Culture medium composition for culturing animal cells to produce recombinant extracellular matrix proteins and method of using the same

Abstract

Provided is a culture medium composition for culturing animal cells to produce recombinant extracellular matrix proteins, a method for producing highly pure recombinant extracellular matrix proteins, and a method for detecting recombinant extracellular matrix protein monomers.

Description

Technical Field

[0001] Disclosed are a culture medium composition for culturing animal cells to produce recombinant extracellular matrix proteins, a method for producing high-purity recombinant extracellular matrix proteins, and a method for detecting recombinant extracellular matrix protein monomers. Background Art

[0002] The extracellular matrix (ECM) is a non-cellular component in an organism formed by various substances secreted by cells to the extracellular space, and it exists in all tissues and organs of organisms. In addition to functions such as cell adhesion and physical support between cells, the extracellular matrix also plays various biological functions such as cell differentiation and growth, and signal transduction and regulation between cells. Since each tissue and organ of multicellular organisms has evolved independently according to its characteristics, the components and functions of the extracellular matrix also vary depending on the type of tissue and cell.

[0003] The extracellular matrix is basically composed of water, proteins, and polysaccharides. Among them, extracellular matrix proteins, according to the functions of the corresponding tissues, self-assemble into molecular scaffolds after adjusting biomechanical properties and compositions, etc. Most extracellular matrix proteins are expressed in trace amounts in tissues and then form multiple bonds in the form of modules to play their roles. Due to the above structural characteristics, there are many technical problems in various aspects in the field of mass production and functional research through the recombination of extracellular matrix proteins. Usually, proteins extracted from animal tissues are used, but the extraction amount is extremely small. Therefore, in order to mass-produce according to the amino acid sequences of the required extracellular matrix proteins, it is necessary to construct a more appropriate and more economical production system.

[0004] On the other hand, it has been reported that the HAPLN (Hyaluronan and proteoglycan link protein) protein in extracellular matrix proteins plays a role in stabilizing the aggregates of hyaluronic acid and proteoglycans in the extracellular matrix and is involved in cell adhesion between cells. Depending on the tissues or organs in which they are expressed, there are four HAPLN proteins in vivo, namely HAPLN1, HAPLN2, HAPLN3, and HAPLN4, and their functions and roles in each tissue or organ are known to be similar.

[0005] Korean Patent Gazette No. 10-1897340 discloses a pharmaceutical composition for improving skin elasticity or wrinkles, which includes HAPLN1 protein as an active ingredient; Korean Patent Publication Gazette Nos. 10-2019-0024727 and 10-2020-0104831 disclose a composition for cartilage regeneration and a composition for treating cartilage-related diseases, both of which include HAPLN1 protein as an active ingredient; recently, Korean Patent Gazette No. 10-2166453 discloses a composition for treating lung diseases, etc., which includes HAPLN1 protein as an active ingredient. HAPLN protein is expected to provide useful functions to humans. Therefore, it is necessary to study methods for culturing, isolating, purifying, and monomer detection, etc., to mass-produce recombinant HAPLN protein, but currently, no research has been conducted on the method for mass-producing such recombinant HAPLN protein. Summary of the Invention

[0006] Technical Problem

[0007] To provide a culture medium composition for culturing animal cells to produce recombinant extracellular matrix protein.

[0008] To provide a method for producing recombinant extracellular matrix protein with high purity.

[0009] To provide a method for detecting monomers of recombinant extracellular matrix protein.

[0010] Technical Solution

[0011] On the one hand, to provide a culture medium composition for culturing animal cells to produce recombinant extracellular matrix protein, which includes a copper compound.

[0012] The term "extracellular matrix (ECM) protein" refers to proteins present in the extracellular matrix. Exemplary types of ECM proteins include collagen, elastin, fibronectin, laminin, vitronectin, tenascin, and HAPLN (Hyaluronan and proteoglycan link protein), etc., but are not limited thereto.

[0013] The term "HAPLN (Hyaluronan and proteoglycan link protein)" is also known as hyaluronic acid and proteoglycan link protein. According to the tissues and organs where it is mainly expressed in the body, there are four types of HAPLN proteins, specifically HAPLN1, HAPLN2, HAPLN3, and HAPLN4. Taking HAPLN1 as an example, the amino acid sequence of the HAPLN protein is recorded in human HAPLN1 Accession No. NP_001875, or mouse HAPLN1 Accession No. NP_038528, etc., but not limited thereto.

[0014] The term "recombinant ECM protein" refers to a protein obtained by expressing DNA encoding an ECM protein in cells, and the ECM protein is made by genetic recombination methods. The genetic recombination can be carried out by conventional methods in the technical field.

[0015] In a specific embodiment, the recombinant extracellular matrix protein can be collagen, elastin, fibronectin, laminin, vitronectin, tenascin, or HAPLN, but not limited thereto.

[0016] The term "recombinant HAPLN protein (recombinant HAPLN, rHAPLN)" refers to a protein obtained by inserting a polynucleotide sequence encoding an HAPLN protein into a vector to construct a recombinant vector, then introducing the recombinant vector into a host cell and expressing it in the cell.

[0017] In a specific embodiment, the recombinant HAPLN protein can be any one protein selected from the group consisting of HAPLN1, HAPLN2, HAPLN3, and HAPLN4.

[0018] The term "recombinant human HAPLN protein (recombinant human HAPLN, rhHAPLN)" refers to a protein obtained by inserting a polynucleotide sequence encoding a human HAPLN protein into a vector to construct a recombinant vector, then introducing the recombinant vector into a host cell and expressing it in the cell.

[0019] The recombinant extracellular matrix protein can be a protein derived from a human or an animal. In a specific embodiment, the recombinant extracellular matrix protein can be a protein derived from a human. The term "animal cell for producing recombinant ECM protein" or "animal cell producing recombinant ECM protein" can refer to an animal cell into which a recombinant vector is introduced to enable the production of a recombinant ECM protein.

[0020] The type of the animal cell is not limited as long as it can produce recombinant ECM protein. The animal cell can be selected from the group consisting of CHO (Chinese Hamster Ovary), VERO, BHK (Baby Hamster Kidney), HeLa, NiH3T3, MDCK (Madin-Darby Canine Kidney), WI38, HEK (Human Embryonic Kidney), hybridoma, and NSO cells. The animal cell can be a CHO cell or a CHO cell variant. The CHO cell can be CHO-K1, CHO-DXB11, CHO-DG44, CHO-S, or CHO-Pro minus.

[0021] The term "copper compound" refers to a compound of copper, and copper is known to have oxidation states of +1, +2, and +3.

[0022] The type of the copper compound included in the culture medium composition is not limited. The copper compound can be a copper(I) compound, a copper(II) compound, or a copper(III) compound. The copper compound can be copper(I) oxide (Cu2O), copper(II) chloride (CuCl2), copper(II) nitrate (Cu(NO3)2), copper(II) oxide (CuO), copper(II) sulfide (CuS), or copper(II) sulfate (CuSO4), but is not limited thereto.

[0023] The medium composition can include the copper compound at a concentration of greater than about 20 μM, greater than or equal to about 30 μM, greater than or equal to about 40 μM, greater than or equal to about 50 μM, for example, greater than 20 μM to about 1000 μM, about 30 μM to about 1000 μM, about 40 μM to about 1000 μM, about 50 μM to about 1000 μM, about 50 μM to about 900 μM, about 50 μM to about 800 μM, or about 100 μM to about 1000 μM. The medium composition can be used to separate ECM proteins that are multiple bonds in a modular form into monomers by including the copper compound at a concentration greater than about 20 μM, particularly greater than or equal to about 50 μM. When the concentration of the copper compound is less than or equal to about 20 μM, the formation of protein polymers increases, thereby the yield of the protein may be reduced. Therefore, the culture medium composition can reduce the formation of recombinant ECM protein polymers by including the copper compound at a concentration greater than about 20 μM, particularly greater than or equal to about 50 μM. In summary, when the culture medium composition is used, ECM proteins can be separated into monomers, thereby solving the problem of difficulty in mass production due to the structural characteristics of ECM proteins combined in a modular form after micro-expression.

[0024] The medium composition may further include common medium components for culturing animal cells. The common medium components may be known or commercially available components required for culturing animal cells to obtain recombinant proteins.

[0025] The medium composition may further include additional additives. The medium composition may further include one or more additives selected from dimethyl sulfoxide (DMSO), glycerol, poloxamer such as poloxamer 188, EDTA, polysorbates such as polysorbate 80, cysteine, glutathione (Glutathione, GSH)), glutathione disulfide (Glutathione disulfide, GSSG) and magnesium chloride (MgCl2), but is not limited thereto.

[0026] In one embodiment, it was confirmed that when copper sulfate was added to the culture medium during the culture of animal cells producing recombinant human HAPLN1 protein, the effect of reducing protein polymer formation was excellent, thereby increasing protein production. Therefore, the culture medium composition including the copper compound can be effectively used to obtain a large amount of recombinant ECM protein from animal cells.

[0027] In a specific embodiment, the culture medium composition may be a culture medium composition for culturing animal cells for the batch production of recombinant HAPLN protein. Specifically, it may be a culture medium composition for culturing animal cells for the batch production of recombinant human HAPLN protein. More specifically, it may be a culture medium composition for culturing animal cells for the batch production of recombinant human HAPLN1 protein.

[0028] In the culture medium composition, the recombinant extracellular matrix protein may be a monomer. The "monomer" and "monomeric protein" are used interchangeably to refer to one of the proteins that make up a multi-protein complex. A complex of two or more polypeptides is called a "multimer" or "oligomer", a complex of two polypeptides is called a dimer, a complex of three polypeptides is called a trimer, and a complex of four polypeptides is called a tetramer. The culture medium composition comprising the copper compound has the effect of reducing the formation of recombinant ECM protein multimers, and thus can be used for the production of monomers of recombinant ECM proteins. That is, the culture medium composition comprising the copper compound can separate the ECM proteins that are multiply-bonded in a modular form into monomers. Therefore, the culture medium composition may be a culture medium composition for culturing animal cells for the production of monomers of recombinant ECM proteins.

[0029] On the other hand, a method for producing a high-purity recombinant extracellular matrix protein is provided, which comprises:

[0030] (1) Culturing animal cells producing recombinant ECM protein in the culture medium composition according to one aspect and obtaining a culture solution; and

[0031] (2) Separating and purifying the recombinant ECM protein from the culture solution.

[0032] The description of the culture medium composition, the recombinant ECM protein, and the animal cells is as described above.

[0033] According to the method for producing recombinant ECM protein, high-purity recombinant ECM protein can be produced in batches. Therefore, the method may be a method for batch-producing recombinant ECM protein, preferably, a method for batch-producing recombinant HAPLN protein, more preferably, a method for batch-producing recombinant HAPLN1 protein, and most preferably, a method for batch-producing recombinant human HAPLN1 protein.

[0034] The cultivation in step (1) can be carried out by methods known in the art. For example, the cultivation can be carried out by fed-batch culture, continuous culture, batch culture, etc. In a specific embodiment, the cultivation in step (1) can be fed-batch culture.

[0035] The term "fed-batch culture" is a culture method in which the culture medium is intermittently supplied, and it refers to a culture method in which the substrate in the culture solution is added at an appropriate rate without leakage, so that the substrate supply amount can be freely controlled.

[0036] The term "continuous culture" refers to a culture method in which a new nutrient medium is continuously supplied while the culture solution containing cells and products is continuously removed.

[0037] The term "batch culture" is a method in which the culture is continuously carried out until all the raw materials and substrates supplied for the first time are completely consumed, and it is a culture method in which the substrate concentration, metabolite concentration, cell concentration, etc. change continuously with time.

[0038] The cultivation in step (1) can be carried out for about 5 days to about 15 days, about 5 days to about 13 days, about 8 days to about 15 days, about 8 days to about 13 days, about 10 days to about 15 days, about 10 days to about 13 days, about 11 days to about 15 days, or about 11 days to about 13 days, but not limited thereto.

[0039] In step (1), the copper compound can be added to the culture medium composition once, twice, or more times during the cultivation of cells.

[0040] In step (1), before culturing the cells, the cultivation can be carried out by adding the copper compound to the culture medium composition. For example, the copper compound can be added to the culture medium composition on the 0th day of culturing the cells.

[0041] In the method for producing the recombinant ECM protein, the recombinant ECM protein can be a monomer. Therefore, the method can be a method for producing a recombinant ECM protein monomer, preferably, a method for producing a recombinant HAPLN protein monomer, more preferably, a method for producing a recombinant HAPLN1 protein monomer, and most preferably, a method for producing a recombinant human HAPLN1 protein monomer.

[0042] Step (2) may include performing chromatography. The chromatography may be any one or more selected from the group consisting of Affinity Chromatography, Anion Exchange Chromatography, Cation Exchange Chromatography, Hydroxyapatite Chromatography, Reversed-phase Chromatography, Size Exclusion Chromatography, Mixed Mode Chromatography, and Hydrophobic Interaction Chromatography.

[0043] Step (2) may include performing anion exchange chromatography.

[0044] The term "Ion Exchange Chromatography (IEC)" refers to a method of separation and analysis that uses an ion exchanger in a stationary phase to perform reversible ion exchange between the stationary phase and the mobile phase, thereby utilizing the difference in affinity of the sample ions for the stationary phase.

[0045] The term "Anion Exchange Chromatography (AEX)" is a type of ion exchange chromatography that uses an anion exchanger with cation functional groups such as amino groups.

[0046] The anion exchange chromatography may include a pre-equilibration step, an equilibration step, a sample loading step, a washing step, and an elution step.

[0047] The anion exchange chromatography may be performed by using a conventional anion exchange resin. Examples of the anion exchange resin may include EMD TMAE (M), EMD TMAE Medcap (M), EMD TMAE Hicap (M), Q, QPX, QPX Hicap, Capto Q, Capto Q ImpRes, Q FF, Q HP, Q XL, 30Q, Adhere Adhere ImpRes 50 HQ 50 XQ 50 PI, Q HyperCel GigaCap Q 650-M GigaCap Q650-S Super Q BioPro Q High Q Q or Q etc., but not limited thereto. Alternatively, depending on the operating conditions and the pI of the protein, a weak anion exchange resin having diethylaminoethyl (DEAE) in the dimethylaminoethyl (DMAE) functional group can also be used. Examples thereof are EMD DEAE EMD DMAE DEAE or DEAE Ceramic F

[0048] The anion exchange chromatography can be carried out in a bind-and-elute mode, but not limited thereto.

[0049] The loading amount of the anion exchange chromatography can be 10 g / L to 50 g / L of resin, but not limited thereto.

[0050] The elution buffer of the anion exchange chromatography can include histidine hydrochloride (His-HCl).

[0051] The elution buffer of the anion exchange chromatography can include histidine hydrochloride (His-HCl) in an amount of about 1 mM to about 1000 mM, about 10 mM to about 800 mM, about 20 mM to about 600 mM, about 40 mM to about 400 mM, about 60 mM to about 200 mM, for example, about 100 mM. When using histidine hydrochloride in the said concentration range, recombinant ECM protein can be separated with excellent purity and yield.

[0052] The elution buffer of the anion exchange chromatography can further include EDTA. The concentration of the EDTA can be appropriately selected by those skilled in the art.

[0053] The elution buffer of the anion exchange chromatography can be pH 4.0 to pH 6.0, pH 4.5 to pH 5.5, for example, pH 5.0, but not limited thereto.

[0054] By performing the anion exchange chromatography, recombinant ECM proteins can be captured. Thus, by performing the anion exchange chromatography, specific recombinant ECM proteins can be specifically separated.

[0055] After performing the anion exchange chromatography in step (2), it may further include performing cation exchange chromatography.

[0056] The term "Cation Exchange Chromatography (CEX)" is a type of ion exchange chromatography that uses an anion exchanger with anionic functional groups such as sulfonic acid groups and carboxyl groups.

[0057] The cation exchange chromatography may include an equilibration step, a sample loading step, a wash I step, a wash II step, a wash III step, and an elution step.

[0058] The cation exchange chromatography can be carried out by using a conventional cation exchange resin. Examples of the cation exchange resin include CPS, CPX, or SP Fast Flow S Resin, SO3(M), Fractogel SE Hicap(M), SP Cellthru BigBead SP, SP XL, SP Big Beads, M-CapⅡSP-550EC, SP A-25, S, SP-550C, SP-650C, 30S, 50HS, 50XS, SP Fast Flow, SP XL, S, SP ImRes, S ImpAct, HR-S, MAX S-r, MAX S-h, S, S, Rapid S, Giga-Cap S-650(M), S HyperCel SP-650M, High S, CM, S Ceramic F, SP, SP ImpRes, SP-650S, SP High Perform, MMC, MMC Imp Res, HCX, High c-Prime, etc., but not limited to this. Alternatively, depending on the operating conditions and the pI of the protein, weak cation exchange resins can also be used. For example, EMD COO(M), CM HP, CM FF, AFCarboxy 650-M, CM, GigaCap CM, CM Ceramic D, or 70.

[0059] The cation exchange chromatography can be carried out in a bind-and-elute mode, but not limited to this.

[0060] The loading amount of the cation exchange chromatography can be 10 g / L to 15 g / L of resin, but not limited to this.

[0061] In the washing II step of the cation exchange chromatography, the washing buffer II can include about 1 mM to about 1000 mM, about 5 mM to about 800 mM, about 10 mM to about 400 mM, about 25 mM to about 200 mM, or about 50 mM to about 150 mM of sodium chloride (NaCl), for example, about 100 mM.

[0062] In the washing III step of the cation exchange chromatography, the washing buffer III can include about 150 mM to about 500 mM, about 150 mM to about 400 mM, about 200 mM to about 500 mM, about 200 mM to about 400 mM, about 300 mM to about 500 mM, or about 300 mM to about 400 mM of sodium chloride (NaCl), for example, about 350 mM.

[0063] The washing buffer II or III of the cation exchange chromatography can further include Tris-HCl, NaAc, EDTA, or a combination thereof.

[0064] The washing buffer II or III for the cation exchange chromatography can have a pH of 5.0 to 8.5, for example, pH 8.0 or pH 5.5, but is not limited thereto.

[0065] The elution buffer for the cation exchange chromatography can include sodium chloride (NaCl) at about 50 mM to about 1000 mM, about 100 mM to about 800 mM, about 200 mM to about 600 mM, about 300 mM to about 500 mM, for example, about 370 mM. The concentration of sodium chloride can be appropriately selected considering the balance between the purity and yield of the product within the said range.

[0066] The elution buffer for the cation exchange chromatography can further include Tris-HCl, EDTA, or a combination thereof.

[0067] The elution buffer for the cation exchange chromatography can have a pH of 7.5 to 8.5, for example, pH 8.0, but is not limited thereto.

[0068] By performing the cation exchange chromatography, protein aggregates, HCP (Host cell protein), and other impurities can be removed.

[0069] The term "aggregate" refers to a form in which multiple substances aggregate together. "Protein aggregate" refers to a form in which proteins accumulate or aggregate, which includes aggregates of abnormal proteins in addition to aggregates of normal proteins. Protein aggregates include the target protein or its state of binding to other proteins, but multimeric proteins refer to the state in which target proteins bind to each other, so there are differences between the two.

[0070] The term "HCP (Host cell protein)" refers to process-related protein impurities produced by the host organism during the preparation and production of biotherapeutics.

[0071] After performing the cation exchange chromatography in step (2), it can further include performing mixed-mode chromatography.

[0072] The term "Mixed-mode Chromatography (MMC)" refers to a chromatography method that utilizes one or more forms of interaction between the stationary phase and the analyte.

[0073] The mixed-mode chromatography can include a pre-equilibration step, an equilibration step, a sample loading step, a washing I step, a washing II step, and an elution step.

[0074] The mixed-mode chromatography can be performed by using a conventional mixed-mode resin. Examples of the mixed-mode resin can include such as adhere, but not limited to this.

[0075] The mixed-mode chromatography can be carried out in a bind-and-elute mode, but not limited to this.

[0076] The loading amount of the mixed-mode chromatography can be 10 g / L to 15 g / L of resin, but not limited to this.

[0077] In the washing II step of the mixed-mode chromatography, the washing buffer II can include arginine at about 200 mM to about 400 mM, about 200 mM to about 350 mM, or about 250 mM to about 350 mM, for example, about 200 mM or about 300 mM.

[0078] The washing buffer II of the mixed-mode chromatography can further include Tris-HCl, EDTA, or a combination thereof.

[0079] The washing buffer II of the mixed-mode chromatography can be at pH 8.5 to pH 9.5, for example, pH 9.0, but not limited to this.

[0080] The elution buffer of the mixed-mode chromatography can include arginine at about 100 mM to about 1000 mM, about 200 mM to about 800 mM, about 300 mM to about 700 mM, about 400 mM to about 600 mM, for example, about 500 mM. The concentration of arginine can be appropriately selected considering the balance between the purity and yield of the product within the said range.

[0081] The elution buffer of the mixed-mode chromatography can further include Tris-HCl, EDTA, or a combination thereof.

[0082] The elution buffer of the mixed-mode chromatography can be at pH 7.5 to pH 8.5, for example, pH 8.0, but not limited to this.

[0083] By performing the mixed-mode chromatography, protein aggregates and HCP can be removed.

[0084] After performing the mixed-mode chromatography in step (2), it can further include performing hydrophobic interaction chromatography.

[0085] The term "Hydrophobic Interaction Chromatography (HIC)" refers to a chromatographic method that utilizes the hydrophobic interaction between the functional groups of the stationary phase and the analyte.

[0086] The hydrophobic interaction chromatography can include an equilibration step, a sample loading step, a washing I step, a washing II step, a washing III step, and an elution step.

[0087] The hydrophobic interaction chromatography can be carried out by using conventional hydrophobic interaction resins. Examples of the hydrophobic interaction resins include, but are not limited to, Butyl-S Sepharose 6 Fast Flow, Capto Octyl, Octyl Sepharose 4 Fast Flow, Phenyl Sepharose 6 Fast Flow (low sub), Capto Butyl, Butyl Sepharose 4 Fast Flow, Phenyl Sepharose High Performance, Capto Phenyl ImpRes, Butyl Sepharose High Performance, Capto Butyl ImpRes, Phenyl Sepharose 6 Fast Flow (high sub), and Capto Phenyl (high sub).

[0088] The hydrophobic interaction chromatography can be carried out in a binding-and-elution mode, but is not limited thereto.

[0089] The loading amount of the hydrophobic interaction chromatography can be 3 g / L to 6 g / L of resin.

[0090] In the washing step II of the hydrophobic interaction chromatography, the washing buffer II can include ammonium sulfate at about 0.1 M to about 1.0 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.2 M to about 1.0 M, about 0.2 M to about 0.8 M, about 0.2 M to about 0.6 M, about 0.2 M to about 0.4 M, about 0.3 M to about 1.0 M, about 0.3 M to about 0.8 M, about 0.3 M to about 0.6 M, or about 0.3 M to about 0.5 M, for example, about 0.4 M.

[0091] The washing buffer II of the hydrophobic interaction chromatography can further include Tris-HCl, EDTA, or a combination thereof.

[0092] The washing buffer II of the hydrophobic interaction chromatography can be at pH 7.5 to pH 8.5, for example, pH 8.0, but is not limited thereto.

[0093] In the washing step III of the hydrophobic interaction chromatography, the washing buffer III can include sodium chloride at about 0.5 M to about 2.0 M, about 0.5 M to about 1.8 M, about 1.0 M to about 2.0 M, about 1.0 M to about 1.8 M, about 1.2 M to about 2.0 M, or about 1.2 M to about 1.8 M, for example, about 1.5 M.

[0094] The washing buffer III for the hydrophobic interaction chromatography may further comprise Tris-HCl, EDTA, or a combination thereof.

[0095] The washing buffer III for the hydrophobic interaction chromatography may have a pH of 7.5 to 8.5, such as pH 8.0, but is not limited thereto.

[0096] The elution buffer for the hydrophobic interaction chromatography may comprise sodium chloride (NaCl) at about 0.1 M to about 1.5 M, about 0.1 M to about 1.2 M, about 0.1 M to about 1.0 M, about 0.1 M to about 0.8 M, about 0.3 M to about 1.5 M, about 0.3 M to about 1.2 M, about 0.3 M to about 1.0 M, or about 0.3 M to about 0.8 M, for example, about 0.5 M. When the sodium chloride concentration is greater than 1.5 M, the recombinant ECM protein may not be eluted.

[0097] The elution buffer for the hydrophobic interaction chromatography may further comprise Tris-HCl.

[0098] The elution buffer for the hydrophobic interaction chromatography may have a pH of 7.5 to 8.5, such as pH 8.0, but is not limited thereto.

[0099] By performing the hydrophobic interaction chromatography, protein multimers and HCP can be removed.

[0100] Step (2) may sequentially include: performing anion exchange chromatography; performing cation exchange chromatography; performing mixed-mode chromatography; and performing hydrophobic interaction chromatography. Step (2) may further additionally include methods known to be capable of separating and purifying recombinant proteins. For example, Harvest and Clarification, Ultrafiltration, Diafiltration, S / D (Solvent / Detergent) virus inactivation, intermediate depth filtration, or a combination of two or more thereof may be further additionally performed. The Harvest and Clarification, Ultrafiltration, Diafiltration, S / D virus inactivation, and Depth Filter can be carried out by conventional methods.

[0101] Step (2) may sequentially include: recovering and clarifying the culture broth obtained from step (1); ultrafiltration and diafiltration; performing anion exchange chromatography; inactivating S / D virus; cation exchange chromatography; performing mixed-mode chromatography; performing hydrophobic interaction chromatography; ultrafiltration and diafiltration; and intermediate depth filtration (Depth Filter).

[0102] On the other hand, a method for detecting a recombinant ECM protein monomer is provided, which includes: performing size exclusion chromatography on a sample including a recombinant ECM protein using a mobile phase including Hydrochloride; and

[0103] Based on the size exclusion chromatography results, analyzing the monomer of the recombinant ECM protein in the sample.

[0104] The method for detecting a recombinant ECM protein monomer may be a method for detecting a recombinant HAPLN protein monomer. Preferably, it may be a method for detecting a recombinant HAPLN1 protein monomer. Most preferably, it may be a method for detecting a recombinant human HAPLN1 protein monomer.

[0105] By including Hydrochloride in the mobile phase, the separation ability of the recombinant ECM protein is improved, and inaccurate peaks in the chromatography can be significantly reduced.

[0106] The type of the Hydrochloride included in the mobile phase is not limited. The Hydrochloride may be arginine hydrochloride (Arg-HCl), aniline hydrochloride, adenine hydrochloride, guanine hydrochloride, guanidine hydrochloride (Gdn-HCl), histidine hydrochloride (His-HCl), or lysine hydrochloride (Lys-HCl), but is not limited thereto.

[0107] The mobile phase may include Hydrochloride at a concentration greater than about 0.5 M, greater than or equal to about 0.8 M, greater than or equal to about 1.0 M, greater than about 0.5 M to about 10.0 M, greater than about 0.5 M to about 8.0 M, greater than about 0.5 M to about 0.4 M, about 0.8 M to about 4.0 M, about 0.8 M to about 3.0 M, about 0.8 M to about 2.0 M, about 0.8 M to about 1.5 M, about 0.8 M to about 1.2 M, greater than or equal to about 1.0 M to about 10.0 M, greater than or equal to about 1.0 M to about 8.0 M, greater than or equal to about 1.0 M to about 4.0 M, greater than or equal to about 1.0 M to about 3.0 M, or about 1.0 M to about 2.0 M. When the concentration of the Hydrochloride is less than or equal to 0.5 M, the separation ability of the recombinant ECM protein may be reduced. When the concentration of the Hydrochloride is less than or equal to 0.5 M, the ability to detect the recombinant ECM protein monomer may be reduced. In one embodiment, it was confirmed that when Hydrochloride was used as an additive to the mobile phase, the recombinant ECM protein monomer could be accurately analyzed even at a low concentration of about 1.0 M.

[0108] The term "Size Exclusion Chromatography (SEC)" is also referred to as "Gel Filtration Chromatography", and it is a method for separating proteins according to size. Different from other forms of chromatography, there is no attraction between the stationary phase and the solute, and the mobile phase simply passes through the porous stationary phase.

[0109] The size exclusion chromatography can be carried out by conventional methods. The size exclusion chromatography can be a size exclusion chromatography for analysis.

[0110] Since this method detects recombinant ECM protein monomers, during the production of recombinant ECM proteins, after each separation and / or purification step, it is possible to accurately analyze the proportion of recombinant ECM protein monomers and other impurities (such as multimers, etc.) in the result, and thus the proportion of recombinant ECM protein monomers can be analyzed. Therefore, the proportion of recombinant ECM protein monomers and other impurities can be analyzed in this analysis step.

[0111] The method for detecting recombinant ECM protein monomers can analyze the proportion of recombinant HAPLN protein monomers, preferably, it can analyze the proportion of recombinant HAPLN1 protein monomers, and most preferably, it can analyze the proportion of recombinant human HAPLN1 protein monomers.

[0112] Beneficial effects

[0113] With the culture medium composition according to one aspect, animal cells for producing recombinant ECM proteins can be cultured in large quantities.

[0114] With the method for producing recombinant ECM proteins according to another aspect, not only can recombinant ECM proteins be separated with high purity, but also monomers of specific recombinant ECM proteins can be specifically separated.

[0115] With the method for detecting recombinant ECM protein monomers according to another aspect, the monomers of recombinant ECM proteins can be analyzed with high accuracy, and thus the proportion of recombinant ECM protein monomers and other impurities can be analyzed. Brief description of the drawings

[0116] Figure 1 It is a chromatogram of AEX linear gradient elution.

[0117] Figure 2 It is a chromatogram of CEX step - wise elution.

[0118] Figure 3 It is a figure showing the SDS_PAGE_NR result of CEX step - wise elution.

[0119] Figure 4 This is a chromatogram of the step-wise elution of MMC.

[0120] Figure 5 It is a figure which shows the SDS_PAGE_NR result of MMC step-wise elution.

[0121] Figure 6A and 6B This is the chromatogram of HIC comparative elution.

[0122] Figure 7 It is a graph showing the SDS_PAGE_NR results of HIC comparative elution.

[0123] Figure 8 The chromatograms show the results of SEC analysis of samples including rhHAPLN1 using phosphate buffer + NaCl, 5 mM EDTA, or 5 mM EDTA + 4MGdn-HCl as the mobile phase.

[0124] Figure 9 : is a chromatogram showing the results of SEC analysis of a sample including rhHAPLN1 using 50 mM PB + 150 mM NaCl + 1 M Arg-HCl pH 6.3 as the mobile phase.

[0125] Figure 10 This is a chromatogram showing the results of SEC analysis of Sample 3 using 50 mM PB + 300 mM NaCl, 0.1 M Arg-HCl, 0.5 M Arg-HCl, 1.0 M Arg-HCl, or 1.0 M Gdn-HCl as the mobile phase.

[0126] Figure 11 This is a chromatogram showing the results of SEC analysis of Sample 3 using 50 mM PB+300 mM NaCl, 0.1 M urea, 0.5 M urea, 1.0 M urea, 2.0 M urea, 4.0 M urea, or 6.0 M urea as the mobile phase.

[0127] Figure 12 The chromatograms show the results of SEC analysis of Sample 3 using 1.0 M Gdn-HCl, 4.0 M urea, or 1.0 M Arg-HCl as the mobile phase.

[0128] Best Mode

[0129] Below, the present invention is further described in detail by examples. However, these examples are only used to exemplify the present invention, and the scope of the present invention is not limited thereto.

[0130] Example 1: Culturing cells for producing recombinant ECM protein

[0131] A vector containing a polynucleotide encoding human HAPLN1 protein in the ECM protein was inserted into CHO-K1 cells to prepare a CHO-K1 cell line producing recombinant human HAPLN1 protein. The cell line with excellent protein production quantity and quality was selected as the MCB (Master Cell Bank).

[0132] The MCB was subcultured and inoculated into a Thermo Hyperforma SUB 250L bioreactor at a concentration of 0.40 ± 0.05×10 6 cells / mL for fed-batch culture.

[0133] 22.36 g of ActiPro TM medium + 0.5846 g of glutamine + 10.00 g of HT Supplement (ThermoFisher Scientific) + 4.29 g of 10N NaOH + 1.80 g of NaHCO3 were used as the basal medium. The culture temperature was set at 36.5°C, the dissolved oxygen (DO) was set at 40.0%, and the pH was set at 7.00 ± 0.20. 1M Sodium Carbonate Monohydrate was used as the pH adjustment solution.

[0134] 181.04 g of HyClone TM Cell Boost 7a + 12.28 g of 10N NaOH of FM020a and 94.60 g of HyClone TM Cell Boost 7b + 105.93 g of 10N NaOH of FM020b were used as the feeding medium (FM). The feeding strategy is shown in Table 1 below.

[0135]

Table 1

[0136]

[0137] 50 μM CuSO4 was used as the feeding additive and added to the bioreactor on the 0th day of fed-batch culture.

[0138] Prepare a glucose supply stock at 400 g glucose / kg. During the third to thirteenth days of fed-batch culture, supply glucose by raising it to 6.0 g / L when the glucose concentration drops below 5.0 g / L.

[0139] When the VCD (Viable Cell Density) reaches 20.00×10 6 cells / mL, change the temperature to 31.0 °C. Harvest the cells on the twelfth day of fed-batch culture or when the survival rate drops below 60%.

[0140] Experimental Example 1: Effect of reducing the formation of recombinant ECM protein multimers according to the type of additive

[0141] When culturing cells for the production of recombinant human HAPLN1 protein, an experiment was conducted to confirm the effect of reducing protein multimer formation according to the type of additive.

[0142] Specifically, cells were cultured by the same method as in Example 1, except that the type, concentration, and supply strategy of the supplied additive were changed separately. The type, concentration, supply strategy of the supplied additive in each experimental group, and the results of cell culture and protein production are shown in Table 2 below.

[0143]

Table 2

[0144]

[0145]

[0146] *DMSO: Dimethyl sulfoxide, PS80: Polysorbate 80, GSH: Glutathione, GSSG: Glutathione disulfide.

[0147] As shown in Table 2, when using the CuSO4 additive, the production titer of recombinant human HAPLN1 protein was the highest at 3.87 g / L, and the yield of recombinant human HAPLN1 protein per cell was also the highest at 28.77 pg / cell / day.

[0148] Therefore, it was confirmed that during the process of culturing cells for the production of recombinant human HAPLN1 protein, when copper compounds such as CuSO4 are used as additives , the effect of reducing protein multimer formation is excellent and the protein yield increases. Specifically, it can be known that when supplying 50 μM concentration of CuSO4 on the 0th day of fed-batch culturing cells , the effect of reducing protein multimer formation and protein yield is the most excellent. Therefore, it can be known that it can be used for the batch production of recombinant human HAPLN1 protein.

[0149] Experimental Example 2: Effect of Recombinant ECM Protein Polymers Formation According to the Reduction of Copper Compound Concentration

[0150] When culturing cells for producing recombinant human HAPLN1 protein, an experiment was conducted to confirm the effect of protein polymers formation according to the reduction of copper compound concentration.

[0151] Specifically, cells were cultured by the same method as in Example 1 except that the concentration of the copper compound was changed separately. The copper compound concentration and protein production results of each experimental group are shown in Table 3 below.

[0152]

Table 3

[0153]

[0154] As shown in Table 3 below, when using CuSO4 with a concentration greater than 20 μM, the production titer of human HAPLN1 protein increased. In particular, Example 1 using a concentration of CuSO4 greater than or equal to 50 μM showed an excellent production titer of human HAPLN1 protein. Therefore, it was confirmed that when using copper compounds such as CuSO4 with a concentration greater than 20 μM, particularly greater than or equal to 50 μM, during the process of culturing cells for producing recombinant human HAPLN1 protein, the effect of reducing protein polymers formation was excellent and the protein yield increased.

[0155] Example 2: Isolation and Purification of Recombinant ECM Protein

[0156] Recombinant human HAPLN1 protein was isolated and purified from the cells cultured according to the above Example 1. Specifically, the isolation and purification of recombinant human HAPLN1 protein were carried out in the following order.

[0157] (1) Harvest and Clarification

[0158] For harvest and clarification, DOHC and A1HC depth filters from Millipore were used. The recommended loading amounts of DOHC and A1HC depth filters are 45 L / m 2 and 90 L / m 2 .

[0159] (2) Ultrafiltration / Diafiltration 1 (UF / DF1)

[0160] The Pellicon 3 (Ultracel, Type C Screen, 30 kDa) from Millipore was selected as the UF / DF1 membrane. The concentration of the loaded sample was less than or equal to 5 g / L in the UF step, and then diafiltration was carried out with a buffer of 50 mM Tris-HCl, 5 mM EDTA, pH 9.0 at a volume greater than or equal to 6 times. The supply flow rate was less than or equal to 300 LMH, and the transmembrane pressure (TMP) was 10 psi to 20 psi. The recommended loading amount was less than or equal to 70 L / m 2 .

[0161] (3) Anion Exchange Chromatography (AEX)

[0162] The Poros 50HQ resin from Life Tech was used as the Capture resin. This step was carried out in a bind-and-elute mode. The recommended protein loading amount was 10 g / L to 50 g / L of resin. The selected elution buffer was a pH 5.0 buffer containing 100 mM His-HCl and 5 mM EDTA. The recommended UV peak collection range was 25 mAU / mm to 75 mAU / mm.

[0163] (4) S / D (Solvent / Detergent) virus inactivation

[0164] S / D virus inactivation was carried out according to the conventional method.

[0165] (5) Cation Exchange Chromatography (CEX)

[0166] This step is carried out in a bind-and-elute mode. CaptoS ImpAct resin from Cytiva (formerly GE Healthcare) is used as the CEX resin. The recommended CEX loading is 10 g / L to 15 g / L of resin. Protein aggregates, HCP (Host cell protein), and other impurities are removed by CEX. The wash buffer II is recommended to be a pH 8.0 buffer containing 50 mM Tris-HCl, 100 mM NaCl, and 5 mM EDTA, and the wash buffer III is recommended to be a pH 5.5 buffer containing 50 mM NaAc, 350 mM NaCl, and 5 mM EDTA. The recommended elution buffer is a pH 8.0 buffer containing 50 mM Tris-HCl, 370 mM NaCl, and 5 mM EDTA. The recommended UV peak collection range is 25 mAU / mm to 50 mAU / mm.

[0167] (6) Mixed-mode Chromatography (MMC)

[0168] This step is carried out in a bind-and-elute mode. Captoadhere resin from Cytiva (formerly GE Healthcare) is used as the MMC resin. The recommended loading is 10 g / L to 15 g / L of resin. To further remove protein aggregates and HCP, the elution buffer used is a pH 8.0 buffer containing 50 mM Tris-HCl, 0.5 M arginine (Arg), and 5 mM EDTA.

[0169] (7) Hydrophobic Interaction Chromatography (HIC)

[0170] This step is carried out in a bind-and-elute mode. ButylSepharose 4 Fast Flow resin from Cytiva (formerly GE Healthcare) is used as the HIC resin. The recommended loading is 3 g / L to 6 g / L of resin. The wash buffer III is recommended to be a pH 8.0 buffer containing 50 mM Tris-HCl and 1.5 M NaCl. The target protein is eluted with high purity using a pH 8.0 buffer containing 50 mM Tris-HCl and 0.5 M NaCl.

[0171] (8) Ultrafiltration / Diafiltration2 (UF / DF2)

[0172] For the UF / DF2 step, Pellicon 3 (Ultracel, Type C Screen, 10 kDa) from Millipore was selected. After the loaded sample was concentrated to 1 g / L to 3 g / L in the UF step, diafiltration was carried out with 6 times the volume or more of a pH 5.0 buffer containing 20 mM NaAc. The UF / DF2 pool concentration was 4.5 g / L to 5.5 g / L. The supply flow rate was less than or equal to 300 LMH, and the transmembrane pressure difference (TMP) was 10 psi to 20 psi. The loading amount was less than or equal to 70 g / m 2 .

[0173] (9) Intermediate Depth Filtration (Int.DF)

[0174] For intermediate depth filtration, an X0SP depth filter from Millipore was selected to remove HCP. The recommended loading amount of the XOSP filter was 400 g / m 2 to 800 g / m 2 .

[0175] (10) Formulation and Bulk Fill

[0176] The concentration of the DS (drug substance) was 2.0 ± 0.2 g / L. The formulation of the formulation buffer was determined by DPD (Drug Product Development) and transferred to DSPD (Downstream Process Development). PS80 and sucrose were added to the VF pool sample at final concentrations of 0.04% (w / v) and 8% (w / v), respectively. Finally, the DS was obtained after 0.2 μm filtration.

[0177] Experimental Example 3: AEX Conditions for Specific Separation of a Specific Recombinant ECM Protein

[0178] Among the ECM proteins, the HAPLN1 protein has a molecular weight of 40 kDa to 50 kDa.

[0179] In the anion exchange chromatography (AEX) step of the above Example 2-(3), experiments were carried out to optimize the AEX conditions to enable specific separation of the recombinant human HAPLN1 protein.

[0180] (1) Elution Conditions

[0181] (1.1) AEX Linear Gradient Comparative Elution

[0182] Materials:

[0183] - Column: Poros 50HQ, 3.024 mL (0.5 cm × 15.4 cm)

[0184] - Loading substance: Concentration 2.64 g / L, pH 9.06, conductivity 1.44 mS / cm

[0185] - Loading amount: 30 g / L resin

[0186] - Column sanitization solution: 1.0 M NaOH

[0187] - Pre-equilibration buffer: 50 mM Tris-HCl, 1 M (NH4)2SO4, 5 mM EDTA, pH 8.0

[0188] - Equilibration / washing buffer I: 50 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, pH 9.0

[0189] - Washing buffer II: 50 mM Tris-HCl, 10 mM NaCl, 5 mM EDTA, pH 9.0

[0190] - Elution buffer: Control: 50 mM Tris-HCl, 80 mM (NH4)2SO4, 5 mM EDTA, pH 8.5; Condition 1 - A: 20 mM His-HCl, pH 7.5, B: 50 mM His-HCl, 30 mM NaCl, 5 mM EDTA, pH 5.8; Condition 2 - A: 100 mM His-HCl, pH 7.0, B: 100 mM His-HCl, 5 mM EDTA, pH 5.5; Condition 3 - A: 100 mM His-HCl, pH 7.0, B: 100 mM His-HCl, 5 mM EDTA, pH 5.0; Linear gradient elution from A to B during 20 CV (Column Volume).

[0191] - Stripping buffer: 50 mM Tris-HCl, 1 M (NH4)2SO4, 5 mM EDTA, pH 8.0

[0192] - Column storage solution: 20% ethanol

[0193] Experimental Procedures:

[0194] The eluate was collected at 25 mAU / mm to 25 mAU / mm. The elution conditions were detected using a chromatogram.

[0195] Results:

[0196] The yields of recombinant human HAPLN1 protein, SEC analysis results, and HCP concentrations for each elution condition are shown in Table 4 below. As shown in Table 4 below, when 100 mM His-HCl was added instead of salt to the elution buffer, the effect of capturing recombinant human HAPLN1 protein was more excellent compared to adding salt to the elution buffer. Additionally, taking the addition of 100 mM His-HCl as an example, the HCP was reduced by 50% compared to the control.

[0197]

Table 4

[0198]

[0199] (1.2) AEX linear gradient elution

[0200] Materials:

[0201] - Column: Poros 50HQ, 3.024 mL (0.5 cm × 15.4 cm)

[0202] - Loaded substance: Concentration 2.64 mg / mL, pH 9.06, conductivity 1.44 mS / cm

[0203] - Loading amount: 30 g / L resin

[0204] - Sanitization solution: 1.0 M NaOH

[0205] - Pre-equilibration buffer: 50 mM Tris-HCl, 1 M (NH4)2SO4, 5 mM EDTA, pH 8.0

[0206] - Equilibration / washing buffer I: 50 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, pH 9.0

[0207] - Washing buffer II: 50 mM Tris-HCl, 5 mM EDTA, pH 9.0

[0208] - Elution buffer: A: 100 mM His-HCl, pH 7.0, B: 100 mM His-HCl, 5 mM EDTA, pH 5.0; Linear gradient elution from A to B was performed during 20 CV (Column Volume).

[0209] - Stripping buffer: 50 mM Tris-HCl, 450 mM (NH4)2SO4, 5 mM EDTA, pH 8.0

[0210] - Storage solution: 20% ethanol

[0211] Experimental Procedures:

[0212] Based on comparing the elution results with a linear gradient, the material after UF / DF1 was loaded onto a Poros 50 HQ column, and the optimal elution buffer was determined using stepwise elution. The optimal elution conditions were confirmed by SEC purity analysis.

[0213] Results:

[0214] Figure 1 It is the chromatogram of AEX linear gradient elution.

[0215] As shown in Table 4 and Figure 1 as shown, 100 mM His-HCl can effectively capture recombinant human HAPLN1 protein without additional salt addition. To adjust the balance between purity and yield, pH 5.0 was selected as the pH for elution.

[0216] Therefore, it can be known that for the specific separation of recombinant human HAPLN1 protein, about 100 mM His-HCl can be selected as the AEX elution buffer. In particular, when 100 mM His-HCl is used, recombinant human HAPLN1 protein can be separated with excellent purity and yield. For example, a pH 5.0 buffer containing 100 mM His-HCl and 5 mM EDTA can be used as the AEX elution buffer.

[0217] Experimental Example 4: CEX Conditions for Removing Recombinant ECM Protein Aggregates, HCP, and Other Impurities

[0218] To remove recombinant human HAPLN1 protein aggregates, HCP, and other impurities, the binding-and-elution mode CEX of the above Example 2-(5) was introduced. Therefore, experiments were conducted to optimize the CEX conditions for removing recombinant human HAPLN1 protein aggregates, HCP, and other impurities.

[0219] (1) Elution Conditions

[0220] Materials:

[0221] - Column: Capto S ImpAct, 2.631 mL (0.5 cm × 13.4 cm)

[0222] - Loaded Material: AEX Eluate, Concentration 11.844 mg / mL, pH 5.52, Conductivity 11.70 mS / cm

[0223] - Loading Amount: 10 mg / L Resin

[0224] - Equilibration / Washing I Buffer: 50 mM NaAc-HAc, 5 mM EDTA, pH 5.5

[0225] - Washing II Buffer: 50 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, pH 8.0

[0226] - Washing III Buffer: 50 mM NaAC, 350 mM NaCl, 5 mM EDTA, pH 5.5

[0227] - Elution Buffer: (A) 50 mM Tris-HCl, 5 mM EDTA, pH 8.0; (B) 50 mM Tris-HCl, 500 mM NaCl, 5 mM EDTA, pH 8.0; Sequential elution: 20% B (100 mM NaCl), 5 CV; 40% B (200 mM NaCl), 5 CV; 60% B (300 mM NaCl), 5 CV; 75% B (375 mM NaCl), 5 CV; 85% B (425 mM NaCl)

[0228] - Stripping Buffer: 50 mM Tris-HCl, 500 mM NaCl, 5 mM EDTA, pH 8.0

[0229] Experimental Procedures:

[0230] Perform sequential elution to confirm the optimal elution conditions. The loading amount is 10 g / L resin, and the eluate is collected at 25 mAU / mm to 25 mAU / mm. Measure the protein concentration of each fraction and calculate the sequential recovery. In addition, analyze the sample purity by SDS_PAGE_NR.

[0231] Results:

[0232] The elution conditions are very important for the quality of the product. The criteria for the optimal conditions are based on the removal of impurities.

[0233] Figure 2 It is the chromatogram of CEX sequential elution.

[0234] Figure 3 It is the figure showing the SDS_PAGE_NR results of CEX sequential elution.

[0235] The experimental results of HCP in CEX sequential elution are shown in Table 5 below.

[0236]

Table 5

[0237]

[0238]

[0239] As Figure 3As shown in Table 5, when using 100 mM NaCl (E01), the most HCP was removed and the loss of the target protein was only 0.7%. The yields of fractions E02 to E04 and fraction E05 were 61.8% and 1.6% respectively. The HMW content increased with the increase of NaCl. For the balance of the purity and yield of the product, the elution buffer is recommended to contain a pH 8.0 buffer of 50 mM Tris-HCl, 370 mM NaCl and 5 mM EDTA.

[0240] Experimental Example 5: MMC Conditions for Removing Recombinant ECM Protein Aggregates and HCP

[0241] The binding-and-elution mode MMC of Example 2-(6) was used to further remove recombinant human HAPLN1 protein aggregates and HCP. Therefore, experiments were conducted to optimize the MMC conditions for removing recombinant human HAPLN1 protein aggregates and HCP.

[0242] (1) Elution Conditions

[0243] Recombinant human HAPLN1 protein aggregates and HCP were removed using Capto adhere. The CEX eluate was loaded onto the Capto adhere column. Based on the elution results of a linear gradient produced with 50 L of the substance, the optimal elution conditions were confirmed by successive elutions.

[0244] Materials:

[0245] - Column: Capto adhere, 2.985 mL (0.5 cm × 15.2 cm)

[0246] - Loaded Substance: CEX eluate, concentration 3.105 mg / mL, HCP 155217 ng / mg, SEC purity 52.9%

[0247] - Loading Amount: 7.5 g / L resin

[0248] - Pre-equilibration Buffer: 50 mM NaAc-HAc, 1 M NaCl, 5 mM EDTA, pH 5.5

[0249] - Equilibration / Washing Buffer I: 50 mM Tris-HCl, 5 mM EDTA, pH 8.0

[0250] - Elution buffer: (A) 50 mM Tris-HCl, 5 mM EDTA, pH 8.0; (B) 50 mM Tris-HCl, 1 M Arg, 5 mM EDTA, pH 8.0; Stepwise elution: 20% B (200 mM Arg), 5 CV; 40% B (400 mM Arg), 10 CV; 50% B (500 mM Arg), 10 CV; 60% B (600 mM Arg), 10 CV; 70% B (700 mM Arg), 10 CV

[0251] - Stripping buffer: 50 mM HAc

[0252] Experimental Procedures:

[0253] The optimal elution conditions were determined by stepwise elution. The CEX eluate was loaded onto a Captoadhere column at 7.5 g / L resin, and the eluate was collected at 25 mAU / mm to 25 mAU / mm. The protein concentration of each fraction was measured, and the stepwise recovery was calculated. The HCP and SDS_PAGE_NR purity were also measured.

[0254] Results:

[0255] Figure 4 It is the chromatogram of stepwise elution of MMC.

[0256] Figure 5 It is the figure showing the SDS_PAGE_NR result of stepwise elution of MMC.

[0257] The experimental results of HCP in stepwise elution of MMC are shown in Table 6 below.

[0258]

Table 6

[0259]

[0260]

[0261] As Figure 5 shown in and Table 6, when 200 mM arginine (peak 1) was used, the most HCP was removed and the loss of the target protein was only 0.2%. The yields of peak 2 and peak 3 were 39.5% and 28.1% respectively. When the concentration of arginine was high, the most HMW was eluted. For the balance of product purity and yield, an elution buffer containing 50 mM Tris-HCl, 500 mM arginine, and 5 mM EDTA at pH 8.0 is recommended. 200 mM arginine can be used for the washing step.

[0262] Experimental Example 6: HIC Conditions for Improving the Purity of Recombinant ECM Protein

[0263] The said Example 2-(7) HIC is used to remove the recombinant human HAPLN1 protein multimer and HCP. Therefore, experiments were conducted to optimize the HIC conditions for removing the recombinant human HAPLN1 protein multimer and HCP.

[0264] (1) Elution conditions

[0265] Materials:

[0266] - Column: Butyl Sepharose 4 Fast Flow, 2.631 mL (0.5 cm × 13.4 cm)

[0267] - Loading substance: 1) MMC eluate, concentration 0.590 mg / mL, pH 8.09, 146.35 mS / cm, SEC purity 68.9%; 2) MMC eluate, concentration 0.543 mg / mL, pH 8.10, 145.82 mS / cm, SEC purity 68.9%

[0268] - Loading amount: 5 g / L resin

[0269] - Equilibration / washing buffer I: 50 mM Tris-HCl, 1 M (NH4)2SO4, 5 mM EDTA, pH 8.0

[0270] - Washing buffer II: 50 mM Tris-HCl, 0.4 M (NH4)2SO4, 5 mM EDTA, pH 8.0

[0271] - Washing buffer III: 1) 50 mM Tris-HCl, 2 M NaCl, 5 mM EDTA, pH 8.0; 2) 50 mM Tris-HCl, 1.5 M NaCl, 5 mM EDTA, pH 8.0

[0272] - Elution buffer: 1) (A) 50 mM Tris-HCl, 2 M NaCl, 5 mM EDTA, pH 8.0; (B) 50 mM Tris-HCl, 5 mM EDTA, pH 8.0; Stepwise elution: 25% B (1.5 M NaCl), 10 CV; 50% B (1 M NaCl), 10 CV; 75% B (0.5 M NaCl), 10 CV; 90% B (0.2 M NaCl), 10 CV; 100% B (0 M NaCl), 10 CV;

[0273] 2) 50 mM Tris-HCl, 0.5 M NaCl, pH 8.0

[0274] - Washing buffer: 50 mM Tris-HCl, 5 mM EDTA, pH 8.0

[0275] Experimental Procedures:

[0276] Sequential elution was performed to determine the washing III and elution conditions. Before loading the MMC eluate onto the HIC column, it was adjusted with ~1 M (NH4)2SO4. The loading amount was 5 g / L resin, and the eluate was collected at 25 mAU / mm to 25 mAU / mm. The protein concentration of each fraction was measured, and the sequential recovery was calculated. The purity was measured using SDS_PAGE_NR.

[0277] Results:

[0278] Figure 6 is a chromatogram of HIC comparative elution.

[0279] Figure 7 It is a figure showing the SDS_PAGE_NR results of HIC comparative elution.

[0280] The results of HIC sequential elution are shown in Table 7 below.

[0281]

Table 7

[0282]

[0283] As Figure 7 shown in and Table 7, the most HMW was removed by washing buffer II, and the target protein was not eluted at pH 8.0 with more than 1.5 M NaCl. Therefore, in Run2, a pH 8.0 buffer containing 50 mM Tris-HCl, 1.5 M NaCl, and 5 mM EDTA was used for washing III, and a pH 8.0 buffer containing 50 mM Tris-HCl, 0.5 M NaCl, and 5 mM EDTA was used for elution. Finally, the yield of the second experiment was 34.8%. Therefore, a pH 8.0 buffer containing 50 mM Tris-HCl, 1.5 M NaCl, and 5 mM EDTA was used as washing buffer III, and a pH 8.0 buffer containing 50 mM Tris-HCl and 0.5 M NaCl was used as elution buffer.

[0284] Example 3: Method for detecting recombinant ECM protein

[0285] Size exclusion chromatography (SEC) was performed using a mobile phase containing hydrochloride to analyze the monomer and other impurities of the recombinant ECM protein in the sample. The specific conditions for size exclusion chromatography (SEC) are as follows.

[0286] -Column: TSKgel G3000SWXL, 7.8×300mm, 5μm Steel (Manuf.TOSOH)

[0287] -Mobile phase: 50mM phosphate buffer (PB), 300mM NaCl, 1M Gdn-HCl or Arg-HCl pH7.5 (±0.5)

[0288] - Detection wavelength: 280nm

[0289] -Flow rate: 1.0mL / min

[0290] - Column temperature: 25±3℃

[0291] - Sample temperature: 5±3℃

[0292] -Injection volume: 100 μg

[0293] Experimental Example 7: Screening of additives for accurate analysis of recombinant ECM proteins

[0294] When using size exclusion chromatography (SEC) to analyze monomers and other impurities of recombinant ECM proteins, experiments were conducted to screen mobile phase additives that can improve accuracy by reducing the occurrence of inaccurate peaks. Specifically, in order to analyze recombinant human HAPLN1 protein monomers and other impurities using size exclusion chromatography (SEC), the accuracy of SEC analysis according to the type and concentration of additives used as mobile phase was confirmed. The intermediate product in the process of separating and purifying the recombinant human HAPLN1 protein according to Example 2 was used as a sample.

[0295] Figure 8 is a chromatogram showing the results of SEC analysis of samples including rhHAPLN1 using phosphate buffer (PB) + NaCl, 5 mM EDTA, or 5 mM EDTA + 4 M Gdn-HCl as the mobile phase.

[0296] Figure 9 The chromatogram shows the results of SEC analysis of a sample including a recombinant human HAPLN1 protein using 50 mM PB + 150 mM NaCl + 1 M Arg-HCl pH 6.3 as a mobile phase.

[0297] like Figures 8 to 9 As shown, when hydrochloride is added to the mobile phase, the peak of the monomer is most obvious.

[0298] Figure 10It is a chromatogram showing the results after SEC analysis using 50 mM PB + 300 mM NaCl, 0.1 M Arg-HCl, 0.5 M Arg-HCl, 1.0 M Arg-HCl, or 1.0 M Gdn-HCl as the mobile phase. The analysis results are shown in Table 8 below.

[0299]

Table 8

[0300] Mobile Phase Monomer (%) HMW (%) LMW (%) 50 mM PB + 300 mM NaCl 22.4 76.9 0.8 0.1 M Arg-HCl 26.1 73.9 ND 0.5 M Arg-HCl 62.0 38.0 ND 1.0 M Arg-HCl 71.8 28.2 ND 1.0 M Gdn-HCl 77.2 22.8 ND

[0301] As Figure 10 shown in and Table 8, as the concentration of Arg-HCl increases, the recombinant human HAPLN1 protein is well separated. In particular, when using 1.0 M Arg-HCl and Gdn-HCl, the separation ability of the recombinant human HAPLN1 protein is excellent.

[0302] Figure 11 It is a chromatogram showing the results after SEC analysis using 50 mM PB + 300 mM NaCl, 0.1 M urea, 0.5 M urea, 1.0 M urea, 2.0 M urea, 4.0 M urea, or 6.0 M urea as the mobile phase known as a monomer detection additive. The analysis results are shown in Table 9 below.

[0303]

Table 9

[0304] Mobile Phase Monomer (%) HMW (%) LMW (%) 50 mM PB + 300 mM NaCl 26.4 73.6 ND 0.1 M Urea 26.4 73.6 ND 0.5 M Urea 26.7 73.3 ND 1.0 M Urea 27.7 72.3 ND 2.0 M Urea 32.1 67.9 ND 4.0 M Urea 75.6 24.4 ND 6.0 M Urea 75.6 24.4 ND

[0305] As Figure 11 shown in and Table 9, as the concentration of urea increases, the recombinant human HAPLN1 protein is well separated. However, different from the hydrochloride salts, when using urea with a concentration greater than or equal to 4.0 M, the recombinant human HAPLN1 protein can be separated.

[0306] Figure 12 It is a chromatogram showing the results after SEC analysis using 1.0 M Gdn-HCl, 4.0 M urea, or 1.0 M Arg-HCl as the mobile phase. The analysis results are shown in Table 10 below.

[0307]

Table 10

[0308]

[0309]

[0310] As Figure 12 shown in and Table 10, it is confirmed that when using hydrochloride salts such as Gdn-HCl and Arg-HCl, the separation ability of the recombinant human HAPLN1 protein is equivalent to or higher than that when using 4.0 M high-concentration urea even at a low concentration of 1.0 M.

[0311] Therefore, it can be known that when SEC analysis is performed using 1.0 M hydrochloride as an additive to the mobile phase, the separation ability of recombinant human HAPLN1 protein is excellent and the inaccurate peaks in the chromatogram are significantly reduced, enabling accurate analysis of the monomer of recombinant human HAPLN1 protein.

[0312] In summary, it was confirmed that the proportion of the monomer of recombinant ECM protein in the sample can be analyzed by size exclusion chromatography using a mobile phase containing hydrochloride.

Claims

1. A method for detecting recombinant extracellular matrix protein monomers, comprising: Perform size exclusion chromatography on a sample containing a recombinant extracellular matrix protein using a mobile phase comprising a hydrochloride at a concentration of 0.5 M to 1.2 M; and Based on the results of the size exclusion chromatography, analyze the monomers of the recombinant extracellular matrix protein in the sample, wherein the recombinant extracellular matrix protein is elastin, fibronectin, laminin, vitronectin, tenascin, or hyaluronan and proteoglycan link protein (HAPLN).

2. The method according to claim 1, wherein, The HAPLN is any one protein selected from the group consisting of HAPLN1, HAPLN2, HAPLN3, and HAPLN4.

3. The method according to claim 1, wherein, The hydrochloride is arginine hydrochloride, aniline hydrochloride, adenine hydrochloride, guanine hydrochloride, guanidine hydrochloride, histidine hydrochloride, or lysine hydrochloride.

4. The method according to claim 1, wherein In the analysis step, analyze the ratio of the recombinant extracellular matrix protein monomers to other impurities.

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