Animal cell, method for producing animal cell, and method for producing target protein

Abstract

The object of the present invention is to provide an animal cell capable of producing a target protein with high productivity, a method for producing the animal cell, and a method for producing a target protein using the animal cell. According to the present invention, there are provided: an animal cell having a gene encoding a target protein and an exogenous gene encoding SNAT2, i.e., an exogenous gene linked to a promoter, wherein the SNAT2 is overexpressed; a method for producing the animal cell; and a method for producing a target protein using the animal cell.

Description

Technical Field

[0001] The present invention relates to an animal cell expressing a target protein, a method for producing the above-mentioned animal cell, and a method for producing a target protein using the above-mentioned animal cell. Background Art

[0002] In the production of biopharmaceuticals such as antibodies, in order to improve the productivity of antibodies, fed-batch culture is generally used, in which the state of cells is improved by adding nutrients to the culture medium. In addition, in the production of biopharmaceuticals such as antibodies, in order to improve the productivity of antibodies, perfusion culture is generally used, in which the culture medium is continuously filtered and discharged, and fresh culture medium containing nutrients is continuously supplied to the culture tank.

[0003] Patent Document 1 discloses a method for producing a polypeptide as a method capable of producing a protein at a high yield, the method comprising culturing cells into which a DNA encoding a desired polypeptide that strongly expresses glutamate-pyruvate aminotransferase is introduced to produce the desired polypeptide.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: International Publication No. WO2009 / 020144 Summary of the invention

[0007] Technical issues to be solved by the invention

[0008] In fed-batch culture, cells can be cultured for a long period of time at a higher cell density than when no nutrients are added. However, due to the accumulation of waste products secreted from the cells, the cell survival rate decreases in the second half of the culture, so there is a problem that the product cannot be increased by extending the culture period.

[0009] Furthermore, in order to culture cells at a high density in the perfusion culture method, it is necessary to recover and supply a medium 1 to 3 times the culture volume every day in order to supply nutrients and discharge waste products to the outside of the system, which increases the culture cost.

[0010] The problem to be solved by the present invention is to provide an animal cell capable of producing a target protein with high productivity. Another problem to be solved by the present invention is to provide a method for producing the above-mentioned animal cell and a method for producing a target protein using the above-mentioned animal cell.

[0011] Means for solving technical problems

[0012] The inventors of the present invention have conducted intensive research to solve the above problems and found that forced expression of the SNAT2 (Sodium-dependent neutral amino acid transporter-2) gene (gene name: SLC38A2) in CHO cells affects protein production, resulting in an increase in antibody productivity (Qp) and an increase in antibody production. The present invention was completed based on the above findings.

[0013] That is, according to the present invention, the following inventions are provided.

[0014] <1> An animal cell comprising a gene encoding a target protein and an exogenous gene encoding SNAT2, that is, an exogenous gene linked to a promoter, wherein the SNAT2 is overexpressed.

[0015] <2> The animal cell according to <1>, wherein

[0016] Overexpression is constitutive.

[0017] <3> The animal cell according to <1> or <2>, wherein

[0018] The exogenous gene encoding SNAT2 has a base sequence having a sequence homology of 90% or more to the base sequence described in SEQ ID NO:1.

[0019] <4> The animal cell according to any one of <1> to <3>, wherein

[0020] The exogenous gene encoding SNAT2 includes the base sequence described in SEQ ID NO:1.

[0021] <5> The animal cell according to any one of <1> to <4>, wherein

[0022] The animal cells are CHO cells.

[0023] <6> The method for producing an animal cell according to any one of <1> to <5>, comprising the step of introducing a gene encoding a target protein and an exogenous gene encoding SNAT2, that is, an exogenous gene linked to a promoter, into the animal cell.

[0024] <7> The method according to <6>, wherein

[0025] The step of introducing a foreign gene encoding SNAT2, that is, a foreign gene linked to a promoter, is performed by electroporation.

[0026] <8> A method for producing a target protein, comprising: culturing the animal cell according to any one of <1> to <5>.

[0027] <9> The method according to <8>, wherein

[0028] The culture was fed-batch culture.

[0029] <10> The method according to <9>, wherein the seeding cell density of the cell culture is 0.2×10 6 cells / mL and above 5×10 6 cells / mL or less.

[0030] <11> The method according to <10>, wherein the viable cell rate during the culture period is 60% or more during the entire period.

[0031] <12> The method according to <8>, wherein

[0032] The culture was perfusion culture.

[0033] <13> The method according to <12>, wherein the seeding cell density of the cell culture is 0.2×10 6 cells / mL and above 1×10 7 cells / mL or less.

[0034] <14> The method according to <13>, wherein the viable cell rate during the culture period is 90% or more during the entire period.

[0035] Effects of the Invention

[0036] According to the animal cell of the present invention, a target protein can be produced with high productivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The graph shows the results of measuring the antibody concentration in the culture supernatant of cells after gene introduction.

[0038] Figure 2 These are the results of measuring the antibody productivity (Qp) per cell in the culture supernatant of cells after gene introduction.

[0039] Figure 3 The results of measuring the cell size after gene introduction are shown.

[0040] Figure 4 The results of integrating the viable cell density during the culture period are shown. DETAILED DESCRIPTION

[0041] Hereinafter, the mode for carrying out the present invention will be described in detail.

[0042] In the present specification, a numerical range expressed using "to" means a range including the numerical values ​​described before and after "to" as the minimum value and the maximum value, respectively.

[0043] [Animal cells]

[0044] The animal cell of the present invention is an animal cell that has a gene encoding a target protein and an exogenous gene encoding SNAT2, that is, an exogenous gene linked to a promoter, and overexpresses the above-mentioned SNAT2.

[0045] In mouse liver, SNAT2 gene expression activates the mTORC1 / S6K pathway (Nature Communications 6, Article number: 7940 (2015)), thus showing the correlation between SNAT2 gene expression and mTOR gene expression. It has been reported that in rat cells, cell proliferation is inhibited and cell size increases by activating mTOR (Genes Dev. 2002 Jun 15; 16 (12): 1472-1487). Moreover, the larger the cell size, the greater the production of protein products (Cytotechnology 34: 59-70, 2000). However, the relationship between SNAT2 gene expression and the production of recombinant proteins is still unclear.

[0046] In Patent Document 1, the decrease in survival rate is slowed down by the introduction of glutamate-pyruvate aminotransferase, but the effect on antibody quality is worrying because the period below 60% is long. In addition, due to the introduction of taurine transfer protein, the antibody productivity (Qp) per cell is reduced, so there is a problem that the yield cannot be increased even if the cell density increases. In contrast, in the present invention, the antibody productivity (Qp) per cell is increased by enhancing the expression of the SNAT2 gene related to protein biosynthesis.

[0047] <Target protein>

[0048] In the present invention, the type of target protein is not particularly limited, for example, it is a recombinant polypeptide chain, a recombinant secretory polypeptide chain, an antigen binding protein, a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, a bispecific antibody, an Fc fusion protein, a fragmented immunoglobulin, a single chain antibody (scFv). The target protein is preferably a human antibody, a humanized antibody, a chimeric antibody or a mouse antibody. As fragmented immunoglobulins, Fab, F(ab')2, Fv, etc. can be cited. The type of antibody is also not particularly limited, and can be any of IgG, IgA, IgD, IgE, IgM, etc., such as IgG1, IgG2, IgG3, IgG4, but when used as medicine, IgG and IgM are preferred.

[0049] Human antibodies include all antibodies having one or more variable regions and constant regions derived from human immunoglobulin sequences. In one embodiment, all variable domains and constant domains are derived from human immunoglobulin sequences (fully human antibodies).

[0050] Humanized antibodies have sequences different from sequences of antibodies derived from non-human species by one or more amino acid substitutions, deletions, and / or additions, so that when administered to human subjects, compared with non-human species antibodies, the possibility of humanized antibodies inducing immune responses is reduced and / or the induction of severe immune responses is reduced. In one embodiment, a specific amino acid in the framework domain and constant domain of the heavy chain and / or light chain of a non-human species antibody is mutated to produce a humanized antibody. In another embodiment, the constant domains from human antibodies are fused to the variable domains of non-human species antibodies.

[0051] Chimeric antibodies refer to antibodies in which variable regions and constant regions of different sources are connected. For example, an antibody composed of the variable regions of the heavy and light chains of a mouse antibody and the constant regions of the heavy and light chains of a human antibody is a mouse / human heterologous chimeric antibody. By connecting the DNA encoding the variable region of a mouse antibody to the DNA encoding the constant region of a human antibody and incorporating it into an expression vector, a product vector expressing a chimeric antibody can be prepared. By culturing the product cells transformed with the above-mentioned vector and expressing the incorporated DNA, the chimeric antibody produced in the culture can be obtained.

[0052] Bispecific antibodies are antibodies that recognize two different antigenic specificities and are produced by chemical methods or cell fusion. As methods for producing bispecific antibodies, the following have been reported: a method of producing by binding two globulin molecules using a cross-linking agent such as N-succinimidyl 3-(2-pyridyldithiol) propionate or S-acetylmercaptosuccinic anhydride, a method of producing by binding Fab fragments of globulin molecules to each other, etc.

[0053] Fc fusion protein refers to a protein having an Fc region, including an antibody.

[0054] Fab has V L 、V H , C L and C H 1 domain monovalent fragment.

[0055] F(ab')2 is a bivalent fragment having two Fab fragments joined by a disulfide cross-link at the hinge region.

[0056] The Fv fragment has a single-arm V L and V H Structural domain.

[0057] Single chain antibody (scFv) is VL Area and V H The regions are joined via a linker (eg, a synthetic sequence of amino acid residues) to form an antibody continuous protein chain, where the linker is of sufficient length to allow the protein chain to fold back on itself to form a monovalent antigen binding site.

[0058] The gene encoding the target protein can be obtained by methods known to those skilled in the art. When the target protein is an antibody, a DNA encoding the L chain and a DNA encoding the H chain of the antibody can be used.

[0059] The DNA encoding the L chain of the antibody and the DNA encoding the H chain can be prepared as follows. mRNA is extracted from a hybridoma, cell, bacteriophage, ribosome, etc. having a gene expressing the antibody. cDNA is prepared from the mRNA by a reverse transcription reaction using a reverse transcriptase. The L chain gene or the H chain gene is amplified by PCR using a primer having a base sequence complementary to the L chain gene or the H chain gene and cDNA, and each gene is obtained by combining with a plasmid for cloning.

[0060] The DNA encoding the fragment of the L chain of the antibody and the DNA encoding the fragment of the H chain can be prepared as follows. mRNA is extracted from a hybridoma, cell, bacteriophage, ribosome, etc. having a gene expressing the antibody. cDNA is prepared from the mRNA by a reverse transcription reaction using a reverse transcriptase. The L chain gene fragment or the H chain gene fragment is amplified by PCR using a primer having a base sequence complementary to the L chain gene fragment or the H chain gene fragment and cDNA, and each gene fragment is obtained by combining with a plasmid for cloning.

[0061] <SNAT2 and SNAT2 gene>

[0062] In the present invention, the source of SNAT2 is not particularly limited, and an exogenous gene encoding SNAT2 derived from a mammal such as a human, monkey, mouse, rat, or hamster can be used. In the present invention, an exogenous gene refers to a gene introduced into an animal cell from the outside. Furthermore, when an endogenous gene is amplified and a gene is introduced, it is also considered an exogenous gene.

[0063] The base sequence and amino acid sequence of human SNAT2 are shown in SEQ ID NOs: 1 and 2 in the sequence listing.

[0064] Human SNAT2 gene base sequence (SEQ ID NO: 1)

[0065] ATGAAGAAGGCCGAAATGGGACGATTCAGTATTTCCCCGGATGAAGACAGCAGCAGCTACAGTTCCAACA

[0066] GCGACTTCAACTACTCCTACCCCACCAAGCAAGCTGCTCTGAAAAGCCATTATGCAGATGTAGATCCTGA

[0067] AAACCAGAACTTTTTACTTGAATCGAATTTGGGGAAGAAGAAGTATGAAACAGAATTTCATCCAGGTACT

[0068] ACTTCCTTTGGAATGTCAGTATTTAATCTGAGCAATGCGATTGTGGGCAGTGGAATCCTTGGGCTTTCTT

[0069] ATGCCATGGCTAATACTGGAATTGCTCTTTTTATAATTCTCTTGACATTTGTGTCAATATTTTCCCTGTA

[0070] TTCTGTTCATCTCCTTTTGAAGACTGCCAATGAAGGAGGGTCTTTATTATATGAACAATTGGGATATAAG

[0071] GCATTTGGATTAGTTGGAAAGCTTGCAGCATCTGGATCAATTACAATGCAGAACATTGGAGCTATGTCAA

[0072] GCTACCTCTCATAGTGAAATATGAGTTGCCTTTGGTGATCCAGGCATTAACGAACATTGAAGATAAAAC

[0073] TGGATTGTGGTATCTGAACGGGAACTATTTGGTTCTGTTGGTGTCATTGGGTGGTCATTCTTCCTTTGTCG

[0074] CTGTTTAGAAATTTAGGATATTTGGGATATACCAGGTGGCCTTTCCTTGTTGTGTATGGTGTTCTTTCTGA

[0075] TTGTGGTCATTTGCAAGAAAATTTCAGGTTCCGTGTCCTGGGAAGCTGCTTTGATAATTAACGAAACAAT

[0076] AAACACCACCTTAACACAGCCAACAGCTCTTGTACCTGCTTTGTCACATAACGTGACTGAAAATGACTCT

[0077] TGCAGACCTCACTATTTTATTTTCAACTCACAGACTGTCTATGCTGTGCCAATTCTGATCTTTTCATTTG

[0078] TCTGTCATCCTGCTGTTCTTCCCATCTATGAAGAACTGAAAGACCGCAGCCGTAGAAGAATGATGAATGT

[0079] GTCCAAGATTTCATTTTTTGCTATGTTTCTCATGTATCTGCTTGCCGCCCTCTTTGGATACCTAACATTT

[0080] TACGAACATGTTGAGTCAGAATTGCTTCATACCTACTCTTCTATCTTGGGAACTGATATTCTTCTTCTCA

[0081] TTGTCCGTCTGGCTGTGTTAATGGCTGTGACCCTGACAGTACCAGTAGTTATTTTCCCAATCCGGAGTTC

[0082] TGTAACTCACTTGTTGTGTGCATCAAAAGATTTCAGTTGGTGGCGTCATAGTCTCATTACAGTGTCTATC

[0083] TTGGCATTTACCAATTTACTTGTCATCTTTGTCCCAACTATTAGGGATATCTTTGGTTTTATTGGTGCAT

[0084] CTGCAGCTTCTATGTTGATTTTTATTCTTCCTTCTGCCTTCTATATCAAGTTGGTGAAGAAAGAACCTAT

[0085] GAAATCTGTACAAAAGATTGGGGCTTTGTTCTTCCTGTTAAGTGGGTGTACTGGTGATGACCGGAAGCATG

[0086] GCCTTGATTGTTTTGGATTGGGTACACAATGCACCTGGAGGTGGCCAT

[0087] Amino acid sequence of human SNAT2 gene (SEQ ID NO: 2)

[0088] MKKAEMGRFSISPDEDSSSYSSNSDFNYSYPTKQAALKSHYADVDPENQNFLLESNLGKKKYETEFHPGT

[0089] TSFGMSVFNLSNAIVGSGILGLSYAMANTGIALFIILLTFVSIFSLYSVHLLLKTANEGGSLLYEQLGYK

[0090] AFGLVGKLAASGSITMQNIGAMSSYLFIVKYELPLVIQALTNIEDKTGLWYLNGNYLVLLVSLVVILPLS

[0091] LFRNLGYLGYTSGLSLLCMVFFLIVVICKKFQVPPCPVEAALIINETINTTLTQPTALVPALSHNVTENDS

[0092] CRPHYFIFNSQTVYAVPILIFSFVCHPAVLPIYEELKDRSRRRMMNVSKISFFAMFLMYLLAALFGYLTF

[0093] YEHVESELLHTYSSILGTDILLLIVRLAVLMAVTLTVPVVIFPIRSSVTHLLCASKDFSWWRHSLITVSI

[0094] LAFTNLLVIFVPTIRDIFGFIGASAASMLIFILPSAFYIKLVKKEPMKSVQKIGALFFLLSGVLVMTGSM

[0095] ALIVLDWVHNAPGGGH

[0096] As the exogenous gene encoding SNAT2, the following can be used:

[0097] (1) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2;

[0098] (2) a gene encoding a protein having the function of SNAT2 and consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence described in SEQ ID NO: 2; or

[0099] (3) A gene encoding a protein having SNAT2 function and consisting of an amino acid sequence having a sequence homology of 85% or more (more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more) to the amino acid sequence described in SEQ ID NO: 2.

[0100] The function of SNAT2 is to activate the mTORC1 / S6K pathway. It is known that in rat cells, cell proliferation is inhibited and cell size increases by activating mTOR.

[0101] As exogenous genes encoding SNAT2, further use can be made of:

[0102] (4) A gene consisting of the base sequence described in SEQ ID NO: 1;

[0103] (5) a gene encoding a protein consisting of a base sequence in which one or more bases are deleted, substituted or added to the base sequence described in SEQ ID NO: 1 and having SNAT2 function;

[0104] (6) a gene encoding a protein consisting of a base sequence that hybridizes under stringent conditions to a complementary sequence to the base sequence described in SEQ ID NO:1 and having SNAT2 function; or

[0105] (7) A gene encoding a protein having a base sequence having 90% or more (more preferably 95% or more, and particularly preferably 98% or more) sequence homology to the base sequence described in SEQ ID NO: 1 and having SNAT2 function.

[0106] The “one or more” in “an amino acid sequence in which one or more amino acids are deleted, substituted or added” preferably means 1 to 20, more preferably 1 to 10, further preferably 1 to 5, and particularly preferably 1 to 3.

[0107] The sequence homology in the present invention refers to the value calculated by the following formula.

[0108] % sequence homology = [(number of identical residues) / (length of the longer alignment)] x 100

[0109] The sequence homology between two amino acid sequences can be determined by any method known to those skilled in the art, and can be determined using the BLAST (Basic Local Alignment Search Tool) program (J. Mol. Biol. 215: 403-410, 1990), etc. The "longer alignment length" of the denominator means that when two alignments are compared, the denominator uses the longer alignment length.

[0110] The “one or more” in “a base sequence in which one or more bases are deleted, substituted or added” preferably means 1 to 20, more preferably 1 to 10, further preferably 1 to 5, and particularly preferably 1 to 3.

[0111] The "under stringent conditions" in "hybridize under stringent conditions" means hybridization under moderate or high stringent conditions, which can be recognized by those skilled in the art. Examples of moderately stringent conditions include the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Vol. 1, 7.42-7.45 Cold Spring Harbor Laboratory Press, 2001. Examples of moderately stringent conditions include hybridization conditions of a pre-wash solution of 5×SSC, 0.5% SDS, 1.0 mmol / L EDTA (pH 8.0) on a nitrocellulose filter, about 50% formamide at about 40 to 50° C., 2×SSC to 6×SSC (or other similar hybridization solutions such as Stark's solution in about 50% formamide at about 42° C.), and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS. Highly stringent conditions can also be easily determined by those skilled in the art, for example, hybridization and / or washing at a higher temperature and / or lower salt concentration than the above-mentioned moderately stringent conditions. For example, the above-mentioned hybridization conditions and washing at 68°C, 0.2×SSC, 0.1% SDS can be cited. Here, the composition of 1×SSC is 150mmol / L NaCl, 15mmol / L sodium citrate, pH 7.4. SDS is sodium dodecyl sulfate, and EDTA is ethylenediaminetetraacetic acid.

[0112] The amino acid sequence and base sequence information of SNAT2 other than human are shown below. The following numbers represent the Gene No. of NCBI (National Center for Biotechnology Information).

[0113] ID: 29642 Rat (Rattus norvegicus)

[0114] ID: 67760 Mouse (Mus musculus)

[0115] ID: 566537 Zebrafish (Danio rerio)

[0116] ID: 338044 Cattle (Bos taurus)

[0117] ID: 417807 Chicken (Gallus gallus)

[0118] ID: 702253 Macaca mulatta

[0119] ID: 101083348 Cat (Felis catus)

[0120] ID: 100525644 Pig (Sus scrofa)

[0121] ID: 101149564 Gorilla gorilla

[0122] For SNAT2 other than human, the same method as human SNAT2 can be used:

[0123] (2A) a gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in a prescribed amino acid sequence and having the function of SNAT2;

[0124] (3A) a gene encoding a protein consisting of an amino acid sequence having a sequence homology of 85% or more (more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more) to a predetermined amino acid sequence and having SNAT2 function;

[0125] (5A) a gene encoding a protein consisting of a base sequence in which one or more bases are deleted, substituted or added to a predetermined base sequence and having SNAT2 function;

[0126] (6A) a gene encoding a protein consisting of a base sequence that hybridizes under stringent conditions to a complementary sequence of a predetermined base sequence and having SNAT2 function; or

[0127] (7A) A gene encoding a protein having a base sequence having a sequence identity of 90% or more (more preferably 95% or more, and particularly preferably 98% or more) to a predetermined base sequence and having SNAT2 function.

[0128] The foreign gene encoding SNAT2 is connected to the promoter.

[0129] As a promoter, there is no particular limitation as long as it can function in the animal cells of the host to express SNAT2. As a promoter, preferably, CMV promoter (cytomegalovirus promoter), EF1α promoter (promoter of human polypeptide chain elongation factor gene), SV40 promoter (simian vacuolating virus 40 promoter), β-actin promoter, MMLV-LTR promoter (long terminal repeat promoter of Moloney murine leukemia virus) or mouse β-globin promoter, more preferably CMV promoter.

[0130] In the animal cell of the present invention, SNAT2 is overexpressed. Overexpression refers to the expression of a gene exceeding the normal expression level in the host. Animal cells in which SNAT2 is overexpressed can be obtained by introducing an exogenous gene encoding SNAT2 into a host and expressing the exogenous gene in the host.

[0131] The expression level of SNAT2 in the animal cell of the present invention is preferably 3 times or more, more preferably 3.5 times or more, further preferably 4 times or more, further preferably 4.5 times or more, further preferably 5 times or more, and particularly preferably 5.5 times or more relative to the animal cell without the exogenous gene encoding SNAT2. There is no upper limit, and it can be 30,000 times or less, or 10,000 times or less.

[0132] The expression level of SNAT2 can be investigated by RT-PCR (reverse transcription-polymerase chain reaction) or the like. The expression level of SNAT2 is preferably implemented by reverse transcription of mRNA and real-time PCR. The expression level of isoSNAT2 is preferably a relative expression level calculated by standardization. Standardization can be performed, for example, by using the expression level of housekeeping genes such as β-actin or HPRT1 as an endogenous control for comparative quantification.

[0133] <Animal cells>

[0134] The cell in the present invention is not particularly limited as long as it is an animal cell. As animal cells, Chinese hamster ovary (CHO) cells, BHK cells, 293 cells, myeloma cells (NS0 cells, etc.), PerC6 cells, SP2 / 0 cells, hybridoma cells, COS cells, 3T3 cells, HeLa cells, Vero cells, MDCK cells, PC12 cells, WI38 cells, etc. can be enumerated. Among the above, CHO cells, BHK cells, 293 cells, myeloma cells (NS0 cells, etc.), PerC6 cells, SP2 / 0 cells, hybridoma cells are particularly preferred, and CHO cells are further preferred. CHO cell product proteins are, for example, widely used in the production of cytokines, coagulation factors and antibodies. It is preferred to use CHO cells lacking dihydrofolate reductase (DHFR), as DHFR-deficient CHO cells, for example, CHO-DG44 can be used.

[0135] [Method for producing animal cells]

[0136] According to the present invention, there is provided a method for producing an animal cell of the present invention, comprising the step of introducing a gene encoding a target protein and an exogenous gene encoding SNAT2, that is, an exogenous gene linked to a promoter, into an animal cell.

[0137] The gene encoding the target protein and the foreign gene encoding SNAT2 are preferably introduced into the host, that is, animal cells, in the form of being incorporated into vectors, respectively.

[0138] As a vector that can be used to introduce a gene into a host, for example, an expression vector derived from a mammal can be used, for example, pCMV6-Entry (manufactured by OriGene), pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17), p5322), pEF, pCDM8 (manufactured by Funakoshi), INPEP4 (manufactured by Biogen-IDEC), etc., but are not particularly limited.

[0139] Furthermore, it is known that mRNA having poly A is stable in cells. The gene encoding the target protein and the exogenous gene encoding SNAT2 may have a poly A signal required for adding poly A to the gene, such as the mouse β-globin poly A signal, the bovine growth hormone poly A signal, the SV40 poly A signal, and the like.

[0140] The method of introducing the gene encoding the target protein and the exogenous gene encoding SNAT2 into the animal cell is not particularly limited and can be carried out by methods known to those skilled in the art. For example, it can be carried out by electroporation, lipofection, calcium phosphate method, DEAE dextran method, a method using cationic liposome DOTAP (manufactured by Roche Life Science) or a method using a viral vector. Among the above, electroporation is preferred.

[0141] When a gene is introduced into an animal cell, the gene is introduced into only a portion of the cells into which the gene is introduced, depending on the type of expression vector used and the gene introduction method. In order to identify and select cells into which the gene has been introduced, for example, a gene encoding a selective marker for antibiotic resistance can be introduced into the host cell together with the target gene. Preferred selective markers include those that confer resistance to drugs, such as G418, hygromycin, and methotrexate.

[0142] In the cells of the present invention, the gene encoding the target protein may be expressed in a transient expression system or a constitutive expression system, but is preferably expressed in a constitutive expression system.

[0143] In the cells of the present invention, the exogenous gene encoding SNAT2 may be expressed in a transient expression system or a constitutive expression system, but is preferably expressed in a constitutive expression system.

[0144] The transient expression system refers to a method of taking a circular plasmid into cells and expressing it by calcium phosphate method, electroporation method, lipofection method, etc. The gene encoding the target protein and the foreign gene encoding SNAT2 are generally present outside the chromosome.

[0145] The constitutive expression system refers to a method in which a circular plasmid or a linear plasmid prepared by restriction enzyme treatment or the like is introduced into a cell by calcium phosphate method, electroporation method, lipofection method, and a part is inserted into the genome of the cell to express the target protein. The expression of the gene encoding the target protein and the exogenous gene encoding SNAT2 can be maintained for a long time. In addition, if the drug resistance gene is introduced into the plasmid, drug selection becomes possible, and cells in which the gene encoding the target protein and the exogenous gene encoding SNAT2 are maintained on the chromosome can be effectively selected.

[0146] [Method for producing target protein]

[0147] According to the present invention, there is provided a method for producing a target protein, comprising the following step: culturing the animal cell of the present invention.

[0148] The target protein can be produced by culturing the animal cells of the present invention. The culturing can be performed according to known methods.

[0149] As the culture medium used in the culture of the animal cell of the present invention, the culture medium used in the culture of the common animal cell can be used. For example, OptiCHO (Lifetechnologies, 12681011) culture medium, Dulbecco's modified Eagle medium (DMEM), Eagle's minimum essential medium (MEM), RPMI-1640 culture medium, RPMI-1641 culture medium, F-12K culture medium, Ham F12 culture medium, Iscob modified Dulbecco's medium (IMDM), McCoy5A culture medium, Leibovitz L-15 culture medium and EX-CELL (trademark) 300 series (JRH Biosciences), CHO-S-SFMII (Invitrogen), CHO-SF (Sigma-Aldrich), CD-CHO (Invitrogen), IS CHO-V (Irvine Scientific), PF-ACF-CHO (Sigma-Aldrich), etc. can be used.

[0150] Serum such as fetal calf serum (FCS) may be added to the culture medium, but the culture is more preferably carried out in a serum-free medium, and most preferably in a complete synthetic medium.

[0151] The culture medium can be supplemented with additional ingredients such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, and hydrolyzates of plant proteins.

[0152] The pH of the medium varies depending on the cells to be cultured, but is generally pH 6.0 to 8.0, preferably pH 6.8 to 7.6, and more preferably pH 7.0 to 7.4.

[0153] The culture temperature is generally 30°C to 40°C, preferably 32°C to 37°C, more preferably 36°C to 37°C, and the culture temperature may be changed during the culture process.

[0154] The culture is preferably carried out in an atmosphere with a CO2 concentration of 0 to 40%, preferably 2 to 10%.

[0155] The culture period is not particularly limited, but is generally 12 hours to 90 days, preferably 24 hours to 60 days, and more preferably 24 hours to 30 days.

[0156] During the culture, the medium may be replaced, aerated, or stirred as necessary.

[0157] The culture of the animal cells of the present invention can be carried out in a culture device (also referred to as a bioreactor) or in a suitable container other than this. As the culture device, a fermenter tank culture device, an airlift culture device, a culture bottle culture device, a stirring bottle culture device, a microcarrier culture device, a fluidized bed culture device, a hollow fiber culture device, a roller bottle culture device, a filling tank culture device, etc. can be used for culture.

[0158] The culture scale is generally 1 L to 20,000 L, preferably 1 L to 10,000 L, more preferably 200 L to 2,000 L, and even more preferably 500 L to 2,000 L.

[0159] The culture may be carried out by any method such as batch culture, fed-batch culture (also referred to as fed-batch culture), or perfusion culture, but preferably, fed-batch culture or perfusion culture is used.

[0160] Batch culture refers to a discontinuous process in which cells are grown in a fixed volume of culture medium for a short period of time and then completely recovered. Cultures grown using the batch method undergo an increase in cell density until a maximum cell density is reached, and then the medium components are consumed, and the density of viable cells declines as the levels of metabolic byproducts (lactate and ammonia, etc.) accumulate. Recovery is typically performed after achieving a maximum cell density (typically 5-10×10 6 cells / mL). Batch processing is the simplest culture method, but the density of viable cells is limited by the ability to utilize nutrients. Once the cells reach the maximum density, the culture decays and the production of the target protein decreases. The accumulation of waste products and nutrient depletion quickly lead to culture decay (typically, about 3 to 7 days), so the production period of the target protein cannot be extended.

[0161] Fed-batch culture is a culture method that improves the batch process by supplying doses (bolus) or continuously supplying culture medium to replenish the consumed culture medium components. That is, in fed-batch culture, the culture form is suspension culture, and additional components are provided in the culture at one or more time points after the start of the culture process. As additional components, nutritional auxiliary components for cells that are consumed in the culture process can be cited, and other auxiliary components (for example, compounds that inhibit the cell cycle) can also be included.

[0162] In fed-batch culture, since additional nutrients are added during the entire culture period, it is possible to achieve higher cell density and high production of target protein compared to batch culture. In fed-batch culture, unlike batch culture, a two-phase culture can be created and continued by manipulating the supply schedule and medium components to distinguish between a cell growth period (growth phase) for achieving a desired cell density and a suspended or slow cell growth period (production phase). Thus, compared to batch culture, fed-batch culture has the potential to achieve higher production of target protein.

[0163] In fed-batch culture, the inoculation cell density of cell culture is generally 0.2×10 6 cells / mL and above 1×10 7 cells / mL or less, preferably 0.2×10 6 cells / mL and above 5×10 6 cells / mL or less, more preferably 0.5×10 6 cells / mL and above 2.5×10 6 cells / mL or less, more preferably 0.5×10 6 cells / mL and above 1.5×10 6 cells / mL or less.

[0164] In the fed-batch culture, the viable cell rate during the culture period is preferably 60% to 100% during the entire period, more preferably 70% to 100%, and even more preferably 75% to 100%.

[0165] Perfusion culture is a culture method that adds fresh culture medium and removes the used culture medium at the same time. It is possible to further improve batch culture and fed-batch culture. Perfusion culture can be performed using, for example, an ATF (Alternating Tangential Flow Filtration) pump or a TFF (Tangential Flow Filtration) pump. According to perfusion culture, more than 1×10 8 cells / mL. A typical perfusion culture is initiated after 1 or 2 days of batch culture, and fresh feed medium is then added to the culture continuously, periodically, and / or intermittently, while spent medium is removed. In perfusion culture, spent medium can be removed while maintaining cell density using methods such as precipitation, centrifugation, or filtration. Perfusion culture can be performed using, for example, an ATF pump or a TFF pump.

[0166] The advantage of perfusion culture is that the culture producing the target protein can be maintained for a longer period of time compared to batch culture or fed-batch culture. However, in order to maintain long-term perfusion culture, especially perfusion culture at a high cell density, it is necessary to prepare, use, store and discard the culture medium. In perfusion culture, a large amount of nutrients is required, and the production cost of the target protein tends to increase compared to batch culture and fed-batch culture. In addition, by selecting the membrane pore size, since the antibody can be recovered outside the system while continuing to culture, the residence time of the antibody in the culture solution is shortened and chemical changes are reduced, thereby maintaining the quality of the antibody at a high level.

[0167] A culture combining fed-batch culture and perfusion culture can also be performed. As an example, fed-batch culture with dosing can be used to maintain the culture of cells in the growth phase, and then perfusion culture can be used to produce the target protein.

[0168] Perfusion can be any form of continuous, staged, intermittent or a combination of these. Animal cells are maintained in culture, and the used culture medium that is removed does not substantially contain cells or may have far fewer cells than the culture. By selecting the membrane pore size, the target protein expressed by cell culture can be maintained or recovered in the culture. In order to prevent the cell density in the culture from becoming too large, the cell density can also be reduced by extracting a portion of the culture solution together with the cells and adding the same amount of fresh culture medium (cell exudation).

[0169] In perfusion culture, the inoculation cell density of cell culture is generally 0.2×10 6 cells / mL and above 3×10 7 cells / mL or less, preferably 0.2×10 6 cells / mL and above 1×10 7 cells / mL or less, more preferably 0.5×10 6 cells / mL and above 1×10 7 cells / mL or less.

[0170] In perfusion culture, the viable cell rate during the culture period is preferably 80% or more, more preferably 85% or more, and even more preferably 90% or more throughout the entire period.

[0171] In perfusion culture, the highest cell density achieved is preferably 2 × 10 8 cells / mL or less, more preferably 1.5×10 8 cells / mL or less, more preferably 1.0×10 8 cells / mL or less.

[0172] The perfusion ratio in the perfusion culture is preferably 0.3 vvd or more and 5.0 vvd or less, and more preferably 0.3 vvd or more and 1.5 vvd or less. Here, vvd means less than or equal to.

[0173] vvd = (volume of fresh medium / working volume of reactor / day, daily supply culture medium volume / culture medium volume)

[0174] The seeding cell density of cell culture and the maximum cell density reached during culture can be determined by measuring the number of cells by a conventional method and dividing the number of cells by the volume of the culture medium.

[0175] The viable cell rate (survival rate) during the culture period is determined by dividing the number of viable cells by (the number of viable cells + the number of dead cells). For example, the number of cells can be measured using Vi-CELL XR (Beckman Coulter).

[0176] The target protein produced by the above-mentioned culture can be purified. The separation and purification of the target protein can be carried out using the separation and purification methods used in common proteins. For example, the target protein can be separated and purified by appropriately selecting and combining chromatographic columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and other electric focusing electrophoresis, but is not limited to these. The concentration determination of the target protein obtained in the above can be carried out by absorbance measurement or enzyme-linked immunosorbent assay (ELISA) and the like.

[0177] Examples of columns used in affinity chromatography include protein A columns and protein G columns. Examples of chromatography other than affinity chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. These chromatography can be performed using liquid chromatography such as HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography).

[0178] In addition, by allowing an appropriate polypeptide modifying enzyme to act on the target protein before or after purification, the target protein can also be modified or the peptide can be locally removed. As the polypeptide modifying enzyme, for example, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, glucosidase, etc. are used.

[0179] [Utilization of target protein]

[0180] When the target protein produced by the method of the present invention has a biological activity useful as a drug, the drug can be produced by mixing the target protein with a pharmaceutically acceptable carrier or additive to prepare a formulation.

[0181] Examples of pharmaceutically acceptable carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymers, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerol, glycerol, propylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, pharmaceutically acceptable surfactants, and the like.

[0182] For example, when used as an injectable preparation, a preparation can be used in which the purified target protein is dissolved in a solvent such as physiological saline, buffer, glucose solution, etc., and an anti-adsorption agent such as Tween 80, Tween 20, gelatin, human serum albumin, etc. is added thereto. Alternatively, the target protein can also be freeze-dried in order to prepare a dosage form that is dissolved and reconstituted before use, and as an excipient for freeze-drying, for example, sugar alcohols such as mannitol and glucose or sugars can be used.

[0183] The method of administering the target protein may be oral administration or parenteral administration, but parenteral administration is preferred. For example, injection (e.g., systemic or local administration based on intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.), nasal administration, pulmonary administration, or transdermal administration may be mentioned.

[0184] The dosage of the target protein can be appropriately selected according to the type of target protein, the type of disease to be treated or prevented, the age of the patient, the severity of the disease, etc. Generally, it is in the range of 0.001 mg to 1000 mg per kg of body weight per time, but is not particularly limited.

[0185] The present invention is further specifically described by the following examples, but the present invention is not limited to the examples.

[0186] Example

[0187] <Example 1> Preparation of animal cells

[0188] A vector encoding the human SNAT2 gene was purchased (OriGene, Cat# RC201892), and the expression cassette (promoter, open reading frame, terminator) of the SNAT2 gene was amplified by polymerase chain reaction (PCR) using primer 1 (sequence number 3) and primer 2 (sequence number 4).

[0189] The amplified SNAT2 expression cassette was inserted into a vector for co-expressing the L chain and H chain of each antibody of antibody 1 (Denosumab, https: / / www.drugbank.ca / drugs / DB06643), antibody 2 (Blosozumab, https: / / www.drugbank.ca / drugs / DB12560) or antibody 3 (anti-MUC1 antibody, JP-T 2016-517691). The construction of the L chain and H chain co-expression vector and its introduction into cells were performed according to Example 2 of JP-T 2016-517691.

[0190] All CHO-DG44 cells were cultured in an incubator at 37°C and 5% CO2 before the introduction of the antibody and SNAT2 genes. The above vectors were introduced into 5×10 host cells using electroporation (Lonza, 4D-Nucleofector). 6 cells, and suspended in 20mL of OptiCHO (Lifetechnologies, 12681011) medium containing HT Supplement (100X) (Lifetechnologies, 11067-030) diluted 100 times, and then inoculated into a T75 flask. One day later, the medium was replaced with OptiCHO (Lifetechnologies, 12681011) medium containing 175nmol / L methotrexate (MTX) (Wako Pure Chemical Corporation) without HT supplement. T75 flasks were observed under a microscope 3 weeks after gene introduction, and the culture volume was expanded to 20mL in sequence from the flask where cell proliferation occurred, inoculated into a 125mL shake flask (Corning), and cultured with shaking at 140rpm.

[0191] By the above method, antibody 1-SNAT2 cells, antibody 2-SNAT2 cells, and antibody 3-SNAT2 cells were established that forcibly expressed the human SNAT2 gene and antibody 1, antibody 2, or antibody 3. In addition, antibody 1 cells, antibody 2 cells, and antibody 3 cells were constructed as a control group by gene introduction using vectors expressing antibody L chains and H chains in the same manner.

[0192] Primer 1: CCGCGGTCATAGCTGTTTCCTGAAC (SEQ ID NO: 3)

[0193] Primer 2: CAGCTATGACCGCGGTTAATGGCCACCTCCAGGTGCATTGTGT (SEQ ID NO: 4)

[0194] <Example 2> Cultivation experiment

[0195] Fed-batch culture experiments were performed using 8 levels each of Antibody 1-SNAT2 cells, Antibody 2-SNAT2 cells, Antibody 3-SNAT2 cells, and Antibody 1 cells, Antibody 2 cells, and Antibody 3 cells.

[0196] The cells were plated at 5×10 5 The cells were suspended in 15 mL of OptiCHO (Lifetechnologies, 12681011) medium at a cell density of 1.3 cells / mL and automatically cultured using an AMBR15 culture apparatus (Sartorius Stedim Japan KK). From the second day to the thirteenth day after the start of culture, a feed medium (Cellboost7a, 7b, GE healthcare) was added every day at a volume ratio of 2% to the initial culture volume. In order to measure the cell density, culture fluid components and antibody concentration over time, sampling was carried out every 3 to 4 days. The culture fluid was recovered on the 14th day after the start of culture, and cells and cell debris were removed using a 0.22 μm depth filter (Merck Millipore). The antibody concentration in the supernatant and the antibody productivity (Qp) per cell were measured by liquid chromatography. As a result, the antibody concentration increased by 121% in Antibody 1, 25% in Antibody 2, and 56% in Antibody 3 ( Figure 1 And Table 1). In addition, Qp increased by 127% in antibody 1, 34% in antibody 2, and 74% in antibody 3 ( Figure 2 and Table 2). It is believed that the expression of SNAT2 activated protein biosynthesis, thereby improving antibody productivity. On the other hand, the results of measuring cell diameters by Vi-CELL (Beckman Coulter, Inc.) surprisingly showed that the sizes of each cell were antibody 1-SNAT2 cells (average 15.8 μm), antibody 2-SNAT2 cells (average 16.3 μm), and antibody 3-SNAT2 cells (average 16.5 μm), and compared with the control group antibody 1 cells (average 16.3 μm), antibody 2 cells (average 15.8 μm), and antibody 3 cells (average 16.2 μm), the cell diameters did not necessarily increase, and showed approximately the same degree ( Figure 3 And Table 3). In addition, the integral viable cell density (IVCD) during the culture period, which showed the proliferation of cells, was the same as or slightly higher than that of the control group ( Figure 4 and Table 4). Figure 1 to Figure 4 In the figure, the left one shows only the antibody, and the right one shows the antibody-SNAT2. This proves that even if SNAT2 is expressed, it does not promote the differentiation of CHO cells, but maintains the cell proliferation capacity required for the establishment of antibody-producing cells and the production of antibodies.

[0197] In addition, the integral viable cell density (IVCD) was measured by the following method. On days 0, 3, 7, 10, and 14, the integral viable cell density (VCD) was measured by Vi-CELL XR (Beckman Coulter), and each value was set as V0, V3, V7, V10, and V14. The integral viable cell density (IVCD) was calculated by the following formula.

[0198] Integrated viable cell density (IVCD) = {(V0+V3)×(3-0) / 2}+{(V3+V7)×(7-3) / 2}+{(V10+V7)×(10-7) / 2}+{(V14+V10)×(14-10) / 2}

[0199] [Table 1]

[0200]

[0201] [Table 2]

[0202]

[0203] [Table 3]

[0204]

[0205] [Table 4]

[0206]

[0207] The antibody productivity per cell Qp [pg / cell / day] was calculated by the following formula.

[0208] [Mathematical formula 1]

[0209]

[0210] Pti [g / L] = concentration of purified Ti on the day of culture

[0211] Xti[cell / day]=viable cell density on culture day ti

[0212] The approximate value of the integral was estimated by finding the area under the growth curve from time t1 to t2 as the area of ​​the trapezoid.

[0213] [Mathematical formula 2]

[0214] Sequence Listing <110> Fujifilm Corporation <120> Animal cell, method for producing animal cell, and method for producing target protein <130> 18F11345W1 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 1518 <212> DNA <213> Human <220> <221> CDS <222> (1)..(1518) <400> 1 atg aag aag gcc gaa atg gga cga ttc agt att tcc ccg gat gaa gac 48 Met Lys Lys Ala Glu Met Gly Arg Phe Ser Ile Ser Pro Asp Glu Asp 1 5 10 15 agc agc agc tac agt tcc aac agc gac ttc aac tac tcc tac ccc acc 96 Ser Ser Ser Tyr Ser Ser Asn Ser Asp Phe Asn Tyr Ser Tyr Pro Thr 20 25 30 aag caa gct gct ctg aaa agc cat tat gca gat gta gat cct gaa aac 144 Lys Gln Ala Ala Leu Lys Ser His Tyr Ala Asp Val Asp Pro Glu Asn 35 40 45 cag aac ttt tta ctt gaa tcg aat ttg ggg aag aag aag tat gaa aca 192 Gln Asn Phe Leu Leu Glu Ser Asn Leu Gly Lys Lys Lys Tyr Glu Thr 50 55 60 gaa ttt cat cca ggt act act tcc ttt gga atg tca gta ttt aat ctg 240 Glu Phe His Pro Gly Thr Thr Ser Phe Gly Met Ser Val Phe Asn Leu 65 70 75 80 agc aat gcg att gtg ggc agt gga atc ​​ctt ggg ctt tct tat gcc atg 288 Ser Asn Ala Ile Val Gly Ser Gly Ile Leu Gly Leu Ser Tyr Ala Met 85 90 95 gct aat act gga att gct ctt ttt ata att ctc ttg aca ttt gtg tca 336 Ala Asn Thr Gly Ile Ala Leu Phe Ile Ile Leu Leu Thr Phe Val Ser 100 105 110 ata ttt tcc ctg tat tct gtt cat ctc ctt ttg aag act gcc aat gaa 384 Ile Phe Ser Leu Tyr Ser Val His Leu Leu Leu Lys Thr Ala Asn Glu 115 120 125 gga ggg tct tta tta tat gaa caa ttg gga tat aag gca ttt gga tta 432 Gly Gly Ser Leu Leu Tyr Glu Gln Leu Gly Tyr Lys Ala Phe Gly Leu 130 135 140 gtt gga aag ctt gca gca tct gga tca att aca atg cag aac att gga 480 Val Gly Lys Leu Ala Ala Ser Gly Ser Ile Thr Met Gln Asn Ile Gly 145 150 155 160 gct atg tca agc tac ctc ttc ata gtg aaa tat gag ttg cct ttg gtg 528 Ala Met Ser Ser Tyr Leu Phe Ile Val Lys Tyr Glu Leu Pro Leu Val 165 170 175 atc cag gca tta acg aac att gaa gat aaa act gga ttg tgg tat ctg 576 Ile Gln Ala Leu Thr Asn Ile Glu Asp Lys Thr Gly Leu Trp Tyr Leu 180 185 190 aac ggg aac tat ttg gtt ctg ttg gtg tca ttg gtg gtc att ctt cct 624 Asn Gly Asn Tyr Leu Val Leu Leu Val Ser Leu Val Val Ile Leu Pro 195 200 205 ttg tcg ctg ttt aga aat tta gga tat ttg gga tat acc agt ggc ctt 672 Leu Ser Leu Phe Arg Asn Leu Gly Tyr Leu Gly Tyr Thr Ser Gly Leu 210 215 220 tcc ttg ttg tgt atg gtg ttc ttt ctg att gtg gtc att tgc aag aaa 720 Ser Leu Leu Cys Met Val Phe Phe Leu Ile Val Val Ile Cys Lys Lys 225 230 235 240 ttt cag gtt ccg tgt cct gtg gaa gct gct ttg ata att aac gaa aca 768 Phe Gln Val Pro Cys Pro Val Glu Ala Ala Leu Ile Ile Asn Glu Thr 245 250 255 ata aac acc acc tta aca cag cca aca gct ctt gta cct gct ttg tca 816 I Have Thr Thr Thr Gln Pro Thr On The Val Pro On The Thr 260 265 270 cat aac gtg act gaa aat gac tct tgc aga cct cac tat ttt att ttc 864 His Asn Val Thr Glu Asn Asp Ser Cys Arg Pro His Tyr Phe Ile Phe 275 280 285 aac tca cag act gtc tat gct gtg cca att ctg atc ttt tca ttt gtc 912 Asn Ser Gln Thr Val Tyr Ala Val Pro Ile Leu Ile Phe Ser Phe Val 290,295,300 tgt cat cct gct gtt ctt ccc atc tat gaa gaa ctg aaa gac cgc agc 960 Cys His Pro Ala Val Leu Pro Ile Tyr Glu Glu Leu Lys Asp Arg Ser 305 310 315 320 cgt aga aga atg atg aat gtg tcc aag att tca ttt ttt gct atg ttt 1008 Arg Arg Arg Met Met Asn Val Ser Lys Ile Ser Phe Phe Ala Met Phe 325 330 335 ctc atg tat ctg ctt gcc gcc ctc ttt gga tac cta aca ttt tac gaa 1056 Leu Met Tyr Leu Leu Ala Ala Leu Phe Gly Tyr Leu Thr Phe Tyr Glu 340 345 350 cat gtt gag tca gaa ttg ctt cat acc tac tct tct atc ttg gga act 1104 His Val Glu Ser Glu Leu Leu His Thr Tyr Ser Ser Ile Leu Gly Thr 355 360 365 gat att ctt ctt ctc att gtc cgt ctg gct gtg tta atg gct gtg acc 1152 Asp Ile Leu Leu Leu Ile Val Arg Leu Ala Val Leu Met Ala Val Thr 370 375 380 ctg aca gta cca gta gtt att ttc cca atc ​​cgg agt tct gta act cac 1200 Leu Thr Val Pro Val Val Ile Phe Pro Ile Arg Ser Ser Val Thr His 385 390 395 400 ttg ttg tgt gca tca aaa gat ttc agt tgg tgg cgt cat agt ctc att 1248 Leu Leu Cys Ala Ser Lys Asp Phe Ser Trp Trp Arg His Ser Leu Ile 405 410 415 aca gtg tct atc ttg gca ttt acc aat tta ctt gtc atc ttt gtc cca 1296 Thr Val Ser Ile Leu Ala Phe Thr Asn Leu Leu Val Ile Phe Val Pro 420 425 430 act att agg gat atc ttt ggt ttt at ggt gca tct gca gct tct atg 1344 Thr Ile Arg Asp Ile Phe Gly Phe Ile Gly Ala Ser Ala Ala Ser Met 435 440 445 ttg att ttt att ctt cct tct gcc ttc tat atc aag ttg gtg aag aaa 1392 Leu Ile Phe Ile Leu Pro Ser Ala Phe Tyr Ile Lys Leu Val Lys Lys 450 455 460 gaa cct atg aaa tct gta caa aag att ggg gct ttg ttc ttc ctg tta 1440 Glu Pro Met Lys Ser Val Gln Lys Ile Gly Ala Leu Phe Phe Leu Leu 465 470 475 480 agt ggt gta ctg gtg atg acc gga agc atg gcc ttg att gtt ttg gat 1488 Ser Gly Val Leu Val Met Thr Gly Ser Met Ala Leu Ile Val Leu Asp 485 490 495 tgg gta cac aat gca cct gga ggt ggc cat 1518 Trp Val His Asn Ala Pro Gly Gly Gly His 500 505 <210> 2 <211> 506 <212> PRT <213> Human <400> 2 Met Lys Lys Ala Glu Met Gly Arg Phe Ser Ile Ser Pro Asp Glu Asp 1 5 10 15 Ser Ser Ser Tyr Ser Ser Asn Ser Asp Phe Asn Tyr Ser Tyr Pro Thr 20 25 30 Lys Gln Ala Ala Leu Lys Ser His Tyr Ala Asp Val Asp Pro Glu Asn 35 40 45 Gln Asn Phe Leu Leu Glu Ser Asn Leu Gly Lys Lys Lys Tyr Glu Thr 50 55 60 Glu Phe His Pro Gly Thr Thr Ser Phe Gly Met Ser Val Phe Asn Leu 65 70 75 80 Ser Asn Ala Ile Val Gly Ser Gly Ile Leu Gly Leu Ser Tyr Ala Met 85 90 95 Ala Asn Thr Gly Ile Ala Leu Phe Ile Ile Leu Leu Thr Phe Val Ser 100 105 110 Ile Phe Ser Leu Tyr Ser Val His Leu Leu Leu Lys Thr Ala Asn Glu 115 120 125 Gly Gly Ser Leu Leu Tyr Glu Gln Leu Gly Tyr Lys Ala Phe Gly Leu 130 135 140 Val Gly Lys Leu Ala Ala Ser Gly Ser Ile Thr Met Gln Asn Ile Gly 145 150 155 160 Ala Met Ser Ser Tyr Leu Phe Ile Val Lys Tyr Glu Leu Pro Leu Val 165 170 175 Ile Gln Ala Leu Thr Asn Ile Glu Asp Lys Thr Gly Leu Trp Tyr Leu 180 185 190 Asn Gly Asn Tyr Leu Val Leu Leu Val Ser Leu Val Val Ile Leu Pro 195 200 205 Leu Ser Leu Phe Arg Asn Leu Gly Tyr Leu Gly Tyr Thr Ser Gly Leu 210 215 220 Serum Leu Leu Cys Met Val Phe Phe Leu Ile Val Val Ile Cys Lys Lys 225 230 235 240 Phe Gln Val Pro Cys Pro Val Glu Ala Ala Leu Ile Ile Asn Glu Thr 245 250 255 Ile Asn Thr Thr Leu Thr Gln Pro Thr Ala Leu Val Pro Ala Leu Ser 260 265 270 His Asn Val Thr Glu Asn Asp Ser Cys Arg Pro His Tyr Phe Ile Phe 275 280 285 Asn Ser Gln Thr Val Tyr Ala Val Pro Ile Leu Ile Phe Ser Phe Val 290 295 300 Cys His Pro Ala Val Leu Pro Ile Tyr Glu Glu Leu Lys Asp Arg Ser 305 310 315 320 Arg Arg Arg Met Met Asn Val Ser Lys Ile Ser Phe Phe Ala Met Phe 325 330 335 Leu Met Tyr Leu Leu Ala Ala Leu Phe Gly Tyr Leu Thr Phe Tyr Glu 340 345 350 His Val Glu Ser Glu Leu Leu His Thr Tyr Ser Ser Ile Leu Gly Thr 355 360 365 Asp Ile Leu Leu Leu Ile Val Arg Leu Ala Val Leu Met Ala Val Thr 370 375 380 Leu Thr Val Pro Val Val Ile Phe Pro Ile Arg Ser Ser Val Thr His 385 390 395 400 Leu Leu Cys Ala Ser Lys Asp Phe Ser Trp Trp Arg His Ser Leu Ile 405 410 415 Thr Val Ser Ile Leu Ala Phe Thr Asn Leu Leu Val Ile Phe Val Pro 420 425 430 Thr Ile Arg Asp Ile Phe Gly Phe Ile Gly Ala Ser Ala Ala Ser Met 435 440 445 Leu Ile Phe Ile Leu Pro Ser Ala Phe Tyr Ile Lys Leu Val Lys Lys 450 455 460 Glu Pro Met Lys Ser Val Gln Lys Ile Gly Ala Leu Phe Phe Leu Leu 465 470 475 480 Ser Gly Val Leu Val Met Thr Gly Ser Met Ala Leu Ile Val Leu Asp 485 490 495 Trp Val His Asn Ala Pro Gly Gly Gly His 500 505 <210> 3 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: Primers <400> 3 ccgcggtcat agctgtttcc tgaac 25 <210> 4 <211> 43 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: Primers <400> 4 cagctatgac cgcggttaat ggccacctcc aggtgcattg tgt 43

Claims

1. An animal cell having: A gene encoding a target protein and an exogenous gene encoding sodium ion-dependent neutral amino acid transporter 2, i.e., SNAT2, connected to a promoter, wherein the SNAT2 is overexpressed, The animal cells are Chinese hamster ovary (CHO) cells, The target protein is a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, a bispecific antibody, an Fc fusion protein or a single-chain antibody.

2. The animal cell according to claim 1, wherein Overexpression is constitutive.

3. The animal cell according to claim 1 or 2, wherein The exogenous gene encoding SNAT2 has a base sequence having a sequence homology of 90% or more to the base sequence described in SEQ ID NO:

1.

4. The animal cell according to claim 1 or 2, wherein The exogenous gene encoding SNAT2 includes the base sequence described in SEQ ID NO:

1.

5. The method for producing an animal cell according to claim 1 or 2, comprising: A step of introducing a gene encoding a target protein and a foreign gene encoding SNAT2 linked to a promoter into animal cells.

6. The method according to claim 5, wherein: The step of introducing the foreign gene encoding SNAT2 linked to the promoter was performed by electroporation.

7. A method for producing a target protein, comprising: Cultivate the animal cell according to claim 1 or 2.

8. The method according to claim 7, wherein: The culture was fed-batch culture.

9. The method according to claim 8, wherein: The inoculation cell density of the cell culture was 0.2 × 10 6 cells / mL and above 5×10 6 cells / mL or less.

10. The method according to claim 9, wherein: The viable cell rate during the culture period was 60% or more throughout the entire period.

11. The method according to claim 7, wherein: The culture was perfusion culture.

12. The method according to claim 11, wherein: The inoculation cell density of the cell culture was 0.2 × 10 6 cells / mL and above 1×10 7 cells / mL or less.

13. The method according to claim 12, wherein: The viable cell rate during the culture period was 90% or more throughout the entire period.

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