A method for producing a culture supernatant containing a target protein with reduced culture impurities.

Abstract

To provide a method for producing a target protein from cell culture without generating impurities in the culture medium. [Solution] This disclosure provides a method for producing a culture supernatant containing a target protein with reduced culture impurities, (1) A step of performing cell culture using a culture medium containing a cell culture scaffold material, (2) A step of collecting the culture supernatant from step (1) above, Includes, The present invention provides a method in which the culture supernatant obtained in step (2) is a culture supernatant with reduced culture impurities, which simplifies the downstream process aimed at reducing culture impurities.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a culture supernatant containing a target protein with reduced culture impurities. [Background technology]

[0002] In recent years, with the development of the bioindustry, development related to manufacturing using cells has been actively pursued. In particular, in the field of biopharmaceuticals, represented by antibody drugs, there is a need to culture cells in large quantities and efficiently produce useful biopharmaceuticals. Generally, biopharmaceuticals (target proteins) are produced by cell culture using animal-derived cell lines, and a representative example of such cells is the Chinese hamster ovary cell (CHO cell).

[0003] One method for mass cell culture is suspension agitation culture using culture vessels such as spinner flasks. However, suspension agitation culture applies strong shear stress to the cells, leading to an increase in dead cells and an increase in impurities such as proteins and nucleic acids derived from dead cells in the culture medium. In order to commercialize biopharmaceuticals for use as medicines, it is necessary to remove impurities from the cell culture medium and purify it to a composition sufficient for human therapeutic use.

[0004] The standard procedure for purifying a target protein from a cell culture medium involves first centrifuging the cell culture medium to remove turbid components by sedimentation. Next, cell debris smaller than approximately 1 μm that cannot be removed by centrifugation is removed by size filtration using a microfiltration membrane. Furthermore, to sterilize the solution, sterile filtration is performed using a filtration membrane with a maximum pore size of 0.22 μm or less to obtain a clear solution containing the target protein (harvest process). Once a clear solution containing the target protein is obtained, the target protein is then separated and purified by a purification process using a combination of multiple chromatography techniques, including affinity chromatography (downstream process). Impurities (nucleic acids, proteins, etc.) produced by cells in the culture medium and dissolved in the culture medium are removed in the downstream process.

[0005] Patent documents 1 and 2 disclose adsorbents capable of removing nucleic acids as impurities from solutions containing biomolecules such as proteins useful as pharmaceuticals, but they do not disclose any methods for simplifying the downstream process. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2006-68590 [Patent Document 2] Japanese Patent Publication No. 2007-54739 [Overview of the project] [Problems that the invention aims to solve]

[0007] While it is important to improve the purity of the target protein by utilizing its physical properties and skillfully combining multiple chromatography processes in the downstream step, the decrease in the recovery rate (yield) of the target protein due to multiple chromatography processes can lead to increased manufacturing costs for pharmaceuticals that use the target protein as a raw material, and ultimately to higher medical costs.

[0008] As described above,although high-density cell culture using suspension agitation culture enables mass production of the target protein,the increase in dead cells causes not only an increase in the concentration of the target protein in the culture liquid but also an increase in the concentration of impurities.As a result,the load on the downstream process increases,not only requiring a long time for the purification process such as column washing,but also shortening the life of the column beads used in chromatography.The deterioration of the column life affects the purification cost and is thus undesirable.

[0009] Also,in the methods disclosed in Patent Documents 1 and 2,although it is possible to remove nucleic acids,the purification process itself cannot be simplified.

[0010] In view of the above-described current state of the prior art,an object to be solved by the present invention is to provide a method for producing a target protein without generating impurities in the culture liquid from cell culture.

Means for Solving the Problems

[0011] As a result of intensive studies and repeated experiments to solve the above problems,the present inventors have surprisingly found that by performing cell culture using a cell culture scaffold material,it is possible to produce a culture supernatant containing a target protein in a state where impurity production is suppressed,and thus have completed the present invention.

[0012] That is,the present invention includes the following aspects.

[0013] [1] A method for producing a culture supernatant with reduced culture impurities containing a target protein,comprising: (1) a step of performing cell culture using a medium containing a cell culture scaffold material; (2) a step of recovering the culture supernatant of the step (1); wherein the culture supernatant obtained in the step (2) is a culture supernatant with reduced culture impurities that enables simplification of the downstream process aimed at reducing culture impurities. ​[2] The method according to [1], wherein the culture impurity is nucleic acid. [3] The culture supernatant with reduced culture impurities has a 35% or greater reduction in the mass of nucleic acids per unit mass of target protein compared to the cell culture supernatant when the cell culture scaffold material is not used, according to the method of [2]. [4] The culture supernatant with reduced culture impurities is the method according to [2], wherein the mass of nucleic acid per mass (mg) of target protein is less than 90 ng / mg. [5] The method according to any one of [1] to [4], characterized in that the simplification of the downstream process is the omission of ion exchange chromatography. [6] The method according to any one of [1] to [5], characterized in that the cell culture scaffold material is a cationic microcarrier. [7] The method according to any one of [1] to [6], wherein the concentration of the cell culture scaffold material in the culture medium at the time of cell seeding is 0.625 g / L or more and 6.25 g / L or less. [8] The method according to any one of [1] to [7], wherein the target protein is a therapeutic protein. [Effects of the Invention]

[0014] The method according to the present invention makes it possible to reduce the amount of impurities per equivalent of the target protein in the culture supernatant and to simplify the downstream process. [Modes for carrying out the invention]

[0015] The embodiments for carrying out the present invention (hereinafter referred to as "this embodiment") will be described in detail below. The embodiments shown below are illustrative examples of methods for realizing the technical concept of the present invention, and are not limited to these examples.

[0016] The method of this embodiment is a method for producing a culture supernatant with reduced culture impurities that contains a target protein such as a biopharmaceutical, (1) A step of performing cell culture using a culture medium containing a cell culture scaffold material, (2) A step of collecting the culture supernatant from step (1) above, It may include the following. The culture supernatant obtained in step (2) is a culture supernatant with reduced culture impurities, which simplifies the downstream process aimed at reducing culture impurities.

[0017] The method according to this embodiment may include the steps of seeding a cell line capable of producing a target protein onto a cell culture scaffold and growing the cells on the cell scaffold.

[0018] In this specification, "culture supernatant with reduced culture impurities" refers to cell culture supernatant obtained by carrying out the method of the present invention, which has a reduced content of culture impurities. For example, it refers to culture supernatant in which impurities such as proteins and nucleic acids derived from dead cells present in the culture medium are reduced compared to conventional suspension agitation culture. In this specification, "culture impurities" refers to proteins, nucleic acids (e.g., DNA, RNA), lipids, and endogenous viruses derived from host cells other than the target protein. In one embodiment of the present invention, a culture supernatant with reduced culture impurities is obtained, but in particular, a culture supernatant with reduced nucleic acids is obtained. This makes it possible to simplify the downstream process described later, which was conventionally aimed at reducing culture impurities contained in the culture supernatant.

[0019] In one embodiment, the culture supernatant with reduced culture impurities provided by the present invention can reduce the mass of nucleic acids per unit mass of target protein by 35% or more, for example, 40% or more, 50% or more, 60% or more, or 70% or more, compared to cell culture supernatant without using the cell culture scaffold material to which the present invention is applied.

[0020] In one embodiment, the culture supernatant with reduced culture impurities provided by the present invention contains less than 90 ng / mg, preferably less than 70 ng / mg, more preferably less than 50 ng / mg, and even more preferably less than 30 ng / mg, of nucleic acids per mass (mg) of target protein.

[0021] In another embodiment, the method of the present invention may further include the step of adding the cell culture scaffold material to which cells are not adhered to the culture supernatant obtained in step (2) and stirring. This is expected to further promote the adsorption of culture impurities contained in the culture supernatant onto the cell culture scaffold, thereby promoting the reduction of culture impurities.

[0022] In one embodiment, Chinese hamster ovary cells (CHO cells) are exemplified as cell lines capable of producing the target protein applied to the cell culture scaffold material, but the cell line is not particularly limited as long as it can proliferate on the cell culture scaffold material. Examples of cell lines include CHO cells, VERO cells, MDBK cells, MDCK cells, BHK-21 cells, HEK293 cells, WI-38 cells, MRC-5 cells, and mesenchymal stem cells (MSCs). These cells can be obtained as standard strains from cell banks such as ATCC, or as improved strains with enhanced productivity and growth capabilities.

[0023] In this embodiment, a cationic microcarrier is exemplified as the cell culture scaffold material, but it is not limited to cationic microcarriers as long as the material, form, and surface properties allow for cell proliferation on the cell culture scaffold material. The cell culture scaffold material may also be a cell adhesion scaffold material (e.g., "Genocel" from Kyoto Medical Engineering Co., Ltd., "BioNOC II" from Esco VacciXcell, "UniTantrix" from Tantti Laboratory Inc.) or a cell adhesion microcarrier (e.g., "PVA Microcarrier" from Kuraray Co., Ltd., "Cytodex 3" from Cytiva, "Synthemax" from Corning).

[0024] When the cell culture scaffold material is a cationic microcarrier, its surface functional group is a diethylaminoethyl (DEAE) group, but it is not particularly limited as long as it is a functional group that imparts cationicity, for example, it may be an imidazolium group, pyridinium group, pyrrolidinium group, piperidinium group, ammonium group, or phosphonium group. The material of the cationic microcarrier is not particularly limited as long as it is a water-insoluble carrier that does not exhibit cytotoxicity, and one embodiment of this is cellulose or dextran. From the standpoint of cell adhesion and proliferation, the surface charge of the cationic microcarrier is preferably 0.90 mmol Cl - / g~1.95 mmol Cl - / g or more, more preferably 0.90 mmol Cl - / g~1.60 mmol Cl - / g, more preferably 0.90 mmol Cl - / g~1.20 mmol Cl - The amount is 1 / g or more. The particle size of the cationic microcarrier is preferably 100-400 μm, more preferably 200-300 μm, and even more preferably 200-280 μm. The cationic microcarrier may be porous or non-porous. In the case of porous microcarrier, the average pore size is preferably 10-70 μm, more preferably 20-40 μm, and the effective surface area is preferably 5000-30000 cm². 2 / g, more preferably 8000~15000cm 2 It is / g.

[0025] In this embodiment, the culture process is not particularly limited in terms of culture conditions, as long as the target protein is produced from the cells. It can be performed by static culture, agitated culture, or shaking culture. However, agitated culture is preferable from the viewpoint of efficiency, and can be appropriately set depending on the type of cells to be cultured and the target protein to be produced. When performing agitated culture using a cell culture scaffold material, such as a cationic microcarrier, it is preferable that the microcarrier concentration in the culture medium at the time of cell seeding is 0.625 g / L to 6.25 g / L, preferably 1.25 g / L to 5.5 g / L, and more preferably 2.5 g / L to 3.75 g / L.

[0026] In this embodiment, the components of the culture medium used in the culture process are not particularly limited and can be appropriately selected depending on the cells to be cultured and the target protein to be produced. In one embodiment, from the viewpoint of avoiding the risk of contamination with animal-derived components, a so-called serum-free medium that does not contain serum or serum-derived proteins is preferred. The serum-free medium may contain proteins or protein hydrolysates derived from plants or microorganisms, or it may contain proteins produced using genetically modified microorganisms or plants, or insulin or growth factors. When using a serum-free medium, cells can be acclimated to the serum-free medium using known methods before being subjected to culture.

[0027] In this embodiment, the "downstream process" refers to the process of separating and purifying the target protein using a purification process that includes affinity chromatography, or a combination of one or more chromatography techniques, performed after the harvesting process, in which the cell culture medium is centrifuged to remove turbid components by sedimentation, then cell debris of approximately 1 μm or less that cannot be removed by centrifugation is removed by size filtration using a microfiltration membrane, and finally sterilization filtration is performed using a filtration membrane with a maximum pore size of 0.22 μm or less to obtain a clear solution of the target protein.

[0028] In this embodiment, impurities produced from cells in the culture medium and dissolved in the culture medium during the downstream process include nucleic acids, proteins, and lipids, but are not limited to these, as long as they are impurities present in the culture medium.

[0029] In this embodiment, the downstream process refers to a purification method performed using any combination of chromatography, such as affinity chromatography, ion exchange chromatography (e.g., cation exchange chromatography, anion exchange chromatography), and size exclusion chromatography. Simplification of the downstream process means reducing the amount of impurities removed by these chromatography methods, reducing the load on the column, or omitting the chromatography process itself.

[0030] In this specification, "target protein" refers to a protein produced by a cell that exhibits a desired function, such as a protein used for therapeutic purposes. The method for expressing the target protein in cells is not particularly limited and may be carried out according to known techniques. For example, cells into which a vector containing the gene expressing the target protein has been transiently introduced using genetic engineering may be used, cells into which the gene has been incorporated into the cell genome and modified to continuously express the target protein may be used, or cells that naturally produce the target protein without the use of genetic engineering techniques may be used.

[0031] The target protein in this embodiment is not limited, but may be, for example, a therapeutic protein. The therapeutic protein may be, for example, a physiologically active protein such as erythropoietin, insulin, or albumin; a cytokine such as tumor necrosis factor α, interleukin 6 (IL-6), interleukin 8 (IL-8), granulocyte colony-stimulating factor (G-CSF), or interferon; an enzyme such as thrombin or trypsin; a monoclonal antibody, a polyclonal antibody, or a fragment thereof (e.g., Fab, Fab', F(ab')2, Fv, scFv, dsFv, bis-scFv, diabody, triabody, or tetrabody). [Examples]

[0032] Hereinafter, the present embodiment will be described more specifically based on examples and comparative examples, but the present invention is not limited to the following examples and comparative examples.

[0033] <(1) Example 1: Production of target protein using cationic porous microcarriers> (1-1) Preparation of medium for scale-up culture Gibco TM L-Methionine-(S,R)-Sulfoximine (manufactured by Cayman Chemical Co., 25 μM) and an anti-clumping agent (「Invitrogen Anti-Clumping Agent」manufactured by Thermo Fisher Scientific, 0.5% (v / v)) were added to Gibco

[0034] (1-2) Scale-up culture 20 ml of the above medium for scale-up culture was added to a 125 ml Erlenmeyer flask (manufactured by Corning, without baffles, polycarbonate). CHO-K1 cells were seeded in the culture flask at a density of 3×10 5 cells / ml, and cultured with shaking at 120 rpm for about 4 days at 37 °C and 5% CO2 using an orbital shaker (「OS-762 for use in incubator」manufactured by Optim Corporation).

[0035] (1-3) Preparation of production culture medium and feed medium Gibco TM Dynamis TM AGT TM L-Methionine-(S,R)-Sulfoximine (manufactured by Cayman Chemical Co., 25 μM) was added to Medium (manufactured by Thermo Fisher Scientific) to prepare a production culture medium.

[0036] Also, Gibco TM CHO CD EfficientFeed TMBLiquid Nutrient Supplement (manufactured by Thermo Fisher Scientific) was used as the feed medium.

[0037] (1-4) Cell counting process Ten μl of the cell suspension obtained from the above-mentioned expansion culture was collected and mixed with ten μl of trypan blue solution. Ten μl of the mixture was added to a hemocytometer (Air Braun Co., Ltd. "Disposable Hemocytometer C-Chip-Medical Neubawell Improved Model"), and the number of viable and dead cells was measured.

[0038] (1-5) Microcarrier preparation process 50 mg of cationic porous microcarrier (BioCradle L, manufactured by Asahi Kasei Corporation) and 5 ml of PBS were added to a 15 ml centrifuge tube and vortexed to suspend the mixture. After swelling at room temperature for at least 1 hour, the mixture was sterilized in an autoclave at 121°C for 15 minutes and allowed to cool to room temperature. Once cooled to room temperature, the PBS was removed carefully, taking care not to remove the microcarrier. Production culture medium prepared in (1-3) above was added to the mixture to a total volume of 5 ml, and the mixture was vortexed again to suspend the mixture.

[0039] (1-6) Cell seeding process A 10 g / L microcarrier suspension prepared in the microcarrier preparation step was added to a 125 ml Erlenmeyer flask (Corning, without baffles, polycarbonate). Production culture medium prepared in (1-3) above was added to the flask until the total volume reached 30 ml, and the mixture was allowed to stand at 37°C under 5% CO2 conditions for at least 1 hour. To a 125 ml Erlenmeyer flask containing the microcarrier suspension that has been left standing for more than 1 hour, add 200 × 10⁻⁶ 5 Cells were seeded after adding the cell suspension prepared in the expansion culture step to a total of cells / ml. At this time, the amount of suspension to be added was calculated using the cell count measured in the cell count measurement step described above. Furthermore, in order to achieve a microcarrier concentration of 1.25 g / l during the cell seeding step, the insufficient production culture medium was added so that the liquid volume in the Erlenmeyer flask became 40 ml. The Erlenmeyer flask containing the cell suspension was placed on an orbital shaker (Optima Co., Ltd. "OS-762 for Incubator Use") set up in an incubator at 37°C and 5% CO2, and shaken at 120 rpm for 5 minutes. After standing for approximately 18 hours, continuous shaking culture was performed at 120 rpm.

[0040] (1-7) Feed addition process In the continuous shaking culture described above, the feed medium prepared in (1-3) above was added on the 4th, 6th, 8th, 10th, and 12th day after sowing. The amount of feed medium added was 8 vol% of the amount of medium in the Erlenmeyer flask at that time.

[0041] (1-8) Clarification process Fourteen days after seeding the cells in the aforementioned cell seeding process, the entire culture medium, including the microcarriers, in the Erlenmeyer flask was transferred to a 50 ml centrifuge tube. After centrifugation at 800 rpm for 5 minutes, the entire culture supernatant was passed through a 0.22 μm filter to obtain a clarified culture supernatant.

[0042] (1-9) Purification process Antibodies were purified from the culture supernatant using the following equipment and columns. • Purification equipment: Cytiva "AKTA" TM "avant 25" • Protein A ligand column: Cytiva "HiTrap" TM MabSelect SuRe LX 1ml

[0043] 16 ml of the culture supernatant clarified above was added to a column equilibrated with equilibration buffer (20 mM NaH2PO4-Na2HPO4, 150 mM NaCl, pH 8.0). After passing 5 CV (column volume) of equilibration buffer through the column, 5 CV of 25 mM Tris-HCl (pH 7.0) was passed through as a washing solution to wash away impurities that did not adsorb to the column. Furthermore, 5 CV of 25 mM acetate buffer (pH 3.4) was passed through as an eluent to elute the target antibody, which was then recovered. Finally, 3 CV of equilibration buffer was passed through to complete the antibody purification.

[0044] (1-10) Determination of antibody concentration The recovered antibody solution was measured using NanoDrop One (Thermo Fisher Scientific), and the antibody concentration was quantified from the absorbance at 280 nm. The amount of antibody contained in the antibody solution was calculated from the volume of the solution and the antibody concentration. Furthermore, the antibody concentration was estimated by dividing this amount of antibody by the volume of the clarified culture supernatant, which was 16 ml. The evaluation criteria for the measurement results are shown below.

[0045] (Evaluation Criteria) A: Antibody concentration 1mg / ml or more B: Antibody concentration less than 0.7-1 mg / ml C: Antibody concentration less than 0.3-0.7 mg / ml D: Antibody concentration less than 0.1-0.3 mg / ml E: Antibody concentration less than 0.1 mg / ml

[0046] (1-11) Nucleic acid purification process The nucleic acids present in the clarified culture supernatant were recovered using the following kit. Nucleic acid purification: "NucleoSpin Gel and PCR Clean-up" manufactured by MACHEREY-NAGEL

[0047] (Preparation of DNA-binding samples) A binding sample was prepared by mixing 6 ml of the clarified culture supernatant with 12 ml of commercially available binding buffer (MACHEREY-NAGEL's "Binding Buffer NTB").

[0048] (Binding DNA) The column included with NucleoSpin Gel and PCR Clean-up was placed in the accompanying collection tube (2 ml), and up to 700 μl of the conjugated sample was loaded. The tube loaded with the conjugated sample was centrifuged at 11,000 × g for 30 seconds. The flow-through was discarded, and the column was returned to the collection tube. The loading and centrifugation of the sample was repeated until all of the remaining conjugated sample passed through the column.

[0049] (Cleaning of silica film) After all the bound samples had passed through the column, 700 μl of Buffer NT3 (diluted to 20% with ethanol) included in the NucleoSpin Gel and PCR Clean-up kit was loaded, and the column was centrifuged at 11,000 × g for 30 seconds. The flow-through was discarded, and the column was returned to the collection tube. Another 700 μl of Buffer NT3 was loaded, and the column was centrifuged at 11,000 × g for 30 seconds. The flow-through was discarded, and the column was returned to the collection tube.

[0050] (Drying of silica film) The collection tube with the above column was centrifuged at 11,000 × g for 1 minute to completely remove Buffer NT3. Then, to completely remove any remaining ethanol, it was allowed to stand at 27°C for 10 minutes.

[0051] (Leaching of nucleic acids) The silica membrane column, after drying, was placed in a new 1.5 ml microcentrifuge tube. 20 μl of Buffer NE (included with NucleoSpin Gel and PCR Clean-up) was added, and the tube was incubated at room temperature (15-27°C) for 1 minute. After incubation, the tube was centrifuged at 11,000 × g for 1 minute, and the eluted nucleic acid solution was collected.

[0052] (1-12) Nucleic acid quantification process (Quantitative method: quantitative PCR) The nucleic acid solution recovered in the nucleic acid purification process described above (1-11) was used to create a series of dilutions of 1 / 10, 1 / 100, and 1 / 1000 to obtain the diluted nucleic acid solutions. A commercially available quantitative PCR reagent kit for CHO cells (QCdetect, manufactured by Fujifilm Wako Pure Chemical Corporation) TM Using the "Residual DNA Detection Kit for CHO Cells," the nucleic acid dilution solution was prepared as a PCR measurement sample according to the kit protocol. qPCR was performed using a QuantStudio 7 Flex real-time PCR system (Thermo Fisher Scientific) for 40 cycles, with each cycle consisting of 95°C for 10 minutes and (95°C for 15 seconds and 60°C for 1 minute). A calibration curve was created from the concentrations of each calibration curve sample, based on the number of PCR cycles (Ct value) at which the target gene amplification curve intersected the threshold at each concentration (copy number) of the target gene calibration curve sample. The concentration of each PCR measurement sample was calculated from the Ct value of the amplification curve of the CHO-derived gDNA for each PCR measurement sample and the aforementioned calibration curve. Among the dilution series, samples that fell between the Ct values ​​of the lowest and highest concentrations of the calibration curve samples were treated as reliable results. The evaluation criteria for the measurement results are shown below.

[0053] (Evaluation Criteria) A: Nucleic acid concentration less than 20 ng / ml B: Nucleic acid concentration less than 20-100 ng / ml C: Nucleic acid concentration less than 100-150 ng / ml D: Nucleic acid concentration less than 150-200 ng / ml E: Nucleic acid concentration 200ng / ml or more

[0054] (1-13) Evaluation of nucleic acid quantity relative to antibody quantity Using the antibody concentration (steps (1-10)) and nucleic acid concentration (steps (1-12)) quantified by the above method, the amount of nucleic acid per unit of antibody contained in the culture supernatant was calculated according to Equation 1 below.

[0055] Nucleic acid amount / antibody amount [ng / mg] = Nucleic acid concentration [ng / ml] / antibody concentration [ng / ml] (Formula 1)

[0056] The evaluation criteria for the measurement results are shown below. (Evaluation Criteria) A: Nucleic acid level / antibody level less than 30 ng / mg B: Nucleic acid amount / antibody amount: less than 30-50 ng / mg C: Nucleic acid amount / antibody amount: less than 50-70 ng / mg D: Nucleic acid amount / antibody amount less than 70-90 ng / mg E: Nucleic acid amount / antibody amount 90ng / mg or more

[0057] Furthermore, the reduction rate was calculated from the nucleic acid amount / antibody amount values ​​in suspension culture shown in Comparative Example 1, according to Equation 2.

[0058] Reduction rate [%] = (Amount of nucleic acid in Comparative Example 1 / Amount of antibody [ng / mg]) / (Amount of nucleic acid in other examples or comparative examples [ng / mg]) ... (Equation 2)

[0059] The evaluation criteria for the reduction rate are shown below. (Evaluation Criteria) A: Reduction rate of 70% or more B: Reduction rate less than 50-70% C: Reduction rate less than 30-50% D: Reduction rate 10-30% E: Reduction rate less than 10%

[0060] The results are shown in Table 1.

[0061] <(2) Example 2: Production of target protein using cationic porous microcarriers> Except for the microcarrier preparation and cell seeding steps, nucleic acid and antibody concentrations were quantified according to the same method as in Example 1. The modified microcarrier preparation and cell seeding steps from Example 1 were performed according to the following methods.

[0062] (2-1) Microcarrier preparation process 100 mg of cationic porous microcarrier (BioCradle L, manufactured by Asahi Kasei Corporation) and 5 ml of PBS were added to a 15 ml centrifuge tube and vortexed to suspend the mixture. After swelling at room temperature for at least 1 hour, the mixture was sterilized in an autoclave at 121°C for 15 minutes and then cooled to room temperature. Once cooled to room temperature, the PBS was removed carefully, taking care not to remove the microcarrier. Production culture medium prepared in (1-3) above was added to the mixture to a total volume of 5 ml, and the mixture was vortexed again to suspend the mixture.

[0063] (2-2) Cell seeding process A 20 g / L microcarrier suspension prepared in the microcarrier preparation step was added to a 125 ml Erlenmeyer flask (Corning, without baffles, polycarbonate). Production culture medium prepared in (1-3) above was added to the flask until the total volume reached 30 ml, and the mixture was allowed to stand at 37°C under 5% CO2 conditions for at least 1 hour. To a 125 ml Erlenmeyer flask containing the microcarrier suspension that has been left standing for more than 1 hour, add 200 × 10⁻⁶ 5 To form cells, the cell suspension prepared in the expansion culture step was added, and the cells were seeded. At this time, the amount of suspension to be added was calculated using the number of cells measured in the cell counting step described above. Furthermore, in order to achieve a microcarrier concentration of 2.5 g / l during the cell seeding step, the insufficient production culture medium was added so that the liquid volume in the Erlenmeyer flask became 40 ml. The Erlenmeyer flask containing the cell suspension was placed on an orbital shaker (Optima Co., Ltd. "OS-762 for Incubator Use") set up in an incubator at 37°C and 5% CO2, and shaken at 120 rpm for 5 minutes. After standing for approximately 18 hours, continuous shaking culture was performed at 120 rpm.

[0064] The antibody amount, nucleic acid quantification, nucleic acid amount per antibody amount, and reduction rate were measured using the same method as in Example 1. The results are shown in Table 1.

[0065] <(3) Example 3: Production of target protein using cationic porous microcarriers> Except for the microcarrier preparation and cell seeding steps, nucleic acid and antibody concentrations were quantified according to the same method as in Example 1. The modified microcarrier preparation and cell seeding steps from Example 1 were performed according to the following methods.

[0066] (3-1) Microcarrier preparation process 150 mg of cationic porous microcarrier (BioCradle L, manufactured by Asahi Kasei Corporation) and 5 ml of PBS were added to a 15 ml centrifuge tube and vortexed to suspend the mixture. After swelling at room temperature for more than 1 hour, the mixture was sterilized in an autoclave at 121°C for 15 minutes and then cooled to room temperature. Once cooled to room temperature, the PBS was removed carefully, taking care not to remove the microcarrier. Production culture medium was then added to the tube to a total volume of 5 ml, and the mixture was vortexed again to suspend the mixture.

[0067] (3-2) Cell seeding process A 30 g / L microcarrier suspension prepared in the microcarrier preparation step was added to a 125 ml Erlenmeyer flask (Corning, without baffles, polycarbonate). Production culture medium prepared in (1-3) above was added to the flask until the total volume was 30 ml, and the mixture was allowed to stand at 37°C under 5% CO2 conditions for at least 1 hour. To a 125 ml Erlenmeyer flask containing the microcarrier suspension that has been left standing for more than 1 hour, add 200 × 10⁻⁶ 5 To form cells, the cell suspension prepared in the expansion culture step was added, and the cells were seeded. At this time, the amount of suspension to be added was calculated using the number of cells measured in the cell counting step described above. Furthermore, in order to achieve a microcarrier concentration of 3.75 g / l during the cell seeding step, the insufficient production culture medium was added so that the liquid volume in the Erlenmeyer flask became 40 ml. The Erlenmeyer flask containing the cell suspension was placed on an orbital shaker (Optima Co., Ltd. "OS-762 for Incubator Use") set up in an incubator at 37°C and 5% CO2, and shaken at 120 rpm for 5 minutes. After standing for approximately 18 hours, continuous shaking culture was performed at 120 rpm.

[0068] The antibody amount, nucleic acid quantification, nucleic acid amount per antibody amount, and reduction rate were measured using the same method as in Example 1. The results are shown in Table 1.

[0069] <(4) Example 4: Production of target protein using cationic porous microcarriers> Except for the microcarrier preparation and cell seeding steps, nucleic acid and antibody concentrations were quantified according to the same method as in Example 1. The modified microcarrier preparation and cell seeding steps from Example 1 were performed according to the following methods.

[0070] (4-1) Microcarrier preparation process 200 mg of cationic porous microcarrier (BioCradle L, manufactured by Asahi Kasei Corporation) and 5 ml of PBS were added to a 15 ml centrifuge tube and vortexed to suspend the mixture. After swelling at room temperature for more than 1 hour, the mixture was sterilized in an autoclave at 121°C for 15 minutes and then cooled to room temperature. Once cooled to room temperature, the PBS was removed carefully, taking care not to remove the microcarrier. Production culture medium was then added to the tube to a total volume of 5 ml, and the mixture was vortexed again to suspend the mixture.

[0071] (4-2) Cell seeding process A 40 g / L microcarrier suspension prepared in the microcarrier preparation step was added to a 125 ml Erlenmeyer flask (Corning, without baffles, polycarbonate). Production culture medium prepared in (1-3) above was added to the flask until the total volume was 30 ml, and the mixture was allowed to stand at 37°C under 5% CO2 conditions for at least 1 hour. To a 125 ml Erlenmeyer flask containing the microcarrier suspension that has been left standing for more than 1 hour, add 200 × 10⁻⁶ 5 To form cells, the cell suspension prepared in the expansion culture step was added, and the cells were seeded. At this time, the amount of suspension to be added was calculated using the number of cells measured in the cell counting step described above. Furthermore, in order to achieve a microcarrier concentration of 5.0 g / l during the cell seeding step, the insufficient production culture medium was added so that the liquid volume in the Erlenmeyer flask became 40 ml. The Erlenmeyer flask containing the cell suspension was placed on an orbital shaker (Optima Co., Ltd. "OS-762 for Incubator Use") set up in an incubator at 37°C and 5% CO2, and shaken at 120 rpm for 5 minutes. After standing for approximately 18 hours, continuous shaking culture was performed at 120 rpm.

[0072] The antibody amount, nucleic acid quantification, nucleic acid amount per antibody amount, and reduction rate were measured using the same method as in Example 1. The results are shown in Table 1.

[0073] <(5) Example 5: Production of target protein using cationic nonporous microcarriers> Except for the microcarrier preparation and cell seeding steps, nucleic acid and antibody concentrations were quantified according to the same method as in Example 1. The modified microcarrier preparation and cell seeding steps from Example 1 were performed according to the following methods.

[0074] (5-1) Microcarrier preparation process 100 mg of cationic non-porous microcarrier (Cytiva "Cytodex 1") and 5 ml of PBS were added to a 15 ml centrifuge tube and vortexed to suspend the microcarrier. After swelling at room temperature for at least 1 hour, the tube was sterilized by autoclaving at 121°C for 15 minutes and then cooled to room temperature. Once cooled to room temperature, the PBS was removed carefully, taking care not to remove the microcarrier. Production culture medium prepared in (1-3) above was added to the tube to a total volume of 5 ml, and the mixture was vortexed again to suspend the microcarrier.

[0075] (5-2) Cell seeding process A 20 g / L microcarrier suspension prepared in the microcarrier preparation step was added to a 125 ml Erlenmeyer flask (Corning, without baffles, polycarbonate). Production culture medium prepared in (1-3) above was added to the flask until the total volume reached 30 ml, and the mixture was allowed to stand at 37°C under 5% CO2 conditions for at least 1 hour. To a 125 ml Erlenmeyer flask containing the microcarrier suspension that has been left standing for more than 1 hour, add 200 × 10⁻⁶ 5To form cells, the cell suspension prepared in the expansion culture step was added, and the cells were seeded. At this time, the amount of suspension to be added was calculated using the number of cells measured in the cell counting step described above. Furthermore, in order to achieve a microcarrier concentration of 2.5 g / l during the cell seeding step, the insufficient production culture medium was added so that the liquid volume in the Erlenmeyer flask became 40 ml. The Erlenmeyer flask containing the cell suspension was placed on an orbital shaker (Optima Co., Ltd. "OS-762 for Incubator Use") set up in an incubator at 37°C and 5% CO2, and shaken at 120 rpm for 5 minutes. After standing for approximately 18 hours, continuous shaking culture was performed at 120 rpm.

[0076] The antibody amount, nucleic acid quantification, nucleic acid amount per antibody amount, and reduction rate were measured using the same method as in Example 1. The results are shown in Table 1.

[0077] <(6) Comparative Example 1: Production of target protein using suspension stirring culture> (6-1) Preparation of culture medium for expansion Gibco TM A culture medium for expanded culture was prepared by adding CD CHO Medium (Thermo Fisher Scientific) + L-Methionine-(S,R)-Sulfoximine (Cayman Chemical Co., 25 μM) and an agglutination inhibitor (Thermo Fisher Scientific "Invitrogen Anti-Clumping Agent", 0.5% (v / v)), and used in the following experiments.

[0078] (6-2) Expanded culture A 125 ml Erlenmeyer flask (without baffles, made of polycarbonate, manufactured by Corning) was used as the culture vessel. 20 ml of the culture medium prepared in (1-1) above was added to a 125 ml Erlenmeyer flask (Corning, no baffles, polycarbonate). 3 × 10⁶ CHO-K1 cells were added to the culture flask. 5Seeds were seeded to a concentration of cells / ml, and cultured using an orbital shaker (Optima Co., Ltd.'s "Incubator OS-762") at 37°C and 5% CO2 conditions with shaking at 120 rpm for approximately 4 days.

[0079] (6-3) Preparation of culture media for production and culture media for feed Gibco TM Dynamis TM AGT TM A culture medium was prepared by adding L-Methionine-(S,R)-Sulfoximine (Cayman Chemical Co., 25 μM) and an agglutination inhibitor (Thermo Fisher Scientific "Invitrogen Anti-Clumping Agent", 0.5% (v / v)) to Medium (Thermo Fisher Scientific), and used in the following experiments.

[0080] Also, Gibco TM CHO CD EfficientFeed TM BLiquid Nutrient Supplement (manufactured by Thermo Fisher Scientific) was used as the feed medium.

[0081] (6-4) Cell counting process 10 μl of the cell suspension obtained from the expanded culture described in (6-2) above was taken and mixed with 10 μl of trypan blue solution. 10 μl of the mixture was added to a hemocytometer (Air Braun Co., Ltd. "Disposable Hemocytometer C-Chip-Medical Neubawell Improved Model"), and the number of viable and dead cells was measured.

[0082] (6-5) Cell seeding process 30 ml of production culture medium was added to a 125 ml Erlenmeyer flask (Corning, no baffles, polycarbonate) and allowed to stand for at least 1 hour under 37°C and 5% CO2 conditions. For the Erlenmeyer flask that has been left standing for more than 1 hour, add 200 × 10 5The cell suspension was added to form cells, and the cells were seeded. At this time, the amount of suspension to be added was calculated using the number of cells measured in the cell counting step described above. Furthermore, the production culture medium that was lacking was added so that the liquid volume in the Erlenmeyer flask became 40 ml. The Erlenmeyer flask containing the cell suspension was placed on an orbital shaker (Optima Co., Ltd. "OS-762 for use in incubators") installed in an incubator at 37°C and 5% CO2 conditions, and cultured with continuous shaking at 120 rpm.

[0083] (6-6) Feed addition process In the continuous shaking culture described above, the feed medium prepared in (6-3) above was added on the 4th, 6th, 8th, 10th, and 12th day after sowing. The amount of feed medium added was 8 vol% of the amount of medium in the Erlenmeyer flask at that time.

[0084] (6-7) Clarification process Fourteen days after seeding the cells in the aforementioned cell seeding process, the entire culture medium, including the microcarriers, in the Erlenmeyer flask was transferred to a 50 ml centrifuge tube. After centrifugation at 800 rpm for 5 minutes, the entire culture supernatant was passed through a 0.22 μm filter to obtain a clarified culture supernatant.

[0085] (6-8) Purification process Antibodies were purified from the culture supernatant using the following equipment and columns. • Purification equipment: Cytiva "AKTA" TM "avant 25" • Protein A ligand column: Cytiva "HiTrap" TM MabSelect SuRe LX 1ml

[0086] 16 ml of the culture supernatant clarified above was added to a column equilibrated with equilibration buffer (20 mM NaH2PO4-Na2HPO4, 150 mM NaCl, pH 8.0). After passing 5 CV (column volume) of equilibration buffer through the column, 5 CV of 25 mM Tris-HCl (pH 7.0) was passed through as a washing solution to wash away impurities that did not adsorb to the column. Furthermore, 5 CV of 25 mM acetate buffer (pH 3.4) was passed through as an eluent to elute and recover the target antibody. Finally, 3 CV of equilibration buffer was passed through to complete the antibody purification.

[0087] (6-9) Determination of antibody concentration The recovered antibody solution was measured using NanoDrop One (Thermo Fisher Scientific), and the antibody concentration was quantified from the absorbance at 280 nm. The amount of antibody contained in the antibody solution was calculated from the volume and antibody concentration. Furthermore, the antibody concentration was estimated by dividing this amount of antibody by the volume of the clarified culture supernatant (16 ml). The results are shown in Table 1.

[0088] (6-10) Nucleic acid purification process The nucleic acids present in the clarified culture supernatant were recovered using the following kit. Nucleic acid purification: MACHEREY-NAGEL's "NucleoSpin Gel an

[0089] (Preparation of DNA-binding samples) A binding sample was prepared by mixing 6 ml of the clarified culture supernatant with 12 ml of commercially available binding buffer (MACHEREY-NAGEL's "Binding Buffer NTB").

[0090] (Binding DNA) The column included with NucleoSpin Gel and PCR Clean-up was placed in the accompanying collection tube (2 ml), and up to 700 μl of the conjugated sample was loaded. The tube loaded with the conjugated sample was centrifuged at 11,000 × g for 30 seconds. The flow-through was discarded, and the column was returned to the collection tube. The loading and centrifugation of the sample was repeated until all of the remaining conjugated sample passed through the column.

[0091] (Cleaning of silica film) After all the bound samples had passed through the column, 700 μl of Buffer NT3 (diluted to 20% with ethanol) included in the NucleoSpin Gel and PCR Clean-up kit was loaded, and the column was centrifuged at 11,000 × g for 30 seconds. The flow-through was discarded, and the column was returned to the collection tube. Another 700 μl of Buffer NT3 was loaded, and the column was centrifuged at 11,000 × g for 30 seconds. The flow-through was discarded, and the column was returned to the collection tube.

[0092] (Drying of silica film) The collection tube with the above column was centrifuged at 11,000 × g for 1 minute to completely remove Buffer NT3. Then, to completely remove any remaining ethanol, it was allowed to stand at 27°C for 10 minutes.

[0093] (Leaching of nucleic acids) The silica membrane column, after drying, was placed in a new 1.5 ml microcentrifuge tube. 20 μl of Buffer NE (included with NucleoSpin Gel and PCR Clean-up) was added, and the tube was incubated at room temperature (15-27°C) for 1 minute. After incubation, the tube was centrifuged at 11,000 × g for 1 minute, and the eluted nucleic acid solution was collected.

[0094] (6-11) Nucleic acid quantification process (Quantitative method: quantitative PCR) The nucleic acid solution recovered in the nucleic acid purification process was diluted in a series of 1 / 10, 1 / 100, and 1 / 1000 dilutions to obtain the nucleic acid dilution solutions. A commercially available quantitative PCR reagent kit for CHO cells (QCdetect, manufactured by Fujifilm Wako Pure Chemical Corporation) TM Using the "Residual DNA Detection Kit for CHO Cells," the nucleic acid dilution solution was prepared as a PCR measurement sample according to the kit protocol. qPCR was performed using a QuantStudio 7 Flex real-time PCR system (Thermo Fisher Scientific) for 40 cycles, with each cycle consisting of 95°C for 10 minutes and (95°C for 15 seconds and 60°C for 1 minute). A calibration curve was created from the concentrations of each calibration curve sample, based on the number of PCR cycles (Ct value) at which the target gene amplification curve intersected the threshold at each concentration (copy number) of the target gene calibration curve sample. The concentration of each PCR measurement sample was calculated from the Ct value of the amplification curve of the CHO-derived gDNA for each PCR measurement sample and the aforementioned calibration curve. Among the dilution series, samples that fell between the Ct values ​​of the lowest and highest concentrations of the calibration curve samples were treated as reliable results.

[0095] The amount of antibody and nucleic acid, as well as the amount of nucleic acid per unit of antibody, were calculated using the same method as in Example 1, and the results are shown in Table 1.

[0096] <(7) Comparative Example 2: Production of target protein using cationic porous microcarriers (low concentration)> Except for the microcarrier preparation step and the cell seeding step, nucleic acid concentration and antibody concentration were quantified according to the same method as in Example 1. The modified microcarrier preparation step and cell seeding step from Example 1 were carried out according to the following method.

[0097] (7-1) Microcarrier preparation process 25 mg of cationic porous microcarrier (BioCradle L, manufactured by Asahi Kasei Corporation) and 5 ml of PBS were added to a 15 ml centrifuge tube and vortexed to suspend the mixture. After swelling at room temperature for more than 1 hour, the mixture was sterilized in an autoclave at 121°C for 15 minutes and then cooled to room temperature. Once cooled to room temperature, the PBS was removed carefully, taking care not to remove the microcarrier. Production culture medium was then added to the tube to a total volume of 5 ml, and the mixture was vortexed again to suspend the mixture.

[0098] (7-2) Cell seeding process A 5 g / L microcarrier suspension prepared in the microcarrier preparation step was added to a 125 ml Erlenmeyer flask (Corning, no baffles, polycarbonate). Production culture medium was then added to the flask until the total volume reached 30 ml, and the flask was left to stand at 37°C under 5% CO2 conditions for at least 1 hour. To a 125 ml Erlenmeyer flask containing the microcarrier suspension that has been left standing for more than 1 hour, add 200 × 10⁻⁶ 5 To form cells, the cell suspension prepared in the expansion culture step was added, and the cells were seeded. At this time, the amount of suspension to be added was calculated using the number of cells measured in the cell counting step described above. Furthermore, in order to achieve a microcarrier concentration of 0.625 g / l during the cell seeding step, the insufficient production culture medium was added so that the liquid volume in the Erlenmeyer flask became 40 ml. The Erlenmeyer flask containing the cell suspension was placed on an orbital shaker (Optima Co., Ltd. "OS-762 for Incubator Use") set up in an incubator at 37°C and 5% CO2, and shaken at 120 rpm for 5 minutes. After standing for approximately 18 hours, continuous shaking culture was performed at 120 rpm.

[0099] The antibody amount, nucleic acid quantification, nucleic acid amount per antibody amount, and reduction rate were measured using the same method as in Example 1. The results are shown in Table 1.

[0100] <(8) Comparative Example 3: Production of target protein using cationic porous microcarriers (high concentration)> Except for the microcarrier preparation step and the cell seeding step, nucleic acid concentration and antibody concentration were quantified according to the same method as in Example 1. The modified microcarrier preparation step and cell seeding step from Example 1 were carried out according to the following method.

[0101] (8-1) Microcarrier preparation process 250 mg of cationic porous microcarrier (BioCradle L, manufactured by Asahi Kasei Corporation) and 5 ml of PBS were added to a 15 ml centrifuge tube and vortexed to suspend the mixture. After swelling at room temperature for more than 1 hour, the mixture was sterilized in an autoclave at 121°C for 15 minutes and then cooled to room temperature. Once cooled to room temperature, the PBS was removed carefully, taking care not to remove the microcarrier. Production culture medium was then added to the tube to a total volume of 5 ml, and the mixture was vortexed again to suspend the mixture.

[0102] (8-2) Cell seeding process A 50 g / L microcarrier suspension prepared in the microcarrier preparation step was added to a 125 ml Erlenmeyer flask (Corning, no baffles, polycarbonate). Production culture medium was then added to the flask until the total volume reached 30 ml, and the mixture was allowed to stand at 37°C under 5% CO2 conditions for at least 1 hour. To a 125 ml Erlenmeyer flask containing the microcarrier suspension that has been left standing for more than 1 hour, add 200 × 10⁻⁶ 5 To form cells, the cell suspension prepared in the expansion culture step was added, and the cells were seeded. At this time, the amount of suspension to be added was calculated using the number of cells measured in the cell counting step described above. Furthermore, in order to achieve a microcarrier concentration of 6.25 g / l during the cell seeding step, the insufficient production culture medium was added so that the liquid volume in the Erlenmeyer flask became 40 ml. The Erlenmeyer flask containing the cell suspension was placed on an orbital shaker (Optima Co., Ltd. "OS-762 for Incubator Use") set up in an incubator at 37°C and 5% CO2, and shaken at 120 rpm for 5 minutes. After standing for approximately 18 hours, continuous shaking culture was performed at 120 rpm.

[0103] The antibody amount, nucleic acid quantification, nucleic acid amount per antibody amount, and reduction rate were measured using the same method as in Example 1. The results are shown in Table 1.

[0104] <(9) Comparative Example 4: Production of target protein using cationic nonporous microcarriers (high concentration)> Except for the microcarrier preparation and cell seeding steps, nucleic acid and antibody concentrations were quantified according to the same method as in Example 1. The modified microcarrier preparation and cell seeding steps from Example 1 were performed according to the following methods.

[0105] (9-1) Microcarrier preparation process 250 mg of cationic, non-porous microcarrier (Cytiva "Cytodex 1") and 5 ml of PBS were added to a 15 ml centrifuge tube and vortexed to suspend the microcarrier. After swelling at room temperature for more than 1 hour, the tube was sterilized in an autoclave at 121°C for 15 minutes and then cooled to room temperature. Once cooled to room temperature, the PBS was removed carefully, taking care not to remove the microcarrier. Production culture medium was then added to the tube to a total volume of 5 ml, and the mixture was vortexed again to suspend the microcarrier.

[0106] (9-2) Cell seeding process A 50 g / L microcarrier suspension prepared in the microcarrier preparation step was added to a 125 ml Erlenmeyer flask (Corning, no baffles, polycarbonate). Production culture medium was then added to the flask until the total volume reached 30 ml, and the mixture was allowed to stand at 37°C under 5% CO2 conditions for at least 1 hour. To a 125 ml Erlenmeyer flask containing the microcarrier suspension that has been left standing for more than 1 hour, add 200 × 10⁻⁶ 5 To form cells, the cell suspension prepared in the expansion culture step was added, and the cells were seeded. At this time, the amount of suspension to be added was calculated using the number of cells measured in the cell counting step described above. Furthermore, in order to achieve a microcarrier concentration of 6.25 g / l during the cell seeding step, the insufficient production culture medium was added so that the liquid volume in the Erlenmeyer flask became 40 ml. The Erlenmeyer flask added with the cell suspension was placed on an orbital shaker (Optima Co., Ltd. "OS-762 for Incubator") installed in an incubator under the conditions of 37°C and 5% CO₂, and shaken at 120 rpm for 5 minutes. Then, after standing for about 18 hours, continuous shaking culture was performed at 120 rpm.

[0107] The antibody amount, nucleic acid quantification, nucleic acid amount per antibody amount, and reduction rate were measured in the same manner as in Example 1. The results are shown in Table 1.

[0108]

Table 1

[0109] [Reference Example] <Verification Experiment of DNA Adsorption (System without Cells)> (Microcarrier Preparation Step) 250 mg of cationic microcarriers and 5 ml of water for injection (Fuso Pharmaceutical Industries, Ltd. "Water for Injection PL Fuso") were added to a 15 ml centrifuge tube and vortexed to suspend. After swelling at room temperature for 1 hour or more, sterilization treatment was performed by autoclaving at 121°C for 15 minutes and cooled to room temperature. When it cooled to room temperature, the water for injection was removed while taking care not to remove the microcarriers. Tris-EDTA buffer (Jena BioScience GmbH "Tris-EDTA Buffer 1x conc. pH7.6") was added thereto so that the total volume became 5 ml, and it was vortexed and suspended again.

[0110] (Preparation of Measurement Samples and Measurement of Nucleic Acid Concentration) Each microcarrier and nucleic acid solution (Takara Bio Inc. "100bp DNA Ladder") prepared in the microcarrier preparation step were added to a 1.5 ml tube (Eppendorf "DNA LoBind Tubes") while diluting with Tris-EDTA buffer to the concentrations shown in Table 2. The 1.5 ml tubes containing microcarriers and nucleic acids were shaken at 37°C and 200 rpm using a constant temperature shaker (M·BR-034P, manufactured by Taitec Co., Ltd.). Supernatants were collected 1, 2, 4, and 8 hours after the start of shaking, and the DNA concentration of each supernatant was measured using a NanoDrop Eight (NanoDrop Eight, manufactured by Thermo Fisher Scientific) with Tris-EDTA buffer as a blank. The measurement results are shown in Table 2. When cationic microcarriers were added and shaken, the nucleic acid concentration in the supernatant was reduced.

[0111] [Table 2] [Industrial applicability]

[0112] According to the present invention, in a method for producing a target protein using cultured cells, it is possible to reduce the amount of impurities per equivalent amount of target protein, thereby simplifying downstream processes such as purification. This makes it possible to reduce the cost of purifying the target protein.

Claims

1. A method for producing a culture supernatant containing a target protein and with reduced culture impurities, (1) A step of performing cell culture using a culture medium containing a cell culture scaffold material, (2) A step of collecting the culture supernatant from step (1) above, Includes, A method wherein the culture supernatant obtained in step (2) is a culture supernatant with reduced culture impurities, which enables simplification of downstream steps aimed at reducing culture impurities.

2. The method according to claim 1, wherein the culture impurity is nucleic acid.

3. The method according to claim 2, wherein the culture supernatant with reduced culture impurities has a reduced mass of nucleic acids per unit mass of target protein by 35% or more compared to the cell culture supernatant obtained without using the cell culture scaffold material.

4. The method according to claim 2, wherein the culture supernatant with reduced culture impurities has a nucleic acid mass of less than 90 ng / mg per mass (mg) of the target protein.

5. The method according to claim 1, characterized in that the simplification of the downstream process is achieved by omitting ion exchange chromatography.

6. The method according to claim 1, characterized in that the cell culture scaffold material is a cationic microcarrier.

7. The method according to claim 1, wherein the concentration of the cell culture scaffold material in the culture medium at the time of cell seeding is 0.625 g / L or more and 6.25 g / L or less.

8. The method according to claim 1, wherein the target protein is a therapeutic protein.

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