KR20150134489A - Composition of cell culture media containing alpha-ketoglutarate for increasing the recombinant protein production and sialylation - Google Patents

Abstract

The present invention relates to a culture medium composition for cell culture comprising alpha ketoglutarate and an increase in recombinant protein production and sialylation enhancement, and more particularly to a culture medium composition comprising alpha-ketoglutarate of the present invention, The culture medium containing no glutamine enhances the rate of non-production of cells and the survival rate of cells, thereby increasing the maximum concentration of the target protein and maintaining the sialylation of the target protein until the second half of the culture, The recombinant protein is significantly increased as compared with the case of using the basic medium. Therefore, the medium of the present invention can be usefully used for industrial use in the related industrial field for producing a therapeutic recombinant protein using animal cells.

Description

[0001] The present invention relates to a composition for cell culture comprising alpha-ketoglutarate and an increased amount of recombinant protein,

The present invention relates to a cell culture medium composition capable of maintaining a high concentration of recombinant protein production and sialydation. More specifically, the present invention relates to a cell culture culture medium containing a recombinant protein-expressing cell line transformed with alpha-ketoglutarate -ketoglutarate is added to a culture medium containing a glutamine-free medium to increase the productivity of a target protein and maintain sialylation.

Mass production of monoclonal antibodies as therapeutic agents for the diagnosis, prophylaxis and treatment of cancer, infectious diseases or autoimmune diseases was developed by Dr. Milstein and Kohler of the United Kingdom in 1975 in the development of mouse hybridoma technology . However, when a monoclonal antibody of a mouse produced using the hybridoma technique is repeatedly administered to the human body, side effects that cause the immune response of the human body have occurred, and the effect has been found to be reduced. Therefore, techniques for humanizing mouse monoclonal antibodies using genetic engineering technology and protein engineering technology have been developed, and humanized antibodies have been developed as pharmaceuticals for the prevention, diagnosis and treatment of various diseases competitively in various advanced countries. In recent years, humanized monoclonal antibodies as well as various recombinant protein drugs have been developed and have shown great clinical effects. In order to clinically test therapeutic recombinant proteins containing such humanized recombinant antibodies and further commercialize them, economic mass production is essential.

Host cells used for producing recombinant protein or recombinant virus drugs include Escherichia coli, yeast, and animal cells. Among these host cells, the proportion of recombinant drugs produced using animal cells as a host is increasing day by day. As such animal cells, Chinese hamster ovary (CHO), baby hamster kidney (BHK) and NS0 myeloma cells are widely used. In order to industrially produce recombinant pharmaceuticals derived from animal cells, animal cells must be cultured. Recently, culture methods using serum-free medium as a method of culturing animal cells have become widely used.

The CHO cell line (CHO dhfr (-)) deficient in dihydrofolate reductase (DHFR), the CHO K1 cell line (subclone from the parental CHO cell, BHK (baby hamster kidney fibroblasts) cell line, and NS0 (murine myeloma) cell line. Among these cell lines, the CHO dhfr (-) cell line is the most widely used host cell line for industrial mass production of therapeutic recombinant proteins. The reason why the CHO dhfr (-) cell line is the most preferred in industry is as follows.

First, posttranslational modification, that is, glycosylation process and phosphorylation process of the protein is similar to human cell, minimizing the immune reaction of the human body, Can be produced. Secondly, floating culture is possible, so it is suitable for high concentration culture and mass production process. Third, it is possible to produce high therapeutic recombinant proteins by using dihydrofolate reductase (DHFR / methotrexate, MTX) gene amplification system. Fourth, since the safety has been verified through long-term studies, it is easy to obtain permission from the regulatory body such as FDA.

The CHO dhfr (-) cell line is transformed by transfection with a vector containing a therapeutic recombinant protein gene to produce a recombinant CHO cell line that produces a therapeutic recombinant protein.

Ammonia significantly affects the glycosylation process, which is a major factor in determining the quality of recombinant proteins. In particular, ammonia accumulated in the medium reduces the stability of the target protein by reducing the sialic acid attached to the end of the target protein glycosylation structure, affects the in vivo half-life, and causes heterogeneity (Gawlitzek et al., 2000; Yang and Butler, 2000). Most of the ammonia accumulated in the medium is known to be produced by the metabolism of glutamine (Schneider et al., 1996). Thus, studies have been routinely conducted to replace glutamine with other medium compositions to prevent the accumulation of ammonia in the medium. Glutamate (Hong et al., 2010), pyruvate (Genzel et al., 2005) and dipeptide (Gillmeister et al., 2009) have been used in place of glutamine, (N) as well as enter the citrate cycle (TCA cycle) through metabolism and perform various roles.

Alpha-ketoglutarate is a currently active cyanide antidote, which binds to cyanide during the decryption of natural cyanide to form alpha-ketoglutarate cyanhydrin, which is then decrypted. N-acetylcysteine is known to restore mitochondrial energy metabolism (Satpute RM, Hariharakrishnan J, Bhattacharya R. Alpha-ketoglutarate and N-acetylcysteine protect PC12 cells from cyanide-induced cytotoxicity and altered energy metabolism. Neurotoxicology 2008; 29: 170-178.), There is no known medium composition using the alpha-ketoglutarate.

Based on the fact that glutamine enters the citric acid circuit, the present inventors have tried to find a substitute for glutamine among the other components contained in the citric acid circuit. As a result, the inventors have found that alpha-ketoglutarate Confirmed that the supplemented glutamine-free medium could enhance production of the target protein and could sustain sialylation, the glutamine-free medium supplemented with the alpha ketoglutarate increased the productivity of the target protein , And sialydation. The present invention has been accomplished on the basis of these findings.

An object of the present invention is to provide a culture medium composition for cell culture which contains alpha-ketoglutarate which can increase the productivity of a target protein and maintain sialydation, but does not contain glutamine And its use.

In order to achieve the above object, the present invention provides a medium composition for cell culture containing alpha-ketoglutarate in a basic medium.

In addition,

1) culturing a transformant containing alpha-ketoglutarate in the basic medium and producing the desired recombinant protein in a medium containing no glutamine, and obtaining a culture; And

2) separating the recombinant protein from the culture medium of step 1).

The present invention relates to a culture medium composition for cell culture which can maintain a high concentration of recombinant protein production and sialydation. The present invention relates to a cell culture medium composition capable of maintaining a high concentration of recombinant protein and sialydation, wherein the cell line transfected with the gene expressing the recombinant protein is alpha-ketoglutarate, As a result of culturing using a medium containing no glutamine, it is possible to increase the non-production rate of the cells and the cell survival rate, thereby increasing the maximum concentration of the target protein, and maintaining the sialylation of the target protein until the latter half of the culture The recombinant protein is remarkably increased as compared with the case of using the conventional basic culture medium. Therefore, the culture medium can be usefully used for industrial use in related industrial fields for producing therapeutic recombinant proteins using animal cells .

FIG. 1 is a graph showing cell growth, cell viability, therapeutic protein concentration, and ammonia concentration when a cell line A (cell line A) producing a therapeutic recombinant protein was batch cultured in a 125 mL flask:
Cell line A control +: Control group containing 4 mM glutamine (Gln) in the basal medium;
Cell line A control -: Control group without glutamine in basal medium; And
Cell line A: Experimental group containing glutamine-free and alpha-ketoglutarate at 4 mM.
FIG. 2 is a graph showing cell growth, cell viability, therapeutic protein concentration, and ammonia concentration when batch-wise cultured cell line B (Cell line B) for the treatment recombinant protein is in a 125 mL flask:
Cell line B control +: Control group containing 4 mM glutamine (Gln) in the basal medium;
Cell line B control -: Control group without glutamine in basal medium; And
Cell line B: Experimental group containing glutamine-free and alpha ketoglutarate at 4 mM.
FIG. 3 is a graph showing cell growth, cell survival rate, therapeutic protein concentration, and ammonia concentration when the cell line A producing the therapeutic recombinant protein (cell line A) was batch-cultured in a 3 L bioreactor:
Cell line A control +: Control group containing 4 mM glutamine (Gln) in the basal medium;
Cell line A control -: Control group without glutamine in basal medium; And
Cell line A: Experimental group containing glutamine-free and alpha-ketoglutarate at 4 mM.
4 is a graph showing cell growth, cell survival rate, therapeutic recombinant protein concentration, and ammonia concentration when the cell line B producing the therapeutic recombinant protein was batch cultured in a 3L bioreactor:
Cell line B control +: Control group containing 4 mM glutamine (Gln) in the basal medium;
Cell line B control -: Control group without glutamine in basal medium; And
Cell line B: Experimental group containing glutamine-free and alpha ketoglutarate at 4 mM.
5 is a graph showing the amount of sialic acid measured after purifying a sample collected in a bioreactor:
(A): Cell line A (Cell line A); And
(B): Cell line B (Cell line B).
Cell line A control +: Control group containing 4 mM glutamine (Gln) in the basal medium;
Cell line A control -: Control group without glutamine in basal medium; And
Cell line A: Experimental group containing glutamine-free and alpha-ketoglutarate at 4 mM.
Cell line B control +: Control group containing 4 mM glutamine (Gln) in the basal medium;
Cell line B control -: Control group without glutamine in basal medium; And
Cell line B: Experimental group containing glutamine-free and alpha ketoglutarate at 4 mM.
Figure 6 is a graph showing the activity of alpha 2,3-sialyltransferase (alpha (2,3) -ST) and sialidase activity measured in a sample collected from a bioreactor:
(A) and (B): a graph obtained by analyzing samples collected in cell line A; And
(C) and (D) are graphs obtained by analyzing samples collected from the cell line B, respectively.
Cell line A control +: Control group containing 4 mM glutamine (Gln) in the basal medium;
Cell line A control -: Control group without glutamine in basal medium; And
Cell line A: Experimental group containing glutamine-free and alpha-ketoglutarate at 4 mM.
Cell line B control +: Control group containing 4 mM glutamine (Gln) in the basal medium;
Cell line B control -: Control group without glutamine in basal medium; And
Cell line B: Experimental group containing glutamine-free and alpha ketoglutarate at 4 mM.

Hereinafter, the terminology of the present invention will be described in detail.

The term " batch culture "of the present invention is a method of continuing cultivation until all the raw material substrates are consumed, and the concentration of the substrate, the concentration of the metabolite product, the concentration of the cells, Culture method.

The term " fed-batch culture "of the present invention is a culture method in which a medium is fed intermittently, wherein the substrate concentration in a culture medium can be arbitrarily controlled, the substrate is added at a proper rate, There is an advantage that the amount can be freely controlled. The fed-batch culture is mainly used when the substrate inhibits cell growth at a relatively low concentration, when production of the desired product is inhibited when the concentration of the specific substrate is increased, or when addition of a specific required substance is required in culturing the auxotroph Means a culture method that can be used when necessary.

Hereinafter, the present invention will be described in detail.

The present invention provides a culture medium for cell culture containing alpha-ketoglutarate in a basic medium.

The above-mentioned basic medium means a known medium used from an animal cell culture medium or a modified medium thereof and is preferably selected from commercially available serum-free medium, endless white medium and chemically defined medium, It is more preferable that the basic medium does not contain glutamine.

The serum-free medium is SFM4CHO medium, Iscove's medium, Ultra-CHO medium (BioWhittaker), or EX-Cell (JHR Bioscience) medium used for culturing host cells widely used for producing recombinant pharmaceuticals (IGF-I), long epidermal growth factor (EGF), sodium selenite, putrescine, and insulin-like growth factor I (Human transfor ming growth factor? 1, hTGF-? 1), ferric chloride, and Pluronic F68. But the present invention is not limited thereto.

The above-mentioned media is preferably a ProCH05 medium, but is not limited thereto, in a serum-free medium containing no animal-derived serum, in particular, a medium in which no protein having a molecular weight of 10 kDa or more is added at all.

Wherein the chemically defined medium is a serum-free medium free from animal-derived components, all components having a known chemical structure, no component of the unknown composition, no protein or hydrolyzate, and the chemically defined medium is PF-CHO (JHR-Bioscience ), A chemically defined medium such as ProCHO 4 CDM (Bio Whittaker) or SMIF 7 (Gibco / BRL-Life Technologies), but is not limited thereto.

When the added concentration of the alpha ketoglutarate is 1 mM or less, it exhibits low cell growth inhibition and low target protein productivity. When the addition concentration is 10 mM or more, the productivity of the target protein is remarkably low. Therefore, It is preferably added at a concentration of 1 to 10 mM, more preferably at a concentration of 1 to 8 mM, and most preferably at a concentration of 4 mM.

The medium composition of the present invention containing the alpha-ketoglutarate and not containing glutamine maintains sialic acid and improves the productivity of the recombinant protein.

For the purposes of the present invention, the recombinant protein refers to a protein expressed in a mammalian host cell genetically engineered to express the protein. The recombinant protein may be the same or similar to the normally expressed protein in the mammalian host cell. In addition, the recombinant protein may be heterologous to the host cell, i. E., Heterologous to the protein normally expressed in the host cell. Or recombinant proteins may be chimeric in that some contain the amino acid sequence that is identical or similar to that normally expressed in the host cell, while the other part is exogenous to the host cell. For the purpose of the present invention, the therapeutic recombinant protein is preferably, but not limited to, thrombopoietin (TPO), erythropoietin (EPO), blood coagulant (tPA), monoclonal antibody or Fc- But is not limited thereto.

The medium composition increases the rate of production of an animal cell line (productivity per unit cell, q _p ) and cell viability.

Preferably, the animal cell line is a cell capable of transfection or transfection of a gene expressing a recombinant protein, and CHO, CHO K1, VERO, BHK, HeLa, Cv1, MDCK, 293, 3T3, myeloma, PC12 and WI38 More preferably a CHO cell, more preferably a CHO (CHO dhfr (-)) cell in which a DHFR gene is deleted.

In a specific example of the present invention, we examined the effect of replacing glutamine with alpha ketoglutarate on the accumulation concentration of ammonia in culture medium and the production of recombinant protein for treatment, in which recombinant CHO cells were treated with glutamine-containing After culturing the cells in a flask scale (125 mL) under the conditions of no glutamine, no glutamine, and containing alpha ketoglutarate (4 mM), the growth and survival rate of CHO cells, Production, Ammonia concentration in culture medium was found to be higher than that of glutamine by alpha-ketoglutarate. The final concentration of target protein was also increased as the cell survival time increased. In addition, it was confirmed that the amount of ammonia accumulated in the medium decreased to 1/5 level of the glutamine containing medium (see FIG. 1 and FIG. 2).

We also conducted the same experiment using a 3L animal cell reactor to see if the effect of alpha ketoglutarate shown in the flask culture was applicable to large scale cultures. As a result, the increase in the amount of the target protein was almost similar in the large-scale culture, and the decrease in the ammonia accumulation concentration in the medium was also the same. Therefore, it was confirmed that it can be applied to a large-scale culture and industrially applicable to a culture process for producing a therapeutic recombinant protein (see FIGS. 3 and 4).

In addition, the present inventors observed how sialylation, one of the glycosylation patterns of the target protein, was changed when carried out using the animal cell reactor. Since sialic oxidation is greatly reduced by the ammonia accumulated in the medium during cell culture, it was confirmed that sialylation is maintained longer as the accumulation of ammonia decreases in the medium in which glutamine is replaced with alpha ketoglutarate (Fig. 5 Reference).

The present inventors also found that alpha-2,3-sialyltransferase (alpha (2,3) -ST), an enzyme involved in sialylation, as a target protein produced in a medium in which glutamine is replaced with alpha ketoglutarate, And sialidase activity were measured. In a sample obtained in a glutamine-free medium containing alpha ketoglutarate, a sample of alpha-2,3-sialyltransferase (alpha (2,3) -ST, an enzyme that links sialic acid) It was confirmed that the activity was maintained high and the sialidase (enzyme for degrading sialic acid) was kept low.

Accordingly, the medium composition containing the alpha-ketoglutarate of the present invention and not containing glutamine increases the rate of non-production of the cells and the survival rate of the cells, thereby increasing the maximum concentration of the target protein, The sialic oxidation of the target protein can be maintained until the second half of the culture, so that the recombinant protein is significantly increased as compared with the case of using the conventional basic medium. Therefore, the culture medium of the present invention can be used for producing a therapeutic recombinant protein And can be usefully used for industrial use in related industrial fields.

In addition,

1) culturing a transformant containing alpha-ketoglutarate in the basic medium and producing the desired recombinant protein in a medium containing no glutamine, and obtaining a culture; And

2) separating the recombinant protein from the culture medium of step 1).

The recombinant protein of step 1) above refers to a protein expressed in a mammalian host cell genetically engineered to express the protein. The recombinant protein may be the same or similar to the normally expressed protein in the mammalian host cell. In addition, the recombinant protein may be heterologous to the host cell, i. E., Heterologous to the protein normally expressed in the host cell. Or recombinant proteins may be chimeric in that some contain the amino acid sequence that is identical or similar to that normally expressed in the host cell, while the other part is exogenous to the host cell. For the purpose of the present invention, the therapeutic recombinant protein is preferably, but not limited to, thrombopoietin (TPO), erythropoietin (EPO), blood coagulant (tPA), monoclonal antibody or Fc- But is not limited thereto.

The medium refers to a known medium used from an animal cell culture medium or a modified medium thereof, and is preferably selected from commercially available serum-free medium, endless white medium and chemically defined medium.

It is preferable that the cell line transformed to express the therapeutic recombinant protein is cultured in a batch culture environment, but it can be used in all the processes for producing therapeutic recombinant proteins such as fed-batch culture and continuous culture.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Examples and Experimental Examples.

< Example 1 > Recombinant Producing Therapeutic Recombinant Proteins CHO  125 of cells mL  Flotation culture in flasks

The present inventors have found that Fc-fusion protein associated with sepsis (4 mM), glutamine-free, glutamine-free, and glutamine-containing glutamate in a basic medium using serum-free medium and 125 mL flask (Corning, Corning, NY) The suspension culture was carried out under the conditions of no-containing and alpha-ketoglutarate-containing medium (4 mM).

Actual culture volume was 50 mL, and 300 nM MTX was added to serum-free medium SFM4CHO (Hyclone, Logan, Utah) for batch culture. The speed of the agitator (Adolf Kuhner AG, Birsfelden, Switzerland) was 110 rpm and the initial cell concentration was 2.0 × 10 ⁵ cells / mL. For further analysis, 1 mL of medium was collected every day.

< Example  2> Recombinant Producing Therapeutic Recombinant Protein CHO  Suspension Culture of Cells in a 3L Animal Cell Reactor

The present inventors performed two recombinant CHO cells producing the same therapeutic recombinant protein as in Example 1 above in a flask using a serum-free medium and a 3 L animal cell reactor (New Brunswick Scientific, Edison, NJ) And culturing was carried out under the same medium conditions as those described above.

The culture was carried out in order to investigate whether the phenomenon of increased productivity of recombinant protein for treatment, productivity of recombinant protein for final treatment and reduction of ammonia production in medium per unit cell in flask culture was applied in large scale culture.

Specifically, the actual culture volume was 2 L, and 300 nM MTX was added to serum-free medium SFM4CHO (Hyclone, Logan, Utah) to perform batch culture. The pH was adjusted to 7.2, the dissolved oxygen amount to 50% and the impeller rotation speed to 50 rpm. The initial cell concentration was 2.0 × 10 ⁵ cells / mL. The medium was recovered in a volume of 50 mL per day for various subsequent analyzes.

< Experimental Example  1> Alpha Ketoglutarate  The Effect of Glutamine Replacement on the Production of Therapeutic Recombinant Proteins and Ammonia Production

The present inventors confirmed how the growth of cells, the cell survival rate, the productivity of therapeutic recombinant proteins, and the production of ammonia changed when glutamine in the medium was replaced with alpha ketoglutarate.

Specifically, the culture was performed under the medium conditions of 4 mM glutamine, 0 mM glutamine, 0 mM glutamine + 4 mM alfa ketoglutarate in the basic serum-free medium, and the experiment was carried out in a 125 mL flask. The concentration of the therapeutic recombinant protein was measured by enzyme-linked immunosorbent assay (ELISA) with the medium collected 1 mL each day, and the concentration of ammonia was measured using YSU7100 (Yellow Springs Instruments, Yellow Springs, OH).

In the enzyme-linked immunosorbent assay, 96-well plates (Nunc) were coated with anti-human IgG (Sigma), and 1% bovine serum (Sigma) was added to PBS containing 0.1% Tween- albumin (Sigma)). After incubation, the reaction mixture was incubated with peroxidase-conjugated goat anti-human IgG (Sigma) conjugated goat anti-human IgG (3,3 ', 5,5'-tetramethyl The color was developed with a solution of tetramethylbenzidine (TMB) (Sigma), and the reaction was stopped using 2.5M H ₂ SO ₄ and the absorbance was measured in a UV ELISA reader.

As a result, as shown in Fig. 1 and Fig. 2, in all of the two cell lines producing the therapeutic recombinant protein, the cell survival rate was maintained at a high level in the culture medium in which glutamine was replaced with alpha ketoglutarate, And the productivity was greatly increased. The concentration of ammonia in the culture medium decreased to 1/5 level (see FIGS. 1 and 2).

< Experimental Example  2> Increased production of recombinant protein and reduction of ammonia production in large-scale culture

In order to confirm that the increase in the productivity of the therapeutic protein and the decrease in the ammonia production in the flask culture of Example 1 were applied to the large-scale culture, as described in Example 2, a 3 L animal cell reactor In the same manner as in Example 1, the production of recombinant protein was increased and the production of ammonia was decreased.

As a result, as shown in FIG. 3 and FIG. 4, even in a large-scale culture with an animal cell reactor (bioreactor), both the increase in production of recombinant protein for treatment and the decrease in ammonia production were similar in both cell lines (cell line A and cell line B) Respectively. Therefore, it was confirmed that the present invention can be applied to cultivation for producing industrial therapeutic recombinant proteins (FIGS. 3 and 4).

< Experimental Example  3> Therapeutic recombinant proteins included Sial oxidation  Confirm

After the sample collected in the bioreactor of Example 2 was purified, the degree of sial oxidation was measured.

Specifically, recombinant CHO cells were cultured in a bioreactor in the same manner as in Example 2, and then analyzed by protein A affinity chromatography according to the protocol of the manufacturer. Fc protein (Fc -fusion protein). The degree of sialylation of the purified protein was then confirmed using an EnzyChrom ^™ sialic acid assay kit (BioAssay Systems, Hayward, Calif.).

As a result, as shown in Fig. 5, it was confirmed that the amount of sialic acid was kept higher until the second half of the culture under the condition that glutamine was replaced with alpha ketoglutarate in both cell line A and cell line B.

These results were confirmed to be related to the activity of enzymes involved in sialylation as the concentration of ammonia in the medium decreased (FIG. 5).

< Experimental Example  4> Measurement of activity of sialic acid-related effect

Activity of the enzyme related to the silicic acid was measured in the cell culture samples separated on the 3rd, 6th and 8th days in the bioreactor of Example 2 above.

Specifically, in order to measure the activity of alpha 2,3-sialyltransferase (alpha (2,3) -ST), an enzyme that binds sialic acid, the separated cells were washed with ice-cold PBS (phosphate-buffered saline) (2,3) -ST activity was measured using a sialyltransferase activity kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions (Hwang and Lee, 2008).

Sialidase activity was measured using Amplex red neuraminidase assay kit (Molecular Probes / Invitrogen, Cergy Pontoise, France) according to the manufacturer's instructions to measure sialidase activity, an enzyme that degrades sialic acid.

As a result, as shown in FIG. 6, in the cell line A and the cell line B, the enzyme alpha 2,3-sialyltransferase (alpha (2,3) -glucosyltransferase ST) up to the second half of culture, and sialidase, an enzyme that degrades sialic acid, was kept low until the second half of culture.

Claims (11)

A culture medium for cell culture comprising alpha-ketoglutarate in a basic medium.
The culture medium for cell culture according to claim 1, wherein the basic medium is serum-free medium, endless white medium or chemically defined medium.
The culture medium for cell culture according to claim 1, wherein the basic medium does not contain glutamine.
The culture medium for cell culture according to claim 1, wherein the alpha ketoglutarate is added at a concentration of 1 to 10 mM.
The culture medium for cell culture according to claim 1, wherein the culture medium composition maintains sialic acid and improves the productivity of the recombinant protein.
6. The method of claim 5, wherein the recombinant protein is selected from the group consisting of thrombopoietin (TPO), erythropoietin (EPO), tissue plasminogen activator (tPA) A monoclonal antibody and an Fc-containing fusion protein. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;
The culture medium for cell culture according to claim 1, wherein the culture medium composition increases the rate of production of an animal cell line (productivity per unit cell, q _p ) and cell viability.
The animal cell line according to claim 7, wherein the animal cell line is any one selected from the group consisting of CHO, CHO K1, VERO, BHK, HeLa, Cv1, MDCK, 293, 3T3, myeloma, PC12 and WI38 cells Wherein the cell culture medium composition is a cell culture medium.
The culture medium for cell culture according to claim 8, wherein the CHO or CHO K1 cell line is CHO (CHO dhfr (-)) cells in which the DHFR gene is deleted.
1) culturing a transformant containing alpha-ketoglutarate in the basic medium and producing the desired recombinant protein in a medium containing no glutamine, and obtaining a culture; And
2) separating the recombinant protein from the culture of step 1).
[Claim 11] The method according to claim 10, wherein the medium of step 1) contains alpha ketoglutarate in a concentration of 1 to 10 mM.

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