TW201731523A - 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

a composition comprising long-acting erythropoietin

The present invention relates to a pharmaceutical composition comprising long-acting erythropoietin and a process for the preparation thereof.

Erythropoietin (EPO), which includes 165 amino acids, is a glycoprotein that promotes differentiation of red blood cells in the bone marrow and produces red blood cells. EPO is produced at most in the kidney and is known to be produced in small amounts in the liver. For example, as shown by chronic renal failure (CRF), loss of renal function typically causes a decrease in EPO production and thus a decrease in associated red blood cells. EPO has been found to have excellent efficacy in the treatment of anemia caused by CRF and other anemia caused by multiple causes, as well as having different hematopoietic functions such as surgical assistance.

In general, polypeptides such as EPO have low stability and are easily degraded and decomposed by proteolytic enzymes, that is, proteases in the blood, thereby being easily eliminated via the kidney or liver. Thus, a polypeptide has a short biological half-life period (half-life). Therefore, in order to maintain the blood concentration and potency of a protein medicine containing a polypeptide as a pharmaceutical composition, the protein medicine needs to be frequently administered to a patient. However, in the protein medicine to be administered, it is a note. In the case of a type of priming formulation, frequent injections to maintain the blood concentration of the active polypeptide may often cause pain in the patient.

In order to increase the half-life of EPO polypeptides, fusion proteins prepared using immunoglobulin (Ig) have been developed. Ig is an important structural component of blood. Human Ig (hIg) can include a variety of classes such as IgG, IgM, IgA, IgD, and IgE.

An immunoglobulin contains four polypeptide chains, and more specifically two heavy chains and two light chains, wherein the chains are combined together via disulfide bonds to form a tetramer. Each chain consists of a variant region and a constant region. The constant region of the heavy chain (the heavy chain constant region) can be subdivided into three or four sites such as CH1, CH2, CH3 and CH4 depending on its isotype. The Ig isotype of the Fc region of the heavy chain constant region may include a hinge, CH2, CH3, and/or CH4 domain.

Regarding half-life, chimeric proteins fused to the Fc region of an immunoglobulin are known to exhibit increased stability and prolonged serum half-life.

Furthermore, it is known that adhesion of sialic acid to the surface of EPO protein can induce prevention of protein degradation in the liver. The sialic acid protects the second galactose gene in the asialoglycoprotein receptor and thus affects the EPO activity (biological activity) in the living body. It is known that sialic acid present in the sugar chain of erythropoietin plays an important role in prolonging the half-life of erythropoietin and, ultimately, affects its efficacy. Furthermore, since the biological activity of erythropoietin has been found to be inactivated by the reaction of sialidase, the function of sialic acid on the biological activity of erythropoietin is more actively studied. In most cases, the sugar used in the manufacture of therapeutic agents has a basis and does not represent the essential efficacy of glycoproteins. Less characters. In particular, for human-derived erythropoietin, the role of sugar is absolutely necessary. In other words, if blood sugar is not included, erythropoietin does not exhibit activity, and the biological activity of EPO is significantly affected depending on the structural composition of the sugar or the content of sialic acid at the terminal.

The sialic acid content in EPO-Fc during host cell expression can be increased by the addition of N-ethinylmannosamine to the culture solution. The EPO-Fc obtained after the above process is usually a mixture of a high sialic acid content EPO-Fc and a low sialic acid content EPO-Fc. Here, EPO-Fc having a high sialic acid content of EPO-Fc having a lower sialic acid content has a longer half life. Therefore, there is a need to develop a technique for selectively producing EPO-Fc having an increased sialic acid content.

In the production of EPO-Fc, it is necessary to culture transfected host cells, express a large amount of EPO-Fc, disintegrate cells, and purify EPO-Fc. Therefore, it is very important to optimize the culture and purification techniques for mass production of high quality EPO-Fc. There is still much room for improvement in the culture and purification techniques that have been developed so far, and it is therefore highly desirable to optimize the EPO-Fc preparation process by developing the above techniques. Korean Patent Publication No. 897938 (registered on May 8, 2009) discloses an immunoglobulin fusion protein.

Accordingly, an object of the present invention is to provide an EPO-Fc fusion protein composition having excellent persistence (biosustainability) in a living body.

Further, another object of the present invention is to provide an EPO-Fc fusion protein composition having a high sialic acid content.

Furthermore, another object of the present invention is to provide an EPO-Fc fusion protein composition having high purity.

Further, it is another object of the present invention to provide an EPO-Fc fusion protein composition having a reduced amount of host cell-derived impurities.

The above object of the present invention is achieved by the following features:

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

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

(3) The EPO-Fc composition according to (1) above, wherein the EPO-Fc has a pI 6.0.

(4) The EPO-Fc composition according to (1) above, wherein the EPO-F has 4.5 pI 5.3.

(5) The EPO-Fc composition according to (1) above, wherein the host cell-derived protein impurity is an agglutinant or a fragment of EPO-Fc.

(6) The EPO-Fc composition according to (1) above, wherein the amount of the host cell-derived protein impurity is 60 ng/mg or less.

(7) The EPO-Fc composition according to (1) above, which further comprises 0.5 ng/mg or less of host cell-derived DNA impurities.

(8) A method of preparing an EPO-Fc composition comprising: at a temperature ranging from 35 ° C to 39 ° C and 6.5 pH The EPO-Fc cell culture solution is flowed at a flow rate of 4 vvd or lower under conditions of 7.5 to prepare a perfusion culture solution; an EPO-Fc pure solution is obtained from the perfusion culture solution; and the EPO-Fc pure solution is adsorbed to the anion exchange resin to prepare Q effluent comprising EPO-Fc having a sialic acid content of 17 mol/mol or more.

(9) The method according to (8) above, wherein the preparation of the Q effluent comprises: adsorption of EPO-Fc to the anion exchange resin; and use of sodium phosphate containing 0.005 to 0.02 M of sodium phosphate, 0.05 to 0.2 M, and 0.02 to 0.1 M sodium chloride with 6.7 pH The buffer of 7.1 elutes the adsorbed EPO-Fc.

(10) The method of (8) above, wherein the step of obtaining an EPO-Fc pure solution comprises passing the perfusion culture solution through a POD filter.

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

(12) The method according to the above (8), wherein the EPO-Fc cell culture solution is inoculated to the incubator in an amount of 2.0 x ^{10 5} cells/mL or more.

(13) The method of (10) above, further comprising, after the perfusion culture solution passes through the POD filter, passing the solution through a column packed with the protein A-binding resin.

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

(15) The method of (8) above, further comprising filtering the Q effluent using a POD filter, an ultrafiltration membrane, and a nanofilter, respectively.

The EPO-Fc composition according to the present invention is characterized in that the normally occurring portion of the sugar chain end is capped with sialic acid, thereby increasing half-life and enhancing patient convenience.

The EPO-Fc composition according to the present invention has a reduced content of host cell-derived protein impurities and DNA impurities, and thus has high EPO-Fc purity.

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

The method for preparing an EPO-Fc composition according to the present invention may significantly improve the attachment of sialic acid to EPO-Fc and enhance the survival of host cells, as well as enhance process efficiency.

The above and other objects, features and other advantages of the present invention will become apparent from the <RTIgt;

Fig. 1 is a view showing the detection results of an EPO-Fc isoelectric focusing inspection.

The inventors of the present invention have completed the present invention based on host cell culture techniques for improving manufacturability and sialic acid content, high sialic acid content EPO-Fc selection techniques, and for removing host cell derived impurities.

The present invention discloses an EPO-Fc composition comprising 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) impurities, and The method for its preparation.

Hereinafter, the present invention will be described in more detail.

The EPO-Fc fusion protein is formed by fusing the Fc region of the human immunoglobulin heavy chain constant region to erythropoietin (EPO). And has a longer half-life than EPO. This increased half-life refers to the biological sustainability of increased efficacy.

Sialic acid is present in the sugar chain of EPO, protecting the second galactose group of the asialoglycoprotein receptor, thus greatly affecting the biological activity of EPO. Further, sialic acid can induce prevention of EPO degradation in the liver and thus increase half-life.

Thus, in addition to the increased half-life due to Fc fusion, attachment of sialic acid contained in the EPO-Fc fusion protein provides increased half-life efficacy.

During the culture of the EPO-Fc host cells, N-ethylmercaptomannosamine (NAM) can be added to the culture solution to increase the sialic acid content of the EPO-Fc. The EPO-Fc obtained after the above process generally comprises a mixture of a high sialic acid content of EPO-Fc and a low sialic acid content of EPO-Fc, wherein a high sialic acid content of EPO-Fc is known to have a lower sialic acid content of EPO- Fc has a longer half-life.

According to a specific example of the present invention, the number of sialic acid attached to EPO-Fc is increased, and the pI value is decreased. Based on this aspect, the high sialic acid content of EPO-Fc can be selectively purified using an anion exchange resin. It is preferred to use an anion exchange resin containing crosslinked agarose. According to a specific example, Q-Sepharose resin manufactured by GE Healthcare Co. can be used.

Selective purification with pI by purification process using anion exchange resin 6 EPO-Fc. In particular, EPO-Fc having a sialic acid content of 17 mol/mol or more can be obtained through this process. According to a specific example, at 4.5 pI In the case of 6.0, an EPO-Fc-containing composition having a sialic acid content of 17 to 28 mol/mol can be obtained. According to another specific example, at 4.5 pI In the case of 5.3, an EPO-Fc-containing composition having a sialic acid content of 20 to 28 mol/mol can be obtained.

Host cell-derived protein (HCP) impurities may be mixed during the manufacture of EPO-Fc. HCP impurities can include, for example, protein impurities, DNA impurities, intrinsic or foreign viruses, and other particles or the like that contain abnormal peptides such as may be derived from different agglutinates and fragments of host cells and other materials.

Elimination of these impurities may be an important process that directly affects the quality of the EPO-Fc fusion protein. When this composition is prepared via a series of processes according to the present invention, the HCP can be adjusted in an amount of 100 ng/mg or less, and preferably 60 ng/mg or less. Host cell-derived DNA impurities may be contained in an amount of 0.5 ng/mg or less.

The method for preparing the EPO-Fc composition of the present invention can be accomplished as follows:

The EPO-Fc composition used to prepare the present invention may comprise: (1) at a temperature in the range of 35 ° C to 39 ° C and 6.5 pH Under conditions of 7.5, the EPO-Fc cell culture solution is flowed at a flow rate of 4 vvd or lower to prepare a perfusion culture solution; (2) an EPO-Fc pure solution is obtained from the perfusion culture solution; (3) the EPO-Fc pure solution is adsorbed to An anion exchange resin was prepared to prepare an EPO-Fc-containing Q effluent having a sialic acid content of 17 mol/mol or more.

The perfusion culture solution of the step (1) can be prepared as follows.

First, the EPO-Fc master cell bank (MCB) can be established from the cell bank (RCB) for EPO-Fc studies, and the EPO-Fc working cell bank (WCB) can be obtained from EPO- The Fc master cell bank (MCB) was established.

The culture for establishing MCB and WCB may be included in a liquid medium of EX-cell CHO DHFR (-) including L-glutamine and methotrexate at 3 to 7% CO ₂ and 37 ° C. The cells are cultured and the product is then dispensed into cryo vials for freezing and storage.

After thawing of the frozen and stored WCB cell line, the thawed cell line is provided and suspended in the medium to proliferate the cells in the medium. For example, the secondary culture can be carried out using a shake flask in a CO ₂ incubator set to 3 to 7% CO ₂ and 37 ° C at a ratio of 1:2 to 1:6 at intervals of 50 to 90 hours. The use of several shake flasks in this culture allows the number of cells cultured to be sufficient to be inoculated into the cell culture incubator.

In this case, EPO-Fc medium can be used. The EPO-Fc medium may include, for example, a powder medium of EX-cell CHO DHFR (-), L-glutamine, methotrexate, and sodium hydrogencarbonate.

When a sufficient number of host cell lines for inoculation are ensured via shake flask culture, the cells can be seeded into a primary incubator for perfusion culture. The main incubator used here can be a 30 liter biological incubator.

After inoculation into the main incubator, a cell concentration of 2.0 x ^{10 5} cells/mL or higher can be obtained.

The culture temperature of the main incubator can range from 35 to 39 ° C or from 36 to 38 ° C.

The acidity of the main incubator can be maintained in the range of 6.5 pH 7.5 (after pH regulation), 6.8 pH 7.2 (after pH control) and so on.

During perfusion culture, if necessary, the culture solution can be sampled and observed under a microscope to monitor the condition of the cells. The process can be analyzed by pH, cell number, cell activity, glucose concentration, glutamine concentration, ammonia concentration, It is implemented by osmotic pressure or the like.

The perfusion rate can be adjusted to 4vvd or lower. The perfusion rate can range from 0 to 4 vvd, 0 to 2 vvd, 1 to 3 vvd, 1 to 2 vvd, and the like.

The perfusion medium used herein may include a medium prepared by adding N-ethylmercaptomannamine in EPO-Fc medium (that is, a medium to which N-ethylmercaptomannamine is added) in EPO-Fc. A medium (manufacturing medium) prepared by adding N-acetylmercaptoamine and glucose to the medium, or both of the mediums may be used in sequence.

For example, a total of 140 to 180 kg of a cell culture solution for EPO-Fc production can be obtained according to a 40-liter perfusion per day culture for 4 days. Repeating the above collection of the cell culture solution once to ten or two to five times provides a more incremental cell culture solution for EPO-Fc production.

The cell culture solution obtained as described above can be a perfusion culture solution of the step (1) of the present invention.

According to the step 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 can adhere to EPO-Fc. Further, the survival of the cells can be modified to enhance the productivity of the EPO-Fc composition.

A method for preparing the EPO-Fc composition of the present invention Further included is (2) obtaining a pure EPO-Fc solution from the perfusion culture solution.

The above steps have a main purpose of removing the components other than the EPO-Fc contained in the perfusion culture solution of the step (1). Such removal can include any conventional method for cell culture. This removal method can be combined with at least one of the filtration and purification processes described above.

After recovering the perfusion culture solution of step (1), this solution can be subjected to various steps for filtration and purification, thereby removing host cell-derived impurities. Such host cell-derived impurities may include, for example, protein impurities, DNA impurities, intrinsic viruses, foreign viruses, and other particles containing anomalous peptides such as different agglutinates and fragments.

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

According to a specific example, in order to remove host cell-derived protein (HCP) impurities or DNA impurities contained in a host cell culture solution for EPO-Fc production, a POD depth filter can be used. POD depth filters are commonly used to remove cells, however, the invention is used to remove proteins.

Host cell-derived protein (HCP) impurities such as protein agglutinates, fragments and other particles can be removed using POD depth filters and filters.

For example, as a POD filter, Millistak+ (MA1HC10FS1) manufactured by Merck Millipore can be used. Further, the filter used herein may be a Sartobran P aseptic grade capsule manufactured by Sartorius Co.

According to another specific example, protein A purification, low pH virus inactivation and/or hydroxyapatite purification, etc., are applicable to EPO-Fc purification.

According to another embodiment, an ultrafiltration unit, i.e., a tangential flow filtration (TFF) membrane system, can be used for concentration and buffer exchange. If conductivity and acidity are replaced by buffers within the reference range, the process of concentration and buffer exchange is completed (reference: conductivity 9.0 ± 1.0 mS/cm, acidity pH 6.9 ± 0.1).

The EPO-Fc pure solution in the step (2) can be prepared as described above.

The method for preparing the EPO-Fc composition of the present invention may comprise (3) adsorbing a pure solution of EPO-Fc to an anion exchange resin to prepare a Q effluent comprising a sialic acid content of 17 mol/mol or more. EPO-Fc.

The above steps have a main purpose of selecting a specific one in which the EPO-Fc contained in the composition has a sialic acid content of 17 mol/mol or more.

This step can be carried out by passing the EPO-Fc pure solution in the step (2) through a column packed with an anion exchange resin containing crosslinked agarose.

According to a specific example, GE Sepharose resin of GE Healthcare can be used, for example, Q Sepharose Fast Flow resin shown in Table 1 below can be used.

The process of preparing the Q effluent may include: equilibrating the 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; The equilibration buffer rebalances the Q-Sepharose resin; and will contain 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 with an acidity of 6.7 pH The effluent from 7.1 was passed through the column to collect the Q effluent of EPO-Fc.

The elution can be repeated as appropriate. For example, when the elution is repeated 4 times, the Q effluents No. 1 to 3 obtained in the first to third purifications are stored in an ultra-low temperature freezer, and after the completion of the fourth elution, all Q effluents No. 1 to 4 can be collected.

Optionally, after elution, a POD filter can be used for filtration. The POD filter used herein may include, for example, Millistak+ (MA1HC10FS1) manufactured by Merck Millipore Co.

In addition to the above process, as needed, a process of installing a filter on the column and a process of sterilizing and washing the Q-Sepharose resin by bringing the resin into contact with the CIP may be further included. According to a specific example, the filter used herein may include, for example, a Sartobran P aseptic grade capsule manufactured by Sartorius Co.

The EPO-Fc contained in the Q effluent obtained in the step (3) may have a pI 6.0 and the sialic acid content was 17 mol/mol or more. For example, EPO-Fc can 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.

The method for preparing the EPO-Fc composition of the present invention may further comprise (4) filtering the Q effluent via a POD filter, an ultrafiltration membrane, and/or a nanofilter, respectively.

According to the above steps, EPO-Fc can have an increased concentration, the buffer can be exchanged, and the virus can be eliminated.

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

The ultrafiltration membranes used herein may include, for example, a tangential flow filtration (TFF) membrane system. For example, after washing the membrane with an injection buffer (interception: 30K), the formulation buffer can be used to equilibrate the membrane. The formulation buffer may contain 0.01 M sodium citrate, 0.1 M glycine and 0.1 M sodium chloride, and has an acidity of pH 6.2 ± 0.2.

After the equilibration is completed, the protein of interest can be concentrated to about 1.5 ± 0.3 mg/mL. If the recovered solution has a protein of about 1.5 ± 0.3 mg / mL For the concentration, buffer exchange can be carried out by continuously adding a buffer for preparing a crude solution in the same volume. If the conductivity and acidity of the finally recovered solution are within its reference range, the concentration and buffer exchange process is completed (reference: conductivity 12.0 ± 2.0 mS/cm, acidity pH 6.2 ± 0.2).

When the concentration of the final recovered solution and the buffer exchange process are complete, a nano filter (PALL (NT6DV20P1G)) can be used for filtration to eliminate viruses that may be derived from host cells or additional materials used in the process.

According to a specific example, after washing the filter for virus filtration, the integrity test of the filter is performed, and the balance can be achieved by passing the buffer for the crude manufacturing through the virus filter. Upon completion of the equilibration, the final recovered solution after completion of the concentration and buffer exchange process can be passed through the filter at a pressure of 30 ± 3 psi. Therefore, the virus-free virus filtrate can be recovered. Once the filtration is complete, the virus filter can be washed with injection buffer and then tested for integrity.

The addition of the polysorbate 20 and the formulation buffer at a concentration of 0.12 g/kg to the virus filtrate allowed the protein concentration to be adjusted to 1.1 ± 0.3 mg/mL, and then a crude filter of EPO-Fc was produced using a sterile filter.

According to a specific example, the formulation buffer may include 0.01 M sodium citrate, 0.1 M glycine, and 0.1 M sodium chloride, and has an acidity of pH 6.2 ± 0.2. According to a specific example, the sterilization filter used herein may be Sartobran P300 manufactured by Sartorius Co. The prepared EPO-Fc crude solution can be dispensed and stored in an ultra-low temperature freezer.

The final EPO-Fc crude solution was obtained by adding the formulation solution to the crude EPO-Fc solution and then filtering through a sterile filter to adjust the protein concentration to 0.5 ± 0.1 mg/mL. The sterilization filter used herein may include, for example, Sartobran P midicap manufactured by Sartorius Co.

Hereinafter, the present invention will be described in more detail with reference to the embodiments. However, the embodiments are merely described to illustrate preferred embodiments of the present invention, and the scope of the present invention is not particularly limited thereto.

Example 1: Preparation of EPO-Fc composition

<Process 1: Preparation of EPO-Fc Master Cell Bank (MCB)>

Adding L-glutamine and methotrexate to EX-cell CHO DHFR(-) liquid medium to form a medium and inoculating the cell bank (RCB) for EPO-Fc study into the medium, increasing by subculture The total number of cells and the total culture volume were used to construct MCB. The cultivation was carried out in a 5.0 ± 2.0% CO ₂ incubator until the culture solution reached a temperature of 37 °C.

<Process 2: Preparation of EPO-Fc Working Cell Bank (WCB)>

Adding L-glutamine and methotrexate to EX-cell CHO DHFR(-) liquid medium to form medium and inoculating MCB prepared in process 1 into medium, increasing total cell number and total culture through subculture Volume to build WCB. The cultivation was carried out in a 5.0 ± 2.0% CO ₂ incubator until the culture solution reached a temperature of 37 °C.

<Process 3: WCB Fusion>

After thawing the frozen and stored WCB in Process 2, the thawed WCB is suspended in EPO-Fc medium (including EX-cell CHO DHFR (-) powder medium, L-glutamine, methotrexate and carbonic acid In sodium hydrogenate, it was then cultivated in a 37 ° C CO ₂ incubator set with 5% CO ₂ .

<Process 4: Culture of shake flask cells>

The culture solution of Process 3 was subcultured at intervals of 64 to 80 hours to ensure the number of cells required to inoculate it into the cell culture incubator. The subculture was carried out at a ratio of 1:3 to 1:4 in a CO ₂ incubator provided with 5% CO ₂ and 37 ° C using EPO-Fc medium.

<Process 5: Manufacturing and culture in a main incubator (30 L biological incubator)>

After a sufficient amount of the culture solution of the process 4 was obtained, the cells were collected and inoculated into a main incubator to reach 2.0 × 10 ⁵ cells/mL. Then, the above solution was cultured using an EPO-Fc medium to which N-ethylmercaptomannamine was further added. Perfusion culture was carried out while maintaining the culture temperature at 37 ± 1 ° C and pH 7.0 ± 0.2 (after pH control). Process detection is performed by sampling the culture solution if necessary. The perfusion rate was adjusted in the range of 0 to 2 vvd based on the results of the process detection. To prepare a culture solution, the medium used was replaced with a new EPO-Fc production medium (prepared by adding N-ethylmercaptoamine and sodium hydrogencarbonate to EPO-Fc medium), and then further subjected to perfusion culture. 140 to 180 kg of the culture solution was collected for about 4 days per day for about 4 days to obtain a sub-batch. The culture was carried out by repeating the above process for a total of 4 times for 16 days until a total of four (4) sub-lots were collected. As a result, a culture solution (perfusion culture solution) for a host cell produced by EPO-Fc was obtained.

<Process 6: Recovery and filtration of culture solution>

Use POD filter (MA1HC10FS1) and filter (Sartobran P-None Bacterial grade capsule) The host cell culture solution for EPO-Fc production of Process 5 was filtered, and then the filtrate of the perfusion culture solution was obtained and stored.

<Process 7: Purification of Protein A>

After making the column packed with Protein A bonding resin in equilibrium with equilibration buffer (0.01 M sodium phosphate) and adsorbing the filtrate of Process 6 to the column, wash buffer (0.7 M L-arginine, pH 5.7 ± 0.05, conductivity 37.0 ± 1.0 mS / cm) clean the column, then use the elution buffer (0.02 M sodium acetate anhydrate, 0.2 M L-arginine, 7.94% (v / v) glycerol, pH 3.7 ± 0.05) The target protein (EPO-Fc) was eluted and then stored.

<Process 8: Low pH virus inactivation>

If the pure solution of Process 7 measures its pH as unsuitable for reference, add pH adjustment buffer (1M sodium hydroxide (NaOH), 10% acetic acid) to adjust the pH (3.7 ± 0.05), followed by this process. The solution was stirred for 2 hours at the same time. After completing the process, the pH was adjusted to about pH 6.9 ± 0.1 using pH adjustment buffer (1 M sodium hydroxide, 10% acetic acid).

<Process 9: Purification of Hydroxyapatite>

After installing a filter (Sartobran P-sterile grade capsule) filled with a stationary phase (hydroxyapatite) and equilibrating it with equilibration buffer (0.01 M sodium phosphate), the pure solution of process 8 is adsorbed to the tube The column was then removed (Sartobran P-sterile grade capsule) and the target protein was eluted using an elution buffer (0.1 M sodium phosphate and 0.1 M L-arginine, pH 6.9 ± 0.1) and stored.

<Process 10: Ultrafiltration Concentration and Buffer Replacement 1>

After the pure solution of Process 9 was concentrated using an ultrafiltration apparatus (membrane: cut off 30K, including 0.5 m ² ), buffer exchange was performed by continuously adding an equilibration buffer (0.01 M sodium phosphate). When the conductivity and pH are within its reference range, the process is complete and the retentate solution of the concentrate and buffer exchange 1 is collected to obtain a pure EPO-Fc solution.

<Process 11: Purification of Q Sepharose>

EPO-Fc pure solution adsorption of process 10 was carried out after installing a filter (Sartobran P-sterile grade capsule) filled with a stationary phase (Q Sepharose) and equilibrating Q Sepharose resin with an equilibration buffer containing 0.01 M sodium phosphate. To the column, then remove the filter, the Sartobran P aseptic grade capsule, and re-equilibrate the Q Sepharose resin, followed by the elution buffer (0.01 M sodium phosphate and 0.1 ML-arginine, 0.05 M sodium chloride, The target protein was eluted at pH 6.9 ± 0.1) to obtain and store the Q effluent of EPO-Fc.

<Process 12: Thawing and Filtration of Effluent>

The stored Q effluent from process 11 is thawed and pooled. The POD filter (MA1HC054H1) was equilibrated using formulation buffer (0.01 M sodium citrate, 0.1 M glycine, 0.1 M sodium chloride, pH 6.2 ± 0.2), and then the filtrate was obtained by passing the Q effluent through a filter.

<Process 13: Ultrafiltration Concentration and Buffer Replacement 2>

After the Q collection filtrate of the process 12 was concentrated using an ultrafiltration apparatus (membrane: 30K, including 0.1 M ² ) to achieve the target protein concentration, a continuous addition of the formulation buffer (0.01 M sodium citrate, 0.1 M glycine, Buffer replacement with 0.1 M sodium chloride, pH 6.2 ± 0.2). When the conductivity and pH are within its reference range, the process is complete and the retention and buffer exchange 2 retention solution is collected.

<Process 14: Nano filter filtration>

Concentration and buffering of Process 13 after the formulation buffer (0.01 M sodium citrate, 0.1 M glycine, 0.1 M sodium chloride, pH 6.2 ± 0.2) was flowed into a nano filter (Pall (NT6DV20P1G)) to equilibrate it. The retentate solution of the liquid exchange 2 flows therein, thereby eliminating viruses that may be present in the concentration and buffer exchange 2 retention solution.

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

0.12 g/kg of polysorbate 20 was added to the virus filtrate obtained in Process 14, and the protein concentration was adjusted using the formulation buffer. Then, sterilization and filtration were carried out using a sterilization and filter (Sartobran P300) to prepare a crude EPO-Fc solution while meeting the crude protein concentration (reference: 1.1 ± 0.3 mg/mL), which was then dispensed into a storage container and The container is stored in an ultra-low temperature freezer.

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

After thawing the crude EPO-Fc solution of Process 15 in a water bath, it was added to the formulation buffer (0.01 M sodium citrate, 0.1 M glycine, 0.1 M sodium chloride, pH 6.2 ± 0.2, polysorbate 20 0.12). g/Kg) To achieve a protein concentration of 0.5 ± 0.1 mg/mL, sterilized and filtered using a sterilization and filter (Sartobran P midicap) to prepare a final crude EPO-Fc solution.

Example 2: Purity detection of EPO-Fc composition

<Detection 1: Test for impurity content of host cell-derived protein (peptide) (HCP)

To test the HCP impurity content in the final crude solution, the following tests were performed using the CHO Host Cell Protein Kit: 1) Dilution and use of reagents a standard solution provided in the cartridge; 2) the test solution is prepared in a diluted state using a dilution buffer; 3) a spiked test solution is prepared by adding a standard solution to the above test solution; 4) in the standard solution, After the test solution and the addition of the test solution are reacted with an anti-CHO (phosphatase conjugate), the mixture is reacted in an anti-CHO coated microtiter plate; and 5) after washing the plate, a PNPP substrate is added thereto, Absorbance was measured using an ELISA plate reader. The samples used here are lot numbers 747R0001 and 747R0002. The test results are summarized in Table 2 below. It can be seen from the test results that the host cell-derived protein impurity content is 60 ng/mg.

<Detection 2: Test for DNA content of DNA derived from host cells>

In order to detect the content of host cell-derived DNA impurities in the final crude solution of EPO-Fc, the following tests were performed: 1) diluting and using the standard reagent provided in the kit as a standard solution; 2) setting the zero calibration provided in the kit The standard agent is diluted and used as a test solution; 3) the test solution is prepared by adding a standard solution to the test sample; 4) the zero calibration standard used herein is the zero calibration standard provided in the kit; 5) by Add the standard solution Zero calibration standard to prepare the zero calibration standard used herein; 6) prepared standard solution, test solution, added test solution, zero calibration standard and zero calibration standard for DNA labeling; and 7) use threshold The system filter unit performs a test stick combination and then reads the test rod to confirm the result. The samples used here were batch Nos. GC1113-PUR-0901-PR, GC1113-PUR-0902-PR, 747R0001, and 747R0002. The test results are summarized in Table 3 below. As can be seen from the test results, the host cell-derived DNA impurity content was 0.5 ng/mg or less, and the maximum was 0.215 ng/mg, and the average was 0.143 ng/mg.

Example 3: Detection of sugar distribution of EPO-Fc in EPO-Fc composition

<Detection 3: Analysis of EPO-Fc glycosylation position>

EPO-Fc refers to a fusion protein in the form of a homodimer, in which two monomers including the EPO and Fc regions combined by the IgD hinge have been passed through the hinge region. The combination of disulfide bonds. There are three N-glycosylation positions (N24, N38 and N83) and one O-glycosylation position (S126) in the EPO sequence. In addition, an additional N-glycosylation position (N261) is present in the IgD CH2 domain of the Fc sequence. It is speculated that EPO-Fc may have a total of eight N-glycosylation sites and two O-glycosylation sites.

The actual glycosylation site has been confirmed by the requesting Protagen.

After treating DTT and iodoacetamide to thereby reduce/alkylate the protein, the protein is treated with PNGase F to remove sugar from the N-glycosylation site. The sugar-free protein and other proteins still containing sugar were treated with trypsin and GluC/trypsin to form a plurality of peptides, and then the size of these peptides was compared using MALDI-MS to determine the N-glycosylation position.

To confirm the O-glycosylation position, after labeling HexNAc, it was treated with PNGas F to remove all sugars in the N-glycosylation position. A plurality of peptides were obtained by treatment with Arg C, trypsin/Glu C and Asp N/Tripsin, and then the size of these peptides was compared using MALDI-MS to determine the O-glycosylation position.

The results of the analysis are summarized in Table 4 below. Three N-glycosylation positions (N24, N38 and N83) and one O-glycosylation position (S126) present in the EPO sequence were identified, and another was found in the IgD CH2 domain of the Fc sequence. N-glycosylation position (N261).

<Detection 4: Analysis of the composition of EPO-Fc monosaccharide>

The sugar chain of the glycoprotein is usually composed of a monosaccharide such as fucose, N-ethyl glucosamine (GluNAc), N-ethyl galactosamine (GalNAc), galactose, mannose, sialic acid or the like. Alternatively, glucose, xylose, mannose-6-phosphate or the like may be included.

Protagen, which accepted the request, analyzed the composition of the monosaccharide. EPO-Fc was hydrolyzed at 4 ° C for 4 hours with 4 M hydrochloric acid (final concentration), then dried by vacuum centrifugation, and the resulting material was dissolved in a third distilled water and subjected to liquid chromatography (high-efficiency anion exchange chromatography using a pulse) Amperometric detection; HPAEC-PAD). The molar ratio of each monosaccharide to glycoprotein moule was estimated by analyzing a standard monosaccharide material having a known concentration under the same conditions and comparing the area between the same and each monosaccharide. The molar ratios of the individual monosaccharides using the standard materials are summarized in Table 5 below. As can be seen from the test results, there were about 2.2 moles of fucose, about 42.1 moles of GluNAc, about 9.3 moles of galactose, about 7.0 moles, respectively, in 1 mole EPO-Fc. Mannose.

Example 4: Detection of sialic acid content of EPO-Fc in EPO-Fc composition

<Detection 5: Isoelectric focusing test of EPO-Fc>

To determine the pI value of the prepared EPO-Fc, the following isoelectric focusing (IEF) gel electrophoresis test was performed: 1) mixing the standard solution and the test solution with the sample buffer (2X) and preparing; 2) The prepared sample was electrophoresed using an IEF pH 3-7 gel; 3) the gel obtained after electrophoresis was fixed using a trichloroacetic acid solution; and 4) the dyeing solution including Brilliant Blue R, methanol and acetic acid mixed together was used. The gel was stained, destained using a mixed bleach solution containing methanol and acetic acid, and dried. The batch number GC1113-PUR-0902-PR is used here. The test results are summarized in Figure 1.

As shown by the test results of Fig. 1, it can be seen that the main band of the produced EPO-Fc is distributed in the range of pI 4.5 to 6.0.

<Test 6: Test for sialic acid content>

In order to detect the sialic acid content of the produced EPO-Fc, the following tests were carried out: 1) preparing a standard solution by diluting N-acetyl thioglycolic acid in distilled water; 2) preparing a test solution in a diluted state by diluting with distilled water; Reheating using a resorcinol reagent to treat the standard solution and the test solution; 4) recovering the reaction product using an extraction solution (1-butanol, butyl acetate); and 5) measuring the absorbance of the recovered reaction product at 580 nm to estimate the test The sialic acid content of the solution was then calculated as the sialic acid content (mol/mol) per 1 mol of EPO-Fc. The samples used here were batch Nos. GC1113-PUR-0901-PR, GC1113-PUR-0902-PR, 747R0001, and 747R0002. The test results are summarized in Table 6 below. As can be seen from the test results, the produced EPO-Fc had a sialic acid content of 20 mol/mol or more.

Claims (15)

An EPO-Fc composition comprising EPO-Fc having a sialic acid content of 17 mol/mol or more, and 100 ng/mg or less of host cell-derived protein impurities. The EPO-Fc composition according to claim 1, wherein the sialic acid content ranges from 20 to 28 mol/mol. The EPO-Fc composition of claim 1, wherein the EPO-Fc has a 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 of claim 1, wherein the host cell-derived protein impurity is an agglomerate or fragment of EPO-Fc. The EPO-Fc composition according to claim 1, wherein the host cell-derived protein impurity is contained in an amount of 60 ng/mg or less. The EPO-Fc composition of claim 1, which further comprises 0.5 ng/mg or less of host cell-derived DNA impurities. A method of preparing an EPO-Fc composition comprising: at a temperature in the range of 35 ° C to 39 ° C and 6.5 pH The EPO-Fc cell culture solution is flowed at a flow rate of 4 vvd or lower to prepare a perfusion culture solution under conditions of 7.5; an EPO-Fc pure solution is obtained from the perfusion culture solution; and the EPO-Fc pure solution is adsorbed to the anion exchange Resin to prepare a Q effluent comprising EPO-Fc having a sialic acid content of 17 mol/mol or more. The method of claim 8, wherein the preparation of the Q effluent comprises: adsorbing the EPO-Fc to the anion exchange resin; and using 0.005 to 0.02 M sodium phosphate, 0.05 to 0.2 M L-fine Amino acid and 0.02 to 0.1 M sodium chloride with 6.7 pH The adsorbed EPO-Fc was eluted with a buffer of 7.1. The method of claim 8, wherein the step of obtaining an EPO-Fc pure solution comprises passing the perfusion culture solution through a POD filter. The method of claim 8, wherein the amount of host cell-derived protein (HCP) impurities in the EPO-Fc composition is 100 ng/mg or less. The method of claim 8, wherein the EPO-Fc cell culture solution is inoculated to the incubator in an amount of 2.0 × 10 ⁵ cells/mL or more. The method of claim 10, further comprising, after passing the perfusion culture solution through the POD filter, passing the solution through a column packed with a protein A bonding resin. The method of claim 8, wherein the EPO-Fc contained in the Q effluent has a pI of 6.0 or less. The method of claim 8, further comprising filtering the Q effluent using a POD filter, an ultrafiltration membrane, and a nanofilter, respectively.