Method for producing recombinant activin A

Abstract

To provide techniques for purifying activin A with improved purity and appropriate dimer structure.SOLUTION: Provided is a method for producing a recombinant activin A containing mature sequence derived from a plant cell, where the method comprises a step of applying a solution containing the recombinant activin A to a cation exchange chromatography for purification, where the cation exchange chromatography uses a salt gradient elution process with a mobile phase comprising a zwitterion-containing buffer of pH 1.5-3.5 and a water soluble organic solvent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to methods for producing recombinant activin A. [Background technology]

[0002] Activin A is a cytokine belonging to the TGF-β (transforming growth factor-β) superfamily. It is involved in development and differentiation as an erythroid differentiation factor and mesoderm-inducing factor, and is a physiologically and industrially useful protein that regulates various functions in various cells.

[0003] Mature activin A is a protein containing a mature region, which is generated by cleavage of proactivin A, which consists of a proregion, a protease cleavage sequence, and a mature region, at the protease cleavage sequence by proprotein convertase (Furin). Mature activin A is a homodimer consisting of 116 amino acid residues of βA chains linked by nine disulfide bonds.

[0004] Patent document 1 discloses the application of cation exchange chromatography using a chaotropic ion gradient elution method to the purification process of activin A as a technology for easily isolating, purifying, and producing highly pure human activin A suitable for pharmaceutical use on an industrial scale.

[0005] Furthermore, Patent Document 2 discloses that when producing recombinant activin A in plants, a technique for avoiding the degradation of activin A by endogenous proteases of the plant other than proprotein convertase (Furin), which would result in a decrease in yield, involves modifying the amino acid sequence in the proregion. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 4055248 [Patent Document 2] International Publication No. 2020 / 100993 Summary of the Invention [Problem to be solved by the invention]

[0007] A primary objective of the present disclosure is to provide techniques for purifying activin A with improved purity and proper dimer structure. [Means for solving the problem]

[0008] To solve the above problems, the present disclosure provides the following [1]-[7]. [1] A method for producing recombinant activin A containing a mature sequence derived from plant cells, comprising: a purification step of purifying the recombinant activin A by subjecting the solution containing the recombinant activin A to cation exchange chromatography; the cation exchange chromatography is performed by a salt gradient elution method using a mobile phase comprising a buffer solution of pH 1.5-3.5 containing a zwitterion and a water-soluble organic solvent; Manufacturing method. [2] The method of [1], wherein the zwitterion is an amino acid selected from proline, glycine, and alanine, taurine, gamma-aminobutyric acid (GABA), or betaine. [3] The method of producing [1] or [2], wherein the pH of the buffer solution is 2.5 to 3.5. [4] The method according to any one of [1] to [3], wherein the concentration of the water-soluble organic solvent in the mobile phase is at least 40% by volume. [5] A manufacturing method according to any one of [1] to [4], further comprising an intermediate treatment step prior to the purification step, in which the solution containing the recombinant activin A is desalted and replaced with a salt-free solvent of pH 1.5-3.5. [6] A manufacturing method according to any of [1] to [5], wherein the purity of the recombinant activin A is 80% or more, the recovery rate is 70% or more, and the monomer ratio of the recombinant activin A under reducing conditions is 80% or more. [7] A method for producing any one of [1] to [6], which does not include a step of refolding the recombinant activin A. [Effects of the Invention]

[0009] The present disclosure provides techniques for purifying activin A with improved purity and proper dimeric structure. DETAILED DESCRIPTION OF THE INVENTION

[0010] Preferred embodiments for carrying out the present disclosure will be described below with reference to the drawings. Note that the embodiments described below are examples of typical embodiments of the present disclosure, and should not be construed as narrowing the scope of the present disclosure.

[0011] The method for producing recombinant activin A containing a mature sequence derived from plant cells according to the present disclosure includes a purification step in which a solution containing recombinant activin A is subjected to cation exchange chromatography to purify the recombinant activin A, and specifically includes the following steps. Pretreatment Affinity purification Protease treatment (pro-region cleavage) Fractionation Desalination Cation exchange chromatography Post-processing Each step will be explained below in order.

[0012] [Preprocessing] In this step, a protein fraction containing recombinant proactivin A derived from plant cells is obtained from a solution containing the recombinant proactivin A.

[0013] In the present disclosure, plant cell-derived recombinant proactivin A may be proactivin A expressed in a plant cell as a host cell, and the plant host cell and the species from which the proactivin A is derived are not particularly limited.

[0014] Examples of host plants include Solanaceae plants (tobacco, tomato, potato, etc.), Gramineae plants (rice, wheat, barley, corn, etc.), Brassicaceae plants (Arabidopsis thaliana, Brassica napus, etc.), Asteraceae plants (lettuce, etc.), and mosses (Marchantia polymorpha, Physcomitrella patens, etc.), with Nicotiana plants being preferred. Examples of Nicotiana plants include Nicotiana benthamiana (N. benthamiana), Nicotiana tabacum (N. tabacum), and Nicotiana excelsior (N. excelsior).

[0015] The species from which proactivin A is derived may be human, monkey, cow, pig, horse, dog, cat, rabbit, mouse, rat, guinea pig, etc. Among these, from the viewpoint of sequence homology, human, mouse, rat, cat, pig, and cow are preferred, and human is particularly preferred.

[0016] Recombinant proactivin A can be expressed in plant cells by conventionally known molecular biological techniques.

[0017] Recombinant proactivin A may consist of a natural amino acid sequence or an artificially modified amino acid sequence. The amino acid sequence of natural human proactivin A consisting of the pro-region, protease cleavage sequence, and mature region is shown in SEQ ID NO: 1, and the amino acid sequence of natural human activin A consisting of the mature region is shown in SEQ ID NO: 2. The amino acid sequence of the mature region of activin A is 100% identical among humans, mice, rats, cats, pigs, and cows.

[0018] Proactivin A consisting of a modified amino acid sequence is not particularly limited, but includes modified human proactivin A consisting of the amino acid sequence set forth in SEQ ID NO: 3. This modified human proactivin A has a His tag sequence at the N-terminus and includes an amino acid sequence modification described in International Publication No. 2020 / 100993 (Patent Document 2) to confer resistance to cleavage by endogenous plant proteases, and an amino acid sequence modification described in Japanese Patent Application Laid-Open No. 2020-156412 to enable cleavage by HRV 3C protease instead of furin. Modified human proactivin A is cleaved by HRV 3C protease to generate modified human mature activin A in which the second leucine is replaced with proline. The amino acid sequence of modified human mature activin A is set forth in SEQ ID NO: 4.

[0019] In the present disclosure, recombinant activin A containing a mature sequence may be recombinant activin A consisting of only the mature sequence, or may be recombinant activin A to which one or more amino acid sequences have been added to the mature sequence. The number of added amino acids is 20-11 or less, preferably 10-6 or less, more preferably 5-3 or less, and particularly preferably 2 or 1. Furthermore, the mature sequence contained in the recombinant activin A is not limited to a naturally occurring amino acid sequence, but may have one or more amino acid substitutions, insertions, and / or deletions. Amino acid substitutions may include, for example, a substitution of the second leucine in the mature sequence described above with a proline. The number of amino acids substituted, inserted, and / or deleted is 20-11 or less, preferably 10-6 or less, more preferably 5-3 or less, and particularly preferably 2 or 1.

[0020] A solution containing recombinant proactivin A can be prepared by crushing and extracting a plant (preferably leaves) that expresses recombinant proactivin A using a general-purpose method.

[0021] Protein fractions containing recombinant proactivin A can be obtained from a solution containing recombinant proactivin A, for example, by applying ammonium sulfate fractionation, gradually changing the concentration of added ammonium sulfate, and fractionating the proteins that precipitate at each concentration. The fractionation method is not limited to ammonium sulfate fractionation, and known techniques can be used. The fractionation procedure may be performed once or twice or more times.

[0022] [Affinity purification] In this step, a solution of protein fractions containing recombinant proactivin A is affinity purified to obtain a recombinant proactivin A solution.

[0023] Affinity purification can be performed using, but is not limited to, a His-tag affinity column. Specifically, for example, an equilibration solution for His-tag affinity purification (20 mM HEPES, 150 mM NaCl, 10% (w / v) glycerol, pH 8.0) is added to the precipitate of a protein fraction containing recombinant proactivin A to dissolve the precipitate. This solution is applied to a His-tag affinity column equilibrated with the equilibration solution for His-tag affinity purification, and the column is then washed with a wash solution for His-tag affinity purification (20 mM HEPES, 150 mM NaCl, 20 mM imidazole, 10% (w / v) glycerol, pH 8.0). Finally, an elution solution for His-tag affinity purification (20 mM HEPES, 150 mM NaCl, 200 mM imidazole, 10% (w / v) glycerol, pH 8.0) is applied, and the elution peak is collected to obtain a recombinant proactivin A solution.

[0024] [Protease treatment] In this step, a protease is added to the recombinant proactivin A solution to cleave the protease cleavage sequence of the recombinant proactivin A, thereby preparing a solution containing recombinant activin A containing the pro-region and the mature region.

[0025] The conditions for the protease treatment are not particularly limited as long as the cleavage of the protease cleavage sequence proceeds sufficiently, but for example, the treatment is performed at room temperature for one day and one night.

[0026] The protease is selected depending on the amino acid sequence of the protease cleavage sequence of the recombinant proactivin A. Furin may be used in the case of a naturally occurring protease cleavage sequence, and for example, HRV 3C protease may be used in the case of a modified protease cleavage sequence.

[0027] [Fraction] In this step, a crude solution of recombinant activin A containing the mature region is obtained from a solution containing the pro-region and recombinant activin A containing the mature region by, for example, applying acetonitrile fractionation. The solution containing the pro-region and recombinant activin A containing the mature region also contains contaminants such as proteases derived from the previous step. By fractionation, the recombinant activin A containing the mature region can be separated from the pro-region and other contaminants. The fractionation method is not limited to acetonitrile fractionation, and any known method can be applied. The fractionation operation may be carried out once or twice or more times.

[0028] [Desalination] In this step, a crude solution of recombinant activin A containing the mature region is desalted and replaced with a salt-free solvent of pH 1.5-3.5.

[0029] Desalting and solvent exchange can be achieved by general methods such as dialysis and ultrafiltration membrane treatment.

[0030] As a salt-free solvent with a pH of 1.5-3.5, hydrochloric acid or glycine buffers can be used. More preferably, the pH of the solvent is 2.5 to 3.5. The concentration of hydrochloric acid is preferably 1-20 mM, more preferably 2-10 mM, especially about 4 mM. The concentration of glycine is preferably 5-100 mM, more preferably 10-25 mM.

[0031] By carrying out desalting and solvent exchange, the purified purity of recombinant activin A containing the mature region is improved.

[0032] [Cation exchange chromatography] In this step, the crude solution of recombinant activin A containing the mature region after desalting and solvent replacement is applied to cation exchange chromatography to obtain a purified recombinant activin A solution.

[0033] Cation exchange chromatography uses a mobile phase containing a buffer solution of pH 1.5-3.5 containing a zwitterion and a water-soluble organic solvent, preferably without chaotropic ions.

[0034] The zwitterion-containing buffer is preferably one in which an amino acid selected from proline, glycine and alanine, taurine, gamma-aminobutyric acid (GABA) or betaine is used as the buffering agent. The concentration of the zwitterion in the buffer is preferably 5-100 mM, more preferably 10-25 mM. The pH of the buffer solution is more preferably 2.5 to 3.5.

[0035] The water-soluble organic solvent is not particularly limited, but is preferably one or more selected from the group consisting of lower alcohols, acetonitrile, dimethyl sulfoxide, and dimethylformamide, and more preferably acetonitrile. The concentration of the water-soluble organic solvent in the mobile phase is at least 40% by volume, preferably 50% by volume or more, and preferably 70% by volume or less to ensure the solubility of the salt.

[0036] The recombinant activin A containing the mature region is eluted from the cation exchange column by a salt gradient elution method. The concentration gradient elution method may be a linear gradient elution in which the concentration is changed linearly or a stepwise elution in which the concentration is changed stepwise. The salts that can be used include sodium chloride, potassium chloride, ammonium sulfate, sodium perchlorate, and the like. The final concentration of the salt depends on the type of salt and the type and concentration of the organic solvent added, but in the case of acetonitrile, for example, it can be set to 250-1000 mM.

[0037] The protein load on the column is preferably 0.1 to 20 mg, more preferably 0.1 to 5 mg, in terms of protein load per unit weight of column resin. Chromatography conditions other than those mentioned above (for example, the diameter, length, and volume of the column, the flow rate of the solution, etc.) can be set appropriately.

[0038] As an example of the specific procedure for this step, first, a crude solution of recombinant activin A is passed through a cation exchange column equilibrated with 20 mM glycine pH 3.0, 40% (v / v) acetonitrile solution, followed by linear gradient elution with 500 mM NaCl solution (0-500 mM NaCl, 50 CV). The resulting elution peak is then fractionated and collected.

[0039] In another example, a crude solution of recombinant activin A is first passed through a cation exchange column equilibrated with 20 mM glycine pH 2.5, 50% (v / v) acetonitrile solution, followed by stepwise washing with 225 mM NaCl solution, followed by stepwise elution with 250 mM NaCl solution, and the eluted fractions are collected.

[0040] Post-processing In this step, the purified solution of recombinant activin A containing the mature region is subjected to solvent substitution with a solvent in which the recombinant activin A can be stably dissolved. For solvent exchange, general-purpose techniques such as dialysis and ultrafiltration can be applied. The type and concentration of buffer and salt used for solvent exchange, as well as pH, can be appropriately set within a range in which recombinant activin A can be stably dissolved. The buffer may be, for example, a glycine buffer (pH 2.5-3.5) or a phosphate buffer (pH 6.0-7.4), preferably a glycine buffer (pH 2.5-3.5). The concentration of the buffer is preferably 0-100 mM, more preferably 10-25 mM. The salt may be, for example, HCl, NaCl, KCl, LiCl, etc. The salt concentration is preferably 0-1000 mM, more preferably 0-500 mM. Note that buffer components also exhibit electrical conductivity and can therefore be considered as salts. Solvent substitution also includes desalting by substituting a solvent with a low salt concentration, such as a hydrochloric acid or glycine buffer solution. The concentration of hydrochloric acid is preferably 1-20 mM, more preferably 2-10 mM, especially about 4 mM. The pH of the glycine buffer is preferably 2.5 to 3.5, and the concentration of glycine is preferably 10 to 25 mM.

[0041] According to the production method of the present disclosure, which comprises the steps described above, recombinant activin A containing the mature region can be purified with improved purity and recovery rate. Furthermore, recombinant activin A that properly maintains the natural dimer structure can be obtained without undergoing a refolding step.

[0042] The purity of the resulting recombinant activin A is, for example, 80% or more, particularly 82% or more. The recovery rate of recombinant activin A is, for example, 70% or more, preferably 75% or more, more preferably 80% or more, and particularly 81% or more.

[0043] <Purity> The purity can be calculated as follows. The purified solution of recombinant activin A is subjected to SDS-PAGE, the gel is stained, and densitometry analysis is performed to calculate the purity as the intensity of the recombinant activin A band among all bands.

[0044] <Recovery rate> The recovery rate can be calculated as follows. The purified solution of recombinant activin A was subjected to SDS-PAGE together with standard solutions of different concentrations, the gel was stained, and densitometry analysis was performed to calculate the amount of recombinant activin A based on a calibration curve prepared from the standard solutions. The recovery rate was calculated by dividing the calculated amount of activin A contained in the purified solution of recombinant activin A by the amount of recombinant activin A before purification.

[0045] Activin A in its native dimeric structure is detected as a monomer by SDS-PAGE under reducing conditions. Therefore, the dimer detected under reducing conditions is a dimer having a structure different from that of the native dimer, and the lower the detection ratio of the dimer to the monomer, the higher the proportion of recombinant activin A in the native dimeric structure in the obtained recombinant activin A. The monomer ratio under reducing conditions of the recombinant activin A obtained by the production method according to the present disclosure is, for example, 80% or more, preferably 90% or more, more preferably 95% or more, and particularly 100%.

[0046] <Monomer ratio> The monomer ratio can be calculated as follows. The purified solution of recombinant activin A was subjected to SDS-PAGE under reducing conditions, the gel was stained, and densitometry analysis was performed to calculate the monomer ratio by dividing the band intensity at the monomer position by the band intensity at the dimer position. [Example]

[0047] [Test Example 1: Expression of recombinant activin A in Nicotiana benthamiana] 1. Vector Construction A vector was constructed for expressing modified human proactivin A (hereinafter simply referred to as "modified proactivin") consisting of the amino acid sequence set forth in SEQ ID NO: 3. The modified proactivin A has a His tag sequence at the N-terminus and includes an amino acid sequence modification described in International Publication No. 2020 / 100993 (Patent Document 2) to confer resistance to cleavage by endogenous plant proteases, and an amino acid sequence modification described in Japanese Patent Application Laid-Open No. 2020-156412 to enable cleavage by HRV 3C protease instead of Furin. The modified proactivin A is cleaved by HRV 3C protease to generate modified human mature activin A (hereinafter simply referred to as "modified activin A") in which the second leucine has been replaced with proline. The amino acid sequence of modified activin A is set forth in SEQ ID NO: 4. The vector was constructed according to the methods described in International Publication No. 2020 / 100993 and Japanese Patent Application Laid-Open No. 2020-156412.

[0048] 2. Preparation of transformants (1) Cultivation of host plants (1-1) Seeding The host plant used was Nicotiana benthamiana, a plant of the Nicotiana genus. Liquid fertilizer for sowing (0.78 g / L from Otsuka House S1 (Otsuka Agritechno Co., Ltd.), 0.25 g / L from Otsuka House 2 (Otsuka Agritechno Co., Ltd.), pH 5.0) was soaked into a urethane mat for hydroponic cultivation, placed in a seedling tray, and Nicotiana benthamiana seeds were sown.

[0049] (1-2) Seedling raising After sowing, the plants were grown in an artificial climate chamber (NC-410HC, Nippon Medical and Chemical Instruments Manufacturing Co., Ltd.) at 28°C under a 16-hour day / 8-hour night light cycle for 12 days.

[0050] (1-3) Cultivation (first half) The seedlings were transplanted into the early cultivation panel. After transplanting, the early cultivation panel was placed in an artificial climate chamber (LH-410SP, Nippon Medical and Chemical Machinery Manufacturing Co., Ltd.) and cultivated for 9 days using the deep flow technique (DFT). The environmental conditions and liquid fertilizer conditions were controlled as follows: <Environmental conditions> - Temperature: 28℃ - Relative humidity: 60-80% - CO2 concentration: 500 ppm - Illumination: Average photosynthetic photon flux density (PPFD): 140-160 μmol / m 2 10 seconds, 24-hour continuous irradiation, three-wavelength fluorescent lamp "Lupica Line" (Mitsubishi Electric Corporation) <Liquid fertilizer conditions> Fertilizer solution A (Otsuka House S1 150 g / L, Otsuka House No. 5 (Otsuka Agritechno Co., Ltd.) 2.5 g / L) and fertilizer solution B (Otsuka House No. 2 100 g / L) were each dissolved in dechlorinated water and mixed in equal amounts for use. The pH was adjusted using pH adjuster Down (Otsuka Agritechno Co., Ltd.) and a 4% KOH aqueous solution. The electrical conductivity (EC) and pH of the liquid fertilizer were adjusted to EC 2.3 mS / cm and pH 6.0 using the "Easy Fertilizer Control Machine 3" (Cem Corporation).

[0051] (1-4) Cultivation (late stage) The plants were removed from the early cultivation panel and transplanted into the late cultivation panel. After transplantation, the late cultivation panel was placed in an artificial climate chamber (LH-410SP, Nippon Medical and Chemical Instruments Manufacturing Co., Ltd.) and cultivated using the DFT method for 7 days (28 days after sowing). The environmental conditions were controlled as follows: <Environmental conditions> - Temperature: 28℃ - Relative humidity: 40-60% - CO2 concentration: 500 ppm - Illumination: Average photosynthetic photon flux density (PPFD): 140-160 μmol / m 210 seconds, 24-hour continuous irradiation, three-wavelength fluorescent lamp "Lupica Line" (Mitsubishi Electric Corporation)

[0052] (2) Preparation of transformants (2-1) Infection by Vacuum Infiltration The vector constructed in "1. Vector construction" above was introduced into Agrobacterium strain AGL1 by electroporation, and Nicotiana benthamiana was infected by agroinfiltration together with the AGL1 strain harboring an expression vector (pP19 / pRI201-AN) containing the gene for a gene silencing suppressor derived from Tomato bushy stunt virus (TBSV P19). Specifically, Nicotiana benthamiana plants 28 days after sowing, obtained in "2.(1) Cultivation of host plants" above, were inverted and submerged in the Agrobacterium solution in a beaker so that all leaves were completely submerged. The beaker was placed in a vacuum desiccator (FV-3P, Tokyo Glass Instruments Co., Ltd.) and left to stand at -0.09 MPa for 1 minute to reduce the pressure. The valve was then quickly opened to restore the pressure. After the pressure had been restored, the plants were returned to an upright position and planted in an artificial climate chamber (LH-410SP, Nippon Medical and Chemical Instruments Co., Ltd.).

[0053] (2-2) Cultivation of infected leaves (expression process) After infection, the plants were cultivated using an artificial climate chamber (LH-410SP, Nippon Medical and Chemical Instruments Manufacturing Co., Ltd.) for 6 days using the DFT method. The environmental conditions were controlled as follows: <Environmental conditions> - Temperature: 20℃ - Relative humidity: 60-80% - CO2 concentration: 500 ppm - Illumination: Average photosynthetic photon flux density (PPFD): 140-160 μmol / m 2 10 seconds, 24-hour continuous irradiation, three-wavelength fluorescent lamp "Lupica Line" (Mitsubishi Electric Corporation)

[0054] Example 1: Production of modified activin A (1) Crushing Tobacco leaves were harvested from the transformed N. benthamiana 6 days after infection and stored frozen at -80°C until extraction. Frozen tobacco leaves were crushed in a crusher (T-25 digital ULTRA-TURRAX (IKA)) while still frozen. The extraction buffer used was 0.1 M sodium phosphate, 0.5 M arginine, 5 mM sodium pyrosulfite, pH 8.0, in an amount twice the weight of the leaves.

[0055] (2) Pretreatment Ammonium sulfate was added to the collected supernatant to make it a 35% saturated ammonium sulfate solution. After stirring for 1 hour at room temperature, the solution was centrifuged at 12,000 g for 15 minutes at room temperature, and the 35% ammonium sulfate fraction supernatant was collected. Ammonium sulfate was added to the supernatant to make it a 60% saturated ammonium sulfate solution. After stirring for 1 hour at room temperature, the solution was centrifuged at 12,000 g for 15 minutes at room temperature, and the 60-90% ammonium sulfate fraction precipitate was collected.

[0056] (3) Affinity purification 50 g of tobacco leaf 60-90% ammonium sulfate fraction precipitate was added to 10 mL of equilibration solution for His-tag affinity purification (20 mM HEPES, 150 mM NaCl, 10% (w / v) glycerol, pH 8.0) to dissolve the precipitate. This solution was loaded onto a HisTrap HP 1 mL column (Cytiva) equilibrated with equilibration solution for His-tag affinity purification at a residence time of 3 min. The column was then washed with wash solution for His-tag affinity purification (20 mM HEPES, 150 mM NaCl, 20 mM imidazole, 10% (w / v) glycerol, pH 8.0). Finally, an elution solution for His-tag affinity purification (20 mM HEPES, 150 mM NaCl, 200 mM imidazole, 10% (w / v) glycerol, pH 8.0) was applied, and the elution peak was collected to obtain a modified proactivin A solution.

[0057] (4) Protease treatment HRV 3C protease was added to the modified proactivin A solution obtained by the above method to cleave the pro-region of the modified proactivin A.

[0058] (5) Fractionation Acetonitrile was added to the pro-domain-modified activin A mixture to a concentration of 30-60%, and the mixture was centrifuged at 10,000 g at room temperature for 10 minutes to recover the supernatant, yielding a crude modified activin A solution.

[0059] (6) Desalination The crudely purified modified activin A was desalted by buffer exchange with 4 mM hydrochloric acid using an ultrafiltration membrane (Amicon Ultra-15 10K membrane (Merck)).

[0060] (7) Cation exchange chromatography The crude modified activin A solution was passed through an SP Sepharose HP (Cytiva) column equilibrated with 20 mM glycine pH 3.0, 40% (v / v) acetonitrile, and linear gradient elution with 500 mM NaCl (0-500 mM NaCl, 50 CV) was performed. The resulting elution peak was fractionated and collected to obtain a purified modified activin A solution.

[0061] (8) Post-processing The purified modified activin A solution was diluted two-fold with a solution of 20 mM glycine, pH 3.0, 250 mM NaCl, and concentrated using an ultrafiltration membrane (Amicon Ultra-4 10K membrane, Merck).

[0062] Comparative Example 1: Study of cation exchange chromatography mobile phase Modified activin A was purified in the same manner as in Example 1, except that the cation exchange chromatography in step (7) of steps (1) to (8) in Example 1 was carried out under the following conditions. (7) Cation exchange chromatography The crude modified activin A solution was passed through an SP Sepharose HP (Cytiva) column equilibrated with 20 mM citric acid, pH 3.0, 40% (v / v) acetonitrile, and subjected to linear gradient elution with 500 mM NaCl (0-500 mM NaCl, 50 CV). The resulting elution peak was fractionated and collected to obtain a purified modified activin A solution.

[0063] Comparative Example 2: Examination of cation exchange chromatography mobile phases Modified activin A was purified in the same manner as in Example 1, except that the cation exchange chromatography in step (7) of steps (1) to (8) in Example 1 was carried out under the following conditions. (7) Cation exchange chromatography The crude modified activin A solution was passed through an SP Sepharose HP (Cytiva) column equilibrated with 20 mM citric acid, pH 3.0, 40% (v / v) acetonitrile, and linear gradient elution with 500 mM NaClO4 (0-500 mM NaCl, 50 CV) was performed. The resulting elution peak was fractionated and collected to obtain a purified modified activin A solution.

[0064] [Comparative Example 3: Examination of fractionation and desalting] The modified activin A was purified in the same manner as in Example 1, except that of steps (1) to (8) in Example 1, the fractionation in step (5), the desalting in step (6), and the cation exchange chromatography in step (7) were changed as follows. (5) Fractionation The pH of the mixture of the pro-domain-modified activin A was adjusted to pH 3.0 with 1 M hydrochloric acid. Acetonitrile was added to a final concentration of 50% (v / v), and the mixture was centrifuged at 10,000 g at room temperature for 10 minutes to recover the supernatant, yielding a crude modified activin A solution. (6) Desalination No desalting was performed. (7) Cation exchange chromatography The crude modified activin A solution was passed through an SP Sepharose HP (Cytiva) column equilibrated with 20 mM citric acid, pH 3.0, and 50% (v / v) acetonitrile. After stepwise washing with 175 mM NaCl, impurities were removed and the column was eluted with 250 mM NaCl. The resulting elution peak was fractionated and collected to obtain a purified modified activin A solution.

[0065] [Comparative Example 4: Examination of fractionation and desalting] The modified activin A was purified in the same manner as in Example 1, except that of steps (1) to (8) in Example 1, the fractionation in step (5), the desalting in step (6), and the cation exchange chromatography in step (7) were changed as follows. (5) Fractionation Acetonitrile was added to the mixture of the pro-domain-cleaved pro-domain and modified activin A to a final concentration of 50% (v / v). 1 M hydrochloric acid was added to adjust the pH to 3.0, and the mixture was centrifuged at 10,000 g at room temperature for 10 minutes. The supernatant was collected to obtain a crude modified activin A solution. (6) Desalination No desalting was performed. (7) Cation exchange chromatography The crude modified activin A solution was passed through an SP Sepharose HP (Cytiva) column equilibrated with 20 mM citric acid, pH 3.0, and 50% (v / v) acetonitrile. After stepwise washing with 175 mM NaCl, impurities were removed and the column was eluted with 250 mM NaCl. The resulting elution peak was fractionated and collected to obtain a purified modified activin A solution.

[0066] [Comparative Example 5: Examination of fractionation and desalting] The modified activin A was purified in the same manner as in Example 1, except that of steps (1) to (8) in Example 1, the fractionation in step (5), the desalting in step (6), and the cation exchange chromatography in step (7) were changed as follows. (5) Fractionation The pH of the mixture of pro-domain-modified activin A was adjusted to pH 2.5 with 1 M hydrochloric acid. Acetonitrile was added to a final concentration of 50% (v / v), and the mixture was centrifuged at 10,000 g at room temperature for 10 minutes, and the supernatant was collected. (6) Desalination No desalting was performed. (7) Cation exchange chromatography The crude modified activin A solution was passed through an SP Sepharose HP (Cytiva) column equilibrated with 20 mM citric acid, pH 2.5, and 50% (v / v) acetonitrile. After stepwise washing with 213 mM NaCl, impurities were removed and the column was eluted with 250 mM NaCl. The resulting elution peak was fractionated and collected to obtain a purified modified activin A solution.

[0067] Test Example 2: Calculation of purity and recovery rate of purified modified activin A and evaluation of dimer structure The purified modified activin A solutions obtained after step (8) in Example 1 and Comparative Examples 1-5 were used as evaluation samples to calculate the purity and recovery rate of modified activin A and to evaluate the dimer structure.

[0068] <Purity> The purity was calculated as follows. The evaluation samples were mixed with an equal volume of non-reducing 2x Laemmli Sample Buffer (Bio-Rad) and heated at 70°C for 10 minutes. Subsequently, they were loaded onto a 10-20% gradient gel e-PAGEL (ATTO) for SDS-PAGE and run at 20-40 mA for 40-90 minutes. After electrophoresis, the gels were stained with Oriole fluorescent dye (Bio-Rad) or CBB (GelCode Blue, ThermoFisher) and photographed using a GelDoc EZ imager (Bio-Rad). The resulting image data was analyzed by densitometry using Image Lab software (Bio-Rad), and the purity of the electrophoresed samples was calculated.

[0069] <Recovery rate> The recovery rate was calculated as follows. The evaluation sample and purified activin A preparation (R&D) were mixed in equal volumes with non-reducing 2x Laemmli Sample Buffer (Bio-Rad) and heated to 70°C for 10 minutes. Subsequently, the gel was loaded onto a 10-20% gradient gel e-PAGEL (ATTO) for SDS-PAGE at 20-40 mA for 40-90 minutes. After electrophoresis, the gel was stained with Oriole fluorescent dye (Bio-Rad) or CBB (GelCode Blue, ThermoFisher) and photographed using a GelDoc EZ imager (Bio-Rad). The resulting image data was analyzed by densitometry using Image Lab software (Bio-Rad). The sample volume was calculated from the standard curve of purified activin A preparation, and the recovery rate of column purification was determined.

[0070] <Evaluation of dimer structure> The dimer structure was evaluated as follows. The evaluation samples were mixed with an equal volume of reducing 2x Laemmli Sample Buffer (Bio-Rad) and heated at 70°C for 10 minutes. Subsequently, they were loaded onto a 10-20% gradient gel e-PAGEL (ATTO) for SDS-PAGE and run at 20-40 mA for 40-90 minutes. After electrophoresis, the gels were stained with Oriole fluorescent dye (Bio-Rad) or CBB (GelCode Blue, ThermoFisher) and photographed using a GelDoc EZ imager (Bio-Rad). The resulting image data was analyzed by densitometry using Image Lab software (Bio-Rad), and the monomer / dimer ratio of the electrophoresed samples was calculated. Activin A in its native dimeric structure is detected as a monomer by SDS-PAGE under the reducing conditions described above. Therefore, the dimer detected here is a dimer having a structure different from the native dimeric structure, and the lower the detected ratio of the dimer to the monomer, the higher the proportion of modified activin A in the native dimeric structure in the obtained modified activin A.

[0071] The results are shown in Table 1.

[0072] [Table 1]

[0073] In Example 1, a highly pure (82.6%) modified activin A having a native dimer structure could be purified with a high recovery rate (81%). In Comparative Example 1, in which a citric acid acetonitrile solution was used instead of a glycine acetonitrile solution for cation exchange chromatography, the recovery rate was lower than in Example 1, and modified activin A having a non-native dimer structure was detected. In Comparative Example 2, in which a citric acid acetonitrile solution was used in place of a glycine acetonitrile solution for cation exchange chromatography and a chaotropic ion (NaClO4) concentration gradient was used in place of a salt concentration gradient, the purity was lower and the recovery rate was significantly lower than in Example 1. Furthermore, in Comparative Example 2, modified activin A having a non-native dimer structure was also detected.

[0074] It was shown that the purification purity could be improved by desalting the crudely purified modified activin A solution and replacing it with a solvent of pH 1.5-3.5 (Example 1) prior to cation exchange chromatography (comparison with Comparative Examples 3-5).

[0075] [Test Example 3: Evaluation of activity of purified modified activin A] The biological activity of the purified modified activin A obtained in Example 1 was measured using the growth inhibition of MPC-11 cells (mouse myeloma) as an index according to the method described in the literature (Journal of Endocrinology (1999) 162, 111-116).

[0076] Purified modified activin A, as well as control activin A (Fujifilm Wako) or activin A (R&D) were added to MPC-11 cells at 1,000-2,000 cells / well and cultured for 3 days in a 5% CO2 incubator at 37°C. Cell Counting Kit-8 (Fujifilm Wako, CK04) was added at 1 / 10 the volume of the culture medium and incubated for 4 hours in a 5% CO2 incubator at 37°C. The absorbance at 450 nm was measured to quantify the cell number. The relative concentration to the solvent control was calculated, and the 50% effective concentration (ED50) was calculated from this value.

[0077] Purified modified activin A was ED 50 The value was lower than 5 ng / mL and was equivalent to that of Activin A manufactured by Fujifilm Wako Co., Ltd. and Activin A manufactured by R&D Co., Ltd., which were used as controls. [Sequence List Free Text]

[0078] SEQ ID NO: 1: Amino acid sequence of human native proactivin A SEQ ID NO: 2: Amino acid sequence of human native activin A SEQ ID NO: 3: Amino acid sequence of modified human proactivin A SEQ ID NO: 4: Amino acid sequence of modified human activin A

Claims

1. A method for producing recombinant activin A containing a mature sequence using a plant cell as a host cell, comprising: an intermediate treatment step in which the solution containing the recombinant activin A is desalted and replaced with a salt-free solvent having a pH of 1.5-3.5; a purification step of purifying the recombinant activin A by subjecting the solution containing the recombinant activin A to cation exchange chromatography; the cation exchange chromatography is performed by a salt gradient elution method using a mobile phase containing a buffer solution of pH 1.5 to 3.5 containing 5 to 100 mM of a zwitterion selected from the group consisting of proline, glycine, alanine, taurine, γ-aminobutyric acid (GABA), and betaine, and 40 to 70% by volume of a water-soluble organic solvent selected from the group consisting of a lower alcohol, acetonitrile, dimethyl sulfoxide, and dimethylformamide; The mature sequence consists of the amino acid sequence of SEQ ID NO: 2, or the amino acid sequence of SEQ ID NO: 2 with one or two amino acid substitutions, insertions and / or deletions. Manufacturing method.

2. The manufacturing method described in claim 1, wherein the mature sequence consists of an amino acid sequence in which the second leucine in the amino acid sequence of sequence number 2 is replaced.

3. The manufacturing method described in claim 2, wherein the mature sequence consists of the amino acid sequence of sequence number 4.

4. A manufacturing method described in any one of claims 1 to 3, wherein the concentration of the zwitterion in the mobile phase is 10-25 mM.

5. A manufacturing method described in any one of claims 1 to 4, wherein the zwitterion is glycine.

6. A manufacturing method described in any one of claims 1 to 5, wherein the water-soluble organic solvent is acetonitrile.

7. A manufacturing method described in any one of claims 1 to 6, wherein the purity of the recombinant activin A is 80% or more, the recovery rate is 70% or more, and the monomer ratio of the recombinant activin A under reducing conditions is 80% or more.

8. The method of any one of claims 1 to 7, which does not include a step of refolding the recombinant activin A.

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