Method of manufacturing a viral vector

Abstract

An object of the present application is to provide a method for producing a viral vector that can increase the production amount of the viral vector. According to the present application, there is provided a method for producing a viral vector, comprising the step of culturing producer cells or packaging cells in a culture solution containing the producer cells or packaging cells at a cell density of 1 x 10 6 producer cells or packaging cells and a histone deacetylase inhibitor.

Description

Technical Field

[0001] This invention relates to a method for manufacturing a viral vector, which includes the step of culturing production cells or packaging cells in a prescribed state. Background Technology

[0002] Adeno-associated virus (AAV) is a straight-stranded single-stranded DNA virus belonging to the Parvoviridae family. The wild-type AAV genome contains replication regulatory genes (Rep genes) and capsid structural genes (Cap genes), and adjacent inverted terminal repeats (ITRs) for viral replication and packaging. AAV vectors can deliver genes into either proliferating or non-proliferating cells, and can achieve long-term expression, especially in non-dividing cells. Furthermore, AAV is considered non-pathogenic and has low immunogenicity. Based on these characteristics, AAV vectors have made progress in clinical applications as gene therapy vectors. Typically, gene therapy AAV vectors contain any therapeutic gene sandwiched between ITRs. Viral vectors, such as AAV vectors, are manufactured by culturing production cells or packaging cells.

[0003] Patent Document 1 describes the following: rAAV particles are generated by culturing cells capable of producing recombinant AAV (rAAV) particles in the presence of a histone deacetylase (HDAC) inhibitor. Non-Patent Document 1 shows that when producing rAAV using HEK293 cells, the titer of the rAAV8 genome is increased by using M344 (a selective HDAC inhibitor). Existing technical documents Patent documents

[0004] Patent Document 1: Japanese Patent Publication No. 2021-533757 Non-patent literature

[0005] Non-patent literature 1: Joseph M. Scarrott et al., Biotechnol. J. 2023:18:2200450 Summary of the Invention The technical problem to be solved by the invention

[0006] Currently, gene therapy drugs based on AAV vectors are on the market, and there is a desire to develop a manufacturing method that can increase the production yield of viral vectors such as AAV vectors. The problem this invention aims to solve is to provide a method for manufacturing viral vectors that can increase the production yield. means for solving technical problems

[0007] As a result of in-depth research conducted by the inventors to solve the aforementioned problems, they discovered that culturing cells at a density of 1×10⁻⁶ in the presence of histone deacetylase inhibitors... 6 A production cell or packaging cell density of more than one cell / mL can increase the production yield of viral vectors. This invention was made based on the above insights.

[0008] According to the present invention, the following invention is provided. <1> A method for manufacturing a viral vector, comprising the following steps: in a container containing cells at a density of 1×102 6 Production or packaging cells are cultured in a medium containing at least one cell / mL of production or packaging cells and a histone deacetylase inhibitor. <2> According to the manufacturing method described in <1>, the histone deacetylase inhibitor is a compound having a benzamide structure or a hydroxyxamic acid. <3> The manufacturing method according to <1>, wherein the histone deacetylase inhibitor is a compound represented by formula (1) or formula (2). R 1 -CONH-R 2 (1) In equation (1), R 1 and R 2 One of them is an aromatic group that can have substituents, R 1 and R 2 The other one is an amino group that can have a substituent, an alkyl group that can have a substituent, an alkenyl group that can have a substituent, an aromatic group that can have a substituent, or an alkoxy group that can have a substituent; R 3 -CONH-OH (2) In equation (2), R 3 It can be an alkyl group that may have substituents or an alkenyl group that may have substituents. <4> The manufacturing method according to <1>, wherein the histone deacetylase inhibitor is a compound represented by formula (1A), formula (1B) or formula (2A). [Chemical Formula 1] In equation (1A), R 10 R represents a halogen, a heterocyclic group that may have substituents, an alkyl group having 1 to 6 carbon atoms that may have substituents, or an amino group that may have substituents. 2 It can be an amino group that may have substituents, an alkyl group that may have substituents, or an aromatic group that may have substituents; [Chemical Formula 2] In equation (1B), R20 R represents an amino group. 1 It can be an alkyl group that may have substituents or an aromatic group that may have substituents; R 31 -R 30 -CONH-OH (2A) In equation (2A), R 30 It represents -(CH2) n - or CH=CH-, where n represents an integer from 2 to 8, R 31 This indicates that it can have a substituent aromatic group or CONH-OH. <5> According to the manufacturing method described in <1>, wherein, in formula (1A), R 10 express bromine, [Chemical Formula 3] , methyl, [Chemical Formula 4] , or CH3-CO-NH-. <6> According to the manufacturing method described in <1>, the histone deacetylase inhibitor is any one of the following compounds. [Chemical Formula 5] <7> The manufacturing method according to any one of <1> to <6> includes the step of perfusion culture of cells. <8> The manufacturing method according to any one of <1> to <6>, wherein the virus is an adeno-associated virus. <9> According to the manufacturing method described in <8>, the production cells or packaging cells are cells in which the Rep gene, Cap gene and accessory genes are integrated into the chromosome. <10> The manufacturing method according to any one of <1> to <6>, wherein the cell is an animal cell. <11> The manufacturing method according to any one of <1> to <6>, wherein the content of histone deacetylase inhibitor in the culture medium is less than 50 μmol / L. <12> The manufacturing method according to any one of <1> to <6>, wherein the culture is carried out for more than 24 hours in a culture medium containing histone deacetylase inhibitor. <13> The manufacturing method according to any one of <1> to <6>, wherein the cell density is 30 × 102 6 Cells / mL or higher. <14> The manufacturing method according to any one of <1> to <6>, wherein the culture is a suspension culture. Invention Effects

[0009] According to the present invention, the production yield of viral vectors can be increased. Attached Figure Description

[0010] Figure 1 The effect of compound-based titer improvement is shown. Figure 2 The results of ddPCR titer determination in high-density cells are shown. Figure 3 The results of cell density measurements in the reactor are shown. Figure 4 The results of AAV titer determination in cell culture media collected over 7 consecutive days are shown. Figure 5 The results of the determination by high performance chromatography are shown. Figure 6 The study demonstrates the improvement in cell culture density and compound-based titer. Figure 7 The effect of the compound on improving titers at different concentrations is shown. Detailed Implementation

[0011] Hereinafter, an example of an embodiment of the present invention will be described. However, the present invention is not limited by the following embodiment, and appropriate modifications can be made to implement it within the scope of the object of the present invention. In this specification, the numerical range indicated by "~" represents the range in which the values ​​before and after "~" are respectively included as the minimum and maximum values.

[0012] <Explanation of Terminology> A promoter is a DNA control region / sequence that enables RNA polymerase to bind and participates in the initiation of transcription of downstream coding or non-coding sequences.

[0013] A vector is a DNA or RNA molecule used to artificially transport foreign genes into other cells. A vector used to express a gene is called an expression vector. If a vector containing the desired foreign gene is introduced into a cell, the foreign gene will be replicated and / or expressed within the cell. Examples of vectors include free-type (e.g., plasmid) vectors and non-free-type vectors. Vectors can be introduced into host cells through methods such as transfection, transduction, cell fusion, and lipid transfection.

[0014] In this specification, a gene refers to a nucleic acid molecule that encodes a polypeptide or RNA, such as cDNA or genomic DNA.

[0015] The method for manufacturing the viral vector of the present invention includes comprising a cell density of 1×10⁻⁶ cells. 6 The steps of culturing production or packaging cells in a culture medium containing at least one cell / mL of production or packaging cells and a histone deacetylase inhibitor.

[0016] In this invention, it is hypothesized that by inserting the gene or a portion thereof required for the expression of the viral vector into the chromosome of the host cell in the production or packaging cell, the acetylation of histones can be enhanced and chromatin structure changes can be induced by inhibiting the activity of histone deacetylase (HDAC) expressed in the cell, thereby increasing the viral vector titer and Full rate (the ratio of the amount of capsid containing the complete gene to the total amount of capsid).

[0017] As a histone deacetylase inhibitor, there are no particular limitations as long as the substance has the function of inhibiting histone deacetylase, but compounds with a benzamide structure or hydroxyxamic acid are preferred.

[0018] Preferred examples of histone deacetylase inhibitors are compounds represented by formula (1) or formula (2). R 1 -CONH-R 2 (1) In equation (1), R 1 and R 2 One of them is an aromatic group that can have substituents, R 1 and R 2 The other one is an amino group that can have a substituent, an alkyl group that can have a substituent, an alkenyl group that can have a substituent, an aromatic group that can have a substituent, or an alkoxy group that can have a substituent; R 3 -CONH-OH (2) In equation (2), R 3 It can be an alkyl group that may have substituents or an alkenyl group that may have substituents.

[0019] As a further preferred example of a histone deacetylase inhibitor, a compound represented by formula (1A), formula (1B) or formula (2A) is provided. [Chemical Formula 6] In equation (1A), R 10 R represents a halogen, a heterocyclic group that may have substituents, an alkyl group having 1 to 6 carbon atoms that may have substituents, or an amino group that may have substituents. 2 It can be an amino group that may have substituents, an alkyl group that may have substituents, or an aromatic group that may have substituents; [Chemical Formula 7] In equation (1B), R 20 R represents an amino group. 1 It can be an alkyl group that may have substituents or an aromatic group that may have substituents; R 31 -R 30 -CONH-OH (2A) In equation (2A), R 30 It represents -(CH2) n - or CH=CH-, where n represents an integer from 2 to 8, R 31 This indicates that it can have a substituent aromatic group or CONH-OH.

[0020] Phenyl is preferred as an aromatic group. As an alkyl group, it is preferred to have 1 to 8 carbon atoms, and more preferably alkyl groups with 1 to 6 carbon atoms. The alkyl group can be straight-chain or branched. As an alkenyl group, an alkenyl group with 2 to 8 carbon atoms is preferred, an alkenyl group with 2 to 6 carbon atoms is more preferred, and an alkenyl group with 2 to 4 carbon atoms is even more preferred. The preferred alkoxy group is one with 1 to 8 carbon atoms, and more preferably one with 1 to 6 carbon atoms. The alkoxy group can be straight-chain or branched. As halogens, fluorine, chlorine, bromine or iodine are preferred.

[0021] Examples of substituents include halogens (e.g., fluorine, chlorine, bromine, or iodine), alkyl groups having 1 to 6 carbon atoms, -NH2, -CONHC6H5, -CONHC6H5(NH2), -NHCOCH3, -NHCOC6H5(CH3), -CONHOH, pyridyl groups, aromatic groups substituted with -CONHOH (e.g., phenyl groups substituted with -CONHOH), and heterocyclic groups that can be substituted. [Chemical Formula 8] wait( (Indicates the bonding location).

[0022] As a heterocyclic group that can be replaced, examples can be given. [Chemical Formula 9] wait( (Indicates the bonding location).

[0023] Especially preferred is that, in formula (1A), R 10 express bromine, [Chemical Formula 10] , methyl, [Chemical Formula 11] , or CH3-CO-NH-.

[0024] Specific examples of histone deacetylase inhibitors include any of the following compounds. [Chemical Formula 12] [Chemical Formula 13] [Chemical Formula 14] [Chemical Formula 15] [Chemical Formula 16] [Chemical Formula 17]

[0025] Preferred examples of histone deacetylase inhibitors include UF010, CI-994, and Resminostat. The content of histone deacetylase inhibitor in the culture medium is preferably below 50 μmol / L, more preferably 0.01 μmol / L to 50 μmol / L, even more preferably 0.05 μmol / L to 50 μmol / L, further preferably 0.1 μmol / L to 50 μmol / L, even more preferably 1 μmol / L to 20 μmol / L, even more preferably 1 μmol / L to 10 μmol / L, even more preferably 3 μmol / L to 10 μmol / L, and particularly preferably 5 μmol / L to 10 μmol / L.

[0026] In this invention, the culture in a culture medium containing a histone deacetylase inhibitor is preferably for 24 hours or more, more preferably 24 hours or more and 240 hours or less, further preferably 24 hours or more and 120 hours or less, and particularly preferably 48 hours or more and 96 hours or less. The upper limit of the period during which the culture medium contains the histone deacetylase inhibitor is not particularly limited, but is generally 240 hours or less, preferably 120 hours or less, and particularly preferably 96 hours or less.

[0027] When using a doxycycline-induced promoter to express exogenous recombinant nucleic acids, the culture medium may contain doxycycline. The concentration of doxycycline is preferably 5 μg / mL or less, more preferably 1 μg / mL or less, even more preferably 700 ng / mL or less, and particularly preferably 500 ng / mL or less. The preferred time for adding doxycycline is 1 hour or more, more preferably 6 hours or more, more preferably 12 hours or more, more preferably 24 hours or more, and more preferably 48 hours or more.

[0028] In this invention, it is preferable to supply a culture medium during cultivation, which may contain doxycycline and histone deacetylase inhibitors. The supplied culture medium refers to the culture medium added during cell culture. The culture method using the supplied culture medium is not particularly limited, but perfusion culture is preferred. That is, the method of the present invention preferably includes perfusion culture of cells.

[0029] The treatment time (period for doxycycline to take effect) is preferably 1 hour or more, more preferably 6 hours or more, more preferably 12 hours or more, more preferably 24 hours or more, and more preferably 48 hours or more.

[0030] In this invention, production cells or packaging cells are used.

[0031] A production cell is a cell that possesses all the genes required for virus production. Preferably, the production cell is a cell that can integrate all the required genes, including the target gene (GOI) contained in the viral vector, into the chromosome.

[0032] Packaging cells are cells that possess a portion of the genes required for virus production. Preferably, these are cells capable of integrating a portion of the genes required for virus production into their chromosomes. Viruses can be produced by transfecting the remaining genes required for virus production into packaging cells that possess a portion of the genes required for virus production.

[0033] Examples of viruses include adeno-associated viruses, lentiviruses, baculoviruses, and retroviruses, with adeno-associated viruses being the preferred choice.

[0034] Adeno-associated virus (AAV) refers to a family of viruses in the Parvoviridae family and the Parvovirus genus, consisting of small, non-enveloped viruses with a single-stranded DNA of approximately 4700 bases and incomplete replication capacity. More than 100 serotypes of AAV are known, and their host range and viral characteristics vary depending on the serotype. Serotype 2 (AAV2) is one of the most extensively studied serotypes with a very broad host range. Serotypes 1 (AAV1), 5 (AAV5), and 6 (AAV6) are serotypes with higher tissue specificity. It can be said that AAV1 has high gene delivery efficiency for muscles, liver, respiratory tract, central nervous system, etc.; AAV5 has high gene delivery efficiency for the central nervous system, liver, retina, etc.; and AAV6 has high gene delivery efficiency for the heart, muscles, liver, etc. In this invention, serotype 2 or serotype 5 is preferred. Serotype 5 is particularly preferred.

[0035] Adeno-associated virus (AAV) genes are genes composed of one or more nucleic acid sequences derived from one or more serotypes of adeno-associated viruses. Preferably, AAV genes are those involved in AAV replication and packaging, and those encoding AAV constituent proteins.

[0036] AAVs are non-enveloped viruses that replicate in the presence of helper viruses such as adenoviruses and herpesviruses. In the production of AAVs for gene therapy or nucleic acid delivery, the classic approach is to co-infect host cells with adenoviruses to replicate the AAVs. Furthermore, the gene responsible for the helper role of the adenovirus is identified, and plasmids integrating this gene are used. For example, recombinant AAVs (rAAVs) can be packaged by simultaneously transfecting cells with plasmids containing the Rep and Cap genes, an adenovirus helper plasmid, and a plasmid containing genes for treatment or prophylaxis.

[0037] The Rep and Cap genes encode proteins involved in viral particle replication and packaging. In the wild type, the Rep gene is expressed from the P5 and P19 promoters. The Cap region expresses VP1, VP2, and VP3. The P40 promoter is a naturally occurring promoter for the Cap gene.

[0038] Adeno-associated virus genes (such as Rep and Cap genes) can be wild-type genes, but as long as the gene performs its original function, it can be a wild-type gene that has been modified by base substitution, deletion, insertion, or addition.

[0039] When altering wild-type adeno-associated virus (AAV) genes (such as the Rep gene and Cap gene) through base substitution, deletion, insertion, or addition, the number of altered bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. The modified AAV gene sequence preferably shows more than 85% sequence homology with the wild-type AAV gene sequence, more preferably more than 90% sequence homology, even more preferably more than 95% sequence homology, and even more preferably more than 98% sequence homology.

[0040] When the Rep and Cap genes are introduced into cells, a vector containing the Rep and Cap genes can be introduced into the cells. There are no particular limitations on the arrangement or orientation of the Rep and Cap genes in the vector, but it is preferable that the Rep and Cap genes have different sequences.

[0041] The Rep gene refers to a region of the AAV genome that encodes viral replication proteins required for replication of viral genomes known to those skilled in the art, or, for example, the Rep gene of human herpesvirus 6 (HHV-6) (known for mediating AAV-2 DNA replication) and its functional homologs. Therefore, the coding region of the Rep gene at least includes genes encoding AAV's REP78 and REP68 (long forms of REP proteins) and REP52 and REP40 (short forms of REP proteins), or their functional homologs. The coding region of the Rep gene used in this invention can be derived from any AAV serotype, but is preferably derived from AAV2. Examples of proteins derived from AAV2 include REP78 and REP68, REP52 and REP40, and ITR.

[0042] The Cap gene refers to the region in the AAV genome that encodes viral capsid proteins known to those skilled in the art. Examples of these capsid proteins are AAV capsid proteins VP1, VP2, and VP3. The Cap gene used in this invention can be derived from any AAV serotype, but is preferably derived from AAV2 or AAV5. AAV5 is particularly preferred.

[0043] As a vector containing the Rep gene and the Cap gene, for example, plasmids, viral nucleic acid sequences, artificially synthesized nucleic acids, etc., plasmids are preferred.

[0044] In this invention, a target gene (GOI) can be introduced into cells. The GOI is preferably introduced in a state sandwiched within an ITR. The GOI is preferably a therapeutic or preventative gene.

[0045] Genes used for treatment or prevention can be genes that are incomplete or missing in the genome of target cells, or genes that encode non-natural proteins with the desired biological or therapeutic effect (e.g., antiviral function), but there are no particular limitations. Specific examples of genes used for treatment or prevention include genes used for the treatment or prevention of inflammatory diseases, autoimmune diseases, chronic and infectious diseases (including AIDS, cancer, neurological diseases, cardiovascular diseases, hypercholesterolemia, etc.), various blood diseases such as anemia and hemophilia, and gene defects (e.g., cystic fibrosis, Gaucher disease, adenosine deaminase (ADA) deficiency, emphysema, etc.).

[0046] Genes used for treatment or prevention can also be antisense oligonucleotides (such as short chain oligonucleotides complementary to sequences surrounding the translation start site (AUG codon) of mRNA) that are useful in antisense therapies for cancer and viral diseases.

[0047] GOI can be linked to a promoter used to express GOI. There are no particular limitations on the promoter used to express GOI, but examples include promoters derived from cytomegalovirus (including enhancers as needed), SV40 early promoters, human elongation factor-1α (EF-1α) promoters, human ubiquitin C promoters, the Rous sarcoma virus LTR promoter (a retrovirus), dihydrofolate reductase promoters, β-actin promoters, and phosphoglycerate kinase (PGK) promoters. Preferably, the GOI and the promoter used to express the gene are nested within an ITR sequence.

[0048] In this invention, it is preferable to introduce a viral helper gene derived from adenovirus into the cell. A viral helper gene is a non-adeno-associated virus gene used for the replication and packaging of adeno-associated virus. As a viral helper gene, genes derived from viruses other than adeno-associated virus can be used. Specific examples of viral helper genes include those derived from adenovirus or herpesvirus, with adenovirus being the preferred source.

[0049] Examples of viral helper genes derived from adenovirus include E1A, E1B, E2A, E4, and VA-RNA. In host cells possessing all or part of the E1 region, the AAV genome is replicated and packaged into the capsid. The regions of the adenovirus genome required for viral particle formation are the E2A, E4, and VA-RNA regions. Regarding the function based on the E4 region, AAV replication requires the E4 34kDa protein encoded by the open reading frame 6 (E4ORF6) of the E4 region. Preferred viral helper genes are the E2, E4, and VA-RNA genes. More preferred are the E2 and E4 genes. The VA-RNA gene is preferably the VA-RNAI gene.

[0050] Viral helper genes derived from adenovirus (such as E1A, E1B, E2A, E4, and VA-RNA) can be wild-type genes. However, as long as the gene performs its original function, it can be a gene that has been modified by base substitution, deletion, insertion, or addition to the wild-type gene.

[0051] When altering the wild-type viral helper gene through base substitution, deletion, insertion, or addition, the number of altered bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. The altered viral helper gene sequence preferably shows at least 85% sequence homology with the wild-type viral helper gene sequence, more preferably at least 90% sequence homology, even more preferably at least 95% sequence homology, and even more preferably at least 98% sequence homology.

[0052] When viral helper genes are introduced into cells, vectors containing viral helper genes can be introduced into cells.

[0053] As a vector containing viral helper genes, for example, plasmids, viral sequences, artificially synthesized nucleic acids, etc., plasmids are preferred.

[0054] Viral helper genes are preferably controlled by promoters, but can also be controlled by promoters that can regulate expression.

[0055] As promoters capable of regulating expression, these are promoters that can regulate the on and off states of expression based on the presence or absence of stimuli that conditionally induce expression. Examples of stimuli that conditionally induce expression include chemical or physical stimuli, but are not particularly limited. Examples of chemical stimuli include endogenous hormones, stress responses, lactose, tetracyclines or their derivatives (e.g., doxycycline), cuminic acid, rapamycin, FKCsA, abscisic acid (ABA), tamoxifen / Cre-loxP (a system using a Cre promoter modified to induce activation via tamoxifen), and riboswitch mechanisms. Examples of physical stimuli include blue light and heat.

[0056] In the above, the promoter capable of expression regulation is preferably a promoter whose expression can be regulated by a drug. As a drug, a tetracycline is preferred, and doxycycline is more preferred. That is, the stimulus for conditionally specific induction of expression can be that the culture medium contains doxycycline.

[0057] Specifically, examples of promoters capable of expression regulation include tetracycline-responsive promoters, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, and metallothionein promoters. Among specific tetracycline-responsive promoters, the Tet on / off system (manufactured by TET Systems GmbH & Co. KG) is an example.

[0058] The promoters that can regulate expression can be promoters that can regulate expression through tetracycline, i.e., Tet-on / off systems, or Tet-on systems.

[0059] In the Tet activation system, the promoter additionally contains at least one Tet operon. By using the Tet operon (tetracycline-controlled transcriptional activation), transcription can be reversibly switched on or off in the presence of an antibiotic, namely tetracycline or one of its derivatives (e.g., doxycycline). Intracellular Tet repressor proteins block expression by binding to the Tet operator (tetracycline operon) sequence introduced into the promoter. Therefore, no gene expression is observed when the Tet repressor is bound to the Tet operator sequence. If tetracycline or doxycycline is added, the Tet repressor is isolated, allowing promoter activity and enabling gene expression. Tet operon systems are widely available, such as the Tet operon used in the pcDNA (trademark) 4 / TO mammalian expression vector available from Invitrogen. In a Tet-enabled system, the Tet operator sequence can be upstream or downstream of the promoter from which expression is to be regulated. To minimize expression leakage when expression is disabled, the Tet operator sequence is preferably downstream of the regulated promoter.

[0060] There are no specific limitations on the specific examples of promoters, but examples that can be cited include promoters derived from cytomegalovirus (CMV promoters) (which may include enhancers as needed), SV40 early promoters, human elongation factor-1α (EF-1α) promoters, human ubiquitin C promoters, Rous sarcoma virus (RSV) LTR promoters, dihydrofolate reductase promoters, β-actin promoters, and phosphoglycerate kinase (PGK) promoters.

[0061] Exogenous recombinant nucleic acids refer to recombinant nucleic acids introduced from outside the cell into the cell. Exogenous recombinant nucleic acids, in the form of a vector containing them, can be introduced into cells through methods such as transfection, transduction, and lipid transfection.

[0062] Transfection refers to the introduction of nucleic acids into eukaryotic cells by crossing the cell membrane through chemical methods (e.g., using reagents such as calcium phosphate or polyethyleneimine), mechanical methods (e.g., electroporation), or physical methods (e.g., bioballistic delivery).

[0063] Transduction refers to the process by which nucleic acids are introduced into eukaryotic cells via a viral vector that crosses the cell membrane.

[0064] Lipid transfection refers to the process by which nucleic acids are introduced into cells through endocytosis or membrane fusion, forming a complex of carriers and positively charged lipids through electrical interactions.

[0065] In animal cells, exogenous recombinant nucleic acids are preferably expressed in constitutive expression systems. Constitutive expression systems refer to methods that express nucleic acids for more than one month. Long-term maintenance of target gene expression can be achieved by inserting plasmids into chromosomes, introducing exogenous vectors, or using artificial chromosomes to introduce exogenous nucleic acids. In this case, if drug resistance genes are introduced into the plasmid, drug-based screening can be performed.

[0066] The recombinant nucleic acid is not particularly limited as long as it is a sequence capable of expressing the virus. A sequence expressing adeno-associated virus (AAV) is preferred. As mentioned above, sequences derived from helper viruses and sequences derived from AAV are known as sequences expressing AAV, but this is not a limitation as long as the sequence can express AAV. Components derived from helper viruses include E1A, E1B, E2A, E4, and / or VA-RNA, but are not limited to these. Sequences derived from AAV include Rep and Cap, but are not limited to these.

[0067] In this invention, preferably at least one of the exogenous recombinant nucleic acids contains a promoter controlled by RNA polymerase III. In this invention, the exogenous recombinant nucleic acid is preferably a nucleic acid encoding E2A, E4, VA-RNA, Rep and Cap.

[0068] At least one of the exogenous recombinant nucleic acids preferably contains a nucleic acid sequence that is conditionally specifically induced for expression. The promoter induced by the conditionally specifically induced nucleic acid sequence is not particularly limited, but a doxycycline-inducible TET promoter is preferred.

[0069] In this invention, it is preferred to use production cells or packaging cells in which the Rep gene, Cap gene, and helper gene are integrated into the chromosome; it is even more preferred to use production cells in which the Rep gene, Cap gene, and helper gene are integrated into the chromosome; it is even more preferred to use production cells in which the Rep gene, Cap gene, helper gene, and GOI are integrated into the chromosome; and it is particularly preferred to use production cells in which the Rep gene, Cap gene, helper gene, and therapeutic or preventive gene are integrated into the chromosome.

[0070] The cells used in this invention are preferably animal cells. There is no particular limitation on the animal cells used, but mammalian cells or insect cells are preferred, and mammalian cells are more preferred. Examples of mammalian cells include, for example, human cells, mouse cells, rat cells, monkey cells, hamster cells, etc., but are not particularly limited. Human cells are preferred. Examples of cells include mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK, Jurkat, NIH3T3, PC12, BHK (juvenile hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS (e.g., COS1 and COS7), QC1-3, HEK293 (kidney cells derived from human fetuses), VERO, PER.C6, HeLa, EB1, EB2, EB3, oncolytic, or hybridoma cell lines. The preferred cells are HEK293 cells and CHO cells, with HEK293 cells being more preferred. The cells used in this invention are preferably cells adapted to growth in suspension culture. Preferably, the cells are HEK293 cells or CHO cells adapted to growth in suspension culture, and more preferably, HEK293 cells adapted to growth in suspension culture.

[0071] The method of the present invention includes the step of culturing cells for production or packaging in a culture medium. Cell culture can be carried out under the usual conditions used for cell culture. The cell density in the viral vector manufacturing process is 1×10⁻⁶. 6 Cells / mL or higher, more preferably 10 × 10 6 Cells / mL or higher, more preferably 20 × 10⁶ 6 Cells / mL or higher, more preferably 30 × 10⁶ 6 Cells / mL or higher, more preferably 50 × 10⁻⁶ 6 Cells / mL or higher, more preferably 70 × 10⁶ 6 Cells / mL or higher, or 100 × 10⁻⁶ 6 Cells / mL or higher. There is no specific upper limit to the cell density, but it is usually 1000 × 10⁻⁶.6 Cells / mL or less.

[0072] In this invention, a cell proliferation process can be performed before the viral vector manufacturing (generation) process.

[0073] For example, cells can be cultured in a medium that does not contain doxycycline or histone deacetylase inhibitors until they reach a density of 1×10⁶ cells before the manufacturing process. 4 cells / mL or higher (more preferably 1×10⁻⁶ cells / mL) 5 cells / mL or higher, more preferably 1×10⁻⁶ 6 cells / mL or higher, more preferably 10 × 10⁻⁶ 6 cells / mL or higher, more preferably 30 × 10⁻⁶ 6 cells / mL or higher, especially preferably 100 × 10⁻⁶. 6 The virus is produced by a proliferation process (cells / mL or higher) followed by a virus manufacturing process using a culture medium containing doxycycline and histone deacetylase inhibitors. There are no particular restrictions on the culture method for the proliferation process, but it can also be carried out in perfusion culture. After the cells have proliferated in perfusion culture, a portion of the cells can be transferred to different containers and the manufacturing process can be carried out.

[0074] There is no particular limitation on the cell culture temperature; it shall be carried out at a temperature at which the cells can survive. The culture temperature is generally 25℃ to 45℃, preferably 30℃ to 42℃, more preferably 35℃ to 40℃, and as an example, 37℃. The CO2 concentration is generally 3-10%, preferably 5-10%, and as an example, it is 8%.

[0075] As a culture medium, batch culture, fed-batch culture, perfusion culture, or shaking culture can be performed. At least a portion of the virus manufacturing process is preferably perfusion culture. For culture, cells are preferably in suspension, and suspension culture is preferred.

[0076] Flasks or bioreactors can be used as culture containers, but there are no particular limitations.

[0077] There is no particular limitation on the culture scale; cells can be cultured in any volume of culture medium, for example, from 1 mL to 5000 L. The lower limit is preferably 1 L, more preferably 10 L, further preferably 50 L, and especially preferably 100 L. The upper limit is preferably 5000 L, more preferably 2500 L, more preferably 1000 L, and especially preferably 500 L.

[0078] Culture can be carried out simultaneously with vibration stirring. The stirring speed during vibration stirring is typically 50 rpm to 200 rpm, preferably 80 to 150 rpm. Stirred culture can be based on rotary stirring using a propeller or similar device within the reactor. The stirring speed during rotary stirring is typically 50 rpm to 200 rpm, preferably 80 to 150 rpm. Furthermore, stirring can be performed by wave-like vibration stirring or by the up-and-down movement of stirring blades, but there are no particular limitations.

[0079] Examples of culture media, not limited to these, include Expi293 Expression Medium (Thermo Fisher Scientific, A1435101), Dulbecco modified Eagle medium (DMEM) containing 10% (vol / vol) fetal bovine serum (FBS), serum-free UltraCULTURE (trademark) medium (Lonza), STEMPRO (trademark)-34SFM medium, Human Endothelial-SFM, GIBCO (registered trademark) FREESTYLE (trademark) 293 expression medium, CD CHOAGT medium, CHO-S-SFM medium, GIBCO (registered trademark) FREESTYLE (trademark) CHO expression medium, CD OPTICHO (trademark) medium, CD CHO medium, CD DG44 medium, SF-900 (trademark) medium, Expi293 (trademark) expression medium, 293 SFM medium, BalanCD (registered trademark) HEK293 medium, and any derivatives or variations thereof. In a particular, non-limiting embodiment, the high-density culture medium may be CD FORTICHO (trademark) medium, CD CHOAGT medium, CHO-S-SFM medium, GIBCO (registered trademark) FREESTYLE (trademark) CHO expression medium, CD OPTICHO (trademark) medium, CD CHO medium, CD DG44 medium, GIBCO (registered trademark) FREESTYLE (trademark) 293 expression medium, Expi293 (trademark) expression medium, LV-MAX (trademark) production medium, FREESTYLE (trademark) F17 expression medium, DYNAMIS (trademark) medium, BalanCD (registered trademark) HEK293 medium, or the same medium or their variants.

[0080] The method for manufacturing a viral vector based on the present invention may include a step of recovering the virus. The viral vector manufactured within the cell where it is produced remains within the cell or is released into the culture supernatant.

[0081] In cases where viral vectors are recovered from cells, samples containing viral vectors can be prepared, for example, by disrupting cells with ultrasound, by freezing and thawing cells, or by contacting cells with an acidic solution or a surfactant. Samples prepared in this way can be used directly or purified as needed. When viral vectors are recovered from culture supernatant, the culture supernatant can be used directly as a sample containing viral vectors, or it can be purified as needed.

[0082] The purification method for purifying viral vectors is not particularly limited; for example, purification methods such as CsCl gradient ultracentrifugation, chromatography, or ultrafiltration can be used. Using these methods, viral vectors can be purified from samples containing them. Furthermore, if necessary, gDNA (genomic DNA) or residual plasmids can be digested by adding MgCl2 and Benzonase to the purified sample as described above.

[0083] The titer of the viral vector produced by the method of the present invention can be determined using conventional methods known to those skilled in the art. For example, the culture medium after culture is recovered, and the cells are disrupted by freeze-thaw cycles, followed by centrifugation to recover the supernatant. The gDNA (genomic DNA) and residual plasmids can be digested by adding MgCl2 and Benzonase (nuclease) to the recovered supernatant. Samples containing the viral vector obtained as described above can be used as ddPCR (Droplet Digital PCR) samples, and the titer of the viral vector can be determined by performing ddPCR.

[0084] Furthermore, the capsid particle titer (vp / mL) can be determined using an ELISA kit, and the Full rate can be calculated based on the ratio to the genomic titer (vg / mL) calculated by ddPCR. Alternatively, the Full / Empty ratio can be determined through analysis using virus extraction, HPLC analysis of purified sample solutions, or isoelectric point capillary electrophoresis, within the same assay system.

[0085] In one example of the present invention, by culturing in the presence of a histone deacetylase inhibitor, the production yield of viral vectors such as AAV can be increased by at least 1.5 times, more preferably by at least 2 times, and even more preferably by at least 3 times compared to the case where no histone deacetylase inhibitor is used.

[0086] In one example of the present invention, in the virus produced during the culture process, the ratio of the amount of capsid containing the complete gene to the total amount of capsid (Full rate) is preferably 5% or more, more preferably 10% or more, further preferably 20% or more, more preferably 25% or more, and especially preferably 30% or more.

[0087] The present invention will be further described in detail through the following embodiments, but the present invention is not limited to the embodiments. Example

[0088] [Materials and Methods] <Cell Maintenance Culture> Plasmids (Sequence No. 1 and Sequence No. 2) were introduced into HEK293 cells (Thermo Fisher Scientific, A49784) adapted for growth in suspension culture, and stable expression lines (AAV production cells) with sequence 1 (containing the reverse tetracycline-controlled transactivator (rtTA) gene, Rep gene, Cap gene, E2 gene, E4 gene, and GFP gene sandwiched between ITR sequences (as GOI), with Rep, Cap, E2, and E4 genes under the control of a promoter with a tetracycline response factor (TRE) sequence) integrated into the cell chromosome were cultured in BalanCD HEK293 medium (FUJIFILM Mirvine Scientific, 551-34231). The cell density was 2 × 10⁻⁶ cells / year. 6 ~30×10 6 The culture was suspended at cells / mL and maintained in a 125mL shaking flask. The culture was incubated at 37°C and 8% CO2 while the medium was stirred at a constant 120 rpm.

[0089] <Perfusion Culture> It will be 0.5×10 6 AAV production cells suspended in 280 mL of BalanCD HEK293 medium (FUJIFILM Irvine Scientific, 551-34231) were seeded into a 0.5 L reactor (BiotT). For perfusion culture, both BalanCD HEK293 medium (FUJIFILM Irvine Scientific, 551-34231) and BalanCD HEK293 Feed (FUJIFILM Irvine Scientific, 550-34245) were used.

[0090] <Plasmid Construction> The complete sequence of the plasmid used is shown in sequence numbers 1–5 of the sequence listing. The sequences were prepared by total synthesis.

[0091] [Table 1]

[0092] <Determination of AAV titer> HEK cell culture medium, after gene introduction, was recovered and cultured at 37°C and 8% CO2 for 72 hours, followed by cell lysis via freeze-thaw at -80°C. The supernatant was then recovered by centrifugation at 13,800×g, and MgCl2 and Benzonase were added to a final concentration of 2 mmol / L. The reaction was carried out at 37°C for 2 hours to digest gDNA (genomic DNA) and residual plasmids. The reacted samples were incubated sequentially at 95°C for 15 minutes, 70°C for 3 minutes, 40°C for 3 minutes, and 20°C for 3 minutes to inactivate Benzonase, serving as ddPCR (Droplet Digital PCR) samples. The ddPCR samples were diluted 50-fold with TE buffer (pH 8.0, 0.05% Pluronic F-68 and containing 10 μg / mL bovine thymus DNA). 10 μL of the ddPCR sample was then added to ice. TM Supermix for Probes (ddPCR probe premix) (nodUTP) (BIORAD), 2 μL of diluted ddPCR sample, primers (Sequence No. 3 and 4, final concentration 900 nmol / L), probe (Sequence No. 5, final concentration 250 nmol / L), and nuclease-free water (Thermo Fisher Scientific, 10977015) were mixed into a tube to a total volume of 22 μL for ddPCR. In ddPCR, droplets were formed from the preparation solution using a droplet forming device and incubated sequentially at 95°C for 10 min (preheating), 94°C for 30 s (transformation), 55°C for 60 s (annealing and extension) for 40 cycles, followed by incubation at 98°C for 10 min. The AAV genomic titer (vg / mL) was calculated using a droplet reader.

[0093] Experiment 1: <Method> AAV production cells were produced at a rate of 1×10 6Cells were seeded at 24-well plates at concentrations of 2.5 μmol / L and 5.0 μmol / L, respectively, with doxycycline (final concentration 0.5 μg / mL) and the Epigenetics Screening Library (CAYMAN, 11076) added. After 72 hours of treatment, cells were recovered and AAV titers were determined.

[0094] <Results> Compounds that improve the titer relative to the titer when DMSO is added were selected, and the titer determination results are presented. Figure 1 The compounds are listed in Table 2. The screening results clearly show that HDAC inhibitors increase titers.

[0095] [Table 2]

[0096] Experiment 2: <Method> AAV-producing cells were cultured in a reactor at a rate of 30 × 10⁻⁶. 6 After culturing cells / mL, 24 mL of culture medium was transferred to a 125 mL flask. Doxycycline (final concentration 5 μg / mL) and CI-994 (CAYMAN, 11076) were added to the cell culture medium to a final concentration of 7.5 μmol / L. Every other day, 140 μL of BalanCDHEK293 Feed (FUJIFILM Irvine Scientific, 550-34245) and 10 μL of 45 w / v% D(+)-glucose solution (FUJIFILM Wako Pure Chemical, 079-05511) were added to each 1 mL of cell culture medium to maintain the cells. After treatment for 72 hours following the addition of the reagents, the cells were recovered and AAV titers were determined.

[0097] <Results> The results of the titer determination are shown in Figure 2 The titer relative to the untreated CI-994 sample (CI-994 conc: 0 μM) was 1.1 × 10⁻⁶. 12 The titer of the sample treated with CI-994 (CI-994 conc: 7.5 μM) was 2.2 × 10⁻⁶ vg / mL. 12 The result of using vg / mL clearly demonstrates that the titer is improved in high-density cell culture media.

[0098] Similarly, titer improvement was observed for the HDAC inhibitors listed in Table 2 other than CI-994.

[0099] Experiment 3: <Method> Similar to Experiment 2, AAV-producing cells were perfused in a reactor at a rate of 30 × 10⁶ cells / year. 6 Cells were cultured at concentrations above 30 cells / mL, and the culture medium was transferred to flasks for AAV production studies. Every 24 hours, 30% of the total cell culture medium was aliquoted from the reactor and redissolved with fresh culture medium, thus maintaining a concentration of 30 × 10⁶ cells / mL for 7 days. 6 Cells were cultured at a density of ≥1000 cells / mL, and 24 mL of the aliquoted cell culture medium was transferred to a 125 mL flask. Doxycycline (5 μg / mL) and CI-994 (CAYMAN, 11076) were added to the cell culture medium to a final concentration of 7.5 μmol / L. Every other day, 140 μL of BalanCD HEK293 Feed (FUJIFILM Irvine Scientific, 550-34245) and 10 μL of 45 w / v% D(+)-glucose solution (FUJIFILM Wako Pure Chemical, 079-05511) were added to each 1 mL of cell culture medium to maintain cell density. After treatment for 72 hours following the addition of the reagents, the cells were recovered and AAV titers were determined.

[0100] <Results> The changes in cell density within the reactor are shown in Figure 3 The titer of AAV produced in cell culture medium aliquots taken every 24 hours is shown in the figure. Figure 4 Under conditions where cell density was maintained at a high level using a reactor while AAV was continuously generated in another culture vessel over 24 hours, the treated CI-994 sample (+CI-994) showed a high titer compared to the untreated CI-994 (-CI-994), thus confirming a titer-improving effect based on the HDAC inhibitor.

[0101] Experiment 4: <Method> For the samples from day 7 of Experiment 3, AAV was extracted and purified using the AAVproPurification Kit (Takara Bio, 6675). The purified sample solution was analyzed using high-performance liquid chromatography (HPLC) to determine the peak areas of the Empty and Full particles of AAV, and the Full rate (the ratio of the amount of capsid containing the complete gene to the total amount of capsid) was calculated. An anion exchange column was used for separation, and a fluorescence detector (excitation: 280 nm, detection: 348 nm) was used for detection.

[0102] <Results> The determination results of untreated CI-994 (-CI-994) and treated CI-994 (+CI-994) obtained by high performance liquid chromatography are shown below. Figure 5 The peak areas were measured, and the ratio of the area of ​​the peaks representing Full particles to that representing Empty particles is shown in Table 3. This demonstrates that CI-994 treatment improves the Full rate.

[0103] [Table 3]

[0104] Experiment 5: <Method> AAV production cells were prepared at a rate of 0.2 × 10⁻⁶. 6 cells / mL, 1.0×10 6 cells / mL, 6.0×10 6 cells / mL, 24×10 6 Cells / mL were seeded in 125 mL flasks. Doxycycline (final concentration 0.5 μg / mL) was added, and after 72 hours of treatment, the cells were recovered and the AAV titer was determined.

[0105] <Results> The higher the cell density of AAV-producing cells, the lower the titer. Specifically, if set to 0.2 × 10⁻⁶... 6 cells / mL up to 1.0 × 10⁻⁶ 6 With a cell density of cells / mL, the titer becomes approximately 1 / 5. If the cell density is 1.0 × 10⁻⁶, the titer becomes approximately 1 / 5. 6 When HDAC inhibitors are added to cells at a density of 0.2 × 10⁶ cells / mL or higher to induce viral production, the decrease in titer is suppressed compared to the absence of HDAC inhibitors, or at a concentration of 0.2 × 10⁶ cells / mL. 6 Using the titer per cells / mL (without HDAC inhibitors) as a baseline, the same or higher titers can be obtained.

[0106] Experiment 6: <Method> AAV production cells were prepared at a rate of 0.2 × 10⁻⁶. 6 cells / mL, 1.0×10 6 cells / mL, 6.0×10 6 cells / mL, 24×10 6 cells / mL, 30×10 6 Cells / mL were seeded in 125mL flasks, and doxycycline (final concentration 0.5μg / mL) was added alone. After 72 hours of treatment, the cells were recovered and the AAV titer was determined. AAV production cells were prepared at a rate of 0.2 × 10⁻⁶. 6 cells / mL, 1.0×10 6 cells / mL, 8.0×10 6 cells / mL, 30×10 6 Cells / mL were seeded in 125mL flasks, and doxycycline (final concentration 0.5μg / mL) and CI-994 (final concentration 7.5μg / mL) were added. After treatment for 72 hours, the cells were recovered and the AAV titer was determined.

[0107] <Results> The higher the cell density of AAV-producing cells, the lower the titer per cell. Figure 6 Specifically, if set to 0.2 × 10⁻⁶ 6 cells / mL up to 1.0 × 10⁻⁶ 6 At a cell density of 1.0 × 10⁻⁶ cells / mL, the titer becomes approximately 1 / 5, but if it is 1.0 × 10⁻⁶ cells / mL... 6 When CI-994 was added to cells at a density of 0.2 × 10⁻⁶ cells / mL or higher to induce viral production, the decrease in titer was inhibited. 6 Using the titer of cells / mL (without CI-994 treatment) as a baseline, titers of the same level can be obtained.

[0108] Experiment 7: <Method> AAV production cells were produced at a rate of 1×10 6 Cells were seeded at concentrations of 1.3 μmol / L in 24-well plates and doxycycline (final concentration 0.5 μg / mL) and various HDAC inhibitors (CI-994, Scriptaid, Resminostat, CBHA) were added to final concentrations of 1.3 μmol / L, 2.5 μmol / L, 5 μmol / L, and 10 μmol / L. After 96 hours of treatment, cells were recovered and AAV titers were determined.

[0109] <Results> Compounds that improve the titer relative to the titer when DMSO is added were selected, and the titer determination results are presented. Figure 7 The concentrations of various compounds above 5 μmol / L showed a particularly high titer-enhancing effect.

[0110] [Inspection] In this technique, it is hypothesized that inserting the gene required for AAV vector expression into the host cell's chromosome (AAV producer cell) induces changes in chromatin structure by inhibiting the activity of histone deacetylase (HDAC) expressed intracellularly, thereby promoting histone acetylation and contributing to increased AAV titer and fullness. Typically, histone acetylation is known to cause chromatin disaggregation; therefore, it is assumed that increased expression of the inserted gene required for AAV production leads to improved titer. Furthermore, it is hypothesized that chromatin disaggregation contributes to improved intact particle size by facilitating increased GOI replication within the cell containing the AAV vector.

[0111] Serial Number 1:

[0112] Serial Number 2:

[0113] Serial Number 3: GGAACCCCTA GTGATGGAGT T Serial Number 4: CGGCCTCAGT GAGCGA Serial Number 5: CACTCCCTCT CTGCGCGCTC G

Claims

1. A method for manufacturing a viral vector, comprising the following steps: in a container containing cells at a density of 1×10⁻⁶ cells... 6 Production or packaging cells are cultured in a medium containing at least one cell / mL of production or packaging cells and a histone deacetylase inhibitor.

2. The manufacturing method according to claim 1, wherein, Histone deacetylase inhibitors are compounds with a benzamide structure or hydroxamic acid.

3. The manufacturing method according to claim 1, wherein, Histone deacetylase inhibitors are compounds represented by formula (1) or formula (2). R 1 -CONH-R 2 (1) In equation (1), R 1 and R 2 One of them is an aromatic group that can have substituents, R 1 and R 2 The other one is an amino group that can have a substituent, an alkyl group that can have a substituent, an alkenyl group that can have a substituent, an aromatic group that can have a substituent, or an alkoxy group that can have a substituent; R 3 -CONH-OH (2) In equation (2), R 3 It can be an alkyl group that may have substituents or an alkenyl group that may have substituents.

4. The manufacturing method according to claim 1, wherein, Histone deacetylase inhibitors are compounds represented by formula (1A), formula (1B), or formula (2A). [Chemical Formula 1] In equation (1A), R 10 R represents a halogen, a heterocyclic group that may have substituents, an alkyl group having 1 to 6 carbon atoms that may have substituents, or an amino group that may have substituents. 2 It can be an amino group that may have substituents, an alkyl group that may have substituents, or an aromatic group that may have substituents; [Chemical Formula 2] In equation (1B), R 20 R represents an amino group. 1 It can be an alkyl group that may have substituents or an aromatic group that may have substituents; R 31 -R 30 -CONH-OH (2A) In equation (2A), R 30 It represents -(CH2) n - or CH=CH-, where n represents an integer from 2 to 8, R 31 This indicates that it can have a substituent aromatic group or CONH-OH.

5. The manufacturing method according to claim 1, wherein, In equation (1A), R 10 express bromine, [Chemical Formula 3] 、 methyl, [Chemical Formula 4] 、 or CH3-CO-NH-.

6. The manufacturing method according to claim 1, wherein, Histone deacetylase inhibitors are any of the following compounds. [Chemical Formula 5] 。 7. The manufacturing method according to any one of claims 1 to 6, comprising the step of perfusion culture of cells.

8. The manufacturing method according to any one of claims 1 to 6, wherein, The virus is an adeno-associated virus.

9. The manufacturing method according to claim 8, wherein, Production or packaging cells are cells in which the Rep gene, Cap gene, and accessory genes are integrated into the chromosome.

10. The manufacturing method according to any one of claims 1 to 6, wherein, The cells are animal cells.

11. The manufacturing method according to any one of claims 1 to 6, wherein, The concentration of histone deacetylase inhibitor in the culture medium is below 50 μmol / L.

12. The manufacturing method according to any one of claims 1 to 6, wherein, Incubate for more than 24 hours in a culture medium containing histone deacetylase inhibitors.

13. The manufacturing method according to any one of claims 1 to 6, wherein, Cell density is 30×10 6 Cells / mL or higher.

14. The manufacturing method according to any one of claims 1 to 6, wherein, The culture is a suspension culture.

Images