JP2012125197A - Method for producing protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for more efficiently producing proteins.SOLUTION: In the method for producing proteins, a desired protein is produced by: subjecting animal cells transformed with a DNA (RRM1 gene or RRM2 gene) encoding ribonucleotide reductase and a DNA encoding the desired protein to semibatch culture; producing and accumulating the protein in the cultured substance; and collecting the protein from the cultured substance. In the method for producing proteins, the animal cells are CHO cells and the protein is an antibody.

Description

  The present invention relates to an efficient protein production method using animal cells.

  In recent years, many methods for producing a target protein have been adopted in which a gene encoding the target protein is introduced into an animal cell and the cell is cultured. Protein production using animal cells has a slower growth rate of animal cells than that using microbial cells, the reagents used in the medium are expensive, and the productivity of the target protein per medium is low. There is a problem. Therefore, various methods for improving the productivity of the target protein in animal cells have been attempted.

  For example, a method for increasing the expression level of mRNA encoding the target protein using a strong promoter such as the SV40 early promoter, a method for amplifying the gene encoding the target protein using the dhfr gene encoding dihydrofolate reductase as a marker gene (Non-Patent Document 1), overexpressing the active form of XBP1 (X box binding protein), which is a transcription factor related to the mechanism of UPR (Unfolded protein response), and the cytoplasmic domain of ATF6 (Activating transcription factor 6) By improving the secretory function of the cell itself (Patent Documents 1 to 3), and further introducing a Bcl-2 family member gene known to suppress apoptosis into the cell. Rate (time until cells die), methods to improve cell density in culture, etc. are known ( Patent Document 2).

However, these methods may not provide sufficient effects depending on the type of target protein, and a more versatile method for improving productivity is required.
Ribonucleotide reductase is an enzyme involved in DNA synthesis and plays a role in converting ribonucleoside diphosphate to deoxyribonucleoside diphosphate (Non-patent Document 3).

Ribonucleotide reductase is composed of two subunits, a large subunit (RRM1) of 85 kDa and a small subunit (RRM2) of 45 kDa, and RRM1 has spindle pole body activity and microtubule centrosome activity during cell division. It is known that it is an essential protein (Patent Document 4).
However, the production method of the target protein using cells that highly express the gene encoding ribonucleotide reductase, and how the high expression of the gene encoding ribonucleotide reductase affects the productivity of the target protein. Is not known.

International Publication No. 04/111194 Pamphlet International Publication No. 05/094355 Pamphlet US Publication No. 2006-0053502 Japanese Patent Laid-Open No. 2002-355048

Mammalian Cell Biotechnology, Ed. Butler, M. IRL Press, P79 Biotechnol.Bioeng.68: 31-43,2000 Biochem Biophys Res Commun. 340: 428-34, 2006

  A method for efficiently producing a foreign protein is provided.

The present application relates to the following (1) to (6).
(1) Animal cells transformed with a DNA encoding a ribonucleotide reductase and a DNA encoding a desired protein are cultured in a medium, the protein is produced and accumulated in the culture, and the protein is extracted from the culture. A method for producing a protein, comprising collecting
(2) The production method according to (1), wherein the DNA encoding ribonucleotide reductase is DNA encoding the RRM1 gene or RRM2 gene.
(3) The production method according to (1), wherein the DNA encoding ribonucleotide reductase is a DNA encoding one protein selected from the group consisting of the following (a) to (d).

(A) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having RRM1 activity; (b) an amino acid sequence represented by SEQ ID NO: 2; A protein having an amino acid sequence having 80% or more homology and having RRM1 activity (c) consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4; And a protein having RRM2 activity (d) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and having RRM2 activity (4) a DNA encoding ribonucleotide reductase, The production method according to (1), which is one DNA selected from the group consisting of the following (a) to (d).

(A) DNA comprising the base sequence represented by SEQ ID NO: 1
(B) a DNA that hybridizes with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 under a stringent condition and encodes a protein having RRM1 activity
(C) DNA comprising the base sequence represented by SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 3 and encodes a protein having RRM2 activity
(5) The production method according to any one of (1) to (4), wherein the animal cell is a CHO cell.
(6) The production method according to any one of (1) to (5), wherein the protein is an antibody or an antibody fragment.

  The present invention provides an efficient protein production method using animal cells.

FIG. 1 shows a schematic diagram of an RRM2 expression vector Tn_pMugneo_RRM2 (7542 bp). In the figure, CMV P / E represents the CMV promoter / enhancer sequence, Neo ^r represents the neomycin resistance gene, and Amp ^r represents the ampicillin resistance gene. FIG. 2 shows a schematic diagram of the transposase expression vector TPEX / pMug (6149 bp). In the figure, the CMV P / E is CMV promoter A promoter / enhancer sequences, Amp ^r respectively represent an ampicillin resistance gene. FIG. 3 shows a schematic diagram of an anti-human influenza M2 antibody expression vector N5LG1_CHX-lox_M2_Z3 (10555 bp). In the figure, CMV P / E represents a CMV promoter / enhancer sequence, CMV P represents a CMV promoter sequence, Neo ^r represents a neomycin resistance gene, and Amp ^r represents an ampicillin resistance gene. FIG. 4 shows the number of living cells in the culture of RRM2-introduced cells and non-introduced cells (mock cells). ▲ indicates the number of RRM2-introduced cells, and △ indicates the number of viable mock cells. FIG. 5 shows the expression level of the anti-human influenza M2 antibody in culture of RRM2-introduced cells and non-introduced cells (mock cells). ▲ indicates the antibody expression level of RRM2-introduced cells, and △ indicates the mock cell antibody expression level. FIG. 6 shows the amount of polymer in the culture solution on day 13 of culture of RRM2-introduced cells and non-introduced cells (mock cells). □ indicates the amount of polymer in the case of RRM2-introduced cells, and ■ indicates the amount of polymer in the case of mock cells.

1. Animal cells used in the production method of the present invention (1) Ribonucleotide reductase gene The ribonucleotide reductase gene encoded by the DNA that transforms the animal cells used in the production method of the present invention is a ribonucleotide reductase derived from a mammal. It refers to a gene, and either or both of the RRM1 gene and RRM2 gene encoding the two subunits constituting ribonucleotide reductase can be used.

As a DNA encoding such a ribonucleotide reductase gene, a DNA encoding a human-derived ribonucleotide reductase gene can be preferably used. Specifically, a human-derived RRM1 gene or a human-derived RRM2 gene can be used. DNA encoding can be used.
The DNA encoding a human-derived RRM1 gene or RRM2 gene refers to a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4 or a homologous protein thereof.

The homologous protein of the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4 is
(A) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having RRM1 activity;
(B) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having RRM1 activity;
(C) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 and having RRM2 activity;
Or (d) A protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and having RRM2 activity.

In the amino acid sequence represented by SEQ ID NO: 2 or 4, one or more amino acids are deleted, substituted or added. One or more amino acids are deleted, substituted or added at any position in the same sequence. May be.
Examples of amino acid positions at which amino acid can be deleted or added include 1 to several amino acids on the N-terminal side and C-terminal side of the amino acid sequence represented by SEQ ID NO: 2 or 4.

  Deletions, substitutions or additions may occur simultaneously, and the amino acids substituted or added may be natural or non-natural. Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.

Examples of amino acids that can be substituted with each other are shown below. Amino acids included in the same group can be substituted for each other.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: asparagine, glutamine Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid Group E: proline, 3 -Hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine As a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 or 4 Use the sequence analysis program described later. 80% or more homology or identity, preferably 90% or more homology or identity, more preferably 95% or more homology with the amino acid sequence represented by SEQ ID NO: 2 or 4. Or a protein having identity, more preferably 98% homology or identity.

The homology of amino acid sequences and base sequences is based on the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90 , 5873 (1993)] and FASTA [Methods Enzymol., 183 , 63 (1990)] by Karlin and Altschul. Can be determined. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215 , 403 (1990)]. When a base sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. Further, when an amino acid sequence is analyzed by BLASTX based on BLAST, parameters are set to score = 50 and wordlength = 3, for example. To obtain gapped alignment, Gapped BLAST can be used as described in Altschul et al. (1997, Nucleic Acids Res. 25: 3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search that detects the positional relationship (Id.) Between molecules and the relationship between molecules that share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs can be used (http://www.ncbi.nlm.nih.gov.).

Specifically, as DNA encoding the RRM1 gene or RRM2 gene derived from human,
(A) DNA comprising the base sequence represented by SEQ ID NO: 1,
(B) a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1 and encodes a protein having RRM1 activity;
(C) DNA consisting of the base sequence represented by SEQ ID NO: 3,
Or (d) a DNA that hybridizes with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 under a stringent condition and encodes a protein having RRM2 activity
Say.

  Here, “hybridize” means that the DNA hybridizes to DNA having a specific base sequence or a part of the DNA under specific conditions. Accordingly, the DNA having the specific base sequence or a part of the base sequence of the DNA is useful as a probe for Northern or Southern blot analysis, or has a length that can be used as an oligonucleotide primer for PCR analysis. May be. Examples of DNA used as a probe include DNA of at least 100 bases, preferably 200 bases or more, more preferably 500 bases or more, but may be DNA of at least 10 bases, preferably 15 bases or more. .

  Methods for DNA hybridization experiments are well known. For example, Molecular Cloning 2nd edition, 3rd edition (2001), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology methods manual, Academic press ( In addition to those described in Molecular), hybridization conditions can be determined and experiments can be performed according to a number of other standard textbooks.

  The above stringent conditions include, for example, a DNA-immobilized filter and probe DNA, 50% formamide, 5 × SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6). ), Incubated in a solution containing 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / L denatured salmon sperm DNA overnight at 42 ° C., for example, in a 0.2 × SSC solution at about 65 ° C. Conditions for washing the filter can be raised, but lower stringent conditions can also be used. Stringent conditions can be changed by adjusting the concentration of formamide (the lower the formamide concentration, the lower the stringency), and changing the salt concentration and temperature conditions. As low stringent conditions, for example, 6 × SSCE (20 × SSCE is 3 mol / L sodium chloride, 0.2 mol / L sodium dihydrogen phosphate, 0.02 mol / L EDTA, pH 7.4), 0.5% Incubate overnight at 37 ° C in a solution containing SDS, 30% formamide, 100 μg / L denatured salmon sperm DNA, and then wash with 50 ° C 1x SSC, 0.1% SDS solution. I can give you. Further, examples of lower stringent conditions include conditions under which hybridization is performed using a solution having a high salt concentration (for example, 5 × SSC) under the low stringent conditions described above, followed by washing.

The various conditions described above can also be set by adding or changing a blocking reagent used to suppress the background of hybridization experiments. The addition of the blocking reagent described above may be accompanied by a change in hybridization conditions to adapt the conditions.
The DNA that can hybridize under the above-described stringent conditions includes, for example, the base represented by SEQ ID NO: 1 or 3 when calculated based on the above-mentioned parameters using the above-described programs such as BLAST and FASTA. DNA having a base sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more of a sequence is at least 80% or more. it can.

  The fact that the DNA that hybridizes with the above-mentioned DNA under stringent conditions encodes a protein having an activity equivalent to the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or 3 indicates that the DNA and the desired protein Confirm that the cells obtained by transforming animal cells with DNA encoding DNA are fed to a fed-batch culture and that the production of the desired protein is improved compared to the parent strain. Can do.

The DNA encoding the RRM1 or RRM2 gene used in the production method of the present application can be obtained by a general method in which mRNA is prepared from cells derived from mammals such as humans or CHO, and cDNA is synthesized with reverse transcriptase. it can. In addition, a cDNA library derived from a cell or tissue in which the RRM1 gene or RRM2 gene is expressed is screened using a DNA probe synthesized based on the nucleotide sequence information represented by SEQ ID NO: 1 or 3. Can be isolated. Furthermore, a commercially available cDNA clone can be purchased and used, and can be artificially synthesized based on the nucleotide sequence represented by SEQ ID NO: 1 or 3.
(2) Ribonucleotide reductase gene expression vector The DNA encoding the ribonucleotide reductase gene of (1) is incorporated into a vector and introduced into a host cell to obtain animal cells used in the production method of the present application. it can.

  Transformation of animal cells with the DNA encoding the ribonucleotide reductase according to the present invention can be performed using an expression vector obtained by incorporating the DNA into a vector. Further, if necessary, an expression vector obtained by operably linking a control sequence operable in a host cell (for example, a promoter sequence and a terminator sequence) and incorporating it into a vector together with DNA encoding ribonucleotide reductase. Can also be used.

Here, operably linked means that the control sequence and the gene according to the present invention are combined so that the gene is expressed under the control of the control sequence in the host into which the gene according to the present invention is introduced. Usually, a promoter can be linked 5 ′ upstream of the gene and a terminator can be linked 3 ′ downstream of the gene.
The promoter used is not particularly limited as long as it exhibits promoter activity in the host cell to be transformed, and the promoter region on the chromosome where the ribonucleotide reductase gene is originally expressed may be used for its expression. However, in the present invention where the host for transformation is an animal cell, the early or late promoter of adenovirus (Ad), the early or late promoter of simian virus (SV40), herpes simplex virus (HSV) Thymidine kinase (tk) gene promoter, rous sarcoma virus, cytomegalovirus, mouse papilloma virus, bovine papilloma virus, avian sarcoma virus, retrovirus, hepatitis B virus, etc. Mammalian promoters such as promoters Down promoter or an immunoglobulin promoter, heat shock protein promoter, and the like can be used.

  The vector for incorporating the DNA encoding the ribonucleotide reductase gene is not particularly limited as long as the vector can incorporate the ribonucleotide reductase gene in a form that can be expressed in the host cell. .1 (manufactured by Invitrogen), pKANTEX93 (International Publication No. 97/10354 pamphlet), pCIneo (Promega), and other forms that can operate on vectors that already contain promoter and terminator sequences that function in animal cells It can be constructed by ligating the gene of ribonucleotide reductase to this.

  For example, a ribonucleotide reductase gene expression unit that can be operated with a CMV early gene promoter and an SV40 late gene terminator is transformed into a plasmid vector pUC18 (Takara Bio) having an E. coli replication origin (ColE1 ori) and an ampicillin resistance gene, pBluescriptII ( It can be constructed by ligating to a plasmid such as Stratagene) or pBR322. Further, if necessary, it may be used in combination with other selection marker genes such as G418 resistance gene which can be operated with promoter and terminator sequences.

  A transposon vector combining a transposon sequence and a transposase can also be used as a vector for incorporating a DNA encoding a ribonucleotide reductase gene. In the case of using a transposon vector, a DNA fragment in which a DNA encoding a ribonucleotide reductase gene to be incorporated is inserted between two corresponding transposon sequences is prepared and introduced into a host cell together with a vector expressing transposase, By expressing the transposase in the host cell, the DNA encoding the ribonucleotide reductase gene can be incorporated into the chromosomal DNA of the host cell.

In addition, human artificial chromosome vectors (Human Artificial Chromosome, HAC; WO 04/031385 pamphlet, WO 08/013067 pamphlet) can also be used according to the purpose.
2. Obtaining animal cells used in the production method of the present invention By introducing the ribonucleotide reductase expression vector described in 1 above and a vector for expressing a desired protein into a host cell, the animal cells used in the production method of the present invention are obtained. Can be acquired.

A vector for expressing a desired protein is obtained by inserting a DNA encoding the desired protein into a vector that can function in a host cell, in the same manner as the ribonucleotide reductase expression vector described above. Can be used.
The ribonucleotide reductase gene and the gene encoding the desired protein may be linked to the same expression vector, or may be inserted into different sites of the same vector and introduced into host cells, or may be separate vectors. May be inserted into a host cell.

When the desired protein is a complex protein consisting of a plurality of polypeptides, each polypeptide may be introduced into the host cell using the same vector, or introduced into the host cell using a plurality of vectors. May be.
A host cell into which a ribonucleotide expression vector and a vector for expressing a desired protein are introduced may be any animal cell as long as it can be used as a host for producing a protein. Specific examples of animal cells that can be used as host cells include human leukemia cells Namalwa cells, HEK293 cells, monkey cell COS cells, Chinese hamster ovary cell CHO cells (Journal of Experimental Medicine, 108, 945 (1958); Proc Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148 , 260 (1997); Proc. Natl. Acad. Sci. USA, 77, 4216 (1980); Proc. Natl. Acad. Sci., 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp. 883-900);), CHO / DG44, CHO-K1 (ATCC CCL-61), DukXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO- S (Life Technologies, Cat # 11619), Pro-3, rat myeloma cell YB2, 3HL.P2.G11.16Ag.20 (also referred to as YB2 / 0), mouse myeloma cell NSO, mouse myeloma cell SP2 / 0-Ag14 Syrian hamster cells BHK or HBT5637 000299).

Furthermore, in the present invention, lectin resistance is acquired in order to control the amount of fucose α-1,6-linked to N-acetylglucosamine present at the reducing end of the N-linked complex-type sugar chain bound to the target protein to be produced. Lec13 [Somatic Cell and Molecular genetics, 12 , 55 (1986)] and CHO cells deficient in the α1,6-fucose transferase gene (WO05 / 35586 pamphlet, WO02 / 31140 pamphlet) It can also be used as a host cell for producing the target protein of the present invention.

When the target protein produced in the present invention is a protein composed of two or more polypeptides and functioning, a host cell into which two or more protein expression vectors are introduced can also be used.
In addition, in the case of using a gene-recombined cell so that a target protein such as an antibody can be produced in advance, the animal cell of the present invention can be obtained by transfecting only a ribonucleotide reductase gene expression plasmid. In addition, when using cells in which the gene has not been recombined, the animal cell of the present invention is obtained by simultaneously transfecting the cell with an expression plasmid carrying the gene of the target protein and a ribonucleotide reductase gene expression plasmid. be able to.

As a method for introducing an expression plasmid into cells, any method can be used as long as it introduces DNA into animal cells. For example, electroporation [Cytotechnology, 3, 133 (1990)], calcium phosphate method (specialized) Kaihei 2-227075), or the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)]. When a human artificial chromosome vector is used as an expression plasmid, a microcell method (Japanese Patent Laid-Open No. 11-313576) can be used as a method for introducing it into a host cell. The expression plasmid introduced into animal cells can be linearized by digestion with a restriction enzyme. However, when an expression vector having a transposase recognition sequence is used, the transposase is not treated with a restriction enzyme. Simultaneously with transfection, the animal cell of the present invention can be constructed.
3. Production method of the present invention (1) Protein produced by the production method of the present invention The target protein produced in the present invention may be any protein as long as it can be expressed in animal cells used in the production method of the protein of the present invention. Specific examples include human serum proteins, peptide hormones, growth factors, cytokines, blood coagulation factors, fibrinolytic proteins, antibodies, and partial fragments of various proteins.

Preferred target proteins produced in the present invention include chimeric antibodies, humanized antibodies, monoclonal antibodies such as human antibodies, Fc fusion proteins, albumin binding proteins, and partial fragments thereof.
The effector activity of the monoclonal antibody produced in the present invention can be controlled by various methods. For example, fucose (also referred to as core fucose) that binds α-1,6 to N-acetylglucosamine (GlcNAc) present at the reducing end of an N-linked complex sugar chain that binds to the 297th asparagine (Asn) of the Fc region of an antibody Control method by controlling the amount of the antibody (WO05 / 035586 pamphlet, WO02 / 31140 pamphlet, WO00 / 61739 pamphlet) or by modifying amino acid residues in the Fc region of the antibody The method of doing is known. The effector activity can be controlled by any method used for the monoclonal antibody produced in the present invention.

Effector activity refers to antibody-dependent activity induced through the Fc region of an antibody. Antibody-dependent cytotoxic activity (ADCC activity), complement-dependent cytotoxic activity (CDC activity), macrophages, dendritic cells, etc. Antibody-dependent phagocytosis (ADP activity) by phagocytic cells is known.
Moreover, the effector activity of an antibody can be increased or decreased by controlling the content of core fucose of the Fc N-linked complex sugar chain of the monoclonal antibody produced in the present invention. As a method of reducing the content of fucose bound to the N-linked complex sugar chain bound to Fc of the antibody, by expressing the antibody using CHO cells deficient in the α1,6-fucose transferase gene, An antibody to which fucose is not bound can be obtained. Antibodies without fucose binding have high ADCC activity. On the other hand, as a method of increasing the content of fucose that binds to the N-linked complex sugar chain that is bound to the Fc of the antibody, the antibody is expressed using a host cell into which an α1,6-fucose transferase gene has been introduced. Thus, an antibody to which fucose is bound can be obtained. An antibody to which fucose is bound has a lower ADCC activity than an antibody to which fucose is not bound.

Furthermore, ADCC activity and CDC activity can be increased or decreased by modifying amino acid residues in the Fc region of the antibody. For example, the CDC activity of an antibody can be increased by using the amino acid sequence of the Fc region described in US Patent Application Publication No. 2007/0148165. In addition, by performing amino acid modification described in US Pat.No. 6,737,056, US Pat.No. 7,297,775, and US Pat. No. 7,317,091, it is possible to increase or decrease ADCC activity or CDC activity. You can also.
(2) Production method of the present invention The animal cells obtainable in 2 above are subjected to fed-batch culture, the desired protein is produced and accumulated in the medium, and the desired protein is collected from the medium. Protein can be produced.

The fed-batch culture performed in the production method of the present invention is also called a semi-batch culture, and is added once or a plurality of times or continuously in a fed-batch medium during the culture period. Accompanied by.
Examples of the culture medium (hereinafter abbreviated as basal medium) used in the production method of the present invention include CHO-S-SFMII, CD-CHO medium, CHO-III-PFM (Invitrogen), EX-CELL. In addition to 325-PF medium (SAFC Biosciences), SFM4CHO medium (HyClone) and other commercially available media used for animal cell culture, saccharides, amino acids, etc. It can also be obtained by blending and preparing.

The pH of the basal medium is carbonates such as NaHCO ₃ , chlorides such as CaCl ₂ 2H ₂ O, sulfates such as MgSO ₄ 7H ₂ O, phosphates such as NaH ₂ PO ₄ 2H ₂ O , Using a buffer such as sodium pyruvate, HEPES (N- [2-hydroxyethyl] piperazine-N '-[2-ethanesulfonic acid]) or MOPS (3- [N-morpholino] -propanesulfonic acid) The pH is adjusted to an appropriate range, preferably pH 6.5 to 7.5, and more preferably around pH 7.0.

On the other hand, if the pH does not fall within the desired range due to the buffering action of the buffer during the culture period, the pH is increased by adding an alkali such as NaOH, or the pH is decreased by increasing the flow rate of CO ₂ blowing. As a result, the pH can be adjusted in the range of 6.5 to 7.5, preferably around pH 7.0.
As the energy source of the cells contained in the basal medium, a monosaccharide such as glucose, mannose, fructose, galactose or maltose, usually about 1000 to 10,000 mg / L can be used, and preferably glucose can be used.

As the other medium components, each component used in a medium for culturing animal cells can be appropriately used. Examples of these components include amino acids, vitamins, lipids, and the like, and trace metal elements, surfactants, growth cofactors, nucleosides, and the like may be added as necessary.
The fed-batch medium added during the culture period may be the same as or different from the basal medium. Examples of such a fed-batch medium include concentrated liquids such as vitamins and metals, and concentrated basal medium. Etc. can be used.

Fed-batch culture is started by adding an appropriate amount of a pre-culture solution containing animal cells used in the production method of the present invention to the basal medium, and then once or a plurality of times depending on the growth of the animal cells. Or continuously with the addition of feed medium.
The culture conditions can be appropriately set as long as the animal cells described in 1 above can grow. For example, the CO ₂ concentration is 0-40%, preferably 2-10%, and the culture temperature is It is carried out at 30-40 ° C, preferably 36-38 ° C, more preferably 37 ° C. In addition, depending on the properties of the desired protein produced by the production method of the present invention, after obtaining a sufficient number of cells by culturing at the above culturing temperature, the culturing is continued at a lower temperature, and the desired protein Can also be produced (Japanese Patent Laid-Open No. 09-075077).

The culture period is not particularly limited as long as a necessary cell amount or protein amount can be obtained. Usually, the culture is performed for a period of about 1-20 days, preferably about 10-15 days.
Purification of the target protein produced and accumulated in the medium by the above culture is performed by separating the target protein and non-target impurities from the culture solution or cell disruption solution containing the protein. Separation can be performed using centrifugation, dialysis, ammonium sulfate precipitation, column chromatography, filters, etc., depending on the difference in the physicochemical properties of the target protein and impurities and the difference in binding strength to the column alone. The protein purification method can be carried out by the method described in Protein Experiment Note (top) Extraction / Separation and Expression of Recombinant Protein, Yodosha, Masato Okada / Kaoru Miyazaki / hen, ISBN 9784897069180.

Preparation of CHO cells expressing anti-human influenza M2 antibody expressing human ribonucleotide reductase subunit RRM2 (1) Preparation of human ribonucleotide reductase expression plasmid Expressing RRM2 gene encoding one of the subunits of human ribonucleotide reductase A plasmid vector was prepared by the following procedure.
A foreign gene expression unit artificially synthesized at the KpnI-NotI site of pBluescripit SK + (Stratagene) (DNA fragment consisting of CMV promoter / enhancer and BGHpA with KpnI on the 5 'side, EcoRV and NotI on the 3' side) To make pMug.

Next, a drug resistance marker expression unit (DNA fragment composed of SV40 ori, neomycin resistance gene, SV40pA) added with the restriction enzyme site was introduced into the EcoRV-NotI site of pMug to prepare pMugneo.
Furthermore, Tn2 transposon recognition sequences (Tol2 R500 and Tol2 L200) were inserted into the NotI and DraIII sites of pMugneo, respectively, to produce Tn / pMugneo.

Next, insert the fragment (fragment containing CMV promoter and intron) obtained by cutting pCI (Promega) with SpeI / SalI into Tn / pMugNeo cut with SpeI / SalI to create Tn / pMugNeo-CI did. A human RRM2 cDNA (purchased from Open biosystems) consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 is added to the XbaI site on the 5 'side and BamHI on the 3' side, and Tn / pMugNeo-CI Was introduced into the XbaI-BamHI site to prepare a human RRM2 expression vector Tn_pMugneo_RRM2 (FIG. 1).
(2) Preparation of transposase expression vector Artificial synthesis of a DNA fragment with the SalI site added to the 5 'side and the BamHI site added to the 3' side of the medaka-derived Tol2 transposase consisting of the nucleotide sequence represented by SEQ ID NO: 7 did. The synthesized DNA fragment was introduced into the SalI-BamHI site of pMug in (1) above to prepare a transposase expression vector TPEX / pMug (FIG. 2).
(3) Preparation of anti-human influenza M2 antibody expression vector An anti-human influenza M2 antibody expression plasmid was prepared by the following procedure. The anti-human influenza M2 antibody Z3G1 expression plasmid vector N5LG1_M2_Z3 (International Publication No. 2006/061723 pamphlet) was digested with SbfI and AscI to prepare a DNA fragment excluding Neo-Exon2.

A resistance gene for cycloheximide (a gene in which proline at position 54 of human L36a ribosomal protein was mutated to glutamine, JP-A-2002-262879) was inserted under the control of a CMV enhancer / promoter with SbfI and AscI sites added by PCR. After introducing the cycloheximide resistance gene expression unit, the lox P site was introduced at both ends of the BspLU11I site. The resulting plasmid was named N5LG1_CHX-lox_M2_Z3 (FIG. 3).
(4) Preparation of CHO cells expressing anti-human influenza M2 antibody Plasmid N5LG1_CHX-lox_M2_Z3, which was digested with the restriction enzyme BspLU11I and linearized, was introduced into CHO cells by the lipofection method. By selecting using a medium (MEM medium supplemented with 10% FBS) containing 3 μg / ml cycloheximide (CHX), CHX resistant cells were obtained. The resulting cells were cultivated by shaking culture in EX-CELL 325-PF medium (SAFC Bioscience) supplemented with L-glutamine to 4 mmol / L, and acclimated to suspension culture, expressing anti-human influenza M2 antibody Cells into which the plasmid was introduced were constructed. The resulting cell was named No20.
(5) Preparation of No20 cells transfected with human ribonucleotide reductase expression plasmid Tn_pMugneo_RRM2
No20 cells (4 × 10 ⁶ cells) suspended in 400 μl PBS buffer, human ribonucleotide reductase expression plasmid Tn_pMugneo_RRM2 or plasmid obtained by removing cDNA of human ribonucleotide reductase from Tn_pMugneo_RRM2 (hereinafter referred to as Mock plasmid) 10 μg and 20 μg of Tol2 transposase expression vector prepared in (2) were co-introduced as circular DNA by electroporation. For electroporation, an electroporator (Gene Pulser XceII system (Bio-Rad)) was used, and a cuvette with a gap width of 4 mm (manufactured by Bio-Rad) was used under the conditions of a voltage of 300 V, a capacitance of 500 μF, and room temperature. Done using.

After the gene introduction by electroporation, the cells were suspended in EX-CELL 325-PF medium (SAFC Biosciences) supplemented with glutamine to 4 mmol / L, and then seeded in a 96-well plate. The 96-well plate on which the cells were seeded was allowed to stand for 3-4 days in a CO ₂ incubator, and then a selective medium (325 PF) containing 500 μg / ml G418 and 3 μg / ml CHX in EX-CELL 325-PF medium. -G418 / CHX) was used, and the cells were statically cultured for one month while changing the medium about every week.

  Cells that can grow on 325PF-G418 / CHX medium are gradually scaled up to culture in 24-well plates, 6-well plates, and 125 ml flasks using 325PF-G418 / CHX medium to reduce human ribonucleotides. 11 clones of No20 cells into which the enzyme expression plasmid Tn_pMugneo_RRM2 was introduced (hereinafter referred to as No20 / Tn_pMugneo_RRM2) and 5 clones of No20 cells into which the Mock plasmid was introduced (hereinafter referred to as No20 / Mock) were obtained.

Antibody productivity evaluation by anti-human influenza M2 antibody-expressing CHO cells expressing human ribonucleotide reductase subunit RRM2 (1) Confirmation of antibody production activity No20 / Tn_pMugneo_RRM2 obtained in Example 1 (5) was EX in a 125 ml flask -Cultivated in CELL325-PF medium for 3 days. The amount of the protein of the anti-human influenza M2 antibody was measured by HPLC for the culture supernatant of the obtained culture solution, and it was confirmed that No20 / Tn_pMugneo_RRM2 had the ability to produce anti-human influenza M2 antibody.

The antibody concentration in the culture supernatant was measured according to the method described in Kuroda et al. (FEMS Yeast Res. 7, (2007), 1307-1316).
(2) Antibody productivity evaluation by fed-batch culture
Fed-batch culture of No20 cells introduced with Tn_pMugneo_RRM2 Fed-batch culture was performed with No20 / Tn_pMugneo_RRM2-introduced cells prepared in (1) and No20 / Mock.

OpticCHOHL medium [CHO CD EFFICIENTFEED A (purchased from Invitrogen) 1000 mL of peptone SE50MAF-UF (DMV International) 5 g and L-glutamine (Wako Pure Chemicals) 0.6 g] and FeedAHL medium [CD OptiCHO ^TM (GIBCO) 1000mL, D-glucose (Wako Pure Chemicals) 22.5g, peptone SE50MAF-UF 16.25g and L-glutamine 2.58g dissolved] mixed 4: 1 Was prepared.

Fed-batch culture was performed by adding 10 mL of each culture solution of No20 / Mock cells and No20 / Tn_pMugneo_RRM2 cells to 30 mL of each prepared basal medium to make a total of 40 mL of conditioned medium.
Every day from the third day of culture, 3% fed-batch medium was added to the culture and cultured for 13 days.
The number of cells in the culture solution at each time point during the culture and the amount of anti-human influenza M2 antibody protein in the culture supernatant were measured. FIG. 4 shows the cell density in the culture solution, and FIG. 5 shows the amount of antibody in the culture supernatant.

As shown in FIG. 4, No20 / Tn_pMugneo_RRM2 had a higher growth rate and highest cell density compared to No20 / Mock. Further, as shown in FIG. 6, it was found that No20 / Tn_pMugneo_RRM2 has a higher antibody-producing ability than No20 / Mock.
Further, Table 1 shows the antibody amount on the 13th day of culture and the polymer ratio (%) shown in the total anti-M2 antibody.

  As shown in Table 1, it was found that No20 / Tn_pMugneo_RRM2 had a lower polymer ratio in addition to a higher anti-M2 antibody production amount than No20 / Mock.

  The present invention provides an efficient protein production method using animal cells.

SEQ ID NO: 5-description of artificial sequence; Tol2-R transposon sequence SEQ ID NO: 6-description of artificial sequence; Tol2-L transposon sequence

Claims (6)

Animal cells transformed with DNA encoding ribonucleotide reductase and DNA encoding a desired protein are cultured in a medium, the protein is produced and accumulated in the culture, and the protein is collected from the culture A protein production method characterized by the above. The production method according to claim 1, wherein the DNA encoding ribonucleotide reductase is DNA encoding the RRM1 gene or the RRM2 gene. The production method according to claim 1, wherein the DNA encoding ribonucleotide reductase is a DNA encoding one protein selected from the group consisting of the following (a) to (d).
(A) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having RRM1 activity; (b) an amino acid sequence represented by SEQ ID NO: 2; A protein having an amino acid sequence having 80% or more homology and having RRM1 activity (c) consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4; And a protein having RRM2 activity (d) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and having RRM2 activity The production method according to claim 1, wherein the DNA encoding ribonucleotide reductase is one DNA selected from the group consisting of the following (a) to (d).
(A) DNA comprising the base sequence represented by SEQ ID NO: 1
(B) a DNA that hybridizes with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 under a stringent condition and encodes a protein having RRM1 activity
(C) DNA comprising the base sequence represented by SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 3 and encodes a protein having RRM2 activity The production method according to any one of claims 1 to 4, wherein the animal cell is a CHO cell. The production method according to any one of claims 1 to 5, wherein the protein is an antibody or an antibody fragment.

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