WO2020122048A1 - Animal cells, animal cell production method, and target protein production method - Google Patents

Abstract

The present invention addresses the problem of providing animal cells capable of producing a target protein with a high yield, a production method for the animal cells, and a target protein production method using the animal cells. The present invention provides: animal cells in which MZB1/pERp1 is overexpressed, said animal cells including a gene that codes for a target protein and a transgene that codes for MZB1/pERp1 and is linked to a promoter; a production method for the mammalian cells; and a target protein production method using the mammalian cells.

Description

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

The present invention relates to an animal cell that expresses a target protein. The present invention relates to a method for producing the above animal cell and a method for producing a target protein using the above animal cell.

In the production of biopharmaceuticals such as antibodies, in order to improve antibody productivity, fed-batch culture is often used in which nutrients are added to the culture medium to improve the cell condition. Further, in the production of biopharmaceuticals such as antibodies, in order to improve the productivity of antibodies, the culture solution is continuously filtered and discharged, while a fresh medium containing nutrient components is continuously supplied to the culture tank. The perfusion culture method is often used.

In Patent Document 1, as a method capable of producing a protein in a high production amount, cells that strongly express alanine aminotransferase and into which a DNA encoding a desired polypeptide is introduced are cultured to obtain a desired polypeptide. Methods for producing the polypeptides have been described, including producing.

International publication WO2009/020144

In fed-batch culture, higher cell density enables long-term cell culture compared to the case where no nutrient is added, but the cell viability in the latter half of the culture is affected by the accumulation of waste products secreted from cells. However, there is a problem that the product cannot be increased by extending the culture period.
In order to cultivate cells at a high density in the perfusion culture method, it is necessary to collect and supply 1 to 3 times the culture volume of the culture medium per day for the purpose of supplying nutrients and discharging waste products to the outside of the system. Since it is necessary, the problem is that the culture cost becomes high.

The present invention has an object to provide an animal cell capable of producing a target protein with high productivity. Another object of 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.

The present inventors have conducted extensive studies in order to solve the above-mentioned problems, and as a result, forcibly expressed the MZB1/pERp1 gene (MZB1 indicates Marginal zone B-and B1-cell-specific protein) in CHO cells, thereby producing a protein. It was possible to improve the antibody productivity (Qp) and the antibody production amount as a result. The present invention has been completed based on the above findings.

That is, according to the present invention, the following inventions are provided.
<1> An animal cell having a gene encoding a target protein and an exogenous gene encoding MZB1/pERp1 which is linked to a promoter, and which overexpresses MZB1/pERp1.
<2> The animal cell according to <1>, in which overexpression is constitutive.
<3> The animal cell according to <1> or <2>, wherein the foreign gene encoding MZB1/pERp1 has a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 1.
<4> The animal cell according to any one of <1> to <3>, in which the foreign gene encoding MZB1/pERp1 contains the base sequence shown in SEQ ID NO: 1.
<5> The animal cell according to any one of <1> to <4>, wherein the animal cell is a CHO cell.
<6><1> to <5> including a step of introducing a gene encoding a target protein and a foreign gene encoding MZB1/pERp1 which is linked to a promoter into an animal cell The method for producing animal cells according to any one of 1.
<7> The method according to <6>, wherein the step of introducing a foreign gene encoding MZB1/pERp1 and linked to a promoter is performed by electroporation.
<8> A method for producing a target protein, which comprises culturing the animal cell according to any one of <1> to <5>.
<9> The method according to <8>, wherein the culture is fed-batch culture.
<10> The method according to <9>, wherein the seeding cell density of the cell culture is 0.2×10 ⁶ cells/mL or more and 5×10 ⁶ cells/mL or less.
<11> The method according to <10>, wherein the viable cell rate during the culture period is 60% or more over the entire period.
<12> The method according to <8>, wherein the culture is perfusion culture.
<13> The method according to <12>, wherein the seeding cell density of the cell culture is 0.2×10 ⁶ cells/mL or more and 1×10 ⁷ cells/mL or less.
<14> The method according to <13>, wherein the viable cell rate during the culture period is 90% or more over the entire period.

According to the animal cell of the present invention, the target protein can be produced with high productivity.

FIG. 1 shows the results of measuring the antibody concentration in the culture supernatant of cells after gene transfer. FIG. 2 shows the results of measuring the antibody productivity (Qp) per cell in the culture supernatant of cells after gene transfer. FIG. 3 shows the results of measuring the cumulative number of viable cells during the culture period.

Hereinafter, modes for carrying out the present invention will be described in detail.
In the present specification, the numerical range indicated by using “to” means a range including the numerical values before and after “to” as the minimum value and the maximum value, respectively.

[Animal cell]
The animal cell of the present invention has a gene encoding a protein of interest and a foreign gene encoding MZB1/pERp1 which is linked to a promoter, wherein the MZB1/pERp1 is overexpressed. Is a cell.

It is thought that MZB1/pERp1 is expressed in the endoplasmic reticulum and promotes the formation of protein disulfide bonds in the mechanism by which cells secrete the useful product of antibodies. Although there is no knowledge that MZB1/pERp1 was overexpressed in CHO cells, it was expressed when B cells were differentiated into plasma cells and is required for the proper production of IgG heavy chain under endoplasmic reticulum stress. It has been reported (Genes & development 28:2014 1165-1178). Further, it has been reported that B cells introduced with pERp1 are important for IgM accumulation and secretion, and increase the IgM production (Immunity. 2010, 24; 33(5):723-735). However, the relationship between MZB1/pERp1 gene expression and recombinant protein production is unknown.

In Patent Document 1, the decrease in the survival rate was moderated by the effect of the introduction of alanine aminotransferase, but the period of 60% or less is long, and there is concern about the effect on antibody quality. Moreover, since the antibody productivity (Qp) per cell is lowered by the introduction of the taurine transporter, there is a problem that the production amount is not linked to the increase in cell density. On the other hand, in the present invention, the antibody productivity (Qp) per cell is improved by enhancing the expression of MZB1/pERp1 in the protein secretory pathway.

<Target protein>
In the present invention, the type of the target protein is not particularly limited, and examples thereof include recombinant polypeptide chain, recombinant secretory polypeptide chain, antigen-binding protein, human antibody, humanized antibody, chimeric antibody, mouse antibody, bispecific antibody, and Fc fusion. Proteins, fragmented immunoglobulins, single chain antibodies (scFv). The target protein is preferably a human antibody, a humanized antibody, a chimeric antibody, or a mouse antibody. Examples of the fragmented immunoimmunoglobulin include Fab, F(ab′) ₂ and Fv. The class of the antibody is not particularly limited, and may be any class such as IgG1, IgG2, IgG3, IgG4 and the like, IgG, IgA, IgD, IgE, IgM, etc., but IgG and IgM are preferable when used as a medicine.

Human antibodies include all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all variable and constant domains are derived from human immunoglobulin sequences (fully human antibodies).
Humanized antibodies are less likely to elicit an immune response and/or elicit a severe immune response when administered to a human subject, as compared to a non-human species antibody. Has a sequence that differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions. In one embodiment, certain amino acids within the heavy and/or light chain framework and constant domains of the non-human species antibody are mutated to produce a humanized antibody. In another embodiment, the constant domain from the human antibody is fused to the variable domain of a non-human species.

A chimeric antibody is an antibody in which variable regions and constant regions that are different from each other are linked. For example, an antibody consisting of the heavy and light chain variable regions of a mouse antibody and the heavy and light chain constant regions of a human antibody is a mouse-human heterologous chimeric antibody. A recombinant vector expressing a chimeric antibody can be prepared by ligating a DNA encoding the variable region of a mouse antibody with a DNA encoding the constant region of a human antibody and incorporating this into an expression vector. By culturing a recombinant cell transformed with the above vector and expressing the incorporated DNA, a chimeric antibody produced in the culture can be obtained.

Bispecific antibodies are antibodies produced by chemical methods or cell fusion that recognize two different antigenic specificities. Bispecific antibodies can be prepared by combining two immunoglobulin molecules using a cross-linking agent such as N-succinimidyl 3-(2-pyridyldithiol) propionate or S-acetylmercaptosuccinic acid anhydride. Methods, methods of binding Fab fragments of immunoglobulin molecules to each other, and the like have been reported.

The Fc fusion protein refers to a protein having an Fc region and includes an antibody.
Fab is a monovalent fragment with V _L , V _H , C _L and C _H 1 domains.
F(ab′) ₂ is a bivalent fragment that has two Fab fragments joined by a disulfide bridge in the hinge region.
The Fv fragment has the single arm _VL and _VH domains of the antibody.
Single chain antibodies (scFv) are antibodies in which the V _L and V _H regions are joined via a linker (eg, a synthetic sequence of amino acid residues) to form a continuous protein chain, wherein the linker is It is long enough to fold the protein chain onto itself, forming a monovalent antigen binding site.

The gene encoding the target protein can be obtained by a method 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.

The DNA encoding the L chain and the DNA encoding the H chain of the antibody can be prepared as follows. MRNA is extracted from hybridomas, cells, phages, ribosomes, etc., which carry a gene expressing an antibody. From this mRNA, cDNA is prepared by a reverse transcription reaction using a reverse transcriptase. Each gene is obtained by amplifying the L chain gene or the H chain gene by PCR using a primer having a complementary nucleotide sequence with the L chain gene or the H chain gene and cDNA, and ligating with a cloning plasmid.

The DNA encoding the L chain fragment and the DNA encoding the H chain fragment of the antibody can be prepared as follows. MRNA is extracted from hybridomas, cells, phages, ribosomes, etc., which carry a gene expressing an antibody. From this mRNA, cDNA is prepared by a reverse transcription reaction using a reverse transcriptase. Each gene fragment is obtained by amplifying the L chain gene fragment or the H chain gene fragment by PCR using a primer and cDNA having a complementary nucleotide sequence to the L chain gene fragment or the H chain gene fragment, and ligating with a cloning plasmid.

<MZB1/pERp1 and MZB1/pERp1 genes>
In the present invention, the origin of MZB1/pERp1 is not particularly limited, and a foreign gene encoding MZB1/pERp1 derived from mammals such as human, monkey, mouse, rat and hamster can be used. In the present invention, a foreign gene refers to a gene introduced into an animal cell from the outside. In addition, even when an endogenous gene is amplified and introduced into a gene, it is regarded as a foreign gene.

The nucleotide sequence and amino acid sequence of human MZB1/pERp1 are shown in SEQ ID NOs: 1 and 2 in the sequence listing.

Base sequence of human MZB1/pERp1 gene (SEQ ID NO: 1)
ATGAGGCTGTCACTGCCACTGCTGCTGCTGCTGCTGGGAGCCTGGGCCATCCCAGGGGGCCTCGGGGACA
GGGCGCCACTCACAGCCACAGCCCCACAACTGGATGATGAGGAGATGTACTCAGCCCACATGCCCGCTCA
CCTGCGCTGTGATGCCTGCAGAGCTGTGGCTTACCAGATGTGGCAAAATCTGGCAAAGGCAGAGACCAAA
CTTCATACCTCAAACTCTGGGGGGCGGCGGGAGCTGAGCGAGTTGGTCTACACGGATGTCCTGGACCGGA
GCTGCTCCCGGAACTGGCAGGACTACGGAGTTCGAGAAGTGGACCAAGTGAAACGTCTCACAGGCCCAGG
ACTTAGCGAGGGGCCAGAGCCAAGCATCAGCGTGATGGTCACAGGGGGCCCCTGGCCTACCAGGCTCTCC
AGGACATGTTTGCACTACTTGGGGGAGTTTGGAGAAGACCAGATCTATGAAGCCCACCAACAAGGCCGAG
GGGCTCTGGAGGCATTGCTATGTGGGGGACCCCAGGGGGCCTGCTCAGAGAAGGTGTCAGCCACAAGAGA
AGAGCTC

Amino acid sequence of human MZB1/pERp1 gene (SEQ ID NO: 2)
MRLSLPLLLLLLGAWAIPGGLGDRAPLTATAPQLDDEEMYSAHMPAHLRCDACRAVAYQMWQNLAKAETK
LHTSNSGGRRELSELVYTDVLDRSCSRNWQDYGVREVDQVKRLTGPGLSEGPEPSISVMVTGGPWPTRLS
RTCLHYLGEFGEDQIYEAHQQGRGALEALLCGGPQGACSEKVSATREEL

As a foreign gene encoding MZB1/pERp1,
(1) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2;
(2) A gene encoding a protein having an MZB1/pERp1 function, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2; or (3) SEQ ID NO: A protein comprising an amino acid sequence having a sequence identity of 85% or more (more preferably 90% or more, particularly preferably 95% or more, most preferably 98% or more) with the amino acid sequence described in 2, and having a MZB1/pERp1 function. A gene encoding
Can be used.

The MZB1/pERp1 function means a function of being expressed in the endoplasmic reticulum and promoting the formation of protein disulfide bonds.

Further as a foreign gene encoding MZB1/pERp1,
(4) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 1;
(5) A gene encoding a protein having the MZB1/pERp1 function, which is composed of the nucleotide sequence of SEQ ID NO: 1 with one or several nucleotides deleted, substituted or added.
(6) A gene encoding a protein having a MZB1/pERp1 function, which comprises a base sequence that hybridizes to the complementary sequence of the base sequence of SEQ ID NO: 1 under stringent conditions; or (7) SEQ ID NO: 1 A gene having a nucleotide sequence having 90% or more (more preferably 95% or more, particularly preferably 98% or more) sequence identity with the nucleotide sequence described in 1. and encoding a protein having MZB1/pERp1 function:
Can also be used.

The "1 or several" in the "amino acid sequence in which one or several amino acids are deleted, substituted or added" is preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5. Mean, particularly preferably 1 to 3.

Sequence identity in the present invention refers to a value calculated by the following formula.
% Sequence identity=[(number of identical residues)/(longer alignment length)]×100
Sequence identity in two amino acid sequences can be determined by any method known to those skilled in the art, and the BLAST ((Basic Local Alignment Search Tool)) program (J. Mol. Biol. 215:403-410, 1990). Etc. can be used to determine. The “longer alignment length” of the denominator means that when the two alignments are compared, the longer alignment length is used as the denominator.

"1 or several" in the "base sequence in which one or several bases are deleted, substituted or added" is preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5. Mean, particularly preferably 1 to 3.

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

Information on the amino acid sequence and nucleotide sequence of MZB1/pERp1 other than human is shown below. The following numbers are Gene No. of NCBI (National Center for Biotechnology Information). Indicates.
ID: 291675 Rat (Rattus norvegicus)
ID: 69816 mouse (Mus musculus)
ID: 510480 cow (Bos taurus)
ID: 100859189 Chicken (Gallus gallus)
ID: 693572 Rhesus monkey (Macaca mulatta)
ID: 101091341 Cat (Felis catus)
ID: 100522482 Pig (Sus scrofa)
ID: 100756072 Chinese Hamster (Cricetulus griseus)
ID: 101130009 Gorilla gorilla

Regarding non-human MZB1/pERp1, as in the case of human MZB1/pERp1,
(2A) a gene encoding a protein having an MZB1/pERp1 function, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in a predetermined amino acid sequence;
(3A) An amino acid sequence having a sequence identity of 85% or more (more preferably 90% or more, particularly preferably 95% or more, most preferably 98% or more) with a predetermined amino acid sequence, and has an MZB1/pERp1 function. Gene encoding a protein;
(5A) a gene comprising a base sequence in which one or several bases have been deleted, substituted or added in a predetermined base sequence, and which encodes a protein having an MZB1/pERp1 function;
(6A) a gene encoding a protein having a MZB1/pERp1 function, which consists of a base sequence that hybridizes to a complementary sequence of the predetermined base sequence under stringent conditions; or (7A) a predetermined base sequence and 90% A gene encoding a protein having a MZB1/pERp1 function having a nucleotide sequence having the above sequence identity (more preferably 95% or more, particularly preferably 98% or more):
May be used.

The foreign gene encoding MZB1/pERp1 is linked to the promoter.
The promoter is not particularly limited as long as it can function in host animal cells and express MZB1/pERp1. The promoters include CMV promoter (cytomegalovirus promoter), EF1α promoter (human polypeptide chain elongation factor gene promoter), SV40 promoter (cyamine virus 40 promoter), β-actin promoter, MMLV-LTR promoter (Molony mouse leukemia). Viral long terminal repeat promoters) or the mouse β-globin promoter are preferred, and the CMV promoter is more preferred.

In the animal cell of the present invention, MZB1/pERp1 is overexpressed. Overexpression means that the expression of a gene exceeds the normal expression level in a host. By introducing a foreign gene encoding MZB1/pERp1 into a host and expressing the foreign gene in the host, an animal cell overexpressing MZB1/pERp1 can be obtained.

The expression level of MZB1/pERp1 in the animal cell of the present invention is preferably 3 times or more, and preferably 3.5 times or more, that of the animal cell having no foreign gene encoding MZB1/pERp1. It is more preferably 4 times or more, still more preferably 4.5 times or more, still more preferably 5 times or more, and particularly preferably 5.5 times or more. Although there is no upper limit, it may be 30,000 times or less, or 10,000 times or less.

The expression level of MZB1/pERp1 can be examined by RT-PCR method (reverse transcription-polymerase chain reaction). The expression level of MZB1/pERp1 is preferably determined by reverse transcription of mRNA and real-time PCR. The expression level of MZB1/pERp1 is preferably a relative expression level calculated by normalization. Normalization can be performed by, for example, comparative quantification using the expression levels of housekeeping genes such as β-actin and HPRT1 as an endogenous control.

<Animal cells>
The cells in the present invention are not particularly limited as long as they are animal cells. Animal cells include Chinese hamster ovary (CHO) cells, BHK cells, 293 cells, myeloma cells (NS0 cells and the like), PerC6 cells, SP2/0 cells, hybridoma cells, COS cells, 3T3 cells, HeLa cells, Vero cells, MDCK cells, PC12 cells, WI38 cells and the like can be mentioned. Among the above, CHO cells, BHK cells, 293 cells, myeloma cells (NS0 cells and the like), PerC6 cells, SP2/0 cells and hybridoma cells are particularly preferable, and CHO cells are more preferable. CHO cells are widely used for the production of recombinant proteins such as cytokines, coagulation factors, and antibodies. It is preferable to use CHO cells deficient in dihydrofolate reductase (DHFR). As the DHFR deficient CHO cells, for example, CHO-DG44 can be used.

[Method for producing animal cell]
According to the present invention, the animal of the present invention, which comprises the step of introducing a gene encoding a protein of interest and a foreign gene encoding MZB1/pERp1 which is linked to a promoter into an animal cell. A method for producing cells is provided.

It is preferable that the gene encoding the target protein and the foreign gene encoding MZB1/pERp1 are introduced into the host animal cell in the form of being incorporated into the vector.

As a vector that can be used for introducing the gene into a host, for example, a mammalian-derived expression vector can be used, and examples thereof include 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) and the like, but not particularly limited thereto.

Also, it is known that mRNA having poly A is stable in cells. The gene encoding the target protein and the foreign gene encoding MZB1/pERp1 are polyA signals necessary for adding polyA to the gene, for example, mouse β globin polyA signal, bovine growth hormone polyA signal, SV40 polyA. It may have signals etc.

The method for introducing the gene encoding the target protein and the foreign gene encoding MZB1/pERp1 into animal cells is not particularly limited, and can be performed by a method known to those skilled in the art. For example, electroporation, lipofection, a calcium phosphate method, a DEAE dextran method, a method using a cationic liposome DOTAP (manufactured by Roche Life Science), or a method using a viral vector can be performed. Of the above, electroporation is preferred.

When a gene is introduced into an animal cell, the gene is introduced into only some of the cells provided for gene introduction, depending on the type of expression vector used and the gene introduction method. To identify and select cells into which the gene has been introduced, for example, a gene encoding a selectable marker for resistance to antibiotics may be introduced into the host cell along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.

In the cell of the present invention, the gene encoding the target protein may be expressed in a transient expression system or may be expressed in a constitutive expression system, but is expressed in a constitutive expression system. Those that are present are preferable.

In the cells of the present invention, the foreign gene encoding MZB1/pERp1 may be expressed in a transient expression system or may be expressed in a constitutive expression system, but is expressed in a constitutive expression system. What has been done is preferable.

The transient expression system is a method in which a circular plasmid is incorporated into cells and expressed by the calcium phosphate method, electroporation method, lipofection method, or the like. The gene encoding the target protein and the foreign gene encoding MZB1/pERp1 are often located extrachromosomally.

In the constitutive expression system, a circular plasmid or a linear plasmid prepared by treatment with a restriction enzyme is incorporated into cells by the calcium phosphate method, electroporation method, lipofection method, etc., and a part is inserted into the genome of the cell. It is a method of expressing a target protein by doing so. It is possible to maintain the expression of the gene encoding the target protein and the foreign gene encoding MZB1/pERp1 for a long period of time. In addition, if a drug resistance gene is introduced into a plasmid, drug selection becomes possible, and it is possible to efficiently select cells in which the gene encoding the target protein and the foreign gene encoding MZB1/pERp1 are maintained on the chromosome. it can.

[Method for producing target protein]
According to the present invention, there is provided a method for producing a target protein, which comprises culturing the animal cell of the present invention.

The target protein can be produced by culturing the animal cell of the present invention. The culture can be performed according to a known method.
As the medium used for culturing the animal cells of the present invention, a medium used for culturing ordinary animal cells can be used. For example, OptiCHO (Lifetechnologies, 12681011) medium, Dulbecco's modified Eagle medium (DMEM), Eagle minimal essential medium (MEM), RPMI-1640 medium, RPMI-1641 medium, F-12K medium, Ham's F12 medium, Iscove modified method Dulbecco's medium (IMDM), McCoy's 5A medium, Leibovitz L-15 medium, and EX-CELL™ 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), and the like can be used.

Serum such as fetal calf serum (FCS) may be added to the medium, more preferably serum-free medium, and most preferably completely synthetic medium.
The medium may be supplemented with additional components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, plant protein hydrolysates.

Although the pH of the medium varies depending on the cells to be cultured, it is generally pH 6.0 to 8.0, preferably pH 6.8 to 7.6, and more preferably pH 7.0 to 7.4.
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.

Cultivation is preferably performed in an atmosphere having a CO ₂ concentration of 0 to 40%, preferably 2 to 10%.
The culture time 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.
In the culturing, medium exchange, aeration and stirring can be added as necessary.

The culturing of the animal cells of the present invention can be carried out in a culturing device (also called a bioreactor) or another suitable container. As the culture device, a fermenter type tank culture device, an air lift type culture device, a culture flask type culture device, a spinner flask type culture device, a microcarrier type culture device, a fluidized bed type culture device, a hollow fiber type culture device, a roller bottle. It can be performed by using a type culture device, a filled-tank type culture device, or the like.

The culture scale is generally 1 L to 20000 L, preferably 1 L to 10000 L, more preferably 200 L to 2000 L, and further preferably 500 L to 2000 L.

For the culture, any method such as batch culture, fed-batch culture (also referred to as fed-batch culture), and perfusion culture may be used, but fed-batch culture or perfusion culture is preferable. ..

Batch culture is a discontinuous method 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 experience an increase in cell density until they reach maximal cell density, then survive as media components are consumed and levels of metabolic byproducts (such as lactate and ammonia) accumulate. Cell density declines. Harvesting is typically performed when maximum cell density (typically 5-10 x 10 ⁶ cells/mL) is reached. The batch process is the simplest culturing method, however, the viable cell density is limited by nutrient availability and once the cells reach maximum density, the culture declines and the production of the protein of interest is reduced. It is not possible to prolong the production period of the protein of interest, as waste product accumulation and nutrient depletion quickly lead to culture decline (typically about 3-7 days).

Fed-batch culture is a culture method that improves the batch process by supplying a medium in a bolus or continuously to supplement the consumed medium components. Thus, in fed-batch culture, the culture form is a suspension culture, providing additional components to the culture at one or more points after the start of the culture process. Additional components include nutritional supplements for cells that are depleted during the culturing process, and may include other supplements (eg, cell cycle inhibiting compounds).

In fed-batch culture, additional nutrients are added throughout the culture period, so there is a possibility that higher cell density and higher production of target protein can be achieved compared to batch culture. In fed-batch cultures, unlike batch cultures, the feeding schedule and medium are used to distinguish the period of cell growth (growth phase) to achieve the desired cell density from the period of aborted or slow cell growth (production phase). By manipulating the components, biphasic cultures can be made and maintained. As a result, the fed-batch culture may be able to achieve a higher production amount of the target protein as compared with the batch culture.

In the fed-batch culture, the seeding cell density of the cell culture is generally 0.2×10 ⁶ cells/mL or more and 1×10 ⁷ cells/mL or less, preferably 0.2×10 ⁶ cells/mL or more 5. ×10 ⁶ cells/mL or less, more preferably 0.5×10 ⁶ cells/mL or more and 2.5×10 ⁶ cells/mL or less, and still more preferably 0.5×10 ⁶ cells/mL or more 1. It is less than or equal to 5×10 ⁶ cells/mL.
In the fed-batch culture, the viable cell rate during the culture period is preferably 60% or more and 100% or less, more preferably 70% or more and 100% or less, and further preferably 75% or more and 100% or less in the entire period. ..

Perfusion culture is a culture method in which fresh medium is added and used medium is removed at the same time, and there is a possibility that batch culture and fed-batch culture can be further improved. Perfusion culture can be performed using, for example, an ATF (Alternating Tangential Flow Filtration) pump or a TFF (Tangential Flow Filtration) pump. With perfusion cultures, it is possible to achieve high cell densities in excess of 1×10 ⁸ cells/mL. A typical perfusion culture begins with a batch culture start-up lasting 1 or 2 days, after which fresh feed medium is added continuously, stepwise, and/or intermittently to the culture to remove spent medium simultaneously. To do. In perfusion culture, methods such as sedimentation, centrifugation or filtration can be used to remove spent medium while maintaining cell density.

The advantage of perfusion culture is that the culture in which the target protein is produced is maintained longer than the batch culture method or the fed-batch culture. However, maintenance of long-term perfusion cultures, especially perfusion cultures at high cell densities, requires media preparation, use, storage and disposal. The perfusion culture requires a large amount of nutrients and tends to increase the production cost of the target protein as compared with batch culture and fed-batch culture. In addition, by selecting the membrane pore size, it is possible to continue culturing while recovering the antibody outside the system.Therefore, the retention time of the antibody in the culture solution is shortened and the chemical changes are reduced to improve antibody quality. Can be kept high. ‥

It is also possible to perform a culture that combines fed-batch culture and perfusion culture. As an example, a fed-batch culture with a bolus feed can be used to maintain a culture of cells in the growing phase, and then a perfusion culture can be used to produce the protein of interest.

The perfusion may be continuous, stepwise, intermittent, or a combination thereof. Animal cells are maintained in culture and the spent media that is removed may be substantially free of cells or have much less cells than culture. The target protein expressed by cell culture can be retained or recovered in the culture by selecting the membrane pore size. In order to prevent the cell density in the culture from becoming excessive, a part of the culture medium may be extracted together with the cells, and the same amount of fresh medium may be added to reduce the cell density (cell bleeding).

In the perfusion culture, the seeding cell density of the cell culture is generally 0.2×10 ⁶ cells/mL or more and 3×10 ⁷ cells/mL or less, preferably 0.2×10 ⁶ cells/mL or more 1. ×10 ⁷ cells/mL or less, more preferably 0.5×10 ⁶ cells/mL or more and 1×10 ⁷ cells/mL or less.
In the perfusion culture, the viable cell rate during the culture period is preferably 80% or more, more preferably 85% or more, and further preferably 90% or more in the entire period.

In perfusion culture, the highest cell density reached is preferably 2×10 ⁸ cells/mL or less, more preferably 1.5×10 ⁸ cells/mL or less, and further preferably 1.0×10 ⁸ cells/mL. It is less than or equal to mL.

The perfusion ratio in the perfusion culture is preferably 0.3 vvd or more and 5.0 vvd or less, more preferably 0.3 vvd or more and 1.5 vvd or less. However, vvd represents the following.
vvd=(volume of fresh medium/working volume of reactor/day, amount of supplied culture fluid/volume of culture fluid per day)

The seeded cell density in cell culture and the highest cell density reached in culture can be determined by measuring the number of cells by a conventional method and dividing the number of cells by the amount of culture solution.
The viable cell rate (viability) during the culture period is obtained by dividing the viable cell number by (viable cell number+dead cell number). For example, the number of cells can be measured using Vi-CELL XR (Beckman Coulter).

The target protein produced by the above culture can be purified. Separation and purification of the target protein may be performed by using the separation and purification methods used for ordinary proteins. For example, a target protein can be obtained by appropriately selecting and combining a chromatography column such as affinity chromatography, a filter, ultrafiltration, salting out, dialysis, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and isoelectric focusing. Can be isolated and purified, but is not limited thereto. The concentration of the target protein obtained as described above can be measured by absorbance measurement, enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay; ELISA), or the like.

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

The target protein can be modified or partially removed by treating it with an appropriate polypeptide-modifying enzyme before or after purification. Examples of the polypeptide modifying enzyme include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, glucosidase and the like.

[Use of target protein]
When the target protein produced by the method of the present invention has a biologically useful biological activity, the target protein is mixed with a pharmaceutically acceptable carrier or additive to prepare a formulation, Can manufacture pharmaceutical products.

Examples of pharmaceutically acceptable carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, 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, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA) , Mannitol, sorbitol, lactose, pharmaceutically acceptable surfactants and the like.

For example, when used as an injectable preparation, the purified target protein is dissolved in a solvent such as physiological saline, a buffer solution, a glucose solution, etc., and an adsorption inhibitor such as Tween 80, Tween 20, gelatin, human serum albumin, etc. Can be used. Alternatively, the target protein may be freeze-dried to obtain a dosage form that is reconstituted before use, and examples of the excipient for freeze-drying include sugar alcohols such as mannitol and glucose, and Sugars can be used.

The method of administering the target protein may be either oral administration or parenteral administration, but parenteral administration is preferred. For example, injection (eg, systemic or local administration by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.), nasal administration, pulmonary administration, transdermal administration and the like can be mentioned.

The dose of the target protein is 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 1 kg of body weight per one time, but is not particularly limited.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.

<Example 1> Preparation of animal cells A vector encoding the human MZB1/pERp1 gene (OriGene, Cat# RC205659) was purchased, and an expression cassette (promoter, open reading frame, terminator) of the MZB1/pERp1 gene was used as a primer 1 ( It was amplified by polymerase chain reaction (PCR) using SEQ ID NO:3) and primer 2 (SEQ ID NO:4).

Antibody 1 (denosumab, https://www.drugbank.ca/drugs/DB06643) or antibody 2 (brosozumab, https://www.drugbank.ca/drugs/DB12560) or antibody 3 (anti-MUC16 antibody, anti-MUC16 antibody). (-5171691) The amplified MZB1/pERp1 expression cassette was inserted into a vector that co-expresses the L chain and H chain of each antibody. Construction of the L-chain and H-chain co-expression vector and introduction into cells were carried out according to Example 2 of JP-T-2016-517691.

All host CHO-DG44 cells before the introduction of the antibody and MZB1/pERp1 gene were cultured in an incubator at 37° C. in a 5% CO ₂ atmosphere. 20 mL of HT Supplement (x100) (Lifetechnologies, 11067-030) diluted with 100 times the above vector was introduced into host cells 5×10 ⁶ cells by electroporation (Lonza, 4D-Nucleofector) and diluted 100-fold. After suspending in OptiCHO (Lifetechnologies, 12681011) medium, it was seeded in a T75 flask. One day later, the medium was replaced with OptiCHO (Lifetechnologies, 12681011) medium containing 175 nmol/L methotrexate (MTX) (Wako Pure Chemical Industries, Ltd.) without HT supplement. Microscopic observation of T75 flask was started from 3 weeks after gene introduction, and the culture volume was expanded to 20 mL sequentially from the flask in which cell growth was observed, seeded in a 125 mL shaking flask (Corning), and shaken at 140 rpm. Cultured.

According to the above method, antibody 1-MZB1 cells, antibody 2-MZB1 cells, antibody 3-MZB1 cells that forcibly express the human MZB1/pERp1 gene and antibody 1 or antibody 2 or antibody 3 were established. In addition, gene transfer was carried out in the same manner using a vector expressing the antibody L chain and H chain to construct a control group of antibody 1 cell, antibody 2 cell, and antibody 3 cell.

Primer-1: CCGCGGTCATAGCTGTTTCCTGAAC (SEQ ID NO: 3)
Primer-2: CAGCTATGACCGCGGTTAGAGCTCTTCTCTTGTGGCTGACACC (SEQ ID NO: 4)

<Example 2> Culture experiment A fed-batch culture experiment was carried out using 8 levels of each of antibody 1-MZB1 cells, antibody 2-MZB1 cells, antibody 3-MZB1 cells and antibody 1 cells, antibody 2 cells, and antibody 3 cells. .
The cells were suspended in 15 mL of OptiCHO (Lifetechnologies, 12681011) medium at a cell density of 5×10 ⁵ cells/mL, and automatic culturing was performed using an AMBR15 culture device (Sartorius Stedim). From the second day to the 13th day from the start of the culture, a feed medium (Cellboost 7a, 7b, GE healthcare) was added every 2% at the initial culture volume ratio every day. Sampling was carried out every 3-4 days in order to measure cell density, culture solution components and antibody concentration over time. The culture medium was collected 14 days after the start of the culture, and cells and cell debris were removed using a 0.22 μm depth filter (Merck Millipore). When the antibody concentration in the supernatant and the antibody productivity per cell (Qp) were measured by liquid chromatography, the antibody concentration was increased by 83% for antibody 1, 24% for antibody 2, and 38% for antibody 3 (Fig. 1). And Table 1). In addition, Qp was increased by 79% for Antibody 1, 40% for Antibody 2, and 55% for Antibody 3 (Fig. 2 and Table 2). On the other hand, integrated viable cell density (IVCD) is almost unchanged for all antibodies (Figure 3 and Table 3). 1 to 3, the left shows only the antibody and the right shows the antibody-MZB1. The expression of MZB1/pERp1 increased the efficiency of the antibody secretory pathway, which is considered to improve the antibody productivity.
The integrated viable cell density (IVCD) was measured by the following method. Viable cell density (VCD) was measured by Vi-CELL XR (Beckman Coulter, Inc.) on days 0, 3, 7, 10 and 14, and the respective values were designated as V0, V3, V7, V10 and V14. The integrated viable cell density (IVCD) is calculated by the following formula.
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}

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

Pti [g/L] = concentration of purified product on culture day ti Xti [cell/day] = viable cell density on culture day ti

The approximate value of the integral was calculated by obtaining the area under the growth curve from time t1 to time t2 as the trapezoidal area.

Claims (14)

1. An animal cell having a gene encoding a protein of interest and a foreign gene encoding MZB1/pERp1 which is linked to a promoter, wherein said MZB1/pERp1 is overexpressed.
2. The animal cell according to claim 1, wherein overexpression is constitutive.
3. The animal cell according to claim 1 or 2, wherein the foreign gene encoding MZB1/pERp1 has a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:1.
4. The animal cell according to any one of claims 1 to 3, wherein the foreign gene encoding MZB1/pERp1 contains the nucleotide sequence set forth in SEQ ID NO: 1.
5. The animal cell according to any one of claims 1 to 4, wherein the animal cell is a CHO cell.
6. 6. The method according to claim 1, further comprising the step of introducing a gene encoding a protein of interest and a foreign gene encoding MZB1/pERp1 which is linked to a promoter into an animal cell. The method for producing an animal cell according to 1.
7. The method according to claim 6, wherein the step of introducing a foreign gene encoding MZB1/pERp1 and linked to a promoter is performed by electroporation.
8. A method for producing a target protein, which comprises culturing the animal cell according to any one of claims 1 to 5.
9. The method according to claim 8, wherein the culture is a fed-batch culture.
10. The method according to claim 9, wherein the seeding cell density of the cell culture is 0.2×10 ⁶ cells/mL or more and 5×10 ⁶ cells/mL or less.
11. The method according to claim 10, wherein the viable cell rate during the culture period is 60% or more over the entire period.
12. The method according to claim 8, wherein the culture is perfusion culture.
13. The method according to claim 12, wherein the seeded cell density of the cell culture is 0.2×10 ⁶ cells/mL or more and 1×10 ⁷ cells/mL or less.
14. The method according to claim 13, wherein the viable cell rate during the culture period is 90% or more over the entire period.

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