Culture medium additives and cell culture media, and their uses

Abstract

This invention provides novel culture medium additives useful for cell culture and related utilization technologies. [Solution] The culture medium additive of this disclosure comprises adenosine-N1-oxide. Another culture medium additive of this disclosure comprises trehalose and is used so that the final concentration of trehalose in the culture medium is 1 mM to 30 mM. Yet another culture medium additive of this disclosure comprises selaginose.

Description

Technical Field

[0001] The present invention relates to a medium additive, a medium for cell culture, and their use.

Background Art

[0002] Conventionally, in cell culture, techniques for adding carbohydrates or nucleosides to the medium are known. For example, Non-Patent Document 1 reports that by adding trehalose to the medium, the formation of aggregates during antibody production by Chinese hamster ovary cells (CHO cells) is suppressed and the antibody production amount increases. Also, Patent Document ① discloses a method for culturing cells for antibody production, which is a method of adding adenosine, a nucleoside, to a medium for cell culture.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Note: There seems to be a formatting error in the original text where "特許文献1" is followed by a blank line. Also, the year in the Non-Patent Document 1 citation seems to be incomplete in the provided text. I've filled in "2014" based on the reference information. If this is incorrect, please adjust accordingly.The cell culture method described in Non-Patent Literature 1 requires acclimatization culture under high trehalose concentration conditions for 40 days, which presents challenges in applying it to existing antibody production processes, including the culture of CHO cells.

[0006] Given the circumstances described above, there was a need for the development of new culture medium additives useful for cell culture and new cell culture methods.

[0007] An object of one aspect of the present invention is to provide a novel culture medium additive useful for cell culture and a technology for using the same. [Means for solving the problem]

[0008] A culture medium additive for a cell culture medium according to one aspect of the present invention contains adenosine-N1-oxide.

[0009] A culture medium additive for cell culture according to another aspect of the present invention comprises trehalose, The trehalose is used in the culture medium so that the final concentration is between 1 mM and 30 mM.

[0010] A culture medium additive for a cell culture medium according to yet another aspect of the present invention comprises selaginose. [Effects of the Invention]

[0011] According to one aspect of the present invention, a novel culture medium additive useful for cell culture and a technology for utilizing the same can be provided. [Modes for carrying out the invention]

[0012] One embodiment of the present invention is described below. However, the present invention is not limited to the configurations described below. The present invention can be modified in various ways within the scope of the claims. The technical scope of the present invention also extends to embodiments or examples obtained by appropriately combining the multiple technical means disclosed herein. In this case, the multiple technical means may be disclosed across multiple embodiments or examples.

[0013] Unless otherwise specified in this specification, the numerical range "A~B" is intended to mean "greater than or equal to A, and less than or equal to B".

[0014] Unless otherwise specified herein, the final concentration of a component in the culture medium refers to the concentration of that component at the start of the culture. Unless otherwise specified herein, the weight ratio of a component in the culture medium to glucose refers to the weight ratio of that component at the start of the culture.

[0015] [1. Culture medium additives] One aspect of the present invention is a culture medium additive for cell culture media. By adding the culture medium additive to the cell culture medium, various advantageous effects are achieved in cell culture. The culture medium additive may be distributed on its own or as a kit combined with other components. An example of such a kit is a kit for preparing cell culture media.

[0016] The cell culture medium contains adenosine-N1-oxide, trehalose, selaginose, or a combination thereof. The culture medium additives may contain other components. The various components contained in the culture medium additives are described individually below.

[0017] [1.1. Adenosine-N1-oxide] In one embodiment, the culture medium additive contains adenosine-N1-oxide. Adenosine-N1-oxide is a derivative of adenosine in which the nitrogen atom at the N1 position is oxidized. The structure of adenosine-N1-oxide is represented by the following formula. [ka]

[0018] Adenosine-N1-oxide is a known compound, its manufacturing method is known to those skilled in the art, and it is also commercially available as a product.

[0019] When using a medium additive containing adenosine-N1-oxide, the lower limit of the final concentration of adenosine-N1-oxide in the medium can be 0.5 μM or more, 1 μM or more, 5 μM or more, 10 μM or more, 25 μM or more, 50 μM or more, 75 μM or more, or 80 μM or more. When using a medium additive containing adenosine-N1-oxide, the upper limit of the final concentration of adenosine-N1-oxide in the medium can be 200 μM or less, 180 μM or less, 150 μM or less, 130 μM or less, or 100 μM or less. Note that the final concentration in the medium is the concentration at the start of the culture.

[0020] When using a medium additive containing adenosine-N-oxide, when the weight of glucose contained in the medium is taken as 1, the lower limit of the weight of adenosine-N1-oxide can be 4.0×10 -6 or more, 9.0×10 -6 or more, 4.0×10 -5 or more, 9.0×10 -5 or more, 2.0×10 -4 or more, 4.0×10 -4 or more, 7.0×10 <{ -4 or more, or 7.5×l0 -4 or more. When using a medium additive containing adenosine-N1-oxide, when the weight of glucose contained in the medium is taken as 1, the upper limit of the weight of adenosine-N1-oxide can be 5.0×10 -3 or less, 4.5×10 -3 or less, 4.0×10 -3 or less, 3.5×10 -3 or less, or 3.0×10 -3 or less. Note that the weight ratio to glucose contained in the medium is the weight ratio at the start of the culture.

[0021] [1.2. Trehalose] In one embodiment, the medium additive contains trehalose. Trehalose is a non-reducing disaccharide having a structure in which two molecules of D-glucose are α,α-1,1-bonded. The structure of trehalose is represented by the following formula. [Chemical formula]

[0022] Trehalose is a well-known compound, its manufacturing method is known to those skilled in the art, and it is also commercially available as a product.

[0023] When using a culture medium additive containing trehalose, the lower limit of the final trehalose concentration in the culture medium is 1 mM or higher, and may be 2 mM or higher, 3 mM or higher, 4 mM or higher, or 5 mM or higher. When using a culture medium additive containing trehalose, the upper limit of the final trehalose concentration in the culture medium may be 50 mM or lower, 40 mM or lower, 30 mM or lower, 25 mM or lower, 24 mM or lower, 23 mM or lower, 22 mM or lower, 21 mM or lower, 20.4 mM or lower, or 20 mM or lower. Note that the final concentration in the culture medium is the concentration at the start of cultivation.

[0024] When using a culture medium additive containing trehalose, if the weight of glucose in the medium is set to 1, the lower limit of the weight of trehalose is 1.0 × 10⁻⁶. -2 The above is 2.0 × 10 -2 The above is 3.0 × 10 -2 The above is 4.0 × 10 -2 or more, or 5.0 x 10 -2 The above is possible. When using a culture medium additive containing trehalose, if the weight of glucose in the medium is set to 1, the upper limit of the weight of trehalose is 20.0 × 10⁻⁶. -1 Below, 15.0 × 10 -1 Below, 10.0 × 10 -1 The following or 6.0 × 10 -1 The following are possible. Note that the weight ratio of glucose in the culture medium is the weight ratio at the start of cultivation.

[0025] [1.3. Selaginose] In one embodiment, the culture medium additive contains selaginose. Selaginose is a non-reducing trisaccharide in which one molecule of D-glucose is linked to trehalose by an α-1,2 bond. The structure of selaginose is represented by the following formula. [ka]

[0026] Selaginose is a well-known compound, its manufacturing method is known to those skilled in the art, and it is also commercially available as a product.

[0027] When using a culture medium additive containing selaginose, the lower limit of the final selaginose concentration in the culture medium may be 1 mM or higher, 2 mM or higher, 3 mM or higher, 4 mM or higher, or 5 mM or higher. When using a culture medium additive containing selaginose, the upper limit of the final selaginose concentration in the culture medium may be 50 mM or lower, 40 mM or lower, 30 mM or lower, or 20 mM or lower. Note that the final concentration in the culture medium is the concentration at the start of cultivation.

[0028] When using a culture medium additive containing selaginose, if the weight of glucose in the medium is set to 1, the lower limit of the weight of selaginose is 1.5 × 10⁻⁶. -2 The above is 3.0 × 10 -2 The above is 5.0 x 10 -2 The above is 6.0 x 10 -2 or more, or 8.0 x 10 -2 The above is possible. When using a culture medium additive containing selaginose, if the weight of glucose in the medium is set to 1, the upper limit of the weight of selaginose is 25.0 × 10⁻⁶. -1 Below, 20.0 × 10 -1 Below, 15.0 × 10 -1 or less or 10.0 × 10 -1 The following are possible. Note that the weight ratio of glucose in the culture medium is the weight ratio at the start of cultivation.

[0029] [1.4. Combinations of ingredients] The components of the culture medium additives described above may be combined. That is, the culture medium additives may include the following combinations. • Adenosine-N1-oxide and trehalose • Adenosine-N1-oxide and selaginose Trehalose and selaginose • Adenosine-N1-oxide, trehalose, and selaginose

[0030] In a culture medium additive containing a combination of two or more components, the concentration of each component and its weight ratio to glucose when using such a culture medium additive may be within the numerical range described above.

[0031] In one embodiment, the culture medium additive further includes, in addition to adenosine-N1-oxide, one or more selected from the group consisting of trehalose and selaginose. Such a combination can further increase the amount of cell products produced by cultured cells and further improve the purity of the cell products.

[0032] [2. Use of culture medium additives] One aspect of the present invention is a cell culture medium containing the culture medium additive described in Section [1]. Another aspect of the present invention is a method for producing cultured cells, comprising the step of culturing cells in such a cell culture medium.

[0033] [2.1. Culture Medium] The final concentrations of adenosine-N1-oxide, trehalose, or selaginose in the cell culture medium may be within the numerical ranges specified in Section [1]. The weight ratio of adenosine-N1-oxide, trehalose, or selaginose content to glucose content in the cell culture medium may be within the numerical ranges specified in Section [1].

[0034] Cell culture media may contain any medium components in addition to the medium additives described in Section [1]. For example, cell culture media may contain basal media or commercially available media. Alternatively, cell culture media may contain additives other than those described in Section [1] (such as glucose, amino acids, vitamins, lipids, cholesterol, growth factors, etc.). Cell culture media may contain nutrients, energy sources, growth factors, etc., necessary for cell survival and / or proliferation. The pH and salt concentration of cell culture media may be adjusted to a range suitable for cell survival and / or proliferation.

[0035] The cell culture medium may be a liquid medium, a semi-solid medium, a solid medium, or a combination of these.

[0036] [2.2. Method for producing cultured cells] Cultured cells can be produced by culturing cells in the cell culture medium described above. The cell culture method is not particularly limited. Examples include liquid culture, solid culture, and three-dimensional culture.

[0037] The cell culture medium described above can be used at any stage of cell culture. For example, the cell culture medium described above may be used at least at the start of cell culture (the medium used at other times is not particularly limited). As another example, the cell culture medium described above may be used at least during the logarithmic growth phase of cell proliferation or thereafter. The cell culture medium described above may also be used as a feed medium added to the culture system during the culture process.

[0038] The type of cells produced in the method for producing cultured cells is not particularly limited and includes, for example, mammalian cells, yeast cells, and bacterial cells. In one embodiment, the cells to be cultured are mammalian cells. In one embodiment, the mammalian cells are Chinese hamster ovary cells (CHO cells). Other examples of cells to be cultured include HEK293 cells (derived from human fetal kidney), HL-60 cells (derived from human leukocytes), HeLa cells (derived from human cervical cancer), and hybridomas.

[0039] The purpose of cell culture is not particularly limited. Examples of culture purposes include the production of cell products (proteins, lipids, sugars, nucleic acids, etc.), maintenance of cell number, proliferation of cell number, cell differentiation, creation of cell aggregates (organoids, cell sheets, etc.), and experiments using cells. As shown in the examples described later, protein production can be improved by using culture medium additives. Therefore, culturing cells for protein production is one example of a preferred method. An example of a protein to be produced is an antibody.

[0040] [3. Effects of culture medium additives] By culturing cells in a cell culture medium containing the culture medium additives described in Section [1], various effects advantageous to cell culture are achieved. One aspect of the present invention is a method for exhibiting various effects, comprising the step of culturing cells in a cell culture medium containing the culture medium additives described in Section [1]. Examples of such effects include one or more of the following. • Promotes the production of cell products. • Improve the purity of cell products. • Improves cell survival and / or proliferation.

[0041] [3.1. Promotion of cell product production] By using culture medium additives, the production of cell products can be promoted. In one embodiment, the cell product is a protein. In another embodiment, the protein is an antibody.

[0042] Whether cell product production is promoted can be confirmed by comparing the system with one or more of the following conditions compared to a system without culture medium additives. • The concentration of cell products in the culture system or a part of it (such as the culture supernatant) increases. • The specific production rate of cell products improves.

[0043] When the cell product is an antibody, a method for measuring the concentration and specific production rate of the cell product is illustrated in the embodiments of this application. Even when producing other types of cell products, those skilled in the art can appropriately measure the concentration and specific production rate.

[0044] [3.2. Improving the purity of cell products] By using culture medium additives, the purity of cell products can be improved. In one embodiment, the cell product is a protein. In another embodiment, the protein is an antibody.

[0045] Whether the purity of the cell products has improved can be confirmed by comparing the system with one or more of the following conditions compared to a system without culture medium additives. • The concentration of host cell-derived proteins in the culture system or a part of it (such as the culture supernatant) decreases. • The concentration of contaminating proteins in the culture system or a part of it (such as the culture supernatant) decreases.

[0046] Here, contaminating proteins are a general term for proteins that are not intended to be produced. Contaminating proteins include proteins derived from cell culture medium, proteins derived from dead cells, and proteins leaked from damaged cells. Therefore, the concentration of contaminating proteins is related not only to the purity of the intended cell products, but also to the viability and health of cells in the culture system.

[0047] A method for quantifying the concentration of host cell-derived proteins when the host cell is a CHO cell is illustrated in the examples of this application. For other types of host cells, a person skilled in the art can appropriately quantify the concentration of host cell-derived proteins. A method for quantifying the concentration of contaminating proteins when antibodies are produced by CHO cells is also illustrated in the examples of this application. Even when the cell product and host cell are different, a person skilled in the art can appropriately quantify the concentration of contaminating proteins.

[0048] [3.3. Improving cell survival and / or proliferation] By using culture medium additives, cell survival and / or proliferation can be improved. In one embodiment, the cells are mammalian cells. In one embodiment, the mammalian cells are CHO cells.

[0049] Whether cell survival and / or proliferation is improved can be determined by comparing the system with one or more of the following conditions compared to a system without culture medium additives. • Cell viability in culture systems improves. • The density of viable cells in the culture system improves. • The cumulative value of the viable cell density in the culture system increases. • The concentration of contaminating proteins in the culture system or a part of it (such as the culture supernatant) decreases. • LDH activity decreases in the culture system or a part of it (such as the culture supernatant).

[0050] Methods for measuring cell viability, viable cell density, cumulative viable cell density, and LDH activity are illustrated in the examples of this application. Even if the methods described in the examples of this application are not directly applicable, those skilled in the art can appropriately measure these parameters. A method for quantifying the concentration of contaminating proteins when antibodies are produced by CHO cells is illustrated in the examples of this application. Even if the cell products and host cells are different, those skilled in the art can appropriately quantify the concentration of contaminating proteins.

[0051] [4. Summary] The present invention includes the following embodiments. [1] A culture medium additive for cell culture media containing adenosine-N1-oxide. [2] The culture medium additive according to [1] further comprises one or more selected from the group consisting of trehalose and selaginose. [3] A culture medium additive according to [1] or [2], used such that the final concentration of adenosine-N1-oxide in the culture medium is 0.5 μM to 200 μM. [4] The content of adenosine-N1-oxide relative to the glucose content in the culture medium is, by weight, 1:(4.0 × 10 -6 ~5.0×10 -3 A culture medium additive described in any one of [1] to [3], used to achieve the following: [5] A culture medium additive for cell culture media, comprising trehalose, used so that the final concentration of trehalose in the culture medium is 1 mM to 30 mM. [6] The ratio of the trehalose content to the glucose content in the culture medium is 1:(1.0 × 10) by weight. -2 ~20.0×10 -1 A culture medium additive as described in [5], used to achieve the following: [7] A culture medium additive for cell culture media containing selaginose. [8] The culture medium additive described in [7], used so that the final concentration of selaginose in the culture medium is 1 mM to 50 mM. [9] The ratio of selaginose content to glucose content in the culture medium is 1:(1.5 × 10⁻¹⁰ by weight). -2 ~25.0×10 -1 A culture medium additive as described in [7] or [8], used to achieve the following:

[10] The aforementioned cells are mammalian cells, and the culture medium additive is one of the following [1] to [9].

[11] The mammalian cells are Chinese hamster ovary cells (CHO cells), as described in

[10] .

[12] A cell culture medium containing any one of the culture medium additives listed in [1] to [9].

[13] A method for producing cultured cells, comprising the step of culturing cells in a cell culture medium containing a culture medium additive described in any one of [1] to [9].

[14] A method for promoting the production of cell products by cells, comprising the step of culturing cells in a cell culture medium containing any one of the culture medium additives described in [1] to [9].

[15] A method for improving cell viability in cell culture, comprising the step of culturing cells in a cell culture medium containing any one of the culture medium additives described in [1] to [9].

[16] A method for reducing the concentration of host cell-derived proteins in cell culture, comprising the step of culturing cells in a cell culture medium containing any one of the culture medium additives described in [1] to [9].

[17] A method for reducing the concentration of contaminating proteins in cell culture, comprising the step of culturing cells in a cell culture medium containing any one of the culture medium additives described in [1] to [9]. [Examples]

[0052] 〔material〕 Examples 1-5 investigated the function of the following compounds as culture medium additives. All of these compounds were prepared by the applicant. Adenosine-N1-oxide (purity: 99.3%) • Trehalose dihydrate (purity: 99.4%) • Selaginose (purity: 98.6%) • Glucosyltrehalose (purity: 97.2%) • Maltosyltrehalose (purity: 98.8%)

[0053] Furthermore, the above-mentioned glucosyltrehalose is a non-reducing trisaccharide in which one molecule of D-glucose is α-1,4 linked to trehalose, and the above-mentioned maltosyltrehalose is a non-reducing tetrasaccharide in which maltose is α-1,4 linked to trehalose.

[0054] [General experimental method (1)] The experimental method used in the example is described below.

[0055] [Preparation of culture media and sample solutions] Various solutions used for cell culture were prepared according to the following procedures. ● Production culture medium 1. To 970 mL of ultrapure water, 33.95 g of cell growth medium ISJ Gp023.2 (Fujifilm Wako Pure Chemical Corporation), 4 g of glucose, and 2 g of sodium bicarbonate were added. 2. Stirred until all components were completely dissolved in ultrapure water. 3. To the obtained solution, 20 mL of 200 mmol / L L-glutamine solution (×100) (Fujifilm Wako Pure Chemical Corporation) and 10 mL of penicillin-streptomycin solution (×100) (Fujifilm Wako Pure Chemical Corporation) were added. 4. The obtained solution was filtered through a filter (pore size: 0.22 μm). The production medium was prepared in this manner. The final glucose concentration in the production medium was 12 g / L. ● Feed medium 1. 45.723 g of cell feed medium ISJ Fp020.1 (Fujifilm Wako Pure Chemical Corporation) and 0.5 g of sodium bicarbonate were added to 242.5 mL of ultrapure water. 2. The mixture was stirred while passing carbon dioxide gas through it until all the components were completely dissolved in ultrapure water. 3. To the obtained solution, 5 mL of 200 mmol / L L-glutamine solution (×100) (Fujifilm Wako Pure Chemical Industries, Ltd.) and 2.5 mL of penicillin-streptomycin solution (×100) (Fujifilm Wako Pure Chemical Industries, Ltd.) were added. 4. The obtained solution was filtered through a filter (pore size: 0.22 μm). The feed medium was prepared in this manner. The final glucose concentration in the feed medium was 60 g / L. ● Glucose stock solution 1. Glucose was dissolved in ultrapure water to prepare a 400 g / L solution. 2. The obtained solution was filtered through a filter (pore size: 0.22 μm). In this way, a glucose stock solution was prepared. ●PBS(-) solution 1. Commercially available PBS(-) powder (Accudia) TM A PBS(-) solution was prepared using D-PBS(-) solution powder (Shimadzu Diagnostics Corporation) by a predetermined method. 2. Unless otherwise specified, the solutions of the various reagents used in the culture tests were prepared using PBS(-) solution as the solvent. Similarly, unless otherwise specified, the prepared solutions were subjected to ultrafiltration (molecular weight cutoff: 10,000) and filter filtration (pore size: 0.22 μm) before use. ● Preparation of cell suspension 1. 20 mL of production medium was added to a 125 mL vent filter cap flask. 2. IgG4-producing CHO cells were added to the flask and pre-cultured for 2-3 days. The culture conditions were 37°C, 150 rpm, and CO2 concentration: 5%. After pre-culture, the cell density increased to 2 × 10⁶. 6 ~10×10 6 The concentration was adjusted to be cells / mL. 3. The culture medium was centrifuged to collect a pellet of living cells. The centrifugation conditions were 20°C, 1500 rpm, and 10 minutes. 4. The pellet was resuspended in the production medium to achieve an appropriate cell density. In this way, a cell suspension was prepared.

[0056] [Measurement of glucose concentration] The measurement was performed using the following procedure. 1. The sampled cell suspension was centrifuged to obtain the culture supernatant. The centrifugation conditions were 20°C, 2,000 rpm, and 2 minutes. 2. The obtained culture supernatant was further centrifuged to obtain the centrifugal supernatant. The centrifugation conditions were 20°C, 14,000 rpm, and 10 minutes. 3. The obtained supernatant was diluted to an appropriate concentration with deionized water. 4. Glucose concentration was measured using the Glucose CII-Test Wako (Fujifilm Wako Pure Chemical Corporation).

[0057] [Calculation of viable cell density and survival rate] The measurement was performed using the following procedure. 1. The sampled cell suspension was diluted as needed with trypan blue stain (0.1% by weight). 2. The number of viable and dead CHO cells was counted using a hemocytometer. 3. The number of viable cells per 1 mL of cell suspension was calculated as the viable cell density (cells / mL). 4. The cell viability was calculated according to the following formula. Viability rate (%) = number of living cells ÷ (number of living cells + number of dead cells) × 100 5. During the test period, the viable cell density was calculated at predetermined points in time according to steps 1-3. From the viable cell density curve obtained in this way, the cumulative value of viable cell density (IVCD) was calculated using trapezoidal approximation according to the following formula. IVCD(pieces / day / mL)=Σ{(t n -t n-1 )×(VCD tn +VCD tn-1 )÷2} During the ceremony, t n -t n-1 : The period (in days) from the calculation of the (n-1) viable cell density to the calculation of the nth viable cell density. VCD tn , VCD tn-1 : Viable cell density calculated on the nth or n-1th time.

[0058] [Measurement of antibody concentration and contamination protein concentration] The measurement was performed using the following procedure. 1. The sampled cell suspension was centrifuged to obtain the culture supernatant. The centrifugation conditions were 20°C, 2,000 rpm, and 2 minutes. 2. The obtained culture supernatant was further centrifuged to obtain the centrifugal supernatant. The centrifugation conditions were 20°C, 14,000 rpm, and 10 minutes. 3. The obtained supernatant was diluted 11-fold with PBS(-) solution. 4. The resulting dilute solution was filtered through a filter (pore size: 0.22 μm). 5. The filtrate was used as a sample and analyzed by HPLC. The analytical conditions were as follows: • Column: POROS TM A 20μm Column (Thermo Fisher Scientific, 2.1mm diameter x 30mm length) ·Eluent A: 50mM Tris-HCl buffer (pH7.0) ·Eluent B: 50mM Tris-HCl buffer (pH3.0) • Elution conditions: The concentration of eluent B was changed according to time as follows. 0% (0 minutes to 0.75 minutes) 0% → 100% (0.75 mins to 0.76 mins) 100% (0.76 min - 2.75 min) ·Flow rate: 4.0mL / min ·Temperature: 30℃ Detection: Wavelength 280nm 6. Antibody concentration, specific production rate of the antibody, and concentration of contaminating proteins were quantified from the chromatogram. To quantify the antibody concentration, the inventors purified IgG4 antibody from the same CHO cell culture medium used in the test and created a calibration curve. The specific production rate was determined by dividing the antibody concentration by the cumulative value of the viable cell density. The concentration of contaminating proteins was expressed by the actual area value of the non-adsorbed component peak.

[0059] [Measurement of LDH activity] Lactate dehydrogenase (LDH) activity was measured using the following procedure. LDH is normally distributed within the cytoplasm and is released extracellularly upon damage to the cell membrane. Therefore, LDH activity serves as an indicator of cells with damaged cell membranes, such as dead cells. 1. The sampled cell suspension was centrifuged to obtain the culture supernatant. The centrifugation conditions were 20°C, 2,000 rpm, and 2 minutes. 2. The obtained culture supernatant was diluted to an appropriate concentration with BSA-50mM potassium phosphate buffer (20 μg / mL, pH 7.0). 3. LDH activity was measured using the Cytotoxicity LDH Assay Kit-WST (Dojin Chemical Research Institute Co., Ltd.).

[0060] [Measurement of host cell-derived protein concentration] The concentration of host cell-derived proteins (HCPs) was measured using the following procedure. Unlike the desired protein produced by the host cell, HCPs are proteins derived from the host cell itself. Therefore, the amount of HCPs serves as an indicator of the purity of the protein intended to be produced by the host cell. 1. The sampled cell suspension was centrifuged to obtain the culture supernatant. The centrifugation conditions were 20°C, 2,000 rpm, and 2 minutes. 2. The obtained culture supernatant was continuously filtered using three types of filters (pore sizes: 0.8 μm, 0.45 μm, and 0.22 μm). 3. The obtained filtrate was diluted to an appropriate concentration with Sample Diluent Buffer (Cygnus Technologies, LLC). 4. The obtained diluted solution was used as a sample, and the HCP concentration in the culture supernatant was measured using the CHO HCP ELISA Kit (Cygnus Technologies, LLC).

[0061] [Statistical processing] For the various measurements taken using the method described above, data was processed using Dunnett's multiple comparison method when comparing the test group with the control group. For comparisons between all groups, including the control group, data was processed using Tukey-Kramer's multiple comparison method.

[0062] [Example 1: Effects of trehalose and selaginose in cell culture] The effects of including trehalose or selaginose as a culture medium additive in cell culture were investigated. The specific procedure is as follows. 1. 19.8 mL of production medium was added to a 125 mL baffled vent filter cap flask, and IgG4-producing CHO cells were seeded. The seeding density was 1.1 × 10⁻⁶. 6 The value was calculated as cells / mL. 2. 0.2 mL of either PBS(-) solution, 1 M trehalose solution, or 1 M selaginose solution was added. This resulted in a concentration of trehalose or selaginose in the culture medium of 10 mM. At this point, assuming the weight of glucose in the culture medium is 1, the weight of trehalose is 2.88 × 10⁻⁶. -1 The weight of selaginose is 4.24 × 10 -1 That was the case. 3. Starting from day 0 at the point of step 2, the cells were cultured with shaking at 37°C, 150 rpm, and a CO2 concentration of 5% until day 10.

[0063] Furthermore, in the culture conditions tested, the system with only PBS(-) solution added (control) showed a cessation of the increase in viable cell density by day 7, and a decrease in viable cell density was observed thereafter. From this, it can be concluded that the cultured cells were in the stationary phase or beyond, at least from day 7 onwards.

[0064] During the culture process, the following procedures were performed at predetermined times. ●Days 3, 4, 5, and 6 • Feed medium was added in an amount equivalent to 8% by volume relative to the culture medium volume at each time point. After adding the feed medium, a 0.6 mL sample of the culture solution was taken, and the glucose concentration in the culture supernatant was measured. After measuring the glucose concentration, glucose stock solution was added to achieve a final glucose concentration of 15 g / L. ● Day 7 • A feed medium was added in an amount equivalent to 16% by volume relative to the culture medium volume. After adding the feed medium, a 0.6 mL sample of the culture solution was taken, and the glucose concentration in the culture supernatant was measured. After measuring the glucose concentration, glucose stock solution was added to achieve a final glucose concentration of 30 g / L. ●Days 3, 5, 7, and 10 The culture medium was sampled, and the items listed in Table 1 were measured.

[0065] [result] The results are shown in Table 1. In the table, the column for weight ratio to glucose represents the weight ratio of each component to glucose at the start of cultivation. [Table 1]

[0066] As can be seen from Table 1, in systems where trehalose or selaginose was added as a culture medium additive, the cumulative value of viable cell density and antibody concentration increased compared to the system where only PBS(-) solution was added. Specifically, compared to the system with only PBS(-) solution, the system with trehalose showed an increase in the cumulative value of viable cell density at day 10 to 112.7% and the antibody concentration at day 10 to 109.5%. Similarly, compared to the system with only PBS(-) solution, the system with selaginose showed an increase in the cumulative value of viable cell density at day 10 to 105.3% and the antibody concentration at day 10 to 110.9%. The specific production rate of antibodies was at a level similar to or higher than that of the system with only PBS(-) solution.

[0067] As can also be seen from Table 1, the systems in which trehalose or selaginose was added as a culture medium additive showed a lower concentration of contaminating proteins compared to the system in which only PBS(-) solution was added. Specifically, compared to the system in which only PBS(-) solution was added, the concentration of contaminating proteins in the system with trehalose was reduced to 91.3% on day 10. Similarly, compared to the system in which only PBS(-) solution was added, the concentration of contaminating proteins in the system with selaginose was reduced to 92.8% on day 10. LDH activity was at a level similar to or higher than that of the system in which only PBS(-) solution was added.

[0068] These results suggest the following effects of including trehalose or selaginose as a culture medium additive: • The cumulative value of viable cell density increases, at least from the stationary phase onward. • At least from the stationary phase onward, the production of cell products increases. • The concentration of interfering proteins decreases, at least from the stationary phase onward.

[0069] [Example 2: Effect of adenosine-N1-oxide on CHO cell culture (1)] The effects of including adenosine-N1-oxide as a culture medium additive in cell cultures were investigated. The specific procedure is as follows. 1. 19.8 mL of production medium was added to a 125 mL baffled vent filter cap flask, and IgG4-producing CHO cells were seeded. The seeding density was 1.2 × 10⁻⁶. 6 The value was calculated as cells / mL. 2. 0.2 mL of either PBS(-) solution or a 5 mM, 7.5 mM, or 10 mM adenosine-N1-oxide solution was added. This resulted in adenosine-N1-oxide concentrations of 50 μM, 75 μM, or 100 μM in the culture medium. At this point, assuming the weight of glucose in the culture medium is 1, the weight of adenosine-N1-oxide is 1.2 × 10⁻⁶. -3 ~2.4×10 -3 That was the case. 3. Cells were cultured in the same manner as in Step 3 and subsequent steps of Example 1, and the items listed in Table 2 were measured.

[0070] [result] The results are shown in Table 2. In the table, the column for weight ratio to glucose represents the weight ratio of each component to glucose at the start of culture. [Table 2]

[0071] As can be seen from Table 2, the antibody concentration was significantly higher in the system to which adenosine-N1-oxide was added as a culture medium additive compared to the system to which only PBS(-) solution was added. Specifically, on day 10, compared to the system to which only PBS(-) solution was added, the antibody concentration in the system to which adenosine-N1-oxide was added increased to 105.1% at a final concentration of 50 μM, 109.7% at a final concentration of 75 μM, and 115.3% at a final concentration of 100 μM. The cumulative value of viable cell density and the specific production rate of antibodies were at the same level or higher than those in the system to which only PBS(-) solution was added. The concentration of contaminating proteins was at the same level or lower than those in the system to which only PBS(-) solution was added.

[0072] These results suggest that including adenosine-N1-oxide as a culture medium additive has the effect of increasing the production of cell products, at least from the stationary phase onward.

[0073] [Example 3: Effects of adenosine-N1-oxide in cell culture (2)] In cell culture, we further investigated the effects of including adenosine-N1-oxide or trehalose as a culture medium additive. The specific procedure is as follows. 1. 19.6 mL of production medium was added to a 125 mL baffled vent filter cap flask, and IgG4-producing CHO cells were seeded. The seeding density was 1.2 × 10⁻⁶. 6 pieces / mL~1.3×10 6 The value was calculated as cells / mL. 2. Either 0.4 mL of PBS(-) solution was added, or 0.2 mL each of PBS(-) solution and 7.5 mM adenosine-N1-oxide solution were added, or 0.2 mL each of PBS(-) solution and 1 M trehalose solution was added. This resulted in a concentration of adenosine-N1-oxide in the medium of 75 μM and a concentration of trehalose in the medium of 10 mM. At this point, assuming the weight of glucose in the medium is 1, the weight of adenosine-N1-oxide is 1.8 × 10⁻⁶. -3 The weight of trehalose is 2.91 × 10⁻⁶. -1 That was the case. 3. Cells were cultured in the same manner as in Step 3 onward of Example 1, and the items listed in Table 3 were measured. However, sampling and measurement were performed only on day 10.

[0074] [result] The results are shown in Table 3. In the table, the column for weight ratio to glucose represents the weight ratio of each component to glucose at the start of culture. [Table 3]

[0075] As can be seen from Table 3, the concentration of host cell-derived proteins was significantly lower in the system in which adenosine-N1-oxide was added as a culture medium additive compared to the system in which only PBS(-) solution was added. Specifically, compared to the system in which only PBS(-) solution was added, the concentration of host cell-derived proteins decreased to 69.5% in the system in which adenosine-N1-oxide was added. In the system in which trehalose was added, a decreasing trend in host cell-derived proteins was observed compared to the system in which only PBS(-) solution was added.

[0076] These results suggest that including adenosine-N1-oxide or trehalose as a culture medium additive contributes to improving the quality of the produced proteins.

[0077] [Example 4: Effects of a combination of adenosine-N1-oxide and trehalose or selaginose in cell culture] The effects of including a combination of adenosine-N1-oxide and trehalose or selaginose as a culture medium additive in cell culture were investigated. The specific procedure is as follows. 1. 19.6 mL of production medium was added to a 125 mL baffled vent filter cap flask, and IgG4-producing CHO cells were seeded. The seeding density was 1.0 × 10⁶. 6 The value was calculated as cells / mL. 2. 0.2 mL of either PBS(-) solution or 7.5 mM adenosine-N1-oxide solution was added. 3. 0.2 mL of either PBS(-) solution, 1 M trehalose solution, or 1 M selaginose solution was added. This resulted in a concentration of adenosine-N1-oxide in the culture medium of 75 μM and a concentration of trehalose or selaginose of 10 mM in the system to which the corresponding solution was added. At this point, assuming the weight of glucose in the culture medium is 1, the weight of adenosine-N1-oxide was 1.8 × 10⁻⁶. -3 The weight of trehalose is 2.91 × 10⁻⁶. -1 The weight of selaginose is 4.39 × 10 -1 That was the case. 4. Cells were cultured in the same manner as in Step 3 and subsequent steps of Example 1, and the items listed in Table 4 were measured.

[0078] [result] The results are shown in Table 4. In the table, the column for weight ratio to glucose represents the weight ratio of each component to glucose at the start of cultivation. [Table 4]

[0079] As can be seen from Table 4, in the system in which a combination of adenosine-N1-oxide and trehalose or selaginose was added as a culture medium additive, the antibody concentration was significantly higher compared to the system with PBS(-) solution alone or the system with adenosine-N1-oxide added. Specifically, on day 10, compared to the system with PBS(-) solution alone as a baseline, the antibody concentration in the system with adenosine-N1-oxide and trehalose added rose to 111.4%. Compared to the system with PBS(-) solution alone as a baseline, the antibody concentration in the system with adenosine-N1-oxide and selaginose added rose to 111.8%. The cumulative value of viable cell density and the specific production rate of antibodies were at or above the same level as the system with PBS(-) solution alone or the system with adenosine-N1-oxide added.

[0080] As can also be seen from Table 4, in the system in which a combination of adenosine-N1-oxide and trehalose or selaginose was added as a culture medium additive, the concentration of contaminating proteins was significantly lower compared to the system with PBS(-) solution alone or the system with adenosine-N1-oxide added. Specifically, on day 10, compared to the system with PBS(-) solution alone as a baseline, the concentration of contaminating proteins in the system with the combination of adenosine-N1-oxide and trehalose was reduced to 87.4%. Compared to the system with PBS(-) solution alone as a baseline, the concentration of contaminating proteins in the system with the combination of adenosine-N1-oxide and selaginose was reduced to 88.0%.

[0081] These results suggest the following effects of including a combination of adenosine-N1-oxide and trehalose or selaginose as a culture medium additive: • At least from the stationary phase onward, the production of cell products increases. • The concentration of interfering proteins decreases, at least from the stationary phase onward.

[0082] [Example 5: Effects of trehalose derivatives in cell culture] The effects of including trehalose or its derivatives as a culture medium additive in cell culture were investigated. The specific procedure is as follows. 1. 19.8 mL of production medium was added to a 125 mL baffled vent filter cap flask, and IgG4-producing CHO cells were seeded. The seeding density was 1.3 × 10⁻⁶. 6 The value was calculated as cells / mL. 2. 0.2 mL of either PBS(-) solution, 1 M or 0.5 M trehalose solution, 1 M or 0.5 M glucosyltrehalose solution, 1 M maltosyltrehalose solution, or 1 M or 0.5 M selaginose solution was added. This resulted in a concentration of trehalose or its derivatives in the culture medium of 5 mM or 10 mM (maltosyltrehalose was 10 mM only). 3. Starting from day 0 at step 2, the cells were cultured with shaking at 37°C, 150 rpm, and a CO2 concentration of 5% until day 7. Based on the same reasoning as in Example 1, the cultured cells from day 7 onward are considered to be in the stationary phase or beyond.

[0083] During the culture process, the following procedures were performed at predetermined times. ●Days 3, 4, 5, and 6 • Feed medium was added in an amount equivalent to 8% by volume relative to the culture medium volume at each time point. After adding the feed medium, a 0.6 mL sample of the culture solution was taken, and the glucose concentration in the culture supernatant was measured. After measuring the glucose concentration, glucose stock solution was added to achieve a final glucose concentration of 15 g / L. ●Days 3, 5, and 7 The culture medium was sampled, and the items listed in Table 5 were measured.

[0084] [result] The results are shown in Table 5. [Table 5]

[0085] As can be seen from Table 5, the antibody concentration was higher in the system to which selaginose was added as a culture medium additive compared to the system to which other trehalose derivatives (glucosyltrehalose or maltosyltrehalose) were added. Specifically, on day 7, compared to the system to which only PBS(-) solution was added as a baseline, the antibody concentration in the system with a final concentration of 10 mM selaginose was 173.5%, while the antibody concentration in the system with a final concentration of 10 mM glucosyltrehalose was 94.8%, and the antibody concentration in the system with a final concentration of 10 mM maltosyltrehalose was only 88.8%.

[0086] These results suggest that selaginose, in particular among trehalose derivatives, is useful as a culture medium additive.

[0087] [General experimental method (2)] The experimental method in Example 6 is described below.

[0088] [Preparation of culture media and sample solutions] Various solutions used for cell culture were prepared according to the following procedures. The prepared solutions were stored at 4°C. ● Production culture medium CHO cell growth medium ISJ Gp023.2 (Fujifilm Wako Pure Chemical Corporation) was prepared according to the manufacturer's specified procedure. The specific procedure was as follows: 1. 16.10 g of CHO cell growth medium ISJ Gp023.2 was added to 450 mL of ultrapure water. The mixture was then stirred for at least 30 minutes to dissolve it in the ultrapure water. 2. Add 1 g of sodium bicarbonate and 2 g of glucose. Then, stir for about 15 minutes. 3. The pH was adjusted to 7.2-7.3 using NaOH solution to dissolve any remaining fine particles. Then, the solution was diluted to 500 mL using ultrapure water. 4. The obtained solution was filtered through a filter (pore size: 0.22 μm) and sterilized. 5. 10 mL of L-glutamine solution (200 mmol / L) was added. 6. To the obtained solution, 5 mL of penicillin-streptomycin solution (×100) was added to prevent microbial growth. The production medium was prepared in this manner. ● Feed medium CHO cell feed medium ISJ Fp020.1 (Fujifilm Wako Pure Chemical Corporation) was prepared according to the manufacturer's specified procedure. The specific procedure was as follows: 1. 45.5 g of CHO cell feed medium ISJ Fp020.1 was added to 200 mL of ultrapure water. The mixture was then stirred for at least 30 minutes to dissolve it in the ultrapure water. 2. 0.55 g of sodium bicarbonate was added. Then, the mixture was stirred for about 15 minutes. 3. The pH was adjusted to 6.9-7.1 using NaOH solution to dissolve any remaining fine particles. Then, the solution was diluted with ultrapure water to a volume of 249 mL. 4. The obtained solution was filtered through a filter (pore size: 0.22 μm) and sterilized. 5. To the obtained solution, 2.49 mL of penicillin-streptomycin solution (×100) was added to prevent microbial growth. The feed medium was prepared in this manner. ● Glucose stock solution 1. D(+)-glucose was dissolved in ultrapure water while heating to obtain a 400 g / L solution. 2. The obtained solution was filtered through a filter (pore size: 0.22 μm) and sterilized. In this way, a glucose stock solution was prepared.

[0089] [Measurement of glucose concentration] The glucose was measured using a glucose measurement kit (Glucose CII-Test Wako, Fujifilm Wako Pure Chemical Industries). For measurement, the sample was diluted 200 times with ultrapure water.

[0090] [Calculation of viable cell density and survival rate] The measurement was performed using the following procedure. 1. The sampled 100 μL cell suspension was diluted 2 to 20 times as appropriate with trypan blue staining solution (0.1% by weight, without sodium azide). 2. The number of viable and dead CHO cells was counted using a hemocytometer. 3. The number of viable cells per 1 mL of cell suspension was calculated as the viable cell density (cells / mL). 4. The cell viability was calculated according to the following formula. Viability rate (%) = number of living cells ÷ (number of living cells + number of dead cells) × 100 5. During the test period, the viable cell density was calculated at predetermined points in time according to steps 1-3. From the viable cell density curve obtained in this way, the cumulative value of viable cell density (IVCD) was calculated using trapezoidal approximation according to the following formula. IVCD(pieces / day / mL)=Σ{(t n -t n-1 )×(VCD tn +VCD tn-1 )÷2} During the ceremony, t n -t n-1 : The period (in days) from the calculation of the (n-1) viable cell density to the calculation of the nth viable cell density. VCD tn , VCD tn-1 : Viable cell density calculated on the nth or n-1th time.

[0091] [Measurement of antibody concentration] The measurement was performed using the following procedure. 1. Standard samples of known concentration were prepared. Specifically, purified IgG antibody was diluted 5, 10, 20, 40, 80, or 160 times with PBS to prepare a dilution series. 2. Standard samples were analyzed by HPLC. The analytical conditions were as follows: • Column: POROS TM A 20μm Column (Thermo Fisher Scientific, 2.1mm diameter x 30mm length) ·Eluent A: 50mM Tris-HCl buffer (pH7.0) ·Eluent B: 50mM Tris-HCl buffer (pH3.0) • Elution conditions: The eluent was switched according to the time as follows. 0 min ~ 0.74 min: A liquid 0.75 minutes to 2.75 minutes: B liquid 2.76 minutes ~ 4.02 minutes: A liquid ·Flow rate: 4.0mL / min ·Temperature: 30℃ ·Injection volume: 10μL Detection: Wavelength 280nm 3. A calibration curve was created based on the measurement results of the standard samples. 4. The culture supernatant of the cell suspension was diluted 10-fold with PBS. The diluted solution was then filtered through a 0.45 μm filter. In this way, a sample for measurement was obtained. 5. Antibody concentration and specific production rate were quantified from the chromatograms of the sample used for measurement. The specific production rate was calculated by dividing the antibody concentration by the cumulative value of the viable cell density.

[0092] [Measurement of host cell-derived protein concentration] The concentration of host cell-derived proteins was measured according to the instructions for the ELISA kit (F550-1-ELISA kit, Cygnus Technologies, LLC). The specific procedure was as follows: 1. The culture supernatant collected from the cell suspension was diluted with the diluent specified in the kit (Sample Diluent Buffer, Cygnus Technologies, LLC). 2. 100 μL of anti-CHO:HRP and 50 μL of sample or CHO hcp STANDARDS were injected into each well of a 96-well plate. Based on prior studies, the samples were diluted 20,000 times before use. 3. The plate was shielded from light and shaken at room temperature for 2 hours. 4. Wash buffer was prepared from wash concentrate (20X). 5. Add 300 μL of wash buffer to each well and wash the wells. Repeat this washing process a total of four times, then drain thoroughly. 6. 100 μL of TMB substrate was added to each well. 7. The samples were left to stand at room temperature for 30 minutes under light protection. 8. 100 μL of Stop solution was added to each well. 9. The concentration was quantified at two different wavelengths (primary wavelength: 450 nm, secondary wavelength: 650 nm). For quantification, two samples were prepared for each experimental condition.

[0093] [Measurement of PLBL2 (Phospholipase B-Like 2) concentration] The concentration of PLBL2 was measured according to the procedure manual for the ELISA kit (Hamster Phospholipase B-like 2 ELISA kit, Immunology Consultants Laboratory, Inc.). The specific procedure was as follows. PLBL2 is a type of host cell-derived protein. 1. The culture supernatant collected from the cell suspension was diluted 500-fold using a diluent concentrate, based on the results of a prior concentration study. 2. 100 μL of sample or standard solution was injected into each well of a 96-well plate. The standard solution was prepared using the PLBL2 calibrator. 3. The plate was shaken at room temperature for 2 hours. 4. Add 300 μL of wash solution to each well and wash the wells. Repeat this washing process a total of four times, then drain thoroughly. The washing method in subsequent steps is the same as in this step. 5. Add 100 μL of detection antibody to each well and shake at room temperature for 20 minutes in the dark. 6. Add 100 μL of HRP-streptavidin to each well and shake at room temperature for 20 minutes in the dark. Then wash the wells. 7. Add 100 μL of TMB substrate to each well and shake at room temperature for 10 minutes in the dark. 8. 100 μL of Stop solution was added to each well. 9. The concentration in PLBL2 was quantified using a wavelength of 450 nm.

[0094] [Quantification of aggregated antibodies] The amount of aggregated antibodies produced by CHO cells was quantified. Agglutinated and non-aggregated antibodies were separated by size exclusion chromatography. The specific procedure was as follows: 1. Based on the antibody concentration measured in advance, the culture supernatant collected from the cell suspension was diluted to 0.8 mg / mL with production medium. This resulted in 500 μL of the diluted solution containing 0.4 mg of IgG. 2. IgG was purified using MonoSpin ProA (GL Sciences). 3. The purified solution was filtered through a filter (pore size: 0.45 μm). In this way, a sample for analysis was obtained. 4. The analytical samples were analyzed by HPLC. The HPLC conditions were as follows: Column: TSKgel G3000SWXL (Inner diameter: 7.8mm x Length: 300mm, Tosoh Corporation) Eluent: 133 mM sodium phosphate + 150 mM sodium chloride (pH 6.6) Detection time: 30 minutes Flow rate: 0.5mL / min Column temperature: 25℃ Injection volume: 50μL Detection: UV (280nm) 5. From the chromatogram, the ratio of the peak area of ​​aggregated antibodies to the total peak area derived from the antibody was calculated. The agglutination rate was calculated according to the following formula. Aggregation rate (%) = Peak area of ​​aggregated antibodies ÷ Total peak area from antibodies × 100

[0095] [Measurement of osmotic pressure] Osmotic pressure was measured using a vapor pressure osmometer (VAPRO (R) 5600 Vapor Pressure Osmometer, Wescor Inc). Standard solutions of 290 mmol / kg, 1,000 mmol / kg, and 100 mmol / kg were used for calibration. OPTIMOLE was used as the standard solution. TM The Osmolality Standard (ELITechGroup Inc.) was used. Osmotic pressure increases due to various substances present in the sample.

[0096] [Example 6: Effects of trehalose concentration in a culture system] The effect of trehalose concentration as a culture medium additive on cell culture was investigated. Specifically, cells were fed cultured in production medium supplemented with trehalose (n=3). Cells were seeded in a flask (volume: 125 mL) containing 20 mL of production medium and cultured with shaking. The seeding density was 1.0 × 10⁻⁶. 6 The concentration was 100 cells / mL. The shaking speed was 150 rpm. The final trehalose concentrations at the start of culture were 0 mM, 5.1 mM, 10.2 mM, 20.4 mM, 40.8 mM, or 81.5 mM. All cultures were performed at 37°C with a CO2 concentration of 5%.

[0097] During cultivation, the following procedures were performed at predetermined times. In the following, the sowing day is considered day 0. ●Days 3, 4, 5, and 6 • 1.5 mL was sampled from each flask, and cell density and glucose concentration were measured. • 8% by volume of feed medium was added to the initial volume of culture medium. A 400 g / L glucose solution was added to achieve a final glucose concentration of 15 g / L. However, if the glucose concentration exceeded 15 g / L at the stage when the feed medium was added, the glucose solution was not added. ● Day 7 • 1.5 mL was sampled from each flask, and cell density and glucose concentration were measured. • 16% by volume of feed medium was added to the initial culture medium volume in two separate additions. A 400 g / L glucose solution was added to achieve a final glucose concentration of 30 g / L. ● Day 10 The entire volume of culture medium in the flask was collected and centrifuged (2,000 rpm, 5 minutes). Then, 15 mL of the supernatant was collected. • 2 mL of the supernatant was collected and centrifuged again (2,000 rpm, 5 minutes). Then, it was filtered through a 0.45 μm filter. The samples were stored at 4°C until the various parameters were measured.

[0098] [result] The results are shown in Table 6. In the table, the column for weight ratio to glucose represents the weight ratio of trehalose to glucose at the start of culture. This weight ratio is based on measured values. [Table 6]

[0099] As can be seen from Table 6, viable cell density and antibody concentration on day 10 of culture improved in the groups with a final trehalose concentration of 5.1–20.4 mM, and significantly decreased in the groups with 40.8 mM or 81.5 mM. These results suggest that trehalose as a culture medium additive, at a certain concentration (e.g., 1–30 mM), contributes to promoting cell survival and the production of cell products.

[0100] As can also be seen from Table 6, the concentration of host cell-derived proteins was maintained or decreased in the groups with a final trehalose concentration of 5.1–20.4 mM, and significantly increased in the groups with 40.8 mM or 81.5 mM. These results suggest that trehalose as a culture medium additive, at a certain concentration (e.g., 1–30 mM), contributes to improving or maintaining the purity of cell products.

[0101] In addition to the above, the specific production rate per cell increased in a manner dependent on the final trehalose concentration. The concentration of PLBL2 decreased in a manner dependent on the final trehalose concentration in the range of 5.1 to 20.4 mM. Furthermore, at a final trehalose concentration of 40.8 mM, the antibody aggregation rate decreased to nearly 70% of that of the control, suggesting that antibody aggregation was suppressed.

[0102] [Example 7: Changes in trehalose content in culture medium over time] During the culture period in Example 6, the trehalose concentration in the culture medium was monitored at predetermined time points. The method for quantifying the trehalose concentration was as follows. 1. Standard samples of known concentration were prepared. Specifically, dilution series of 0.02% by weight, 0.04% by weight, 0.1% by weight, 0.5% by weight, or 1.0% by weight were prepared from the standard product Trehalose SG (Nagase Vita Co., Ltd.) using ultrapure water (concentrations are converted to anhydrous trehalose values). 2. Standard samples were analyzed by HPLC. The analytical conditions were as follows: • Column: SUGAR KS-801 (Resonac Co., Ltd., inner diameter: 8.0mm x length: 300mm) ·Eluent: Ultrapure water • Detection time: 30 minutes ·Flow rate: 0.5mL / min Column temperature: 80°C ·Injection volume: 20μL • Detection: RI 3. A calibration curve was created based on the measurement results of the standard samples. 4. The culture supernatant from days 0 to 7 was diluted three-fold with ultrapure water and filtered through a 0.45 μm filter. In this way, samples for measurement from days 0 to 7 of culture were obtained. 5. The culture supernatant on day 10 was heated at 100°C for 10 minutes, and then centrifuged at 4°C for 10 minutes (15,000 rpm) to remove proteins. Further dilution was performed three-fold with ultrapure water to remove salt. The mixture was then filtered through a 0.45 μm filter. In this manner, a measurement sample was obtained on day 10 of culture. 6. For the chromatograms of each measurement sample, the measured values ​​of trehalose concentration in the culture medium were calculated based on the calibration curve.

[0103] [result] Table 7 shows the measured trehalose concentrations calculated by HPLC analysis. The theoretical values ​​in the table are estimated concentrations at each time point, assuming that the trehalose added on day 0 of culture is not degraded or metabolized during the culture period. In this case, trehalose is diluted only by the addition of feed medium and glucose stock solution. [Table 7]

[0104] As can be seen from Table 7, the measured and theoretical values ​​of trehalose concentration in the culture supernatant were in close agreement from day 3 to day 7 of culture. The result on day 10 of culture shows a large difference from the measured value, which is thought to be due to the dilution of the culture supernatant during the desalting process. From these results, it is suggested that under the culture conditions described in Example 6, trehalose was not actively consumed by CHO cells, and that there was almost no abiotic degradation in the culture medium. Combined with the results of Example 6, it is suggested that even without supplementing trehalose during culture, a constant amount of trehalose added at the start of culture is always present in the culture medium and has a certain effect on the cultured cells. [Industrial applicability]

[0105] This invention can be used for cell culture and other applications.

Claims

1. A culture medium additive for cell culture media containing adenosine-N1-oxide.

2. The culture medium additive according to claim 1, further comprising one or more selected from the group consisting of trehalose and selaginose.

3. The culture medium additive according to claim 1, used such that the final concentration of adenosine-N1-oxide in the culture medium is 0.5 μM to 200 μM.

4. The content of adenosine-N1-oxide relative to the glucose content in the culture medium is 1:(4.0 × 10) by weight ratio. －６ ~5.0 x 10 －３ The culture medium additive according to claim 1, used in such a manner as described above.

5. Contains trehalose, A culture medium additive for cell culture media, used so that the final concentration of trehalose in the culture medium is 1 mM to 30 mM.

6. The ratio of the trehalose content to the glucose content in the culture medium is 1:(1.0 × 10) by weight. －２ ~20.0 x 10 －１ The culture medium additive according to claim 5, used in such a manner as described above.

7. A culture medium additive for cell culture media containing selaginose.

8. The culture medium additive according to claim 7, which is used so that the final concentration of selaginose in the culture medium is 1 mM to 50 mM.

9. The content of selaginose relative to the glucose content in the culture medium is 1:(1.5 × 10) by weight ratio. －２ ~25.0 x 10 －１ The culture medium additive according to claim 7, used in such a manner as described above.

10. The culture medium additive according to claim 1, 5, or 7, wherein the cells are mammalian cells.

11. The culture medium additive according to claim 10, wherein the mammalian cells are Chinese hamster ovary cells (CHO cells).

12. A cell culture medium comprising the culture medium additive according to claim 1, 5, or 7.

13. A method for producing cultured cells, comprising the step of culturing cells in a cell culture medium containing the culture medium additive according to claim 1, 5, or 7.

14. A method for promoting the production of cell products by cells, comprising the step of culturing cells in a cell culture medium containing the culture medium additive according to claim 1, 5, or 7.

15. A method for improving cell viability in cell culture, comprising the step of culturing cells in a cell culture medium containing the culture medium additive according to claim 1, 5, or 7.

16. A method for reducing the concentration of host cell-derived proteins in cell culture, comprising the step of culturing cells in a cell culture medium containing the culture medium additive according to claim 1, 5, or 7.

17. A method for reducing the concentration of contaminating proteins in cell culture, comprising the step of culturing cells in a cell culture medium containing the culture medium additive according to claim 1, 5, or 7.

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