Method for purifying follicle stimulating hormone

Abstract

The present invention relates to a method for purifying follicle stimulating hormone (FSH) in high yield and high purity, said method comprising an immunoaffinity chromatography (IAC) step.

Description

TECHNICAL FIELD

[0001] The present invention relates to a method of purifying follicle stimulating hormone with high yield and high purity. BACKGROUND

[0002] Various different protein therapeutics, which can be used as various therapeutic agents, have not only been applied to research on the preparation of recombinant proteins, but also to their clinical application and commercialization.

[0003] One of these recombinant proteins, i.e., follicle stimulating hormone (FSH), is a hormone produced by gonadotropes in the anterior pituitary, released into the bloodstream, and acts together with luteinizing hormone (LH) to control oocyte maturation in females and spermatogenesis in males. Human FSH is used to treat anovulatory women, to stimulate multiple follicle development (superovulation), and to prepare for assisted conception (e.g., IVF, ICSI, GIFT, and CIFT). In addition, human FSH is used to stimulate follicle maturation in women with low or no FSH production, and to stimulate spermatogenesis in men with oligospermia.

[0004] Since FSH is important in the treatment of reproductive disorders, there is a demand for providing recombinant FSH with high purity and high specific activity.

[0005] FSH treatment requires repeated injections, and a high-purity FSH preparation can be administered subcutaneously, can be self-administered by a patient, thereby enabling to improve the convenience and compliance of the patient.

[0006] International Publication No. WO 2006 / 051070 A1 relates to a method of purifying FSH, the method comprising the steps of: 1) performing dye affinity chromatography, 2) performing hydrophobic interaction chromatography; and 3) performing reverse phase chromatography; and a method of purifying FSH, the method comprising the steps of: 1) performing anion exchange chromatography, 2) performing dye affinity chromatography, 3) performing hydrophobic interaction chromatography, 4) performing reverse phase chromatography, and 5) performing anion exchange chromatography.

[0007] International Publication No. WO 2005 / 063811 A1 relates to a method of purifying recombinant human FSH, the method comprising the steps of: 1) performing ion exchange chromatography; 2) performing immobilized metal ion chromatography; and 3) performing hydrophobic interaction chromatography (HIC).

[0008] International Publication No. WO 2007 / 065918 A2 relates to a method of purifying FSH or FSH mutants, the method comprising the steps of: dye affinity chromatography; weak anion exchange chromatography; hydrophobic interaction chromatography; and strong anion exchange chromatography; the method can be performed in any order.

[0009] Therefore, there is a continuous need for new methods for purifying recombinant FSH and FSH variants. Specifically, dye affinity chromatography is primarily used as the capture step in FSH purification. While dye affinity chromatography has the advantage of excellent target protein recovery by removing culture medium components from the culture medium, it also has the disadvantage of reduced ability to remove impurities derived from host cells. These impurities are diverse and highly complex, making it impossible to identify a single type. Consequently, there is a high risk of alteration in the quality of the target protein, and these alterations are difficult to control. Furthermore, complex purification steps are required after the capture step to further remove a large number of impurities, leading to decreased yield.

[0010] Therefore, the inventors have strived to develop a purification method for obtaining FSH with high purity and high yield. They discovered that by effectively arranging the sequence of the purification process and specifying the number of washes and the washing solvent in the washing step of the immunoaffinity chromatography (IAC) purification process, the process efficiency can be improved, while simultaneously increasing the yield and maximizing impurity removal, thus completing this invention. Summary of the Invention

[0011] [Technical Issues]

[0012] The purpose of this invention is to provide a method for purifying follicle-stimulating hormone (FSH), the method comprising the step of performing immunoaffinity chromatography (IAC).

[0013] [Technical Solutions]

[0014] The invention will be described in more detail below.

[0015] Furthermore, the corresponding descriptions and embodiments disclosed in this invention can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this invention are within the scope of this invention. Moreover, the scope of this invention is not limited by the specific descriptions below.

[0016] Furthermore, those skilled in the art can identify or confirm multiple equivalents of specific aspects of this application described herein using only conventional experiments. Moreover, such equivalents are intended to be included in this application.

[0017] One aspect of the invention provides a method for purifying follicle-stimulating hormone (FSH), the method comprising an immunoaffinity chromatography (IAC) step, wherein the immunoaffinity chromatography (IAC) includes a washing step, wherein the washing is performed at least three times.

[0018] The purification method of the present invention is a method for purifying follicle-stimulating hormone (FSH) with high yield and high purity.

[0019] Specifically, the purification method may be a purification method performed in the following order, wherein the method includes: (a) performing immunoaffinity chromatography (IAC); (b) performing hydrophobic interaction chromatography (HIC); and (c) performing anion exchange chromatography (AEX).

[0020] The purification method of the present invention may be a purification method in which the chromatography in each step (a) to (c) is performed only once, and no further chromatography is performed.

[0021] The additional chromatography is selected from one or more of size exclusion chromatography, dye affinity chromatography, reversed-phase chromatography and cation exchange chromatography, but is not limited thereto.

[0022] The importance of the purification method of the present invention lies in the fact that even if the chromatography in each step (a) to (c) is performed only once, it can remove host cell-derived proteins (i.e. impurities) and improve the purification yield.

[0023] When used herein, the term "follicle-stimulating hormone (FSH)" is a hormone produced and released into the bloodstream by gonadotropic cells in the anterior pituitary gland. FSH works in conjunction with luteinizing hormone (LH) to control oocyte maturation in women and spermatogenesis in men. FSH and LH belong to a group of heterodimeric glycoproteins, which consist of two non-covalently linked α and β chains encoded by separate genes. Both the α and β chains are glycosylated. The α-subunit consists of 92 amino acid residues, while the β-subunit consists of 111 amino acid residues, with each subunit having two potential asparagine-linked glycosylation sites.

[0024] Human FSH is used to treat anovulatory women, stimulate multiple follicle development (superovulation), and prepare for assisted conception (e.g., IVF, ICSI, GIFT, and CIFT). Additionally, human FSH is used to stimulate follicle maturation in women with low or no FSH production, and to stimulate spermatogenesis in men with oligospermia.

[0025] In a typical treatment regimen for ovulation induction, patients receive FSH or a variant thereof daily via injection at a rate of approximately 75 IU FSH / day to approximately 450 IU FSH / day for approximately 6 to approximately 12 days. In a typical treatment regimen for controlled ovarian hyperstimulation, patients receive FSH or a variant thereof daily via injection at a rate of approximately 150 IU FSH / day to approximately 600 IU FSH / day for approximately 6 to approximately 12 days.

[0026] In other words, FSH treatment requires repeated injections, therefore high-purity FSH preparations are essential.

[0027] To this end, the inventors have attempted to improve the yield, maximize the removal of host cell-derived proteins (i.e., impurities), and maximize impurity removal by effectively arranging the sequence of the purification process and specifying the number of washes and the wash solvent in the washing steps of the immunoaffinity chromatography (IAC) purification process, thereby simplifying the number of purification steps and improving the efficiency of the process operation by optimizing the procedure steps in column washing.

[0028] Each step of the method for purifying follicle-stimulating hormone will be described in detail below. First, step (a) is the immunoaffinity chromatography (IAC) step.

[0029] When used herein, the term "immunoaffinity chromatography (IAC)" refers to a method in which an antibody against a physiologically active substance to be purified is prepared and covalently bonded to a solid-phase support; a sample containing the physiologically active substance to be purified is added to the antibody-carrier complex to allow the physiologically active substance to adsorb onto the antibody; and an eluent is added to it in a suitable manner to obtain the target substance by elution. In this invention, immunoaffinity chromatography for FSH purification refers to an exchange resin that utilizes a principle to selectively capture FSH using a column comprising an FSH-specific protein (i.e., a protein that binds to the α or β subunits of FSH), and is prepared by linking the FSH-specific protein to a suitable matrix using a protein having similar structural, physical, and chemical characteristics.

[0030] Immunoaffinity chromatography used for FHS purification includes CaptureSelect. TM FHS resin (Thermo Fisher Scientific), etc., but not limited to, and generally any resin used for immunoaffinity chromatography and specifically bound to FSH can be used.

[0031] The advantage of this method is that it can obtain physiologically active substances more quickly and quantitatively compared to conventional methods (such as gel filtration, ion exchange chromatography, etc.).

[0032] For the purposes of this invention, the immunoaffinity chromatography may be performed using one or more steps selected from the equilibration step, sample injection step, washing step, elution step, and resin cleaning step.

[0033] To utilize the principle of immunoaffinity, a buffer solution with a pH range of 7 to 8 can be used, and Tris with a molar concentration of 10 mM to 50 mM can be used; in embodiments, buffer solutions such as Tris, PBS, 3-morpholinylpropane-1-sulfonic acid (MOPS), sulfonates, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), TES, phosphates and / or isopropanol can be used, but the buffer solutions are not limited thereto.

[0034] The equilibration step refers to the process of creating an environment (e.g., suitable pH, salt concentration, etc.) within the column to allow FSH contained in the culture medium to attach to the chromatography column.

[0035] For the purposes of this invention, in step (a) above, impurities, including host cell-derived proteins other than FSH, in various proteins adsorbed onto the column can be removed by performing the washing step once or multiple times, particularly by performing the washing step three or more times, but the number of times the washing step is performed is not limited thereto.

[0036] The washing step refers to the process of removing impurities, including host cell-derived proteins, from the various proteins adsorbed onto the column, in addition to FSH. This removes not only host cell-derived proteins but also culture medium components. This embodiment incorporates a multi-step washing process. Specifically, a buffer solution with a pH range of 7 to 8 can be used, and this pH range can be applied to all equilibration, washing, and elution steps.

[0037] In one embodiment, in the first cleaning step, impurities that are non-specific or weakly specific to the immunoaffinity chromatography resin, or impurities that are relatively weakly hydrophobic, can be removed. Specifically, in the presence of salt, the salt can exist in an ionic state, and these ions surround the protein, such that the total charge may be close to zero, thus placing it in a hydrophobic state. Non-specific impurities attached to the resin in this state can be removed in the cleaning step by using these impurities to detach in the absence of salt or under very low salt concentration conditions. In the first cleaning step of the present invention, Tris with a molar concentration of 2 mM to 50 mM can be used, particularly Tris with a molar concentration of 2 mM to 10 mM, but the Tris used are not limited to these. In addition, the cleaning solution used for the first cleaning step can be selected from Tris, PBS, 3-morpholinylpropane-1-sulfonic acid (MOPS), sulfonates, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), TES, phosphates, and / or isopropanol, but the cleaning solution is not limited to these.

[0038] In the second washing step, impurities with relatively weak ion-binding affinity to the immunoaffinity chromatography resin can be removed. Specifically, the resin may be polar, and the oppositely polar portions of the protein can bind to the resin. In this state, when Na+, as a strongly polar ion... + and Cl - When Na enters a non-specific material attached to a resin, an exchange phenomenon may occur, wherein... + and Cl - It can adhere to the resin, thereby allowing the non-specific impurities to detach from the resin. In the second cleaning step of the present invention, Tris with a molar concentration of 10 mM to 50 mM can be used, particularly Tris with a molar concentration of 10 mM to 30 mM, but the Tris used is not limited to this. In addition, the salt used can be in the range of 0.5 M to 4.5 M or less, 0.5 M to 4.0 M or less, 0.5 M to 3.5 M or less, 0.5 M to 3.0 M or less, 0.5 M to 2.5 M or less, 0.5 M to 2.0 M or less, 0.5 M to 1.5 M or less, 1.0 M to 4.5 M or less, 1.0 M to 4.0 M or less, 1.0 M to 3.5 M or less, and 1.0 M to 3.0 M or less, particularly in the range of 0.5 M to 1.5 M. In one embodiment, the salt used may include sodium sulfate, sodium chloride, ammonium sulfate, ammonium chloride and / or magnesium chloride, more specifically sodium chloride, but the salt used is not limited thereto.

[0039] In the third cleaning step, impurities with relatively weak ion-binding affinity to immunoaffinity chromatography resins compared to FSH can be removed, and Tris with a molar concentration of 10 mM to 50 mM, especially Tris with a molar concentration of 10 mM to 30 mM, can be used, but the Tris used is not limited to this. Furthermore, the salt concentration used can be in the range of 0.001M to 1.0M, 0.001M to 0.75M, 0.001M to 0.5M, 0.005M to 1.0M, 0.005M to 0.75M, 0.005M to 0.5M, 0.01M to 1.0M, 0.01M to 0.75M, 0.01M to 0.5M, 0.1M to 1.0M, 0.1M to 0.75M, and 0.1M to 0.5M, particularly in the range of 0.1M to 0.3M, but the concentration range of the salt used is not limited to this. In one embodiment, the salt used may include sodium sulfate, sodium chloride, ammonium sulfate, ammonium chloride, and / or magnesium chloride, more specifically magnesium chloride, but the salt used is not limited to these. Typically, magnesium chloride is the salt used to elute proteins from resin, and it is used at a high concentration of 1.5M to 2.5M. However, in the third step of this invention, a low salt concentration of 0.1M to 0.5M is used, and the principle used in this third washing step is to remove proteins that are not specifically bound to the resin while maintaining the binding between the antibody to the target protein to be obtained in this invention and the resin.

[0040] For the purposes of this invention, after three or more washing steps in immunoaffinity chromatography, the content of host cell protein (HCP) can be in the range of 0.01 ppm to 350 ppm, 0.1 ppm to 340 ppm, 1 ppm to 330 ppm, 3 ppm to 320 ppm, 5 ppm to 300 ppm, and more particularly below 250 ppm, but not limited thereto. Furthermore, after three or more washing steps, the purification yield can be above 60%, but the purification yield is not limited thereto.

[0041] In one embodiment of the invention, it was confirmed that the purification yield of host cell proteins varied with the type of washing solvent and the number of washes in the first purification (IAC) step (Example 2). Specifically, it was confirmed that the HCP content was lowest when at least three washes were performed using a specific type and concentration of washing solvent. In particular, it was confirmed that the HCP content decreased to below 4,000 ppm after the first wash step, below 500 ppm after the second wash step, and below 250 ppm after the third wash step.

[0042] The elution step refers to the step of recovering FSH bound to the resin. Magnesium chloride (MgCl2) can be used to counteract the immunoaffinity between FSH and the resin, and specifically, the elution conditions can be carried out in a buffer solution with a pH range of 7 to 8 at a salt concentration of 1.5 M to 2.5 M. In one embodiment, the salt used may include sodium sulfate, sodium chloride, ammonium sulfate, ammonium chloride, and / or magnesium chloride, and more specifically may include magnesium chloride, but the salt used is not limited thereto.

[0043] In addition, as another method for eluting FSH, a glycine buffer solution with a pH of 2.5 to 3.5 can be used, but the buffer solution used is not limited to this.

[0044] The resin cleaning (CIP) step is used to completely remove residual FSH, microorganisms, or other impurities remaining in the column to prevent residue. The elution buffer used may include citric acid, acetic acid, phosphoric acid, PAB, urea, guanidine hydrochloride and / or isopropanol, NaOH and / or ethanol, etc., and specifically, acetic acid with a concentration of 0.1M to 1M can be used, but the elution buffer is not limited to these.

[0045] In the method used to purify FSH, step (b) above is a step of performing hydrophobic interaction chromatography (HIC).

[0046] Hydrophobic interaction chromatography is an exchange resin that utilizes the reversible interaction between proteins and the hydrophobic surfaces of the medium. Proteins bind to the column under high ionic strength conditions (i.e., high salt concentrations), and as the ionic strength gradually decreases, these proteins are differentially separated from each other.

[0047] For the purposes of this invention, the hydrophobic interaction chromatography can be performed using one or more steps selected from the sample introduction step, equilibration step, washing step, elution step, and resin cleaning step.

[0048] When used in this document, "hydrophobic interaction chromatography (HIC)" refers to a separation method that utilizes the hydrophobic interactions between a matrix having hydrophobic functional groups (e.g., phenyl, octyl, (iso)propyl, butyl, ethyl, etc.) and certain molecules.

[0049] Specifically, this chromatography can be performed using HIC resins with relatively weak hydrophobic surfaces (compared to the much stronger hydrophobic surfaces of reversed-phase resins). Proteins with hydrophobic surface properties typically adhere to resins containing ether, phenyl, butyl, or hexyl groups. In this invention, the resin used in the hydrophobic interaction chromatography can be a resin in which the functional groups are selected from phenyl, octyl, (iso)propyl, butyl, and ethyl groups, but the resin is not limited thereto, and any resin commonly used in hydrophobic interaction chromatography can be used.

[0050] The equilibration step in the hydrophobic interaction chromatography refers to the step of creating a certain environment (e.g., suitable pH, salt concentration, etc.) within the column to allow FSH contained in the eluent obtained from immunoaffinity chromatography to adhere to the column. Specifically, the salt concentration used can be in the range of 1.5M to 2.5M, and in one embodiment, sodium sulfate, sodium chloride, ammonium sulfate, and / or ammonium chloride can be used. Furthermore, a buffer solution with a pH of 7 to 8 can be used, and Tris with a molar concentration of 10 mM to 50 mM can be used. The above conditions can be applied to all equilibration, washing, and elution steps.

[0051] In the sample introduction step, an elution buffer obtained by immunoaffinity chromatography can be used.

[0052] For the purposes of this invention, a balanced buffer solution is used in the cleaning step to remove impurities that are not specifically bound to the resin.

[0053] The elution step refers to the step of recovering FSH bound to the resin. To suppress the hydrophobic interaction between FSH and the resin, a salt-free or low-salt environment may be required. Furthermore, a buffer solution with a pH in the range of 7 to 8 can be used, and Tris with a molar concentration of 10 mM to 50 mM can be used, but the buffer solution is not limited to these.

[0054] The resin cleaning (CIP) step is used to completely remove residual FSH, microorganisms, or other impurities remaining in the column to prevent residue. The residual FSH can be removed using pure water, or alternatively, sodium hydroxide (NaOH), phosphoric acid, etc., but the invention is not limited to these methods.

[0055] For the purposes of this invention, step (b) aims to further improve the purity of FSH by further removing impurities (such as host cell proteins) that may not have been removed in step (a). In this step, host cell proteins can be removed more effectively by using a filtration device capable of removing host cell-derived proteins, or by using a hydrophobic reaction of an immunoaffinity chromatography method that differs from the separation mechanism of step (a).

[0056] When used herein, the term "host cell protein (HCP)" refers to a protein distinct from FSH, and generally refers to a protein derived from the host cell. For antibodies or proteins that can be used as pharmaceuticals, HCPs are preferably excluded from the original antibody or protein formulation. The host cell protein to be removed is a concept that includes all impurities other than the FSH to be purified, and these can include not only the host cell protein itself, but also DNA derived from the host cell, factors used for cell growth, etc. Therefore, when host cell proteins are removed, only the target protein to be purified can be purified to a high degree of purity.

[0057] In the above method for purifying FSH, step (c) is an anion exchange chromatography (AEX) step.

[0058] Ion exchange chromatography refers to an exchange resin that utilizes the reversible interaction of net charge between the medium and the protein surface (i.e., the difference in ionic strength).

[0059] Representative strong ion exchange groups include Q and SP, while representative weak ion exchange groups include DEAE, ANX, and CM. Since each has a different pH range carrying sufficient charge, selectivity is possible. Ion exchange resins are classified into negative ion exchange resins and positive ion exchange resins, and specifically, negative ion exchange resins are used in this invention.

[0060] When used herein, the term “anion exchange chromatography (AEX)” refers to chromatography using a column packed with anion exchange resin, and in the steps described above, anion exchange chromatography can further remove impurities, particularly host cell proteins, and can selectively separate subtypes with desired isoelectric points.

[0061] The anion exchange resin refers to a synthetic resin that is added to different aqueous solutions to exchange its anions with specific anions in the aqueous solution, and the anion exchange column can adsorb proteins carrying anions at a point above their isoelectric point. In the case of FSH according to the present invention, the third purification step can be carried out in such a way that, when using a neutral pH buffer, the protein (FSH) attaches to the anion exchange resin due to its low isoelectric point, and after washing, the target protein (i.e., FSH) is separated upon passing through an elution buffer.

[0062] As anion exchange resin, commonly used anion exchange resins in the art can be used, but the anion exchange resin is not limited to these. Specifically, Q Sepharose, quaternary aminoethyl, quaternary ammonium (Q), etc., can be used, and more specifically, Q Fast Flow can be used. TM .

[0063] For the purposes of this invention, the anion exchange chromatography can be performed using one or more steps selected from the sample introduction step, equilibration step, washing step, elution step, and resin cleaning step.

[0064] The equilibration step refers to the process of creating an environment within the column (e.g., suitable pH, salt concentration, etc.) to allow FSH obtained from hydrophobic interaction chromatography to adhere to the column. Buffer solutions with a pH in the range of 7 to 8 can be used, and Tris solutions with a molar concentration of 10 mM to 50 mM can be used, but the buffer solutions are not limited to these. The buffer solution can be applied to both the equilibration step and the elution step, excluding the washing step.

[0065] The cleaning step refers to the removal of impurities adsorbed within the column. Furthermore, subtypes other than those with the desired isoelectric point can be selectively removed. Buffer solutions with a pH in the range of 5 to 6 can be used, and acetates with a molar concentration of 1 mM to 100 mM, particularly 10 mM to 50 mM, can be used, but the buffer solutions are not limited thereto. Additionally, in one embodiment, acetates, citrates, etc., can be used, but the buffer solutions used are not limited thereto.

[0066] The elution step refers to the step of recovering FSH bound to the resin. Elution conditions may be conditions with a salt concentration of 0.05M to 0.2M in a buffer solution with a pH range of 7 to 8. In one embodiment, sodium sulfate, sodium chloride, ammonium sulfate, and / or ammonium chloride may be used, but the salts used are not limited to these.

[0067] The resin cleaning (CIP) step is used to completely remove any remaining residual FSH, microorganisms, and other impurities from the column to prevent residue. In one embodiment, sodium hydroxide (NaOH), phosphoric acid, etc., can be used, but are not limited to these.

[0068] The host cell protein to be removed is a concept that includes all impurities except for the FSH to be purified, as described above. These impurities can include not only the host cell protein itself, but also DNA derived from the host cell, factors used for cell growth, etc. Therefore, when the host cell protein is removed, only the target protein to be purified can be purified to a high degree of purity.

[0069] According to the present invention, the FSH purification method (i.e., the three-step column process) in steps (a) to (c) makes it possible to ultimately purify FSH with high purity and high yield, from which impurities, especially host cell proteins, are efficiently removed.

[0070] In this invention, following steps (a) to (c), any process selected from concentration and dialysis and filtration may be performed, but the invention is not limited thereto. Furthermore, a virus filtration process and a process of exchanging the buffer solution with a storage buffer solution (UF / DF) may be included, but the invention is not limited thereto.

[0071] After the final purification, the content of the host cell protein (HCP) can be in the range of 0.001 ppm to 50 ppm, particularly 0.01 ppm to 40 ppm, 0.1 ppm to 30 ppm, 1 ppm to 30 ppm, 3 ppm to 25 ppm, particularly 5 ppm to 20 ppm, and even more particularly 0.01 ppm to 12 ppm, but the content of the host cell protein is not limited thereto. In embodiments of the invention, it was confirmed that the content of the host cell protein was reduced to below 200 ppm after the first purification, below 50 ppm after the second purification, and below 15 ppm after the third purification (Examples 3-2). Specifically, it was shown that by using only three steps—immunoaffinity chromatography, hydrophobic interaction chromatography, and anion exchange chromatography—in sequence, a host cell protein content of less than 12 ppm could be achieved, thus confirming the excellent effect of the invention (Examples 3-2).

[0072] The above method can be performed by equilibrating the column with a buffer solution of pH 7 or lower before sample injection in steps (a) to (c). The buffer solution can be any or more of Tris, PBS, 3-morpholinylpropane-1-sulfonic acid (MOPS), sulfonates, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), TES, and phosphates, more specifically Tris, but the buffer solution is not limited thereto.

[0073] Furthermore, in steps (a) to (c) of the above method, washing with a washing solution with a pH of 4 or higher and 8 or lower can be performed once or multiple times. This is to remove major impurities from the culture medium and improve sample purity. The washing solution may be a washing solution comprising any one or more salts selected from sodium phosphate, potassium chloride, magnesium chloride, potassium phosphate, sodium chloride, Tris, 3-morpholinylpropane-1-sulfonic acid (MOPS), PIPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), citrate, acetate, succinate, sodium citrate, sodium acetate, sodium succinate, sodium sulfate, ammonium sulfate, ammonium chloride and / or magnesium chloride, but the washing solution is not limited thereto.

[0074] FSH separated using the purification method of the present invention can refer to FSH with a purity of 90% or higher, particularly 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher, more particularly 99% or higher, but the purity of FSH is not limited to these. When used herein, the term "purity" refers to pure FSH from which impurities have been removed. For example, if the purity is 92%, the remaining 8% refers to impurities. Furthermore, purity can simply represent the purity of the substance separated from the eluent, but the final percentage of purity may vary depending on the purity of the sample.

[0075] In addition, the purity of FSH can be analyzed by HPLC, SDS-PAGE, etc. after it is purified from the eluent, but the analytical methods are not limited to these.

[0076] In this embodiment, it was confirmed that the final yield was the best and the content of host cell protein (HCP) was the lowest when the process was performed in the order of IAC->HIC->AEX, which is the purification method of the present invention. These results not only confirm that the purification efficiency increases with the number of chromatography steps performed, but also show that the purification efficiency varies between different steps depending on how the process is performed, even though the same three types of chromatography (i.e., immunoaffinity chromatography, hydrophobic interaction chromatography, and anion exchange chromatography) are used in each step.

[0077] Furthermore, FSH purified by the purification method of the present invention can be used as a therapeutic protein. When used herein, the term "therapeutic protein," as a general term for proteins commonly used in biomedicine, refers to proteins with various physiological activities. These physiological activities refer to the activities that regulate gene expression and physiological function to correct abnormalities caused by the deficiency or over-secretion of substances involved in functional regulation in the body, and may include general protein therapeutic agents.

[0078] [Beneficial Effects]

[0079] According to the purification method of the present invention, by effectively arranging the sequence of the purification process, the efficiency of the process operation can be improved, and the yield of FSH can be increased at the same time, while maximizing the ability to remove impurities. Attached Figure Description

[0080] Figure 1 The results of purity analysis performed by SE-HPLC are shown.

[0081] Figure 2 The results of oxide content analysis performed by RP-HPLC are shown. Detailed Implementation

[0082] The invention will be described in more detail below with reference to the following embodiments. However, these embodiments are merely illustrative and the scope of the invention is not limited thereto.

[0083] Example 1: Purification process

[0084] Example 1-1: Sequence of the purification process

[0085] [Table 1]

[0086]

[0087]

[0088] As described above, to optimize the sequence of purification steps and processes for FSH, the FSH protein was purified using 15 different purification methods. Depending on the experiment, purification was performed up to step 1, step 2, or step 3, and the purified solution was collected at each step.

[0089] Since the purified solution from each step is used as a loading sample for the next step, pretreatment can be performed as needed (e.g., dilution or buffer exchange for the AEX process, salt addition for the HIC process, etc.). All basic process conditions used for IAC, HIC, and AEX purification are used in the same manner.

[0090] Examples 1-2: Purification conditions for each step

[0091] The buffers and specific process conditions used in each purification process are summarized in Tables 2 through 4 below.

[0092] [Table 2] IAC Process

[0093] Step Buffer used Injection volume (CV) Equilibration 20 mM Tris (pH 7.6) 7 Injection Culture broth or purified solution from previous step - Equilibration 20 mM Tris (pH 7.6) 5 Wash 1 5 mM Tris (pH 7.6) 7 Wash 2 20 mM Tris (pH 7.6) / 1 M NaCl 5 Wash 3 20 mM Tris (pH 7.6) / 0.2 M MgCl2 7 Elution 20 mM Tris (pH 7.6) / 2 M MgCl2 7 CIP 0.5 M acetic acid 5

[0094] [Table 3] HIC Process

[0095] Step Buffer used Injection volume (CV) Equilibration 20 mM Tris (pH 7.6) / 2 M NaCl 5 Injection Culture broth or purified solution from previous step - Equilibration 20 mM Tris (pH 7.6) / 2 M NaCl 3 Elution 20 mM Tris (pH 7.6) 10 Regeneration WFI 5 CIP 0.5 N NaOH 5

[0096] [Table 4] AEX Procedure

[0097] Step Buffer used Injection volume (CV) Equilibration 20 mM Tris (pH 7.6) 5 Injection Culture broth or purified solution from previous step - Equilibration 20 mM Tris (pH 7.6) 3 Wash 30 mM sodium acetate (pH 5.6) 10 Equilibration 20 mM Tris (pH 7.6) 5 Elution 20 mM Tris (pH 7.6) / 0.1 M NaCl 10 CIP 0.5 N NaOH 5

[0098] Example 2: Comparison of HCP content based on the washing step in the IAC purification process

[0099] Example 2-1: IAC Cleaning Procedure

[0100] If the HCP removal capacity increases with the number of washes during the IAC purification process, the final purification efficiency will be excellent. Under this assumption, the inventors sought to determine the optimal conditions for the number of washes, the type of wash solution, etc., during the IAC purification process. Specifically, the wash solvents used in the IAC purification process are shown in Table 5 below, and the wash steps are shown in Table 6 below.

[0101] [Table 5]

[0102]

[0103]

[0104] [Table 6]

[0105] Test number Wash step of IAC 1 Wash 1 2 Wash 2 3 Wash 3 4 Wash 1 + Wash 2 5 Wash 1 + Wash 3 6 Wash 2 + Wash 3 7 Wash 1 + Wash 2 + Wash 3

[0106] Example 2-2: Comparison of HCP content based on the washing methods used in the IAC purification process

[0107] As a result of the IAC purification process performed under the conditions of Example 2-1, the purification yield shown below was confirmed to vary with the type of washing solvent and the number of washing cycles.

[0108] [Table 7]

[0109]

[0110]

[0111] [Table 8]

[0112] Test number Condition variation of second wash Purification yield (%) HCP (ppm) Load Culture broth (HCCF) 8933549.5 1 0.2 M NaCl 66.7 316.2 2 1.0 M NaCl 68.2 187.0 3 3.0 M NaCl 66.5 179.0 4 4.0 M NaCl 64.9 181.4

[0113] [Table 9]

[0114] Test number Condition variation of third wash Purification yield (%) HCP (ppm) Load Culture broth (HCCF) 8933549.5 5 0.005 M MgCl2 68.6 320.2 6 0.01 M MgCl2 70.4 203.4 7 0.1 M MgCl2 69.2 189.4 8 0.3 M MgCl2 70.3 191.0 9 0.5 M MgCl2 60.8 190.2

[0115] Specifically, as shown in Table 7, it was confirmed that the HCP content was lowest when three or more washes were used in the IAC purification process; furthermore, even with the same number of washes, the HCP content varied depending on the wash solution used. Additionally, as shown in Tables 8 and 9, it was confirmed that when the second and / or third wash solutions were used at various concentrations, the HCP content was lowest at specific concentrations of the second and / or third wash solutions. Specifically, it was confirmed that when the wash solution used in the second wash step was sodium chloride with a salt concentration in the range of 1 M to 4 M, the purification yield was 60% or higher, and the HCP content was 250 ppm or lower; furthermore, when the wash solution used in the third wash step was magnesium chloride with a salt concentration in the range of 0.01 M to 0.5 M, the purification yield was 60% or higher, and the HCP content was 250 ppm or lower.

[0116] These results indicate that, in the purification method of the present invention, the number of washes and the type and concentration of the wash solvent are the most important factors in the IAC purification process.

[0117] Example 3: Confirmation of purification results according to the order of purification process

[0118] The inventors attempted to further confirm the purification results based on the order in which IAC, HIC, and AEX were used in the purification process.

[0119] Example 3-1: Yield Results

[0120] The Octet Qk analyzer was used to quantitatively analyze proteins in culture medium and purification solutions. The Octet Qk method is a quantitative analysis method using an antigen / antibody reaction, and only the target protein can be quantified. The analytical method is as follows:

[0121] The biosensor was stabilized using Kinetics buffer (Pall-ForteBio).

[0122] Immobilization was performed using Kinetics buffer and biotinylated anti-FSH antibody.

[0123] Quantify each sample that has been appropriately diluted with Kinetics buffer.

[0124] Regeneration was performed using a buffer solution (20 mM Tris-HCl (pH 7.6) and 1.7 M MgCl2).

[0125] Neutralize using Kinetics buffer.

[0126] Subsequently, the purified solution from each step was quantified and the yield was analyzed.

[0127] The final yields, in order of the purification process, are summarized in Table 10.

[0128] [Table 10]

[0129]

[0130]

[0131] As a result, it was confirmed that the final yield was best when the process was performed in the order of IAC->HIC->AEX (Case 1), which is the purification method of the present invention. This result confirms that the purification efficiency varies between different processes depending on the order in which the processes are performed, even though the same type of chromatography (i.e., immunoaffinity chromatography, hydrophobic interaction chromatography, and anion exchange chromatography) is used in each process, and performing the process in the order of IAC->HIC->AEX is the most important factor.

[0132] Example 3-2: Conditions and results for HCP analysis

[0133] As a pretreatment step for HCP content analysis, the purified solution was concentrated with pure water and the buffer was exchanged using an Amicon centrifugal filter and centrifuged. Quantitative analysis of proteins in the buffer-exchanged sample was performed using the Octet Qk analyzer from Example 3-1. HCP content analysis was performed using the concentrated and buffer-exchanged purified solution. The HCP analysis was performed using the "CHO ​​Host Cell Protein Generation 3" kit (Cygnus). The analytical method is as follows.

[0134] Each sample was diluted appropriately with dilution buffer so that the HCP content in the sample fell within the range of the standard.

[0135] Anti-CHO antibody:HRP was loaded into each well at a rate of 100 μL.

[0136] Standards and each sample were loaded into each well at a rate of 50 μL per well and allowed to react at 24 ± 4 °C for 2 hours at a speed of 400 rpm to 600 rpm.

[0137] Discard the reactants and wash four times with washing buffer.

[0138] TMB substrate was loaded into the wells at a rate of 100 μL per well and allowed to react at 24 ± 4 °C for 30 minutes.

[0139] The reaction was terminated by adding a stop solution to each well in an amount of 100 μL.

[0140] The HCP content for each purification process was calculated in ppm using the quantitative values ​​of the target protein and the measured HCP content.

[0141] [Table 11]

[0142]

[0143] As a result, it was confirmed that the HCP content was lowest when the process was performed in the order of IAC->HIC->AEX (test 3), which is the purification method of the present invention. Furthermore, it was confirmed that the HCP content was lower when multiple types of chromatography were used compared to using a single chromatography. These results confirm that the purification efficiency not only increases with the number of chromatography steps, but also varies between different steps depending on the order in which the steps are performed, even though the same three types of chromatography (i.e., immunoaffinity chromatography, hydrophobic interaction chromatography, and anion exchange chromatography) are used in each step.

[0144] Specifically, it can be observed that the HCP content can differ by 3,000 times or more between purification processes performed in the order of HIC->AEX->IAC (Experiment 8) and purification processes performed in the order of IAC->HIC->AEX, i.e., the purification method of the present invention, even though the same three types of chromatography (i.e., immunoaffinity chromatography, hydrophobic interaction chromatography, and anion exchange chromatography) are used in both processes. These results indicate that the process sequence is the most important factor in the purification method of the present invention.

[0145] Example 3-3: Results of purity analysis by SE-HPLC

[0146] An attempt was made to analyze the purity of FSH by SE-HPLC. A mobile phase of pH 7 was prepared by adding acetonitrile to a sodium phosphate solution. The column was then equilibrated and injected. Purity was confirmed based on the peaks obtained after sufficient flow through the mobile phase. The purity of each purification step is shown in [the table / image / image]. Figure 1 middle.

[0147] like Figure 1 As shown in the figure, the purity of the main peak (i.e., the dimer peak) increases as the purification process continues.

[0148] Examples 3-4: Results of oxide content analysis by RP-HPLC

[0149] An attempt was made to analyze the oxide content of FSH by RP-HPLC. A mobile phase (B) at pH 2.5 was prepared by adding acetonitrile and potassium phosphate to a potassium phosphate mobile phase (A). The column was then equilibrated and injected. The oxide content was confirmed by peaks obtained when the two mobile phases were passed through them under concentration gradient conditions. The oxide content for each purification step is shown in [the table / image / image]. Figure 2 middle.

[0150] As in Figure 2 As shown in the figure, it is confirmed that as the purification process continues, the purity increases while the oxide peak decreases.

[0151] In summary, those skilled in the art will understand that the present invention can be embodied in other specific forms without altering its technical concept or essential characteristics. Therefore, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of the invention. Rather, the invention is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method for purifying follicle-stimulating hormone (FSH), the method comprising an immunoaffinity chromatography (IAC) step, wherein the immunoaffinity chromatography (IAC) includes a washing step, wherein the washing is performed at least three times. The cleaning solution in the second cleaning step contains 10 mM to 30 mM Tris and sodium chloride with a concentration between 1 M and 3 M. The cleaning solution in the third cleaning step contains 10 mM to 30 mM Tris and magnesium chloride with a salt concentration in the range of 0.1 M to 0.3 M. The follicle-stimulating hormone (FSH) purified through the third washing step has a purity of less than 250 ppm HCP. The cleaning step described herein is performed using a cleaning solution with a pH of 7.

6. The method for purifying follicle-stimulating hormone (FSH) is performed in the following order, the method comprising: (a) Perform immunoaffinity chromatography (IAC); (b) Perform hydrophobic interaction chromatography (HIC); and (c) Perform anion exchange chromatography (AEX).

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