Method for purifying ophthalmic protein drugs

Abstract

The present invention relates to a method for purifying an ophthalmic protein drug having an isoelectric point within a specific range by applying cation exchange chromatography or anion exchange chromatography. According to the present invention, a high-purity ophthalmic protein drug can be produced more economically by applying a triple or quadruple chromatography process.

Description

Technical Field

[0001] The present invention relates to a method for purifying aflibercept used as an active ingredient of an ophthalmic protein drug. According to the present invention, aflibercept having an isoelectric point value within a specific range can be purified by performing cation exchange chromatography or anion exchange chromatography, thereby improving the quality and yield of the ophthalmic protein drug. Background Art

[0002] Angiogenesis is believed to be involved in the pathogenesis of a variety of diseases such as solid tumors, proliferative retinopathy, age-related macular degeneration (AMD) and rheumatoid arthritis. Vascular endothelial growth factor (VEGF) is one of the factors required for angiogenesis, expressed in human cancers, and also plays a key role in tumor neovascularization. It is also known that high concentrations of VEGF in the ocular fluid of patients with diabetes and other ischemic retinopathy are highly correlated with angiogenic activity (Aiello LP, et al., N Engl J Med 1994; 331: 1480-1487), and lead to the localization of growth factors in the choroidal neovascular membrane of patients with age-related macular degeneration (AMD) (Amin R1, et al. Invest Ophthalmol Vis Sci. 1994 Jul; 35 (8): 3178-88). Therefore, anti-VEGF antibodies or VEGF inhibitors may be promising candidates for the treatment of solid tumors and intraocular angiogenesis-related diseases.

[0003] Aflibercept, one of the VEGF inhibitors, is a recombinant fusion protein consisting of the vascular endothelial growth factor (VEGF) binding portion from the extracellular domains of human VEGF receptors 1 and 2, fused to the Fc portion of human IgG1 immunoglobulin. Aflibercept has been marketed in the United States and Europe under the trade name Eylea TM Approved to treat wet macular degeneration.

[0004] By using gene recombination technology, Aflibercept can be expressed in animal cells such as CHO cells and NS0 cells. Aflibercept exists in the form of dimers and exhibits biological activity. It is known that the expressed dimer exhibits a form in which a glycan structure is bound to a total of ten asparagine residues and negatively charged sialic acid is contained in the terminal part of each glycan structure, and therefore, the Aflibercept substance expressed by cell culture exhibits a complex form having an isoelectric point value in a wide range of pH 6.0-10 due to the presence of various contents of sialic acid sugars.

[0005] In order to use protein drugs produced using gene recombination technology on the human body, very strict safety verification procedures and effectiveness verification procedures are required, and therefore, these procedures require an effective purification process that can achieve purification at an extremely high purity level. That is, the culture produced by cell culture contains various contents of sialic acid sugars and impurities from various processes, and therefore, a process is required to purify therefrom only proteins having isoelectric point values ​​distributed within the standard settings for strict safety verification and effectiveness verification. In addition, such proteins can only be used industrially when economic efficiency is ensured by ensuring a sufficient purification yield.

[0006] Conventional purification process schemes (or sequences) for antibody drugs or Fc fusion proteins generally include a chromatography step for testing the ability of the antibody molecule to preferentially (preferentially) bind or immobilize on the solid phase of a chromatography column when compared to the binding strength or retention strength of various impurities. These chromatography processes are characterized by performing a series of chromatography methods under predetermined conditions, including various techniques such as protein A or protein G solid phase affinity chromatography, solid phase metal affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, size exclusion gel filtration chromatography, mixed mode or multimodal chromatography, and hydroxyapatite chromatography. However, the specific conditions for performing chromatography to purify aflibercept (or aflibercept biosimilar) substances of high purity have not yet been disclosed. Summary of the invention

[0007] Technical issues

[0008] The present inventors attempted to complete a series of processes for developing a high-purity drug that can be used for humans by effectively removing polymers, endotoxins, heterologous proteins and nucleic acids, impurity analogs in the form of high isoelectric points, etc. from aflibercept produced by cell culture using animal cell lines.

[0009] The present inventors excluded size exclusion chromatography which would negatively affect the yield due to the limited injection amount of the sample, and attempted to complete a process having an advantage of being unlikely to denature the protein due to the non-use of an organic solvent or the like.

[0010] Specifically, based on the fact that aflibercept contains a total of ten N-linked glycan structures, which is much more than that of general antibody drugs, and that negatively charged sialic acid is contained in the terminal portion of each glycan structure, and therefore, due to the presence of various contents of sialic acid sugars, aflibercept exhibits a complex form having isoelectric point values ​​in a wide range of pH 6.0-10, the present inventors attempted to provide a technology for purifying a substance having an isoelectric point value only within a very specific range (pH 6.0-8.3) with high purity by performing anion exchange chromatography and cation exchange chromatography under specific conditions.

[0011] Technical Solution

[0012] According to one aspect of the present invention, there is provided a cation exchange chromatography method, the method comprising the steps of: (a) loading the aflibercept mixture onto a cation exchange resin equilibrated with a buffer solution of pH 4.5-6.6;

[0013] (b) washing with a buffer solution containing 0-40 mM sodium chloride or potassium chloride at pH 4.5-6.6; and

[0014] (c) under the condition of pH 4.5-6.6, by eluting with a buffer solution containing 40-120 mM sodium chloride or potassium chloride at a higher concentration than that in step (b), aflibercept having an isoelectric point in the range of 6.0-8.3 is collected, thereby purifying aflibercept having an isoelectric point in the range of 6.0-8.3.

[0015] The buffer solution used for column equilibration, washing and elution can be sodium acetate or sodium phosphate buffer.

[0016] Furthermore, the ligand used for the cation exchange resin may be a carboxymethyl (CM) group.

[0017] According to another aspect of the present invention, there is provided an anion exchange chromatography method, the steps of which include:

[0018] (a) loading the aflibercept mixture onto an anion exchange resin equilibrated with a buffer solution of pH 7.5-9.0;

[0019] (b) washing with a buffer solution containing 0-20 mM sodium chloride or potassium chloride at pH 7.5-9.0; and

[0020] (c) Aflibercept having an isoelectric point in the range of 6.0-8.3 can be purified by eluting with a buffer solution containing 20-150 mM sodium chloride or potassium chloride at a higher concentration than that in step (b) under the condition of pH 7.5-9.0 to collect aflibercept having an isoelectric point in the range of 6.0-8.3.

[0021] The buffer solution used for column equilibration, washing and elution can be sodium phosphate or Tris buffer.

[0022] Additionally, the ligand used in the anion exchange resin may be a quaternary ammonium (Q) group.

[0023] Prior to performing ion exchange chromatography on the aflibercept mixture, at least one selected from the group consisting of protein A affinity chromatography, mixed-mode or multimodal chromatography, and desalting ultrafiltration may be applied.

[0024] Furthermore, aflibercept having an isoelectric point in the range of 6.0-8.3 can be purified by performing a cation exchange chromatography method and an anion exchange chromatography method in combination.

[0025] According to yet another aspect of the present invention, there is provided a method for formulating the final refined Aflibercept to have any one of the following three compositions suitable for intravitreal injection:

[0026] (a) a composition of 10 mM sodium acetate, 7% sucrose, and 0.03% polysorbate 20, wherein the composition has a pH of 5.5;

[0027] (b) a composition of 10 mM sodium acetate, 6.5% sucrose, 15 mM sodium chloride, 0.03% polysorbate 20, wherein the composition has a pH of 5.5; and

[0028] (c) a composition of 50 mM sodium acetate, 6% sucrose, and 0.03% polysorbate 20, wherein the composition has a pH of 5.5.

[0029] The present invention as described above will be described in detail.

[0030] One aspect of implementing the present invention is a method of purifying aflibercept, which is a target protein expressed by cell culture from Chinese hamster ovary (CHO) cells, which are transgenic animal cells, with high yield and high purity.

[0031] Since the injectable amount of the sample in size exclusion chromatography is limited by the industrial production scale, resulting in a negative impact on the yield, size exclusion chromatography is excluded in the present invention. In addition, reversed phase chromatography, which may denature proteins due to the use of organic solvents, and hydrophobic chromatography, which may denature proteins due to the use of high concentrations of salt, are also excluded.

[0032] According to a preferred embodiment of the present invention, specific conditions for performing cation exchange chromatography for refining highly pure substances having only isoelectric point values ​​within a specific range while removing impurities in the form of relatively high isoelectric point analogs from aflibercept substances having various isoelectric points caused by heterogeneity in the content of sialic acid present in the terminal of the glycan structure are applied once or more.

[0033] According to another preferred embodiment of the present invention, specific conditions for performing anion exchange chromatography for refining highly pure substances having only isoelectric point values ​​within a specific range while removing impurities in the form of relatively high isoelectric point analogs from aflibercept substances having various isoelectric points caused by heterogeneity in the content of sialic acid present in the terminal of the glycan structure are applied once or more.

[0034] According to another preferred embodiment of the present invention, specific conditions for performing cation exchange chromatography and specific conditions for performing anion exchange chromatography may be applied together.

[0035] In embodiments, prior to carrying out the purification method of the present invention, Protein A solid phase affinity chromatography, mixed mode or multimodal chromatography and ultrafiltration (dialysis) may be additionally performed.

[0036] Beneficial Effects

[0037] Features and advantages of the present invention are summarized as follows.

[0038] (a) The present invention provides a novel purification method, which can effectively purify aflibercept (or aflibercept biosimilar) substances having only isoelectric point values ​​within the range of pH 6.0-8.3.

[0039] (b) The present invention provides a purification method that can overcome the difficulties caused by the physicochemical properties of aflibercept having a wide range of isoelectric point distribution and produce aflibercept in large quantities on an industrial scale.

[0040] (c) The present invention provides a purification method for producing aflibercept in large quantities with high yield even without performing ultrafiltration, which corresponds to a buffer exchange step that causes a decrease in yield between each chromatography process.

[0041] (d) According to the present invention, impurity proteins are effectively removed by subjecting aflibercept substance expressed by cell culture to a three- or four-time chromatography process, thereby obtaining aflibercept suitable for intravitreal administration and intraocular administration to patients suffering from various diseases such as wet age-related macular degeneration and diabetic macular edema. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a graph showing the results of performing multimodal chromatography using Capto adhere resin. The fractions obtained during loading and washing are indicated in the bottom of the figure.

[0043] Figure 2 The results of Coomassie staining of isoelectric point electrophoresis of aflibercept fractions having isoelectric point values ​​within the range of pH 6.0-8.3, which is included in the purification method according to the present invention by using cation exchange chromatography, are shown.

[0044] Lane 1: pI marker (SERVA)

[0045] Lane 2: Aflibercept internal standard

[0046] Lane 3: Cation exchange resin chromatography fraction (Example 1-1)

[0047] Lane 4: Cation exchange resin chromatography fraction (Example 1-2)

[0048] Lane 5: Cation exchange resin chromatography fractions (Examples 1-3)

[0049] Lane 6: Cation exchange resin chromatography fractions (Examples 1-4)

[0050] Lane 7: Cation exchange resin chromatography fractions (Examples 1-5)

[0051] Figure 3 The results of Coomassie staining of isoelectric point electrophoresis of aflibercept fractions having isoelectric point values ​​within the range of pH 6.0-8.3, which is included in the purification method according to the present invention by using anion exchange chromatography, are shown.

[0052] Lane 1: pI marker (SERVA)

[0053] Lane 2: Aflibercept internal standard

[0054] Lane 3: Anion exchange resin chromatography fraction (Example 2-1)

[0055] Lane 4: Anion exchange resin chromatography fraction (Example 2-2)

[0056] Lane 5: Anion exchange resin chromatography fraction (Example 2-3)

[0057] Lane 6: Anion exchange resin chromatography fractions (Example 2-4)

[0058] Lane 7: Anion exchange resin chromatography fractions (Example 2-5)

[0059] Lane 8: Anion exchange resin chromatography fractions (Example 2-6)

[0060] Figure 4 The results of Coomassie staining of isoelectric point electrophoresis of a washing fraction and an elution fraction of an anion exchange chromatography containing an elution sample loaded with a cation exchange chromatography in the purification method according to the present invention are shown.

[0061] Lane 1: marker (SERVA)

[0062] Lane 2: Aflibercept internal standard

[0063] Lane 3: Wash fraction from anion exchange resin chromatography

[0064] Lane 4: elution fraction from anion exchange resin chromatography

[0065] Lane 5: Sample loaded onto anion exchange resin chromatography (eluate from cation exchange resin chromatography). DETAILED DESCRIPTION

[0066] The purification method for aflibercept of the present invention will be described in detail through steps and examples. These steps or examples are provided to illustrate the present invention, and the scope of the present invention is not limited thereto.

[0067] Step 1: Obtaining a pretreatment solution from a culture of animal cells expressing aflibercept

[0068] First, a pretreatment solution is obtained by removing animal cells from a culture of animal cells expressing aflibercept. The animal cells expressing aflibercept are preferably mammalian cells, rodent cells, avian cells or insect cells, more preferably Chinese hamster ovary (CHO) cells, VERO cells, baby hamster kidney (BHK) cells and NS0 cells, and most preferably CHO cells.

[0069] Animal cells expressing aflibercept can be cultured by various methods known in the art. For example, at least one selected from the group consisting of batch culture, fed-batch culture, repeated fed-batch culture, continuous culture, and perfusion culture among CHO cell culture methods known in the art can be performed.

[0070] Since various proteins, sugars, and fats are generally incorporated into the culture of animal cells, a pretreatment step for removing impurities is required before performing column chromatography in order to improve the purification efficiency in the purification method according to the present invention.

[0071] Preferably, the pretreatment step may be performed by various methods known in the art such as depth filtration and membrane filtration.

[0072] Step 2: Purification of Aflibercept from Pre-treatment Cultures

[0073] Prior to performing cation exchange chromatography or anion exchange chromatography for refining aflibercept having only isoelectric point values ​​within a specific range, the pretreated culture may be purified by at least one method selected from the group consisting of affinity chromatography, mixed mode or multimodal chromatography, and desalting ultrafiltration, but is not limited thereto.

[0074] For monoclonal antibodies and Fc fusion proteins, the industry standard for purification processes generally includes a purification process comprising several steps. In the present example, the pre-treated culture was initially purified using protein A affinity chromatography. This is a purification step in which an affinity ligand called protein A that binds to the Fc region of the Fc fusion protein or antibody is applied. The pre-treated culture was loaded onto a protein A column, washed, and then eluted with an acetate or citrate buffer solution at about pH 3, thereby obtaining a pre-purified aflibercept fraction.

[0075] Protein aggregates or high molecular weight protein species are one type of major impurities that need to be removed from biopharmaceuticals containing Fc fusion proteins or antibody molecules. For example, protein aggregates and other contaminants need to be removed from biopharmaceuticals before the product is used for diagnosis, treatment or other applications. In the examples of the present invention, mixed mode or multimodal chromatography is used, and therefore, this chromatography is performed by loading the pre-purified fractions on mixed mode or multimodal resins without any ultrafiltration process.

[0076] Multimodal chromatography in which two or more sites that are distinct from each other but work together to interact with the target protein can be advantageously applied. Protein aggregates and other impurities in the culture are removed from the elution pool to obtain a higher purity of the aflibercept substance.

[0077] The mixed mode or multimodal resin used for purification of aflibercept in the present invention is commercially available, and an example thereof is Capto MMC TM (GE Healthcare), Capto stick TM (GE Healthcare), MBIHyperCel TM (Pall) et al. Compared with conventional ion exchangers, Capto adhere TM The multimodal resin used in the embodiments of the present invention is Capto adhere. TM , but not limited to this.

[0078] The mixed mode or multi-mode chromatography is performed by a column equilibration step, a loading step and an elution step, and the mixed mode or multi-mode chromatography may further include one or more washing steps after the loading step and before the elution step. The conditions for performing the chromatography may be controlled so that a high-purity aflibercept substance may be obtained without binding to the resin during the loading step and the washing step, or a high-purity aflibercept substance may be obtained in the elution step after first binding to the resin. In an example of the present invention, a column (GE Healthcare, XK16 / 20) was filled with 10 mL of Capto adhere (GE Healthcare) resin, and then the column was equilibrated with 50 mM sodium acetate buffer solution (pH 5.5) containing 20 mM sodium chloride, and the solution (equivalent to the equilibration buffer solution) that flowed out during the loading step and the washing step and was not bound to the resin was collected (e.g. Figure 1), so that protein aggregates and other impurities can be effectively removed, and it is possible not to perform an ultrafiltration process and ion exchange chromatography, the ultrafiltration process is performed to exchange the buffer solution of the loaded sample to load the loaded sample in the next step.

[0079] Step 3: Refining Aflibercept with an Isoelectric Point Value within a Specific Range

[0080] Prior to performing cation exchange chromatography or anion exchange chromatography for refining aflibercept having only isoelectric point values ​​within a specific range, the pretreated culture may be purified by at least one method selected from the group consisting of affinity chromatography, mixed mode or multimodal chromatography, and desalting ultrafiltration, but is not limited thereto.

[0081] In order to analyze the isoelectric point pattern, isoelectric focusing was used. After preparing 20 μg of each standard solution and test solution, each solution was loaded onto Novex pH 3-10IEF Gel 1.0 mm, 10 wells (Life Technologies) and electrophoresed at 100 V, 200 V, and 500 V for 1 hour, 1 hour, and 30 minutes, respectively. The gel was solidified with 12% trichloroacetic acid for 30 minutes, washed 3 times with ultrapure water for 10 minutes each time, stained with Coomassie staining solution, and then observed.

[0082] Hereinafter, performing cation exchange chromatography (Example 1), performing anion exchange chromatography (Example 2), and performing cation exchange chromatography and anion exchange chromatography in combination will be described separately.

[0083] <Example 1: Performing cation exchange chromatography>

[0084] (1) Cation exchange chromatography

[0085] Cation exchange chromatography was performed on the aflibercept sample solution collected by protein A affinity chromatography or the aflibercept sample solution collected from protein A affinity chromatography and multimodal chromatography performed together to purify only the aflibercept species having isoelectric point values ​​within a specific range.

[0086] Examples of the charged functional groups of the cation exchange resin used in the present invention may be carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), etc., and examples of the carrier of the cation exchange resin may be polystyrene, polystyrene / divinylbenzene, cellulose, dextran, agarose, and Toyopearl. Examples of the cation exchanger used in the present invention particularly have carboxymethyl (CM).

[0087] Any buffer solution that can have a buffering capacity can be used as the buffer solution used in the cation exchange chromatography process of the present invention, and examples thereof include sodium citrate, sodium acetate, sodium phosphate, potassium phosphate, and glycine-HCl.

[0088] Various buffer solutions can be used as balancing solutions, particularly sodium acetate and sodium phosphate buffer solutions, and the pH used is 4.5-6.6. Various buffer solutions can be used as washing solutions, particularly sodium acetate and sodium phosphate buffer solutions, and the pH used is 4.5-6.6, and the buffer solution can contain low concentrations of sodium chloride or potassium chloride (a concentration range of 0-40mM, which means that sodium chloride or potassium chloride is not included, or if present, sodium chloride or potassium chloride is included in a concentration range greater than 0-40mM). Various buffer solutions can be used as elution solutions, particularly sodium acetate and sodium phosphate buffer solutions, and the pH used is 4.5-6.6. It has been demonstrated that, along with the increase of the salt concentration of sodium chloride or potassium chloride, proteins with lower pI values ​​are first eluted sequentially. In order to purify materials with an isoelectric point of 8.3 or less, elution is preferably carried out with a salt concentration of about 120mM or less.

[0089] When performing cation exchange chromatography, the present inventors used a solution in the range of pH 4.5-6.6 during the equilibration step, the washing step, and the elution step. The reason is that under conditions below pH 4.5, aflibercept substances with low sialic acid content are collected, while under conditions above pH 6.6, aflibercept substances with isoelectric point values ​​within a specific range (pH 6.0-8.3) are lost, resulting in reduced purification efficiency.

[0090] The steps will be described below with reference to specific examples of the present invention. 1) NaCl salt gradient elution and 2) pH gradient elution as preliminary tests for finding the appropriate pH and salt concentration for pI cleavage were performed by using a Tricon column (GE healthcare, diameter 0.5 cm, height 10 cm) filled with 2 mL of CM-Sepharose FF (GE healthcare), which is a weakly acidic cation exchange resin.

[0091] First, for the NaCl salt gradient elution test, the column was equilibrated with 10 column volumes (CV) of a 50 mM sodium acetate (pH 5.5) equilibration buffer solution. The aflibercept sample solution obtained by performing protein A affinity chromatography or Capto adhere multimodal chromatography in step 2 as described above was diluted with each equilibration buffer solution, and the pH of the sample solution was adjusted to be the same as that of the equilibration buffer solution. Thereafter, the sample solution was loaded onto the column at a flow rate of 0.3 mL / min. After washing the column with the same 5 CV equilibration buffer solution, the elution buffer (50 mM sodium acetate, 0.5 M sodium chloride, pH 5.5) was allowed to flow from 0% to 100% for 30 CV, thereby eluting the protein by increasing the salt concentration, and 16 fractions were collected by an automatic fraction collector. The collected fractions were analyzed by isoelectric electrophoresis, and the results demonstrated that, as the salt concentration of sodium chloride increased, proteins with lower pI values ​​were sequentially eluted first. In order to purify a substance having an isoelectric point of 8.3 or less, elution is preferably performed at a salt concentration of about 120 mM or less.

[0092] For the pH gradient elution test, the column was equilibrated with 10CV of an equilibration buffer solution (20mM sodium acetate, 20mM sodium phosphate, pH 5.5), and the aflibercept sample solution was treated by the same method as in the salt concentration gradient elution test. After washing the column with 5CV of an equilibration buffer solution, the elution buffer (20mM sodium acetate, 20mM sodium phosphate, pH 8.5) was allowed to flow from 0% to 100% for 30CV, thereby eluting the protein by increasing the pH, and 20 fractions were collected by an automatic fraction collector. The collected fractions were analyzed by isoelectric electrophoresis, and the results demonstrated that, as the pH increased, proteins with lower pI values ​​were sequentially eluted first. As a result of performing a stepwise pH elution condition test to explore more detailed pH conditions, it was found that the equilibration step and the washing step of the cation exchange chromatography were preferably performed under the condition of pH 6.6 or less, and for the reproducibility of future scale-up, the salt concentration increasing elution method was selected to accomplish more precise elution conditions as shown in Table 1, which can achieve a more stable process compared to the pH elution method.

[0093] According to the conditions of each example in Table 1, the resin was equilibrated with the equilibration buffer at a flow rate of 150 cm / h. Then, the aflibercept sample solution obtained by performing the protein A affinity chromatography or Capto adhere multimodal chromatography in step 2 as described above was diluted with each equilibration buffer solution, and then its pH was adjusted to be the same as the pH of the equilibration buffer solution. Thereafter, the sample solution was loaded onto a column filled with CM-Sepharose FF resin at a flow rate of 150 cm / h. Thereafter, according to the conditions of each example on Table 1, a washing step was performed using a washing buffer solution at a flow rate of 150 cm / h, thereby removing aflibercept substances having a relatively higher isoelectric point value, and then an elution buffer solution was flowed at a flow rate of 150 cm / h, thereby collecting only aflibercept substances having an isoelectric point value within a specific range (pH 6.0-8.3). The resin used in the test was regenerated with a 0.5N sodium hydroxide solution, re-equilibrated with each equilibration buffer solution, and then reused.

[0094]

Table 1

[0095] Detailed example conditions for CM-Sepharose cation exchange chromatography

[0096]

[0097] (2) Cation exchange chromatography results

[0098] Use CM-Sepharose FF (GE Healthcare) resin test various equilibrium conditions, washing conditions and elution conditions as a kind of weak cation exchange resin.Mainly use sodium acetate and sodium phosphate buffer solution as equilibrium solution, and the pH used is 4.5-6.6.Mainly use sodium acetate and sodium phosphate buffer solution as washing solution, and the pH used is 4.5-6.6, and buffer solution preferably comprises sodium chloride or potassium chloride (the concentration range of 0-40mM, which means not comprising sodium chloride or potassium chloride, or if present, then comprises sodium chloride or potassium chloride with the concentration range greater than 0mM to 40mM).Use the pH used in the elution solution of sodium acetate and sodium phosphate buffer solution to be 4.5-6.6, comprise the sodium chloride or potassium chloride of higher concentration (the concentration range of 40-120mM) in the elution solution. The sodium chloride salt concentration in the elution solution is preferably 40-100 mM, and the respective equilibration steps, loading steps, washing steps and elution steps shown in Table 1 are performed to effectively purify the aflibercept substance having the most similar isoelectric point value range (pH 6.0-8.3) to the standard aflibercept.

[0099] Table 2 and Figure 2The isoelectric point analysis results in demonstrate that chromatographic purification using CM-Sepharose FF resin can be effectively utilized by following the detailed conditions for performing chromatography on Table 1 in comparison with the standard aflibercept.

[0100] The present inventors have found that during the washing step and the elution step, by obtaining fractions corresponding to a volume unit of 1 column volume (CV) from the washing liquid and the eluent using an automatic fraction collector, and when each of these fractions is analyzed by isoelectric point analysis, and then the washing fraction and the elution fraction designated as having an isoelectric point range most similar to that of the standard aflibercept are collected, it is easier to ensure the similarity of the isoelectric point distribution. This fractionation method can be selectively used in a cation exchange chromatography process.

[0101]

Table 2

[0102] Purification results by cation exchange chromatography

[0103] Example Isoelectric point analysis results IEF gel loading amount (μg) 1-1 Figure 2 Lane 3 in 20 1-2 Figure 2 Lane 4 in 20 1-3 Figure 2 Lane 5 20 1-4 Figure 2 Lane 6 20 1-5 Figure 2 Lane 7 20

[0104] <Example 2: Performing anion exchange chromatography>

[0105] (1) Anion exchange chromatography

[0106] The aflibercept sample solution collected by protein A affinity chromatography or the aflibercept sample solution collected from protein A affinity chromatography and multimodal chromatography performed together was subjected to anion exchange chromatography to purify only the aflibercept species having isoelectric point values ​​within a specific range.

[0107] Examples of the charged functional groups with the anion exchange resin used in the present invention may be amines or amino groups, more preferably primary amines, secondary amines, tertiary amines or quaternary amines, and most preferably tertiary amines or quaternary amines, and examples of the carrier of the anion exchange resin may be polystyrene, polystyrene / divinylbenzene, cellulose, dextran, agarose and Toyopearl. Examples of the anion exchanger used in the present invention particularly have diethylaminoethyl (DEAE) ligands and quaternary ammonium (Q) ligands.

[0108] Any buffer solution that can have a buffering capacity can be used as the buffer solution used in the anion exchange chromatography process of the present invention, and examples thereof include sodium phosphate, potassium phosphate, HEPES, glycine-HCl, and Tris.

[0109] Various buffer solutions can be used as balancing solutions, particularly sodium phosphate and Tris buffer solutions, and the pH can be in the range of 7.5-9.0. Various buffer solutions can be used as washing solutions, particularly sodium phosphate and Tris buffer solutions, and the pH used is 7.5-9.0, and the buffer solution can contain low concentrations of sodium chloride or potassium chloride (a concentration range of 0-20mM, which means that sodium chloride or potassium chloride is not included, or if present, sodium chloride or potassium chloride is included in a concentration range greater than 0 to 20mM). Various buffer solutions can be used as elution solutions, particularly sodium phosphate and Tris buffer solutions. It has been demonstrated that, as the salt concentration of sodium chloride or potassium chloride increases, proteins with lower pI values ​​are first eluted sequentially. In order to purify materials with an isoelectric point of 8.3 or less, elution is preferably performed with a salt concentration of about 150mM or less.

[0110] When performing anion exchange chromatography, the present inventors used a solution in the range of pH 7.5-9.0 during the equilibration step, the washing step, and the elution step. The reason is that under conditions less than pH 7.5, aflibercept substances having isoelectric point values ​​within a specific range (pH 6.0-8.3) are lost, resulting in reduced purification efficiency, while under conditions greater than pH 9.0, aflibercept substances having a low sialic acid content are collected.

[0111] The steps will be described below with reference to specific examples of the present invention. 1) NaCl salt gradient elution and 2) pH gradient elution as preliminary tests for finding the appropriate pH and salt concentration for pI cleavage were performed by using a Tricon column (GE healthcare, diameter 0.5 cm, height 10 cm) filled with 2 mL of Q-Sepharose FF (GE healthcare), which is a strongly basic anion exchange resin.

[0112] First, for the NaCl salt gradient elution test, the column was equilibrated with 10 column volumes (CV) of a 20 mM Tris (pH 8.5) equilibration buffer solution. The aflibercept sample solution obtained by performing the protein A affinity chromatography or Capto adhere multimodal chromatography in step 2 as described above was diluted with each equilibration buffer solution, and then the pH of the sample solution was adjusted to be the same as the pH of the equilibration buffer solution. Thereafter, the sample solution was loaded onto the column at a flow rate of 0.3 mL / min. After washing the column with 5 CV of the same equilibration buffer solution, the elution buffer (20 mM Tris, 0.5 M sodium chloride, pH 8.5) was allowed to flow from 0% to 100% for 30 CV, thereby eluting the protein by increasing the salt concentration, and 13 fractions were collected by an automatic fraction collector. The collected fractions were analyzed by isoelectric electrophoresis, and the results demonstrated that, as the salt concentration of sodium chloride increased, proteins with higher pI values ​​were sequentially eluted first. In order to purify a substance having an isoelectric point of 8.3 or less, elution is preferably performed at a salt concentration of about 150 mM or less.

[0113] For the pH gradient elution test, the column was equilibrated with 10CV of an equilibration buffer solution (20mM sodium acetate, 20mM Tris, pH 8.5), and the aflibercept sample solution was treated by the same method as in the salt concentration gradient elution test. After washing the column with 5CV of an equilibration buffer solution, the elution buffer (20mM sodium acetate, 20mM Tris, pH 5.5) was allowed to flow for 30CV at 0% to 100%, thereby eluting the protein by decreasing the pH, and 25 fractions were collected by an automatic fraction collector. The collected fractions were analyzed by isoelectric point electrophoresis, and the results demonstrated that, as the pH value decreased, proteins with higher pI values ​​were sequentially eluted first. It was found that, since the aflibercept protein having a relatively low isoelectric point was not adsorbed on the column under the equilibration condition and the washing condition of pH 7.5 or less, it was preferred to perform chromatography under the condition of pH 7.5 or more. For reproducibility in future scale-up, the salt concentration increasing elution method was selected to achieve more precise elution conditions as shown in Table 3 , which can achieve a more stable process compared to the pH elution method.

[0114] According to the conditions of each example in Table 3, the resin was equilibrated with the equilibration buffer at a flow rate of 150 cm / h. Then, the aflibercept sample solution obtained by performing the protein A affinity chromatography or Capto adhere multimodal chromatography in step 2 as described above was diluted with each equilibration buffer solution, and then its pH was adjusted to be the same as the pH of the equilibration buffer solution. Thereafter, the sample solution was loaded onto a column filled with Q-Sepharose FF resin at a flow rate of 150 cm / h. Thereafter, according to the conditions of each example on Table 3, a washing step was performed using a washing buffer solution at a flow rate of 150 cm / h, thereby removing aflibercept substances having a relatively higher isoelectric point value, and then an elution buffer solution was flowed at a flow rate of 150 cm / h, thereby collecting only aflibercept substances having an isoelectric point value within a specific range (pH 6.0-8.3). The resin used in the test was regenerated with a 1N sodium hydroxide solution, re-equilibrated with each equilibration buffer solution, and then reused.

[0115]

Table 3

[0116] Detailed example conditions for anion exchange chromatography on Q-sepharose

[0117]

[0118] (2) Cation exchange chromatography results

[0119] Various equilibrium conditions, washing conditions and elution conditions are tested using Q-Sepharose FF (GE Healthcare) resin as a strongly basic anion exchange resin. Sodium phosphate and Tris buffer solution can be used as equilibrium solution in particular, and the pH used can be in the range of 7.5-9.0. Sodium phosphate and Tris buffer solution can be used as washing solution, and the pH used can be in the range of 7.5-9.0, and the buffer solution preferably comprises sodium chloride or potassium chloride of low concentration (concentration range of 0-20mM). Sodium phosphate and Tris buffer solution can be used as elution solution in particular. In order to purify the material with an isoelectric point of 8.3 or less, elution is preferably performed with a salt concentration of 20mM or greater, a salt concentration of 20mM or greater is higher than the salt concentration in the washing step and is less than 150mM, and more preferably, under the condition of pH 7.5-8.5, the sodium chloride salt concentration in the elution solution is 20-90mM. Even at a salt concentration of 90 mM or greater, aflibercept within the target isoelectric point range could be purified, but as the shape of the elution peak became relatively sharp, the difference in isoelectric point values ​​between the collected fractions tended to decrease.

[0120] The various equilibration steps, sample loading steps, washing steps and elution steps shown in Table 3 were performed to effectively purify the aflibercept substance having the closest isoelectric point value range (pH 6.0-8.3) to the standard aflibercept. Figure 3 The isoelectric point analysis results in demonstrate that chromatographic purification using Q-Sepharose FF resin can be effectively utilized by following the detailed conditions for performing chromatography on Table 3 in comparison with the standard aflibercept.

[0121] The present inventors have found that it is easier to ensure the similarity of isoelectric point distribution when fractions of a volume unit corresponding to 1 column volume (CV) are obtained from the washing liquid and the eluent by using an automatic fraction collector during the washing step and the elution step, and when each of these fractions is analyzed by isoelectric point analysis, and then the washing fraction and the elution fraction designated as having an isoelectric point range most similar to that of the standard aflibercept are collected. This fractionation method can be selectively used in an anion exchange chromatography process.

[0122]

Table 4

[0123] Purification results by anion exchange chromatography

[0124] Example Isoelectric point analysis results IEF gel loading amount (μg) 2-1 Figure 3 Lane 3 in 20 2-2 Figure 3 Lane 4 in 20 2-3 Figure 3 Lane 5 20 2-4 Figure 3 Lane 6 20 2-5 Figure 3 Lane 7 20 2-6 Figure 3 Lane 8 20

[0125] <Example 3: Combination of cation exchange chromatography and anion exchange chromatography>

[0126] Aflibercept sample solutions collected by protein A affinity chromatography or from protein A affinity chromatography and multimodal chromatography performed together were subjected to a combined application of cation exchange chromatography and anion exchange chromatography to purify only aflibercept substances having isoelectric point values ​​within a specific range.

[0127] That is, according to the methods of Examples 1 and 2, chromatography using CM-Sepharose FF resin as a cation exchange resin is first performed and then chromatography using Q-Sepharose FF resin as an anion exchange resin is performed, or chromatography using Q-Sepharose FF resin as an anion exchange resin is first performed and then chromatography using CM-Sepharose FF resin as a cation exchange resin is performed, so that the aflibercept substance having only an isoelectric point value within a specific range (pH 6.0-8.3) can be purified more easily than when ion exchange chromatography is performed alone, and the purification efficiency can also be improved.

[0128] A combined application is described in detail below, wherein chromatography is performed with CM-Sepharose FF resin as the cation exchange resin and then chromatography is performed with Q-Sepharose FF resin as the anion exchange resin. For sequential chromatography applications, it is advantageous to reduce the concentration of each salt in the wash buffer and the elution buffer to 0-30 mM. A column (GE Healthcare, XK16 / 20) filled with 10 mL of CM-Sepharose FF resin is equilibrated with an equilibration buffer (50 mM sodium acetate, pH 5.5) at a flow rate of 1 mL / min. Then, the aflibercept sample solution obtained by performing protein A affinity chromatography or Capto adhere multimodal chromatography in step 2 as described above is diluted with the equilibration buffer and then its pH is adjusted to the same pH as the equilibration buffer. Thereafter, the sample solution is loaded onto the column filled with CM-Sepharose FF resin at a flow rate of 1 mL / min. After the equilibration buffer solution was allowed to flow for 3 column volumes (CV), a washing step was performed using a washing buffer solution at a flow rate of 1 mL / min to remove aflibercept substances having a relatively high isoelectric point value, and then fractionation was performed using an automatic fraction collector while an elution buffer solution (20 mM sodium phosphate, 50 mM sodium chloride, pH 6.5) was allowed to flow at a flow rate of 1 mL / min. After only aflibercept substances having an isoelectric point value within a specific range (pH 6.0-9.0) were collected, the sample was diluted 10 times with the equilibration buffer solution in the Q-Sepharose chromatography process and adjusted to pH 8.5. A column filled with 10 mL of Q-Sepharose FF resin (GE Healthcare, XK16 / 20), which is a strongly basic anion exchange resin, was equilibrated with an equilibration buffer solution (10 mM Tris, pH 8.5) at a flow rate of 1 mL / min. Then, the aflibercept sample solution prepared by cation exchange chromatography was loaded onto the column at a flow rate of 1 mL / min. After that, a washing step was performed using a 10 mM Tris (pH 8.5) washing buffer solution containing 20 mM sodium chloride at a flow rate of 1 mL / min to remove aflibercept substances having a relatively high isoelectric point value, and then an elution buffer solution (10 mM Tris, 120 mM sodium chloride, pH 8.5) was flowed at a flow rate of 1 mL / min to collect only aflibercept substances having an isoelectric point value within a specific range (pH 6.0-8.3).

[0129] The purification yield of aflibercept having an isoelectric point value within a specific range by the combined application of cation exchange chromatography and anion exchange chromatography in Example 3 showed a value higher by about 15%-30%, as compared to cation exchange chromatography or anion exchange chromatography performed alone, and as shown from Figure 4As can be seen from the isoelectric point analysis results in , it is possible to produce aflibercept substances with very similar isoelectric point distributions compared to the standard.

[0130] Step 4: Prepare the final stock solution by recipe

[0131] In order to prepare a stock solution by finally formulating an aflibercept solution having an isoelectric point of pH 6.0 to 8.3, the aflibercept solution was concentrated using Vivaspin (Sartorius, MWCO 50 kDa), and then replaced with a formulation buffer solution collected by purification by cation exchange chromatography, anion exchange chromatography, or combined cation / anion exchange chromatography by repeated addition. Finally, polysorbate 20 was added to a content of 0.03%, thereby preparing a final stock solution containing aflibercept at a concentration of approximately 40 mg / mL. For the composition for preparing the buffer solution, a composition of 10 mM sodium acetate, 7% sucrose, 0.03% polysorbate 20 (pH 5.5) can be selected, or as a composition with increased salt concentration to improve long-term stability, a composition of 10 mM sodium acetate, 6.5% sucrose, 15 mM sodium chloride, 0.03% polysorbate 20 (pH 5.5) or a composition of 50 mM sodium acetate, 6% sucrose, 0.03% polysorbate 20 (pH 5.5) can be selected. Sterile filtration is performed using a 0.22 μm PES membrane.

[0132] As explained above, the present invention provides a method for purifying aflibercept having an isoelectric point value within a specific range by performing cation exchange chromatography or anion exchange chromatography under specific conditions thereof, and ultimately provides an advantage that a high-purity ophthalmic protein drug can be more economically produced by a process including steps 1 to 4.

[0133] Although the present invention has been described with reference to specific illustrative embodiments, it will be appreciated by those skilled in the art that the present invention may be implemented in other specific forms without departing from the technical spirit or essential characteristics of the present invention. Therefore, the above-described embodiments should be interpreted as exemplary, rather than limiting the present invention. The scope of the present invention is not limited by the detailed description as set forth above, but by the appended claims of the present invention, and it should also be understood that all changes or modifications derived from the definition and scope of the claims and their equivalents fall within the scope of the invention.

Claims

1. A method for purifying aflibercept having an isoelectric point in the range of 6.0-8.3 by performing anion exchange chromatography after cation exchange chromatography, wherein the step of cation exchange chromatography comprises the following steps: (a-1) loading the aflibercept mixture onto a cation exchange resin equilibrated with a buffer solution of pH 4.5-6.6; (a-2) washing with a buffer solution containing 0-40 mM sodium chloride or potassium chloride at pH 4.5-6.6; as well as (a-3) collecting aflibercept having an isoelectric point in the range of 6.0-8.3 by eluting with a buffer solution containing 40-120 mM sodium chloride or potassium chloride at a higher concentration than that in step (a-2) under the condition of pH 4.5-6.6; The anion exchange chromatography step comprises the following steps: (b-1) loading the aflibercept having an isoelectric point in the range of 6.0-8.3 in step (a-3) onto an anion exchange resin equilibrated with a buffer solution of pH 7.5-9.0; (b-2) washing with a buffer solution containing 0-20 mM sodium chloride or potassium chloride at pH 7.5-9.0; as well as (b-3) collecting aflibercept having an isoelectric point in the range of 6.0-8.3 by eluting with a buffer solution containing 20-150 mM sodium chloride or potassium chloride at a higher concentration than that in step (b-2) under the condition of pH 7.5-9.0, wherein the buffer solution in steps (a-1), (a-2) and (a-3) is at least one selected from the group consisting of sodium acetate and sodium phosphate buffers, wherein the buffer solution in steps (b-1), (b-2) and (b-3) is at least one selected from the group consisting of sodium phosphate and Tris buffer, Wherein the ligand used for the cation exchange resin is a carboxymethyl (CM) group, and The ligand used in the anion exchange resin is a quaternary ammonium (Q) group.

2. A method for purifying aflibercept having an isoelectric point in the range of 6.0-8.3 by performing cation exchange chromatography after anion exchange chromatography, wherein the step of anion exchange chromatography comprises the following steps: (b-1) loading the aflibercept mixture onto an anion exchange resin equilibrated with a buffer solution of pH 7.5-9.0; (b-2) washing with a buffer solution containing 0-20 mM sodium chloride or potassium chloride at pH 7.5-9.0; as well as (b-3) collecting aflibercept having an isoelectric point in the range of 6.0-8.3 by eluting with a buffer solution containing 20-150 mM sodium chloride or potassium chloride at a higher concentration than that in step (b-2) under conditions of pH 7.5-9.0; The cation exchange chromatography step comprises the following steps: (a-1) loading the aflibercept having an isoelectric point in the range of 6.0-8.3 in step (b-3) onto a cation exchange resin equilibrated with a buffer solution of pH 4.5-6.6; (a-2) washing with a buffer solution containing 0-40 mM sodium chloride or potassium chloride at pH 4.5-6.6; as well as (a-3) collecting aflibercept having an isoelectric point in the range of 6.0-8.3 by eluting with a buffer solution containing 40-120 mM sodium chloride or potassium chloride at a higher concentration than that in step (a-2) under the condition of pH 4.5-6.6, wherein the buffer solution in steps (a-1), (a-2) and (a-3) is at least one selected from the group consisting of sodium acetate and sodium phosphate buffers, wherein the buffer solution in steps (b-1), (b-2) and (b-3) is at least one selected from the group consisting of sodium phosphate and Tris buffer, Wherein the ligand used for the cation exchange resin is a carboxymethyl (CM) group, and The ligand used in the anion exchange resin is a quaternary ammonium (Q) group.

3. The method of claim 1 or 2, wherein at least one selected from the group consisting of protein A affinity chromatography, mixed-mode or multimodal chromatography, and desalting ultrafiltration is applied prior to performing cation exchange chromatography on the aflibercept mixture.

4. The method of claim 1 or 2, wherein at least one selected from the group consisting of protein A affinity chromatography, mixed-mode or multimodal chromatography, and desalting ultrafiltration is applied prior to performing anion exchange chromatography on the aflibercept mixture.

5. The method of claim 1 or 2, wherein the buffer solution in step (b-3) comprises 20-90 mM sodium chloride or potassium chloride.

6. The method of claim 1 or 2, wherein the buffer solution in step (b-3) comprises 20-90 mM sodium chloride or potassium chloride and has a pH of 7.5-8.5.

Images