JP7276679B2 - Useful Substance Specific Production Rate Accelerator and Viability Decrease Suppressor in Cells - Google Patents

Description

The present invention provides an agent for promoting the specific production rate of a useful substance in cells, comprising a component having protein kinase inhibitory activity; A cell culture medium containing an agent for suppressing decrease in viability, which is used for producing a useful substance; A method for producing a useful substance, comprising the step of culturing cells using a cell culture medium containing the agent for promoting the specific production rate or the agent for suppressing decrease in viability. etc.

Proteins and peptides are artificially produced using gene recombination technology. Especially in the pharmaceutical field, proteins useful in medicine (eg, humanized antibodies, erythropoietin, etc.) are produced using cultured cells.
Various attempts have been made for the purpose of improving the production of useful substances. It is disclosed that the increase can increase the amount of protein produced.

JP 2013-247927 A

Techniques for increasing the cell density, such as the method described in Patent Document 1, are widely known for improving the production of useful substances such as antibodies. However, as a result of intensive studies by the inventors of the present application, it was found that if the cell density is excessively increased, impurities other than useful substances such as cell fragments also increase, which may increase the load on the purification process. In addition, when the cell density is high, the amount of nutrient components used in the medium is also required to be increased accordingly. Thus, it was found that the method of increasing the cell density does not lead to a reduction in the total cost of production of useful substances.
Therefore, the inventors of the present application focused on the fact that cell proliferation and the production rate of useful substances per cell (specific production rate) tended to be a trade-off, and suppressed cell proliferation to lower the cell density. On the other hand, the idea of improving the production rate of useful substances per cell (that is, the specific production rate), which is completely opposite to the idea of increasing the cell density described in Patent Document 1, was conceived.
In addition, during the later stages of cell culture, nutrient depletion, accumulation of toxic metabolites, and various other factors usually cause intracellular transmission of growth-suppressive and apoptosis-inducing signals. As a result, cell death occurs in the later stages of cell culture, resulting in decreased cell viability. Decrease in cell viability due to cell death is a negative factor that lowers the final production amount of useful substances. In addition, as cell death progresses, useful substances may be partially degraded by proteases leaked from the cells, resulting in structural changes. In addition, the intracellular glycosylation function may not work sufficiently in cells undergoing cell death. This results in either no glycosylation or incomplete glycosylation of the useful substance. For reasons related to target product yield and quality, there is a need to develop methods to prevent cell viability loss during the later stages of culture.
The present invention provides an agent for promoting the specific production rate of a useful substance in cells, comprising a component having protein kinase inhibitory activity; A cell culture medium containing an agent for suppressing decrease in viability, which is used for producing a useful substance; A method for producing a useful substance, comprising the step of culturing cells using a cell culture medium containing the agent for promoting the specific production rate or the agent for suppressing decrease in viability. etc.

As a result of intensive studies based on the above idea, the inventors of the present application have found that a component having protein kinase inhibitory activity can suppress cell proliferation at a certain rate and reduce cell density, and at the same time, inhibit the production of useful substances such as proteins in cells. The inventors have found that the production rate (specific production rate) per cell can be improved, and have completed the present invention based on this finding.
In addition, the component having protein kinase inhibitory activity suppresses the decrease in cell viability in the latter stage of cell culture, improves the total production of useful substances, and increases the production rate (specific production rate) of useful substances in the latter stage of cell culture. The inventors have found that the reduction is suppressed, and have completed the present invention.
The present invention provides a useful substance specific production rate enhancer in cells containing a component having protein kinase inhibitory activity.
The present invention provides an agent for suppressing decrease in viability in cells containing a component having protein kinase inhibitory activity.
One aspect of the present invention provides a cell culture medium containing a component having protein kinase inhibitory activity and used for producing useful substances.
As one aspect of the present invention, a useful substance comprising a step of culturing cells using a cell culture medium containing a specific production rate promoter or viability reduction suppressor containing a component having protein kinase inhibitory activity. A manufacturing method is provided.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an animal cell culture medium containing, as an active ingredient, a component having protein kinase inhibitory activity, which can improve the specific production rate of useful substances such as proteins in cells. The cell culture medium of the present invention, which contains a component having protein kinase inhibitory activity, improves the specific production rate of useful substances regardless of the morphology and type of cells, the presence or absence of transformation, and the like. In addition, it suppresses the decrease in cell viability in the later stage of cell culture. That is, the useful substance specific production rate enhancer and viability reduction suppressor provided by the present invention, a cell culture medium containing the specific production rate enhancer or viability decrease inhibitor, and a useful substance using the medium can improve the specific production rate of useful substances in cells. In addition, it is possible to suppress the decrease in cell viability in the latter stage of cell culture, and it is possible to apply it in fields such as clinical diagnostic drug production, biopharmaceutical production, regenerative medicine, and cell therapy.

FIG. 2 is a diagram showing antibody production rate (specific production rate) per cell in media containing components having protein kinase inhibitory activity in Examples 1 and 2. FIG. FIG. 6 is a diagram showing antibody production rate (specific production rate) per cell in media containing components having protein kinase inhibitory activity in Examples 3 to 6. FIG. FIG. 10 is a diagram showing cell viability during culture in a medium containing a component having protein kinase inhibitory activity in Example 7. FIG. FIG. 10 is a diagram showing antibody concentrations in media (day 16 of culture) containing components having protein kinase inhibitory activity in Example 7. FIG. FIG. 10 shows cell viability during culture in media containing components with protein kinase inhibitory activity in Examples 8 and 9. FIG. FIG. 10 is a diagram showing antibody concentrations in media (day 16 of culture) containing components having protein kinase inhibitory activity in Examples 8 and 9. FIG.

The present invention provides an agent for promoting the specific production rate of a useful substance in cells, comprising a component having protein kinase inhibitory activity; A cell culture medium containing an agent for suppressing decrease in viability, which is used for producing a useful substance; A method for producing a useful substance, comprising the step of culturing cells using a cell culture medium containing the agent for promoting the specific production rate or the agent for suppressing decrease in viability. etc.

In the present invention, a component having protein kinase inhibitory activity suppresses cell proliferation at a certain rate to reduce cell density, and at the same time, it is possible to improve the production rate per cell of useful substances such as proteins in cells. can. As a result, it is possible to reduce the number of cells required to obtain the useful substance production amount while maintaining (or increasing) the useful substance production amount per culture batch. As the cell density is reduced, impurities other than useful substances, such as cell fragments, can be reduced, and the burden of the process of purifying useful substances can be reduced. Also, the amount of nutrients used in the medium can be reduced. As a result, it is possible to reduce the total cost of producing useful substances.

In addition, in the present invention, the component having protein kinase inhibitory activity can suppress the death of cells in the later stage of cell culture and suppress the decrease in cell viability. As a result, the production rate (specific production rate) of the useful substance in the latter stage of cell culture can be prevented from decreasing, and the total production amount of the obtained useful substance can be improved. In addition, it is possible to suppress quality deterioration of the obtained useful substance. More specifically, it suppresses the deterioration of the intracellular glycosylation function in the later stage of cell culture. As a result, incomplete sugar chain modification can be suppressed.

In the present invention, ingredients with protein kinase inhibitory activity are used. In the present invention, any component can be used without particular limitation as long as it has protein kinase inhibitory activity. The component having protein kinase inhibitory activity may be used by purchasing a commercially available product, or by appropriately synthesizing or extracting from a natural product. It is also possible to use a combination of two or more components having multiple protein kinase inhibitory activities.

Although the details of the mechanism by which the component with protein kinase inhibitory activity improves the specific production rate of useful substances are unknown, for example, protein kinase inhibition may have stopped or suppressed cell cycle transition. In this case, it is presumed that the cell cycle stops at the G1 phase or the G2 phase of the cell cycle, suppressing cell proliferation and improving the ability to synthesize protein in the cell. It is also possible that other mechanisms promoted gene transcription and extracellular secretion of proteins.

In addition, the details of the mechanism by which a component having protein kinase inhibitory activity suppresses the decrease in viability in cells is unknown. One possible reason could be changes in cell community composition during culture. Staurosporine, violacein, and the like are known to cause cell death by inducing apoptosis within a certain concentration range. Cells used for production of useful substances are usually used after being made to have a single gene by an operation called cloning. However, many cells are known to undergo various gene mutations due to repeated culturing. This gene mutation can also occur during cell bank production, subculturing, and expansion culture after cloning. As a result, when culturing for the production of useful substances, there are cases where the cell clusters have some variation even though it is not desired. By adding staurosporine, violacein, etc. at a low concentration that does not cause significant cell death in this state, it is possible that cells that are difficult to induce apoptosis can predominantly survive and proliferate in the culture medium. (changes in cell community composition) and may have contributed to the decreased viability in the latter half of culture.

In the present invention, "suppressing cell proliferation" means the effect of decreasing the cell proliferation rate (growth rate) itself and the effect of decreasing the value of the maximum attainable cell density of cells. The effect of the present invention may be attributed to any effect of inhibition of cell proliferation.
The specific production rate is an index showing the substance production rate per cell. A specific production rate is calculated by a calculation method used in the field of cell culture or microorganism culture. As an example, there is a method of creating a scatter diagram with the cumulative number of cells on the horizontal axis and the substance concentration on the vertical axis, and calculating the specific production rate from the slope of the scatter diagram. More specifically, "Practical Basics of Useful Microorganism Culture from Test Tube to Industrial Scale; NTS Co., Ltd.", "Dutton RL, Scharer JM, Moo-Young M. Cytotechnology. 1999 Jan. 29(1) , p1-10” can be referred to.
As a method for calculating the specific production rate in the present invention, any method other than the calculation methods exemplified above may be used as long as it indicates the substance production rate per cell. It is also possible to calculate the substance production rate per a certain number of cells instead of the substance production rate per cell, for example, "substance production rate per cell to the power of 10".

In addition, in the present invention, "to suppress a decrease in cell viability" means that the cell viability in a medium containing a component having protein kinase inhibitory activity is reduced to that in a medium containing no component having protein kinase inhibitory activity. It means 10% or more, preferably 15% or more higher than the rate at the latter stage of culture or at the end of culture.
The survival rate can be determined by the number of viable cells/total number of cells in the culture medium. Various methods can be used to measure the number of cells in the culture medium. For example, after staining the cells with trypan blue, the number of viable cells and the total number of cells in the culture medium can be measured using a hemocytometer.

As a component having protein kinase inhibitory activity, a component having serine/threonine protein kinase inhibitory activity or the like is preferable, and a component having protein kinase A inhibitory activity or a component having protein kinase C inhibitory activity is more preferable.

Examples of ingredients having protein kinase inhibitory activity include violacein, staurosporine, erlotinib, imatinib, sunitinib, lapatinib, gefitinib, sorafenib, nilotinib, toceranib, bosutinib, neratinib, vatalanib, regorafenib, cabozantinib, imatinib mesylate, dasatinib , canertenib, enzastaurin, midostaurin, saphingol, perifosine, vandetanib, recentin, pazopanib, axitinib, lestaurtinib, semaxinib, SU-14813, flavopiridol, seliciclib, BMS-387032, AT-7519, R-547, AG- 024322, P-276-00, ZK-304709, PD-0325901, ARRY-142886 and the like.

Examples of components having serine/threonine protein kinase inhibitory activity include vemurafenib, hesperadin, MK0457, ZM447439, staurosporine, violacein and the like.
Examples of components having protein kinase A inhibitory activity include cAMP-Rp, KT5720, (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, violacein and the like.
Examples of components having protein kinase C inhibitory activity include bisindolylmaleimide, staurosporine, sphingosine, phloretin, phlorizin, ganglioside, palmitoylcarnitine, quercetin, polymyxin B, trifluoperazine, violacein, and the like.

Violacein or staurosporine is preferred among the components having protein kinase inhibitory activity described above.

The component having protein kinase inhibitory activity described above can be used to improve the specific production rate of useful substances in cells. Preferably, use of a component having protein kinase inhibitory activity is provided in order to improve the specific production rate when producing useful substances using cells in vitro. A component having protein kinase inhibitory activity should improve the production rate (specific production rate) of a useful substance per cell, regardless of its mechanism.

The component having protein kinase inhibitory activity described above can be used to suppress the decrease in cell viability in the later stage of cell culture. Preferably, use of a component having protein kinase inhibitory activity is provided in order to suppress a decrease in viability of cells during the production of useful substances using cells in vitro. A component having protein kinase inhibitory activity should suppress the decrease in cell viability in the later stage of cell culture, regardless of the mechanism.

A component having protein kinase inhibitory activity can be used to improve the specific production rate of a useful substance by adding it to a cell culture medium. In addition, by adding it to a medium for thin culture, it can be used to suppress a decrease in cell viability in the latter stage of cell culture. In a preferred embodiment, by adding a component having protein kinase inhibitory activity to the cell culture medium, it is possible to improve the specific production rate of useful substances and suppress the decrease in cell viability in the latter stage of cell culture. Therefore, a component having protein kinase inhibitory activity can be used alone as a useful substance specific production rate enhancer or cell viability reduction suppressor, which is a medium additive factor. It can also be used together with substances that contribute to promoting the production of other useful substances (eg proteins such as transferrin and insulin) and/or low-molecular-weight compounds (eg glucose, phosphate, selenite). That is, the useful substance-specific production rate enhancer is not particularly limited as long as it contains a component having protein kinase inhibitory activity, and may or may not contain other substances.

The promoter provided by the present invention (for example, the specific production rate promoter of a useful substance) or the cell viability reduction suppressor is determined by the amount of the component having protein kinase inhibitory activity added to the medium volume (protein kinase inhibitory activity in the medium The concentration of the component having ) may be 0.01 nM or more, preferably 0.05 nM or more, and more preferably 0.1 nM or more. In addition, it may be 2.0 μM or less, preferably 1.5 μM or less, and more preferably 1.2 μM or less.

The amount of the component having protein kinase inhibitory activity to be added to the medium may be such that the specific growth rate reduction rate is in the range of 5 to 75%, preferably in the range of 25 to 70%. The specific growth rate reduction rate can be calculated by the following formula. When A is the specific growth rate in the state without the component having protein kinase inhibitory activity, and B is the specific growth rate in the state with the component having protein kinase inhibitory activity, the specific growth rate reduction rate (%) = (1- B/A)×100.

The specific growth rate is an index showing changes in cell number over a certain period of time. A specific growth rate is calculated by a calculation method used in the field of cell culture or microorganism culture. For example, by measuring the cell concentration at two different points (phases) during the logarithmic growth phase, it can be calculated by the following formula. Specific growth rate = {ln (m2/m1)}/( t2-t1). In addition, the unit of culture time may be seconds, minutes, hours, days, etc., as long as it is a unit representing time, and the unit of cell concentration is cells/mL, cells/L, mg/mL, g. Any unit such as /L may be used as long as it represents the cell concentration. It should be noted that the specific growth rate can be obtained using a method other than the above calculation method, as long as it indicates a change in the number of cells over a certain period of time.

The specific addition amount of the component having protein kinase inhibitory activity, for example, when violacein is used, may be in the range of 0.5 μM to 1.5 μM, preferably in the range of 0.6 μM to 1.4 μM. A range of 0.7 μM to 1.3 μM is more preferred, and a range of 0.8 μM to 1.2 μM is particularly preferred. Also, when staurosporine is used, it may be in the range of 0.1 nM to 10 nM, preferably in the range of 0.1 nM to 7 nM, more preferably in the range of 2 nM to 5 nM. The above concentration range is merely an example, and can be adjusted as appropriate according to the component having protein kinase inhibitory activity to be added to the medium. The specific production rate enhancer or viability decrease suppressor may be liquid or solid at room temperature. For example, the protein kinase can be added to buffers such as phosphate buffered saline, phosphate buffer, acetate buffer, citrate buffer, Tris buffer, HEPES buffer, MOPS buffer and solvents such as dimethylsulfoxide (DMSO). Dissolving and/or suspending a component having inhibitory activity and adjusting the component having protein kinase inhibitory activity in the medium within the above range as a specific production rate or viability reduction inhibitor provided by the present invention. can be done.

The medium to which the component having protein kinase inhibitory activity is added may be, for example, MEM, DMEM, HamF12, RPMI1640, Fisher's medium, or a mixture thereof. By adding a component having protein kinase inhibitory activity as described above to these basal media, media useful for producing useful substances can be prepared. It can also be used by adding it to a serum-free medium, protein-free medium, or chemically-defined medium that is intended to be used without serum by adding growth factors or serum substitutes. Examples of these media include EX-CELL 302, EX-CELL 325-PF, EX-CELL CD CHO, etc. manufactured by SAFC Biosciences, SFM II, CHO-III-PFM, CD CHO, etc. manufactured by Life Technologies. , IS-CHO CD manufactured by Irvine Scientific, BalanCD Growth A Medium, etc., but are not limited thereto. In addition, a mixed medium in which two or more of such serum-free medium, protein-free medium, or chemically-defined medium are mixed at an arbitrary ratio can also be used in the same manner.

A medium containing a component having protein kinase inhibitory activity, in addition to the component having protein kinase inhibitory activity, includes, for example, inorganic salts (e.g., sodium salts, potassium salts, calcium salts), carbohydrates (e.g., sugars such as glucose). , amino acids (e.g., glutamine and isoleucine), vitamins (e.g., riboflavin, thiamine), fatty acids/lipids (e.g., steroids such as cholesterol), proteins/peptides (e.g., albumin, transferrin, L-alanyl-L-glutamine, etc.) dipeptides), trace elements (eg magnesium, copper, iron) and combinations thereof. In one embodiment, the liquid medium containing a component having protein kinase inhibitory activity is, in addition to the component having protein kinase inhibitory activity, 0.1 to 5 g/L of glucose, 0.1 to 0.5 g/L of CaCl ₂ , 1-10 g/L NaCl, 0.001-0.3 g/L L-arginine.HCl, 0.001-0.3 g/L L-cysteine.2HCl, 0.001-0.3 g /L L-histidine.HCl.H ₂ O, 0.001-0.3 g/L L-isoleucine, 0.001-0.3 g/L L-leucine, 0.001-0.3 g/L of L-lysine.HCl, 0.001-0.3 g/L L-methionine, 0.001-0.3 g/L L-phenylalanine, 0.001-0.3 g/L L-threonine, 0 0.001-0.3 g/L L-tryptophan, 0.001-0.3 g/L L-tyrosine.2Na.2H ₂ O, and 0.001-0.3 g/L L-valine. Further, as one embodiment, the medium may be in the form of a powder that has the above composition when dissolved in water. A medium containing such a component having protein kinase inhibitory activity as described above can be used for producing useful substances. In addition, the components described above (eg, glucose, etc.) may be added to the medium during culture.

Specifically, useful substances produced by a method comprising a culturing step of culturing cells that produce useful substances in a medium containing a component having protein kinase inhibitory activity, and a purification step of isolating and purifying the useful substances produced from the cells. can be manufactured. For example, when an antibody is produced as a useful substance, the antibody can be produced by a method comprising culturing antibody-producing cells in a medium containing a component having protein kinase inhibitory activity and purifying the antibody. Antibody purification steps include, for example, methods using affinity column chromatography. In addition, in the above culture step, so-called fed-batch culture (fed-batch culture) may be performed in which medium components are added during culture. In this case, the medium to be added may or may not contain a component having protein kinase inhibitory activity.

In the present invention, the timing of addition of the component having protein kinase inhibitory activity to the medium is not particularly limited. Components with kinase inhibitory activity may be added to the medium.

As used herein, the useful substance is not particularly limited as long as it is useful for pharmaceuticals, agricultural chemicals, foods, and other chemical industries. Preferred examples include bioactive proteins such as antibodies, enzymes (urokinase etc.), hormones (insulin etc.), cytokines (interferon, interleukin, erythropoietin, G-CSF, GM-CSF etc.), peptides and the like. Antibodies are, for example, murine, humanized or human monoclonal antibodies. The class of immunoglobulin is not particularly limited, but is, for example, IgG (eg, IgG1, IgG2). A useful substance can be a recombinant protein that is the expression product of a foreign gene.

In the present specification, the cells are not particularly limited as long as they can be used for producing useful substances such as recombinant proteins, CHO cells, BHK cells, HepG2 cells, rodent myeloma cells (e.g., SP2/O cells, NSO mammalian cells such as mouse myeloma cells); animal cells such as insect cells such as silkworm cells and fruit fly cells; plant cells such as Arabidopsis thaliana cells; It is mentioned as. When producing antibodies as useful substances, hybridomas obtained by cell fusion of animal cells (preferably mammalian cells) such as CHO cells, SP2/O cells or NSO cells can be employed as antibody-producing cells. can. Among them, animal cells are preferred, mammalian cells are more preferred, and CHO cells are even more preferred.

EXAMPLES The present invention will be further described with reference to examples below, but the present invention is not limited to these examples.

Confirmation of effects of components having protein kinase inhibitory activity (Examples 1 and 2)
A CHO cell line (ATCC CRL-12445) transfected with an IgG gene and secreting and producing an IgG antibody was used, and cells adapted and suspended in BalanCD Growth A Medium (manufactured by Irvine Scientific) were used in the experiment. 30 ml of Balan CD Growth A Medium was added to a 125 ml Erlenmeyer flask, the suspended cells were seeded at 1.0×10 ⁶ cells/ml, and shaken at 120 rpm at 37° C. in a 5% CO ₂ environment. It was cultured for 14 days.
At the start of culture, a component having protein kinase inhibitory activity (Violacein: manufactured by Sigma-Aldrich) was added to the medium to a concentration of 0.9 μM (Example 1) or 1.0 μM (Example 2). On the other hand, as a control (no violacein added), a medium in which DMSO was added at the start of culture was used.
The culture medium was sampled on days 0, 2, 4, 7, 9, 11 and 14 of culture, and the number of viable cells and IgG antibody concentration in the culture medium were measured to determine the specific antibody production rate in CHO cells. The results are shown in FIG. Table 1 shows the specific growth rate reduction rate (%) calculated by the following calculation, where A is the specific growth rate of the control (violacein-free) and B is the specific growth rate of the violacein-added control. Specific growth rate decrease (%) = (1-B/A) x 100.

Confirmation of effects of ingredients having protein kinase inhibitory activity (Examples 3 to 6)
A CHO cell line (ATCC CRL-12445) transfected with an IgG gene and secreting and producing an IgG antibody was used, and cells adapted and suspended in BalanCD Growth A Medium (manufactured by Irvine Scientific) were used for the experiment. 35 ml of Balan CD Growth A Medium was added to a 125 ml Erlenmeyer flask, the suspended cells were seeded at 1.0×10 ⁶ cells/ml, and shaken at 120 rpm at 37° C. in a 5% CO ₂ environment. It was cultured for 12 days. In order not to deplete the glucose in the medium, a 45% (w/v) glucose solution was used on the 7th and 10th days of the culture so that the glucose concentration in the medium was 5.0 g/l. added. At the start of culture, a component having protein kinase inhibitory activity (staurosporine: manufactured by Sigma-Aldrich) was added to the medium at 2 nM (Example 3), 3 nM (Example 4), 4 nM (Example 5), 5 nM (Example It was added so as to obtain the concentration of 6). On the other hand, as a control (no staurosporine added), a medium in which DMSO was added at the start of culture was used. The culture medium was sampled on days 0, 4, 7, 9 and 12 of culture, and the number of viable cells and IgG antibody concentration in the culture medium were measured to determine the specific antibody production rate in CHO cells. The results are shown in FIG. Table 1 shows the specific growth rate reduction rate (%) calculated by the following calculation, where A is the specific growth rate of the control (no staurosporine added) and B is the specific growth rate with the addition of staurosporine. . Specific growth rate decrease (%) = (1-B/A) x 100.

Method for measuring IgG concentration Using BLItz manufactured by Pall ForteBio, the IgG concentration in the cell culture medium was measured according to the method described in the attached instruction manual.

Method for measuring the number of viable cells Using a Countess Automated Cell Counter manufactured by Life Technologies, the number of viable cells was measured according to the method described in the instruction manual.

From Table 1 and FIGS. 1 and 2, it can be seen that addition of components having protein kinase inhibitory activity (violacein, staurosporine) to the medium suppresses cell proliferation and increases the relative production of useful substances such as proteins in cells. It turns out that speed can be improved.

Confirmation of effect of component having protein kinase inhibitory activity in fed-batch culture (Example 7)
A CHO cell line (ATCC CRL-12445) transfected with an IgG gene and secreting and producing an IgG antibody was used, and cells adapted and suspended in BalanCD Growth A Medium (manufactured by Irvine Scientific) were used for the experiment. 50 ml of the cell solution prepared by adjusting the concentration of the suspended cells to 1.0×10 ⁶ cells/ml with Balan CD Growth A Medium was added to a 250 ml Erlenmeyer flask, and the mixture was stirred at 37° C., 5% CO ₂ environment, 120 rpm. cultured for 16 days with shaking at .
At the start of culture, a component having protein kinase inhibitory activity (Violacein: manufactured by Sigma-Aldrich) was added to the medium to a concentration of 0.9 μM (Example 7). On the other hand, as a control (no violacein added), a medium in which DMSO was added at the start of culture was used.
On the 4th, 6th, 7th, 8th, 10th, 12th and 14th days of culture, BalanCD Feed I Medium (manufactured by Irvine Scientific) was added in an amount of 5% (v/v) of the culture solution. In addition, 0.9 μM violacein was added to the BalanCD Feed I Medium in advance so that the addition of the BalanCD Feed I Medium would not dilute the violacein in the culture medium.
The culture medium was sampled on days 0, 2, 4, 6, 7, 8, 10, 12, 14 and 16 of culture, and the number of viable cells, cell viability, and IgG antibody concentration in the culture medium were measured. FIG. 3 shows changes in cell viability during culture, and FIG. 4 shows IgG antibody concentrations on day 16 of culture.

Confirmation of effect of component having protein kinase inhibitory activity in fed-batch culture (Examples 8 and 9)
A CHO cell line (ATCC CRL-12445) transfected with an IgG gene and secreting and producing an IgG antibody was used, and cells adapted and suspended in BalanCD Growth A Medium (manufactured by Irvine Scientific) were used in the experiment.
30 ml of the cell solution prepared by adjusting the concentration of the suspended cells to 1.0×10 ⁶ cells/ml with BalanCD Growth A Medium was added to a 125 ml _Erlenmeyer flask. cultured for 16 days with shaking at .
At the start of culture, a component having protein kinase inhibitory activity (staurosporine: manufactured by Sigma-Aldrich) was added to the medium at concentrations of 2 nM (Example 8) and 3 nM (Example 9). On the other hand, as a control (no staurosporine added), a medium in which DMSO was added at the start of culture was used.
On the 4th, 5th, 7th, 9th, 11th and 14th days of culture, BalanCD Feed I Medium was added in an amount of 5% (v/v) of the culture solution. In addition, 2 nM or 3 nM of staurosporine was previously added to the BalanCD Feed I Medium so that violacein in the culture medium would not be diluted by the addition of the BalanCD Feed I Medium.
The culture medium was sampled on days 0, 2, 4, 5, 7, 9, 11, 14 and 16 of culture, and the number of viable cells, cell viability and IgG antibody concentration in the culture medium were measured. FIG. 5 shows changes in cell viability during culture, and FIG. 6 shows IgG antibody concentrations on day 16 of culture.

From the results of FIGS. 3 to 6, it was found that the addition of components having protein kinase inhibitory activity (violacein, staurosporine) to the medium suppresses the decrease in cell viability in the later stage of culture, and at the same time inhibits the production of proteins, etc. in the cells. It has been found that the total production of useful substances can be improved.

The accelerator, medium and production method provided by the present invention can improve the specific production rate of useful substances in cells, and can be used in the fields of clinical diagnostic drug production using antibodies, antibody drug production, regenerative medicine, cell therapy, etc. can be applied in

Claims (8)

A useful substance specific production rate accelerator in cells, comprising a component having serine/threonine protein kinase inhibitory activity,
The added amount of the component having serine/threonine protein kinase inhibitory activity relative to the medium volume is 0.5 μM to 1.5 μM violacein or 0.1 nM to 10 nM staurosporine. A cell viability reduction inhibitor comprising a component having serine/threonine protein kinase inhibitory activity,
The added amount of the component having serine/threonine protein kinase inhibitory activity relative to the medium volume is 0.5 μM to 1.5 μM violacein or 0.1 nM to 10 nM staurosporine. 3. The agent according to claim 1, wherein the useful substance is an IgG antibody. The agent according to any one of claims 1 to 3, wherein the cells are transformed cells. The agent according to any one of claims 1 to 4, wherein the cells are CHO cells. A method for producing a useful substance, comprising a step of culturing cells using the agent according to any one of claims 1 to 5. 7. The method according to claim 6, wherein the useful substance is an IgG antibody and/or the cell is a transformed cell. 8. The method of claim 6 or 7, wherein the cells are CHO cells.

Images