completed, filtration may be conducted using a nano-filter (PALL (NT6DV20P1G)) in order to eliminate viruses possibly derived from the host cells or additional materials used in the process.

[93] According to one embodiment, after washing a filter for virus filtration, an integrity test for the filter was conducted, and the equilibration may be accomplished by passing

WO 2017/061780

PCT/KR2016/011169 a buffer for crude production through the virus filtration filter. After completing the equilibration, a finally recovered solution after the completion of concentration and buffer change processes may be passed through the filter under a pressure of 30+3 psi. Therefore, a virus filtrate free from virus could be recovered. After completing the filtration, the virus filtration filter may be washed using an injection buffer, and then, the integrity test may be conducted.

[94] Adding polysorbate 20 in a concentration of 0.12 g/Kg to the virus filtrate and also a formulation buffer thereto may enable a protein concentration to be adjusted to 1.1+0.3 mg/mL, then, using a sterilization filtration filter may produce an EPO-Fc crude solution.

[95] According to one embodiment, the formulation buffer may include 0.01M sodium citrate, 0.1 M glycine and 0.1 M sodium chloride, and have acidity of pH 6.2+0.2. According to one embodiment, the sterilization filtration filter used herein may be Sartobran P300 manufactured by Sartorius Co. The prepared EPO-Fc crude solution may be dispensed and stored in an ultra-low temperature freezer.

[96] A protein concentration was adjusted to 0.5+0.1 mg/mF by adding a formulation buffer to the EOP-Fc crude solution, followed by filtering through a sterilization filtration filter, resulting in a final EPO-Fc crude solution. Such a sterilization filtration filter used herein may include, for example, Sartobran P midicap manufactured by Sartorius Co.

[97] Hereinafter, the present invention will be described in more details with reference to examples. However, these examples are proposed for illustrating preferred embodiments of the present invention only and the scope of the present invention is not be particularly limited to the same.

[98] Example 1: Preparation of EPO-Fc composition [99] <Process 1: Preparation of EPO-Fc master cell bank (MCB)>

[100] After adding F-glutamine and methotrexate to an EX-cell CHO DHFR(-) liquid medium to form a medium and inoculating a cell bank for EPO-Fc studying (RCB) onto the medium, a total cell number and a total culturing volume were increased through subculture to thus build MCB. The culture was conducted in 5.0 + 2.0% CO2 incubator until the culture solution reached a temperature of 37°C.

[101] <Process 2: Preparation of EPO-Fc working cell-line bank (WCB)>

[102] After adding F-glutamine and methotrexate to an EX-cell CHO DHFR(-) liquid medium to form a medium and inoculating the MCB prepared in process 1 onto the medium, a total cell number and a total culturing volume were increased through subculture to thus build WCB. The culture was conducted in 5.0 + 2.0% CO2 incubator until the culture solution reached a temperature of 37°C.

[103] <Process 3: WCB fusion>

WO 2017/061780

PCT/KR2016/011169 [104] After thawing WCB frozen and stored in process 2, the thawed WCB was suspended in an EPO-Fc culture medium (including EX-cell CHO DHFR(-) powder medium, Lglutamine, methotrexate and sodium bicarbonate), followed by culturing the same in a CO2 incubator set up with 5% CO2 at 37°C.

[105] <Process 4: Culture of shake flask cells>

[106] The culture solution of process 3 was subjected to subculture at an interval of 64 to 80 hours to ensure the desired number of cells enough to inoculate the same onto a cell incubator. The subculture was conducted in a ratio of 1:3 to 1:4 using the EPO-Fc culture medium in a CO2 incubator set up with 5% CO2 and at 37°C.

[107] <Process 5: Production and culture in a main incubator (30L bio-incubator)>

[108] After gaining a sufficient amount of the culture solution of process 4, cells were collected and inoculated onto the main incubator to reach 2.0xl0⁵ cells/mL. Then, the above solution was cultured using an EPO-Fc culture medium including Nacetylmannosamine further added thereto. While maintaining a culturing temperature of 37±1°C and pH 7.0+0.2 (after pH control) during the culture, perfusion culture was conducted. If necessary, process examination was conducted by sampling the culture solution. According to results of the process examination, a perfusion rate was controlled in a range of 0 to 2 vvd. For production of a culture solution, the used medium was changed by a new EPO-Fc production medium (prepared by adding Nacetylmannosamine and sodium bicarbonate to the EPO-Fc culture medium), followed by further conducting perfusion culture. 140 to 180 kg of culture solution was collected at about 40 L per day for 4 days to obtain one sub-lot. By repeating the above procedures total 4 times for 16 days, the culture was conducted until collecting a total four (4) sub-lots. As a result, a culture solution (perfusion culture solution) of host cells for EPO-Fc production was obtained.

[109] <Process 6: Recovery of cultured solution and filtration>

[110] The host cell culture solution for EPO-Fc production of process 5 was filtered using a POD filter (MA1HC10FS1) and a filtration filter (Sartobran P-sterile grade capsule), then, a filtrate of the perfusion culture solution was obtained and stored.

[111] <Process 7: Purification of protein A>

[112] After making a column filled with a protein A-bonded resin in equilibrium state using an equilibration buffer (0.01M sodium phosphate) and adsorbing the filtrate of process 6 to the column, the column was washed using a washing buffer (0.7M LArginine, pH 5.7+0.05, conductivity 37.0+1.0 mS/cm), followed by eluting the purposed protein (EPO-Fc) using an elution buffer (0.02 M sodium acetate anhydride, 0.2 M L-arginine, 7.94%(v/v) glycerol, pH 3.7+0.05), then storing the same.

[113] <Process 8: Low pH virus inactivation>

[114] If the pure solution of process 7 is not proper to a reference as a result of measuring

WO 2017/061780 PCT/KR2016/011169 pH thereof, a pH adjusting buffer (1M sodium hydroxide (NaOH), 10% acetic acid) was added thereto in order to adjust pH (3.7+0.05), followed by carrying out this process while stirring the solution for 2 hours. After completing the process, the pH was adjusted to reach about pH 6.9+0.1 using the pH adjusting buffer (1M sodium hydroxide (NaOH), 10% acetic acid).

[115] <Process 9: Purification of hydroxyapatite>

[116] After mounting a filtration filter (Sartobran P-sterile grade capsule) on a column filled with a fixing phase (hydroxyapatite) and equilibrating the same with an equilibration buffer (0.01M sodium phosphate), the pure solution of process 8 was adsorbed to the column, then, the filtration filter (Sartobran P-sterile grade capsule) was removed, followed by eluting the purposed protein using an elution buffer (0.1 M sodium phosphate and 0.1 M L-arginine, pH 6.9+0.1), then storing the same.

[117] <Process 10: Ultrafiltration concentration and buffer change 1>

[118] After concentrating the pure solution of process 9 using an ultrafiltration device (Membrane: Cut off 30K, including 0.5m2), buffer change was conducted by continuously adding the equilibration buffer (0.01M sodium phosphate). When the conductivity and pH came within a reference range thereof, the process was completed and a concentration and buffer change 1 retentate solution was collected to obtain an EPOFc pure solution.

[119] <Process 11: Purification of Q Sepharose>

[120] After mounting a filtration filter (Sartobran P-sterile grade capsule) on a column filled with a fixing phase (Q Sepharose) and equilibrating an Q Sepharose resin with an equilibration buffer including 0.01M sodium phosphate, the EPO-Fc pure solution of process 10 was adsorbed to the column, then, the filtration filter, that is, Sartobran Psterile grade capsule was removed and the Q Sepharose resin was re-equilibrated, followed by eluting the purposed protein using an elution buffer (0.01M sodium phosphate and 0.1 M L-arginine, 0.05M sodium chloride, pH 6.9+0.1) to obtain and store Q effluent of EPO-Fc.

[121] <Process 12: Thawing and filtration of effluent>

[122] The stored Q effluent of process 11 was thawed and subjected to pooling. The POD filter (MA1HC054H1) was equilibrated using a formulation buffer (0.01M sodium citrate, 0.1 M glycine, 0.1 M sodium chloride, pH 6.2+0.2), followed by passing the Q effluent through the filter to obtain a filtrate.

[123] <Process 13: Ultrafiltration concentration and buffer change 2>

[124] After concentrating the Q pooling filtrate of process 12 to reach a purposed protein concentration using an ultrafiltration device (Membrane: Cut off 30K, including 0.1 M2), buffer change was conducted by continuously adding a formulation buffer (0.01M sodium citrate, 0.1 M glycine, sodium phosphate, 0.1 M sodium chloride, pH

WO 2017/061780 PCT/KR2016/011169

6.2+0.2). When the conductivity and pH came within a reference range thereof, the process was completed and a concentration and buffer change 2 retentate solution was collected.

[125] <Process 14: Nano-filter filtration>

[126] After flowing a formulation buffer (0.01M sodium citrate, 0.1 M glycine, 0.1 M sodium chloride, pH 6.2+0.2) into a nano-filter (Pall (NT6DV20P1G)) to make equilibrium, the concentration and buffer change 2 retentate solution of process 13 was flowed into the same, to thus eliminate viruses possibly present in the concentration and buffer change 2 retentate solution.

[127] <Process 15: Preparation of EPO-Fc crude solution>

[128] 0.12 g/kg of polysorbate 20 was added to the virus filtrate obtained in process 14 and a protein concentration was adjusted using a formulation buffer. Then, sterilization and filtration were conducted using a sterilization and filtration filter (Sartobran P300) to prepare an EPO-Fc crude solution while matching to a crude protein concentration (reference: 1.1+0.3 mg/mL), followed by dispensing the same in a storage container and storing the container in an ultra-low temperature freezer.

[129] <Process 16: Preparation of EPO-Fc final crude solution>

[130] After thawing the EPO-Fc crude solution of process 15 in a water bath and adding a formulation buffer (0.01M sodium citrate, 0.1 M glycine, 0.1 M sodium chloride, pH 6.2+0.2, polysorbate 20 0.12 g/Kg) to the same to reach a protein concentration of 0.5+0.1 mg/mL, sterilization and filtration were conducted using a sterilization and filtration filter (Sartobran P midicap) to thus prepare a final EPO-Fc crude solution.

[131] Example 2: Purity examination of EPO-Fc composition [132] Examination 1: Test of host cell derived protein (peptide) ('HCP') impurity content >

[133] In order to examine the HCP impurity content in the final crude solution, the following test was conducted using a CHO host cell protein kit: 1) a standard solution provided in the kit was diluted and used; 2) a test solution was prepared in a diluted state using a dilution buffer; 3) a spiked test solution was prepared by adding the standard solution to the above test solution; 4) after reacting the standard solution, test solution and spiked test solution with Anti-CHO (phosphatase conjugate), this mixture was reacted in an anti-CHO coated microtiter plate; and 5) after washing the plate, a PNPP substrate was added thereto and absorbency was measured using an ELISA plate reader. The samples used herein were batch Nos. 747R0001 and 747R0002. Results of the examination are summarized in Table 2 below. From the test results, it could be seen that the content of host cell-derived protein impurity was 60 ng/mg.

[134] [Table 2]

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PCT/KR2016/011169

Content of host cell-derived protein (peptide) impurity
Lot. No.	: Content of protein (ng/mg)
747R0001	58.1
747R0002	57.8
Mean	57.95

[136] Examination 2: Test of host cell-derived DNA impurity content>

[137] In order to examine the content of host cell-derived DNA impurity in the DPO-Fc final crude solution, the following test was conducted: 1) a high calibrator provided in the kit was diluted and used as a standard solution; 2) a zero calibrator provided in the kit was diluted and used as a test solution; 3) a spiked test solution was prepared by adding the standard solution to a test sample; 4) a zero calibrator used herein was the zero calibrator provided in the kit; 5) a spiked zero calibrator used herein was prepared by adding the standard solution to the zero calibrator; 6) the prepared standard solution, test solution, spiked test solution, zero calibrator and spiked zero calibrator were subjected to DNA labeling; and 7) stick binging was conducted using a threshold system filter unit, then, stick was read to confirm the result. The samples used herein were batch Nos. GC1113-PUR-0901-PR, GC1113-PUR-0902-PR, 747R0001 and 747R0002. Results of the examination are summarized in Table 3 below. From the test results, it could be seen that the content of host cell-derived DNA impurity was 0.5 nm/mg or less with a maximum of 0.215 ng/mg and a mean of 0.143 ng/mg.

[138] [Table 3]

Content of host cell-derived DNA impurity
Lot. No.	DNA content (ng/mg)
GC1113-PUR-0901	; 0.198
GC1113-PUR-0902	0.215
747R0001	: Below detection limit
747R0002	: 0.015
Mean	: 0.143
Standard deviation (SD)	: 0.111

[140] Example 3: Sugar profile examination of EPO-Fc in EPO-Fc composition [141] Examination 3: Analysis of EPO-Fc glycosylation position >

[142] EPO-Fc refers to a fusion protein in a form of homo-dimer wherein two monomers including EPO and Fc regions combined by an IgD hinge have been combined by a disulfide bond in a hinge region. There are three N-glycosylation positions (N24, N38, and N83) and one O-glycosylation position (S126) in EPO sequence. Also, a further Nglycosylation position (N261) is present in an IgD CH2 domain of Fc sequence. It is presumed that EPO-Fc may have a total eight N-glycosylation positions and two Oglycosylation positions.

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PCT/KR2016/011169 [143] Actual glycosylation position has been confirmed by Protagen Co. who received a request.

[144] After treating DTT and iodoacetamide to thus reduce/alkylate the protein, the protein was treated using PNGase F to remove sugar in N-glycosylation position. The protein free of the sugar and the other protein still with sugar were treated using trypsin and GluC/trypsin to form a plurality of peptides, followed by comparing sizes of these peptides using MALDI-MS, thereby determining an N-glyosylation position.

[145] In order to confirm an O-glycosylation position, after marking HexNAc, the same was treated with PNGas F to remove all sugars in the N-glycosylation position. By treatment using Arg C, Trypsin/Glu C and Asp N/Tripsin, a plurality of peptides were obtained, followed by comparing sizes of these peptides using MAFDI-MS, thereby determining the O-glycosylation position.

[146] Analyzed results are summarized in Table 4 below. Three N-glycosylation positions (N24, N38, and N83) and one O-glycosylation position (S126) present in EPO sequence were identified, and a further N-glycosylation position (N261) was also found in the IgD CH2 domain of Fc sequence.

[147] [Table 4]

Examination of EPO-Fc glycosylation position and glycosylation level
Expected glycosylation position	Confirmation of glycosylation	Glycosylation level
N24	Confirmed	High
N38	Confirmed	High
N83	Confirmed	High
S126	Confirmed	Medium
N261	Confirmed	Medium

[149] Examination 4: Analysis of constitutional composition of EPO-Fc monosaccharide>

[150] The sugar chain of the glycoprotein generally consists of monosaccharide such as fucose, N-acetylglucosamine (GluNAc), N-acetylgalactosamine (GalNAc), galactose, mannose, sialic acid, etc. Alternatively, glucose, xylose, mannose-6-phosphate, or the like, may also be included.

[151] The constitutional composition of monosaccharide was analyzed by Protagen Co. who received a request. After hydrolyzing EPO-Fc with 4M hydrochloric acid (final concentration) at 100 °C for 4 hours, then drying the same through a vacuum centrifuge, the resulting material was dissolved in third distilled water and analyzed through liquid chromatography (high performance anion-exchange chromatography with pulsed amperometric detection; HPAEC-PAD). By analyzing a standard

WO 2017/061780

PCT/KR2016/011169 monosaccharide substance having known concentration under the same condition and comparing areas between the same and each monosaccharide, a molar ratio of each monosaccharide to mole of glycoprotein was estimated. The molar ratios of respective monosaccharides using the standard substance are summarized in Table 5 below. From the test results, it could be seen that about 2.2 mole of fucose, about 42.1 mole of GluNAc, about 9.3 mole of galactose, about 7.0 mole of mannose, respectively, are present in 1 mole of EPO-Fc.

[152] [Table 5]

Analysis of constitutional composition of EPO-Fc monosaccharide
Sample	Amount of monosaccharide (pmol/10 pi)	Molar ratio of monosaccharide per 1 mol of protein	Relative amounts
Fucose	26.12	2.23	4%
GlcN	493	42.05	69%
Galactose	109.53	9.34	15%
Mannose	82.42	7.03	12%

[154] Example 4: Examination of sialic acid content of EPO-Fc in EPO-Fc composition [155] Examination 5: Isoelectric focusing test of EPO-Fc>

[156] In order to determine a pi value of the prepared EPO-Fc, An isoelectric focusing (IEF) gel electrophoresis test was conducted as follows: 1) each of standard solution and test solution was mixed with a sample buffer (2X) and prepared; 2) the prepared sample was subjected to electrophoresis using IEF pH 3-7 gel; 3) the gel obtained after completing electrophoresis was fixed using a trichloroacetic acid solution; and 4) the gel was dyed using a dyeing solution including Brilliant Blue R, methanol and acetic acid mixed together, decolorized using a bleaching solution including methanol and acetic acid mixed together, and dried. The same used herein was batch No. GC1113-PUR-0902-PR. Results of the examination are summarized in FIG. 1.

[157] As shown in the test results in FIG. 1, it could be seen that main bands of the produced EPO-Fc are distributed in a range of pi 4.5 to 6.0.

[158] Examination 6: Test of sialic acid content>

[159] In order to examine the sialic acid content of the produced EPO-Fc, the following test was conducted: 1) a standard solution was prepared by diluting N-acetylneuraminic acid in distilled water; 2) a test solution was prepared in diluted state using distilled water; 3) the standard solution and test solution were treated using a resorcinol reagent then heated; 4) a reaction product was recovered using an extracting solution (1-butanol, butyl acetate); and 5) absorbency of the reaction product recovered was measured at 580 nm to estimate sialic acid content of the test solution, then, the sialic

WO 2017/061780

PCT/KR2016/011169 acid content (mol/mol) per 1 mole of EPO-Fc was calculated. The samples used herein were batch Nos. GC1113-PUR-0901-PR, GC1113-PUR-0902-PR, 747R0001 and 747R0002. Results of the examination are summarized in Table 6 below. From the test results, it could be seen that the produced EPO-Fc has sialic acid content of 20 mol/ mol or more.

[160] [Table 6]

Sialic acid content of produced EPO-Fc
Lot. No.	Resulted value (mol/mol)
GC1113-PUR-0901	21.462
GC1113-PUR-0902	21.168
747R0001	22.783
747R0002	22.821
Mean Standard deviation (SD)	22.058 0.867

WO 2017/061780

PCT/KR2016/011169 [Claim 1] [Claim 2] [Claim 3] [Claim 4] [Claim 5] [Claim 6] [Claim 7] [Claim 8] [Claim 9] [Claim 10] [Claim 11] [Claim 12]

Claims (1)

1. Claims

   An EPO-Fc composition, comprising EPO-Fc having a sialic acid content of 17 mol/mol or more, and host cell-derived protein impurity in an amount of 100 ng/mg or less.

   The EPO-Fc composition according to claim 1, wherein the sialic acid content ranges from 20 to 28 mol/mol.

   The EPO-Fc composition according to claim 1, wherein the EPO-Fc has pi < 6.0.

   The EPO-Fc composition according to claim 1, wherein the EPO-Fc has 4.5 < pi < 5.3.

   The EPO-Fc composition according to claim 1, wherein the host cellderived protein impurity is an aggregate or fragment of EPO-Fc.

   The EPO-Fc composition according to claim 1, wherein the host cellderived protein impurity is contained in an amount of 60 ng/mg or less. The EPO-Fc composition according to claim 1, further comprising host cell-derived DNA impurity of 0.5 ng/mg or less.

   A method for preparing an EPO-Fc composition, comprising: flowing an EPO-Fc cell culture solution at a flow rate of 4 vvd or less under conditions of a temperature ranging from 35°C to 39°C and 6.5 < PH < 7.5 to prepare a perfusion culture solution;

   obtaining an EPO-Fc pure solution from the perfusion culture solution; and adsorbing the EPO-Fc pure solution to an anion exchange resin to prepare a Q effluent that includes EPO-Fc having a sialic acid content of 17 mol/mol or more.

   The method according to claim 8, wherein the preparation of Q effluent includes: adsorbing the EPO-Fc to the anion exchange resin; and eluting the adsorbed EPO-Fc using a buffer which contains 0.005 to 0.02 M sodium phosphate, 0.05 to 0.2 M L-arginine and 0.02 to 0.1 M sodium chloride, and has 6. 7< pH <7.1.

   The method according to claim 8, wherein the step of obtaining of EPO-Fc pure solution includes passing the perfusion culture solution through a POD filter.

   The method according to claim 8, wherein an amount of host cellderived protein (HCP) impurity in the EPO-Fc composition is 100 ng/ mg or less.

   The method according to claim 8, wherein the EPO-Fc cell culture

   WO 2017/061780 [Claim 13] [Claim 14] [Claim 15]

   PCT/KR2016/011169 solution is inoculated in an amount of 2.0xl0⁵ cells/mL or more onto an incubator.

   The method according to claim 10, further comprising, after passing the perfusion culture solution through the POD filter, passing the above solution through a column filled with a protein A-bonded resin.

   The method according to claim 8, wherein the EPO-Fc contained in the Q effluent has pi of 6.0 or less.

   The method according to claim 8, further comprising filtering the Q effluent using a POD filter, ultrafiltration membrane and nano-filter, re spectively.

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   PCT/KR2016/011169

   1/1

   [Fig. 1] 1 2 3 b.9 **··«·. 6.0 -”~ 1. Protein Marker 2. GC1 113-PUR-0902-PR 5.3¾^^¾ 5.28111· 3. 747R0002 j· ⁴·⁵ι^^Β 4.2.1^^¾ 3.5.