High Eukaryotic Cell Density Transient Transfection Process for the Production of Recombinant Virus Vectors

Abstract

The present disclosure provides a process for producing recombinant viral vectors in eukaryotic cells. In particular, the present disclosure is a process for transient transfection of eukaryotic cells for producing recombinant viral vectors, the process comprising the step of transiently transfecting cells in a cell culture, wherein the cell culture has a surprisingly high cell density of at least 25 MVC / mL. After the transfection step, an optional cell recovery step can be performed depending on the type of host cell, the exogenous nucleic acid(s), and the transient transfection method used. After the transfection step, or after an optional cell recovery step, the production step of the recombinant viral vector starts at a surprisingly high cell density of at least 20 MVC / mL, either by limiting dilution after the transfection step or without performing dilution.

Description

【Technical Field】 【0001】 The present disclosure relates to the fields of transient transfection of cells and production of recombinant viral vectors. More specifically, the present disclosure relates to a high cell density transient transfection process for the production of recombinant viral vectors. 【Background Art】 【0002】 Transient transfection is a gene transfer technique widely used for producing biopharmaceuticals, which are molecules or molecular aggregates having biological activity. Instead of integrating into the genome of host cells, exogenous nucleic acid(s) is introduced into host cells for only a limited period. After transient transfection, cells incorporating the target nucleic acid(s) express the transfected genetic material and can enter the production stage of biopharmaceuticals, which can be proteins, polymers, viruses, virus-like particles, extracellular vesicles, etc. In contrast to the years required to establish a stable production cell line, transient transfection requires only a few days and is highly versatile for host cell lines, transfection vectors, and target products. Due to such advantages, transient transfection is an optimal candidate for the production of macromolecules such as recombinant proteins and viral vectors when intensive screening and characterization of target products and their production process parameters are performed in drug discovery research and preclinical stages, or when the expression pattern contains toxic moieties. However, in the biopharmaceutical industry, insufficient productivity of molecules such as viral vectors has always been reported due to bottlenecks in the production process such as low production yields and high production costs. 【0003】 In transient transfection, plasmid DNA is one of the common genetic materials introduced into host cells. Plasmid DNA is a small, circular, double-stranded DNA molecule, and in some cases, it is necessary to co-transfect multiple plasmids to produce a specific product. For example, for the production of recombinant adeno-associated virus vectors (rAAV) in the fields of gene therapy and cell therapy, the multi-plasmid transient transfection of HEK293 (human embryonic kidney 293) is currently the most widely used method [Xiao, et al. "Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus". J Virol 72 (1998):2224-2232; Grimm, et al. "Novel tools for production and purification of recombinant adeno associated virus vectors". Hum Gene Ther 9 (1998):2745-2760; Matsushita, et al. "Adeno-associated virus vectors can be efficiently produced without helper virus". Gene Ther 5 (1998):938-945]. Co-transfection is usually more difficult because all gene elements need to be introduced simultaneously, but due to technical constraints, it is sometimes the only or preferred option. Furthermore, the biosynthesis of recombinant viruses is very complex at a very high level and actually means the biosynthesis and accurate assembly of a capsid consisting of a total of 60 viral proteins, and the encapsulation of the target genome into the capsid.This manufacturing process is well known to have what is called the so-called cell density effect, where efficiency decreases as cell density increases in cell reference units [Maranga, L., et al. 2005. "Characterization of changes in PER.C6(TRADE MARK) cellular metabolism during growth and propagation of a replication-deficient adenovirus vector". Biotechnology and bioengineering, 90(5), pp. 645-655; Henry, O. et al. 2005. "Metabolic flux analysis of HEK-293 cells in perfusion cultures for the production of adenoviral vectors". Metabolic engineering, 7(5-6), pp. 467-476].Similar observations have also been reported for the production of rAAV by insect cells using a baculovirus-based expression system [Joshi et al., 2021. "Advancements in molecular design and bioprocessing of recombinant adeno-associated virus gene delivery vectors using the insect-cell baculovirus expression platform". Biotechnology Journal, 16(4), p.2000021.; Meghrous et al., 2005. "Production of recombinant adeno-associated viral vectors using a baculovirus / insect cell suspension culture system: From shake flasks to a 20-L bioreactor". Biotechnology progress, 21(1), pp.154-160.; Urabe et al., 2002. "Insect cells as a factory to produce adeno-associated virus type 2 vectors". Human gene therapy, 13(16), pp.1935-1943]. 【0004】 The manufacturing process of recombinant viral vectors is typically divided into three steps: the first step to obtain the desired number of cells, i.e., the step by cell expansion, the transient transfection step, and the manufacturing step. The cells that are rapidly increasing in the culture vessel(s) are first prepared for transient transfection, which is usually done by setting the density of the growing cells to about 600,000 to 800,000 viable cells per milliliter (0.6 - 0.8 MVC / mL) one day before transfection. On the day of transfection, the cell density reaches about 1 - 1.5 MVC / mL from cell growth and preferably grows exponentially. By this operation, the cells serving as the host for transfection can be obtained under culture conditions that are advantageous for transfection efficiency with a high cell survival rate. 【0005】 When performing transfection at a high cell density, the high-cell-density cell suspension can be obtained by concentrating a low-cell-density cell culture or by culturing the cells at a high density. After transient transfection, the cells into which the target nucleic acid(s) has been incorporated can express the transfected genetic material and enter the manufacturing stage. In the case of transfection at a high cell density, a dilution step is usually performed after transfection to return the cells to a low-cell-density culture to enable manufacturing. The time point at which the dilution is performed and the resulting cell density after dilution greatly depend on the type of host cell, the transient transfection method used, the experimental equipment, and the available control system. After a certain manufacturing period (typically 48 - 72 hours post-transfection (hpT)), the manufactured recombinant viral vector is harvested, and the manufacturing titer is carefully examined before proceeding to the next step of manufacturing, i.e., purification, etc. 【0006】 There are various commercially available non-viral transient transfection methods, such as physical methods and chemical methods. Chemical transfection is a widely adopted method that uses transfection reagents to form a complex with the target nucleic acid and delivers the target nucleic acid into the host cell through the cell membrane via the formed complex. Among the various commercially available transfection reagents, polyethyleneimine (PEI) is a stable cationic polymer, and this cationic polymer condenses negatively charged DNA into PEI-DNA complex microparticles. Subsequently, these complex microparticles bind to the cell surface and enter the cell through endocytosis / phagocytosis. The transfection process mediated by PEI is a popular technology because it is relatively low-cost and easy to operate. According to the state-of-the-art technology, chemical transfection for molecular manufacturing is generally carried out at a cell density of 5MVC / mL or less, and is operated at 1 - 2MVC / mL in the standard protocol. 【0007】 Another common physical transfection technique is electroporation, which can perform the transient transfection process at high cell densities (up to 100MVC / mL). This is done by concentrating the cell culture to a very high target cell density, usually by centrifugation, immediately before electroporation. After electroporation, the cell suspension is diluted to a low cell density of 5MVC / mL or less, such as 1 - 2MVC / mL, and enters the production stage. However, the application of this technology is limited by the operation scale and cost. Electroporation is used at a laboratory scale of less than 6 mL per batch, generally less than 1 mL. There are also larger-scale solutions, such as the flow electroporation technology commercially provided by Maxcyte, which can process cell suspensions of up to 100 mL at 100MVC / mL per batch. However, in the production stage, it is necessary to redilute to a low cell density of 5MVC / mL or less, such as 1 - 2MVC / mL, within 1 hour after electroporation. 【0008】 Therefore, the prior art methods are plagued by many drawbacks such as low production yields, being limited to small operation scales, and high production costs. The object of the present invention is to provide a process for transient transfection of cells and production of recombinant viral vectors that can potentially be used on a large scale to eliminate these drawbacks and reduce costs. 【Summary of the Invention】 【Problems to be Solved by the Invention】 【0009】 The present disclosure provides a process or method for producing recombinant viral vectors in eukaryotic cells. Herein, the process may also be referred to as a method, that is, the terms are used interchangeably herein. 【0010】 In particular, the present disclosure relates to a process for transient transfection of eukaryotic cells for producing recombinant viral vectors, which process includes the step of transiently transfecting cells in a cell culture, and the cell culture has a surprisingly high cell density of at least 25MVC / mL. 【Means for Solving the Problems】 【0011】 After the transfection step, an optional cell recovery step or process can be performed depending on or by the type of host cell, the exogenous nucleic acid(s), or the foreign plasmid DNA(s), and the transient transfection method used. After the transfection step, or after an optional cell recovery step or elapsed time, the production step of the recombinant viral vector starts at a surprisingly high cell density of at least 20MVC / mL with or without restricting dilution after the transfection step. The method includes a) obtaining a cell culture having a cell density of at least 25 million viable cells per milliliter; b) transiently transfecting the cells in the cell suspension with exogenous plasmid DNA or multiple exogenous plasmid DNAs required for producing the recombinant virus vector, i.e., co-transfecting with multi-plasmid DNA; c) optionally, recovering the cells for a period according to the type of host cell, exogenous plasmid DNA(s), and the transient transfection method used. During the cell recovery period, the transfected cells are maintained in a culture medium or solution having a large surface beneficial for gas exchange such as oxygen exchange or air exchange, and the transfected cells are normally kept in a stationary state, or essentially in a stationary state, and normally kept in a stationary state on a sufficiently large surface. This time is normally more than 1 minute and varies depending on the type of host cell, exogenous plasmid DNA(s), and the transient transfection method used; d) After the transfection step b) and the optional cell recovery step c), the manufacturing stage is started after restricting the dilution of the transfected cells or without dilution to start the manufacturing stage from a cell density of at least 20 million viable cells per milliliter after the transfection step. 【0012】 That is, provided herein is a process for producing a recombinant vector in a eukaryotic cell, the method comprising: a) obtaining a cell suspension having a cell density of at least 25 million viable cells per milliliter; b) performing transient transfection on the cells in the cell suspension with a single type of exogenous plasmid DNA or multiple different types of exogenous plasmid DNAs to obtain transfected cells; c) optionally, performing cell recovery for a certain period, wherein during the cell recovery period, the transfected cells are kept essentially in a stationary state on the surface; and d) After the transfection of step b) and / or after the optional cell recovery step c), restricting or not performing at all the dilution of the transfected cells present in the cell suspension, and then starting the production of the recombinant vector from a culture of transfected cells having a cell density of at least 20 million viable cells per milliliter Also disclosed is the above process, which comprises. 【0013】 The surface of step c) above is usually the surface of a container or vessel, etc., as described elsewhere in this specification. 【0014】 The desired recombinant viral vector is harvested some time after transfection, typically between 24 and 240 hours post-transfection (hpT), although longer production times are also envisaged, as disclosed elsewhere in this specification. 【0015】 By partially or completely eliminating the dilution step of returning to a low cell density of 5 MVC / mL or less, for example 1 - 2 MVC / mL, after transfection, the manufacturing process is significantly simplified. The same container can be used for cell culture, transfection, and production from the start of inoculation of the host cells until the final harvest of the desired recombinant viral vector. Therefore, the manufacturing footprint is largely minimized, enabling the entire manufacturing process to be scaled up for large-scale industrial production. 【Brief Description of the Drawings】 【0016】 【Figure 1】Figure 1 shows the titer of rAAV capsids / mL produced when varying the ratios of cell number to plasmid DNA and plasmid DNA to PEI (polyethyleneimine) in the transient transfection process of the present invention performed at 50 MVC / mL. White bars: experimental control, cell density at transfection in two different media was 1 MVC / mL; black bars: groups 3 to 12, cell density at transfection was 50 MVC / mL, and the ratios of cell number (1 million cells), plasmid DNA (μg), and PEI (μg) were as follows. In the groups of 1:1:1, 1:1:2, 1:1:4, and 1:1:8, the ratio of plasmid DNA to cell number was 1 μg DNA per MVC, and the ratios of DNA (μg):PEI (μg) were 1:1, 1:2, 1:4, 1:8, respectively. In the groups of 1:2:2, 1:2:4, and 1:2:8, the plasmid DNA was 2 μg DNA per million cells, and the ratios of DNA (μg):PEI (μg) were 1:1, 1:2, and 1:4, respectively. In the groups of 1:4:4, 1:4:8, 1:4:16, the plasmid DNA was 4 μg DNA per million cells, and the ratios of DNA (μg):PEI (μg) were 1:1, 1:2, and 1:4, respectively. 【Figure 2】 Figure 2 shows the production titer of rAAV capsids / mL at 72 h pT in the transient transfection process performed at 50 MVC / mL when varying the incubation time of the cationic polymer and DNA, i.e., 5 minutes, 10 minutes, 15 minutes, 0 minutes ("rapid mixing"), and when adding the DNA to the cell suspension and then immediately adding the cationic polymer to the cell suspension without pre-mixing the cationic polymer and DNA ("direct transfection"). 【Figure 3】Figure 3 shows the production titer of rAAV capsid / mL after the transient transfection process of the present invention performed at 100MVC / mL, and then AAV production harvested at 72 hpT was diluted at different cell densities using different medium exchange frequencies; from left to right: 1) diluted to 10MVC / mL and used as an experimental control for cell density diluted outside the present invention, medium exchange at 24 hpT and 48 hpT; 2) diluted to 10MVC / mL and used as an experimental control for cell density diluted outside the present invention, one medium exchange at 48 hpT; 3) diluted to 30MVC / mL, medium exchange at 18 hpT, 42 hpT, and 66 hpT; 4) diluted to 30MVC / mL, medium exchange at 24 hpT and 48 hpT. 【Figure 4】 Figure 4 shows the production of rAAV viral vector by the transient transfection process of the present invention at a cell density of 64MVC / mL in a perfusion bioreactor culture with a working volume of 220 mL. Black circles: viable cell density (MVC / mL); white circles: viability (%); white squares: transfection efficiency (GFP signal %). 【Figure 5】 Figure 5 shows the production of rAAV viral vector by the transient transfection process of the present invention using flow electroporation at a cell density above 80MVC / mL in a perfusion bioreactor culture with a working volume of 200 mL. Black circles: viable cell density (MVC / mL); white circles: viability (%); white squares: transfection efficiency (GFP signal %). 【Figure 6】Figure 6 shows the production of rAAV viral vectors carried out by the transient transfection process of the present invention at 50 MVC / mL in perfusion bioreactor cultures with a working volume of 200 mL. The transfection process was carried out in a pseudo-perfusion mini-bioreactor, i.e., a 50 mL container with a vent cap, in a 37 °C CO2 incubator, and two control cultures with viable cell densities of 2 MVC / mL and 15 MVC / mL are also shown. Figure 6A shows the long-term production in the bioreactor, where the viable cell density during the production stage was up to 120 MVC / mL; black circles and solid line: viable cell density; white circles: cell viability; white squares and dotted line: transfection efficiency as GFP expression; white diamonds: production titer viral genomes / mL; experimental control cultures in the mini-bioreactor transfected at a cell density of 1 MVC / mL (circles filled with the left pattern) and their viral genome production / mL (black diamonds on the left), and experimental control cultures in the mini-bioreactor transfected at a cell density of 15 MVC / mL (circles filled with the right pattern) and their viral genome production / mL (black diamonds on the right) are presented in the same graph for convenience, but no time scale is applied to these. Figure 6B shows a consistently high infectivity titer over a long period, which is demonstrated using a transduction assay demonstrated by GFP expression (black bars) in infected cells of the produced viral vectors in supernatant samples recovered from the bioreactor runs in Figure 6A at 3 days, 6 days, and 7 days post-transfection (3 dpT, 6 dpT, and 7 dpT). For comparison, a reference value (Ref) is shown, which was transfected in a shaking flask at a low cell density of 1 MVC / mL, a common practice in rAAV production, and the supernatant was recovered 3 days after transfection. 【Figure 7】Figure 7 shows several transient transfection manufacturing processes of rAAV viral vectors carried out using the present invention at a cell density of 50 MVC / mL. Two cell lines were used: HEK293F and Virus Production Cell 2.0 (VPC2.0). Two different chemical transfection methods, the cationic polymer PEI and the cationic lipid-based transfection reagent (VPT), were used, and three transfection media, Virus Production Medium (VPM), FreeStyle293 (FS293), and BalanCD HEK (BCD), were used. The cell density and viability at 3 dpT harvested are shown in Figure 7A, and the transduction assay performed using the supernatant from the harvested 3 dpT is shown in Figure 7A, and the related manufacturing titers are shown in Figure 7B. 【Mode for Carrying Out the Invention】 【0017】 Definitions and Abbreviations Terms not specifically defined herein should be given the meaning that would be ascribed by one of ordinary skill in the art in light of the present disclosure and the context. As used herein, unless otherwise indicated, the following terms have the indicated meanings. 【0018】 As used herein, the term "transient transfection" means that the introduced nucleic acid is potentially present in the cell only for a limited period and is not necessarily integrated into the cell genome. Transfected cells then potentially express the transiently transfected gene for a finite period, typically limited to several days. A common model of transfection is to express the green fluorescent protein, GFP, as the gene of interest. The production of GFP can be easily quantified based on its fluorescence in cells that have been successfully transfected. "Transfection efficiency" indicates the proportion of cells expressing GFP among all cells. In the present disclosure, the gene encoding GFP is used as the gene of interest or cargo gene to be delivered by AAV used for gene therapy. 【0019】 As used herein, the term "cell suspension" refers to a solution of cells that are not adhered to a static support. Such cells typically grow as a culture in a mixture that ensures nutrient supply to the cells in a stirred tank bioreactor or a stirred culture vessel. A cell suspension is obtained by cell growth, concentration procedures, and / or dilution procedures in a culture. Here, this term is used interchangeably with terms or expressions such as "cell culture suspension" and "suspension containing cells". 【0020】 As used herein, the term "cell density" means the concentration of cells in a given volume and quantifies the biomass from the perspective of cell concentration. Thus, in this specification, the term "cell density" is used interchangeably with expressions such as "concentration of cells". A commonly used unit is 1 million viable cells per milliliter (MVC / mL), which is widely used for eukaryotic cells. The quantification of cells can also be expressed as a cell volume fraction. For example, in the present disclosure, in a cell suspension with an average cell diameter of 17 - 22 μm and a cell density of 25 MVC / mL, the cells occupy at least 6% (volume / volume) of the culture volume. 【0021】 Here, when "restricted or no dilution is performed" as disclosed herein, this means that the starting cell density for the production of the recombinant vector is at least 20 million viable cells per milliliter, meaning that dilution is restricted, as compared to conventional methods where dilution to a low cell density, 5 MVC / mL or less, typically 1 - 2 MVC / mL, is commonly performed to initiate the production of the recombinant vector. 【0022】 As used herein, the term "perfusion" is a process of continuously exchanging the culture medium in a reactor or container while retaining cells within a bioreactor or culture vessel using a dedicated device connected to the bioreactor or culture vessel, at a point in time when it is considered suitable for a particular process. Medium exchange removes spent medium, including cellular waste and by-products, while supplying fresh medium to the cells. This process is known in the art, and any variations of this process that can be applied to the present disclosure by those skilled in the art are similarly applicable. 【0023】 As used herein, the term "pseudo-perfusion" refers to a process whose objective is comparable to a perfusion process, i.e., a process in which spent medium is completely or partially removed and replaced with fresh medium at a suitable time and rate for a particular process, but the operation is carried out by centrifuging or sedimenting the cells. This procedure can be performed manually or automated using a robotic handler or its derivatives. 【0024】 As referred to herein, "perfusion bioreactor culture" means cell culture carried out in a bioreactor according to the present disclosure in a perfusion process. 【0025】 Riedl et al. previously reported non-viral transfection of human T lymphocytes where the transfection process was carried out up to 40MVC / mL [Riedl, et al. "Non-Viral Transfection of Human T Lymphocytes." Processes 2018, 6, 188]. Since the diameter of naïve T cells is about 5 - 7μm, this number should not be directly considered as such. When human T lymphocyte cells account for 6% of the culture volume, the cell density of these cells is 530MVC / mL, while 6% of the culture volume of cells with a diameter of 17 - 22μm corresponds to a density of 25MVC / mL as described in the present disclosure. The term "cell recovery" as used herein means an optional period after transfection, enabling cell recovery from the transfection process, improving cell viability, and further promoting the production of the target product of interest. In gene transfer by non-viral transient transfection, no matter what method is used, foreign DNA has to overcome several barriers in order to successfully express the target gene it carries. This process involves a certain degree of disruption of the host cell membrane to allow the introduction of foreign DNA into the cell, then the transport of the DNA towards the nuclear membrane within the cytoplasmic compartment, and finally crossing the nuclear membrane. Only after a sufficient amount of intact DNA enters the cell nucleus does gene expression occur. This is also the reason why all transfection methods are considered to be partially harmful to cells. 【0026】 During transfection, the cell viability decreases significantly. Therefore, in non-viral gene transfer methods such as electroporation, cell recovery may be employed after transfection. However, this is not generally used in transfection methods that use chemical reagents. This is because the cell density employed in chemical transfection is generally very low, below 5MVC / mL, typically 1 or 2MVC / mL. Therefore, the cells do not undergo stress such as nutrient and oxygen utilization limitations that commonly occur in high-density cell suspensions. 【0027】 In the present disclosure, since the transient transfection process is based on high cell density, an optional cell recovery process is employed in two embodiments. In the two embodiments where the cell recovery process is applied after transfection, the cell suspension transfected at high cell density is transferred to a container with a large surface area where the transfected cell suspension can spread well so that sterile air / oxygen can be utilized. Cell recovery is not an essential step in the present disclosure, and the cell recovery time can vary within a wide range depending on the transfection method used, cell type, plasmid DNA, etc. 【0028】 Embodiment 4 of this specification is an example of AAV production without a cell recovery process. 【0029】 In addition to the above definitions, the following abbreviations are used in this specification and the exemplary embodiments. When an abbreviation used in this specification is not defined, the abbreviation has a generally accepted meaning. 【0030】 【Table A】 【0031】 Detailed Description The present disclosure provides a process for transiently transfecting eukaryotic cells at high cell density to produce recombinant viral vectors in high yields. Such processes are used in the large-scale production of recombinant vectors, rationalizing and reducing the costs commonly associated with such processes. Any particular process step or situation discussed in the context of any method or process herein is equally applicable to any other process or method disclosed herein. 【0032】 A method or process as disclosed herein is an in vitro method or process, and the cells used in such method or process are isolated from their natural environment. Thus, cells "obtained" or "provided" in a method etc. as disclosed herein are not directly obtained from their natural environment. 【0033】 Here, more specifically, a process for producing a recombinant vector in eukaryotic cells, the method comprising: a) obtaining a cell suspension having a cell density of at least 25 million viable cells per milliliter; b) performing transient transfection on the cells in the cell suspension with a single exogenous plasmid DNA or multiple different exogenous plasmid DNAs to obtain transfected cells; c) optionally, performing cell recovery for a certain period, wherein during the cell recovery period, the transfected cells are kept essentially stationary on the surface, the above steps; d) after the transfection of step b) and / or the optional cell recovery step c), restricting or not performing at all the dilution of the transfected cells present in the cell suspension, and then starting the production of the recombinant vector from a culture of transfected cells having a cell density of at least 20 million viable cells per milliliter is provided. 【0034】 "Manufacturing", which may also be referred to as the "manufacturing stage" in this specification, relates to the manufacture of recombinant vectors. The surface is preferably the surface within a receptacle or container that can exchange a large amount of oxygen. 【0035】 There is also provided a process in which the cell suspension in step a) has a cell density or concentration of at least 30 million cells per milliliter. There is also provided a process in which the cell suspension in step a) has a cell density or concentration of at least 50 million cells per milliliter. There is also provided a process in which the cell suspension in step a) has a cell density or concentration of at least 80 million cells per milliliter. There is also provided a process in which the cell suspension in step a) has a cell density or concentration of at least 100 million cells per milliliter. Naturally, the manufacturing in step d) that starts after the transfection in step b) or after step c) can start at a cell density or concentration closer to 80 million cells per milliliter, for example, 75 million cells per milliliter or 78 million cells per milliliter, when the cell suspension in step a) has a higher cell density or concentration, such as 80 million cells per milliliter. 【0036】 Similar reasoning also applies when comparing other examples of cell density or cell concentration disclosed herein, namely the initial cell density or concentration in step a) and the density or concentration in step d). As described above, the terms cell density and cell concentration may be used interchangeably in this specification. 【0037】 There is also provided a process in which the process includes a medium exchange that is performed at one or more time points starting from the time when the manufacturing in step d) is started. Such a medium exchange can be performed at different time points, for example, 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours after the start of manufacturing, and / or 48 hours after, but is not limited thereto. 【0038】 There is also provided a process including perfusion. 【0039】 The process is also provided, wherein the medium exchange is carried out by perfusion when the production or production stage in step d) is started. Thus, the medium exchange is carried out during the production in step d). The perfusion can be carried out in a perfusion bioreactor culture as exemplified herein. 【0040】 Here, as described elsewhere herein, it is beneficial not to perform dilution, or to limit or partially perform dilution before the start of the production in step d). 【0041】 Also provided is a process in which no dilution is carried out to produce a cell density lower than 20MVC / mL before the start of the production in step d). 【0042】 The medium exchange and perfusion process has been shown to be particularly relevant for maintaining a high cell density after the transfection process and obtaining a high production yield of the recombinant vector after the production stage. 【0043】 Instead of perfusion bioreactor culture, a pseudo-perfusion process can also be used. 【0044】 The production in step d) of the process described herein can be carried out for a period of at least 24 hours, for example, at least 48 hours or 72 hours, or 4, 5, 6, 7, 8, 9 or 10 days. As shown in the examples, the yield of the recombinant vector can benefit from a longer production period. 【0045】 The cell suspension in step a) may be made such that the cells of the suspension account for at least 6% of the culture volume, and the cell suspension is selected from the group consisting of cell cultures, microbial fermentations, cell suspensions derived from cell tissues, and cell suspensions derived from blood. 【0046】 Here, the eukaryotic cell can be selected from the group consisting of mammalian cells, human cells, avian cells, insect cells, and plant cells. More specifically, the cells can be CHO, CHO-DBX11, CHO-DG44, CHO-S, CHO-K1, Vero, BHK, HeLa, COS, MDCK, HEK-293, HEK-293T, HEK-293S, HEK-293F, L293, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0, CRL7030, HsS78Bst cells, PER.C6, SP2, SP0, hybridoma, MRC-5, MDCK, WI-98, CAP, EB66, HUVEC, AGE1.CR, CR, cells of Trichoplusia ni, cells of Spodoptera Frugiperda, SF9, SF21, Hi5, mesenchymal stem cells, endothelial cells, induced pluripotent stem cells, primary cells, cells of Nicotiana tabacum, BY2, cells of Nicotiana benthamiana, cells of Oriza sativa, cells of Arabidopsis thaliana, and cells of Daucus carota. 【0047】 The cells used in the present disclosure do not contain cells involving the destruction of human embryos. Thus, such cells are excluded from the present disclosure and do not form part of the present disclosure. 【0048】 Here, the recombinant viral vector can be a viral vector, a virus, a virus-like particle (VLP), or any combination thereof, or an aggregate derived therefrom, or an aggregate that associates with a small molecule having a size of less than 1000 kDa derived therefrom. More specifically, in the present specification, recombinant adeno-associated virus (AAV) viral vector particles can be produced, and optionally, the transfection in step b) of the process is performed using an (AAV) transient transfection system that includes co-transfection of at least three plasmids. This is exemplified in the experimental items, and this system is well known to those skilled in the art. 【0049】 The cell density in step a) of this process can be obtained by adjusting the cell density of the culture suspension by concentrating the cell content of the cell suspension and / or by culturing the cells in the culture suspension. Alternatively, the cell density in step a) can be adjusted by concentrating the cells in the suspension, such as by centrifugation, filtration, microfiltration, precipitation, acoustic sedimentation, or specific binding of cells, or by diluting the cells in the suspension. Further, the cell density in step a) of this process may be adjusted by culturing, and the culture may be batch culture, fed-batch culture, perfusion culture, chemostat, or a combination of at least two of these culture modes, and the culture can be carried out in combination with cell concentration and / or in combination with cell dilution. All of these are procedures well known in the art. 【0050】 Here, the transient transfection may include chemical transfection using a chemical reagent selected from the group consisting of cationic polymers such as DEAE-dextran, polybrene, polyethyleneimine and their derivatives, dendrimers, calcium phosphate, polycations; cationic lipids including liposomes and non-liposome transfection reagents and lipid nanoparticles. The polyethyleneimine may be a synthesized cationic polymer having a linear or branched topology, and the molecular weight may be in the range of 1 kDa to 1,000 kDa. Further, in this specification, the transfection may include physical transfection methods from the group consisting of microinjection method, optical transfection method, biolistic transfection method (also known as particle bombardment method), electroporation method, iron oxide nanoparticle delivery method (also known as magnetofection method), sonoporation method, laser irradiation method, and electric field-induced molecular vibration method. 【0051】 The present disclosure more specifically also provides a method for producing AAV viral vectors by eukaryotic cells. In particular, the present disclosure relates to a process for transient transfection of eukaryotic cells for the production of AAV viral vectors, including the step of transiently transfecting cells in a cell suspension, the cell suspension having a cell density of at least 25MVC / mL. After transfection, depending on the type of host cell, plasmid DNA, and the transient transfection method used, an optional cell recovery process may be performed. After transfection or an optional cell recovery period, the production of AAV viral vectors is initiated at a cell density of at least 20MVC / mL, and the dilution step after transfection is restricted or not performed at all. 【0052】 High cell density is one of the important factors in a biomanufacturing process to generate high expression levels of the product of interest. A very high cell density can significantly increase the volumetric yield of the product of interest being produced. However, to date, for the production of viral vectors by transient transfection processes, transfection at very high cell densities followed by production at very high cell densities has not been reported. This is due to limitations related to cell density and the vulnerability of cells during transfection. It is difficult to maintain cell culture at high cell densities, especially above 50MVC / mL, while the cells are still producing and maintaining an average good viability. These are the reasons why transient production of viral vectors is not performed at cell densities in this range. As described above, all transfection methods are harmful to cells. 【0053】 An important factor for achieving high cell density culture is an average high viability, which is very difficult to maintain in the transient transfection process. In high cell density culture, since the number of cells to be transfected increases significantly, the amount of plasmid DNA and, in the case of chemical transfection, the amount of chemical transfection reagent also increase significantly, which raises concerns about toxicity to cells. As a result, cell viability is impaired and the yield of viral vectors decreases. 【0054】 Note that after high cell density transfection, the step of diluting to a low density is considered inefficient and a drawback from a process perspective. This not only hinders the rationalization of the process, but also increases the amount of medium used, further greatly increasing the overall manufacturing footprint and causing unnecessary storage and transportation problems. Prior to the present disclosure, Backliwal et al. reported that HEK-293 cells adapted to suspension were transfected in situ with 25 kDa linear polyethyleneimine at a concentration of up to 20 MVC / mL in a complex medium, and then produced at a lower cell density (1 MVC / mL) [Backliwal et al., "High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI". Biotechnol Bioeng. 2008 Feb 15;99(3):721-7]. Blackstock et al. also reported a transfection process for producing virus-like particles at a higher transfection cell density of 15-25 MVC / mL [Blackstock et al., "Comprehensive Flow Cytometry Analysis of PEI-Based Transfections for Virus-Like Particle Production". Research, 2020, Article ID1387402]. 【0055】 In their research, the cells are centrifuged and set to a cell density of 1 MVC / mL 3 hours after transfection, which means that the transfected cell suspension is diluted to 1 MVC / mL at which the manufacturing stage is initiated. The dilution step after transfection is currently necessary for high cell density transfection processes. Dilution is particularly mentioned when using physical transfection methods such as electroporation. In the electroporation method, high cell density has the advantage of a short physical distance between the host cell and plasmid DNA, but the cells need to be diluted after electroporation to initiate the manufacturing stage. Currently, the highest reported cell density after electroporation is 10 MVC / mL [Steger et al. "CHO-S antibody titers > 1 gram / liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines". J Biomol Screen. 2015 Apr;20(4):545-51]. 【0056】 The present disclosure provides a method comprising the step of transiently transfecting cells in a cell suspension, wherein the cell suspension has a cell density of at least 25 MVC / mL. After transfection, an optional cell recovery process can be performed if necessary. After transfection or after an optional cell recovery period, the production of viral vectors is initiated at a cell density of at least 20 MVC / mL with the dilution step performed after transfection being limited or not performed at all. 【0057】 In other words, the present disclosure a) obtaining a cell suspension having a cell density of at least 25 million viable cells per milliliter; b) performing transient transfection with exogenous plasmid DNA or co - transfection with multiple exogenous plasmid DNAs, i.e., multi - plasmid DNA, which is necessary for producing a recombinant viral vector, on the cells in the cell suspension; c) optionally, a step of cell recovery for a certain period, where the recovery period depends on the type of host cell, the exogenous plasmid DNA(s), and the transient transfection method used. During the cell recovery period, the transfected cells are kept in a stationary state on a surface of sufficient size. This period can vary widely depending on the type of host cell, the exogenous plasmid DNA(s), and the transient transfection method used; d) after the transfection step b) and / or the optional cell recovery step c), starting the production stage from a cell density of at least 20 million viable cells per milliliter without restricting or without diluting the transfected cells at all; A method or process is provided that includes the above steps. 【0058】 In some embodiments of the process of the present disclosure, the cell culture suspension has a cell density of at least 30 million cells per milliliter. 【0059】 In some embodiments of the process of the present disclosure, the cell culture suspension containing cells has a cell density of at least 50 million cells per milliliter. 【0060】 In some embodiments of the process of the present disclosure, the cell culture suspension has a cell density of at least 80 million cells per milliliter. 【0061】 In some embodiments of the process of the present disclosure, the cell culture suspension has a cell density of at least 100 million cells per milliliter. 【0062】 In some embodiments of the processes of the present disclosure, the cell culture suspension is made such that the cells occupy at least 6% of the culture volume, and the cell culture suspension is selected from the group consisting of cell cultures, microbial fermentations, cell suspensions derived from cell tissues, and cell suspensions derived from blood. 【0063】 Cells useful in the processes of the present disclosure are typically eukaryotic cells such as mammalian cells, human cells, avian cells, insect cells, and plant cells. 【0064】 Typically, the cells are selected from the group consisting of CHO, CHO-DBX11, CHO-DG44, CHO-S, CHO-K1, Vero, BHK, HeLa, COS, MDCK, HEK-293, HEK-293T, HEK-293S, HEK-293F, L293, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0, CRL7030, HsS78Bst cells, PER.C6, SP2, SP0, hybridomas, MRC-5, MDCK, WI-98, CAP, EB66, HUVEC, AGE1.CR, CR, cells of Urtica dioica, cells of Spodoptera litura, SF9, SF21, Hi5, primary T cells, Jurkat cells, mesenchymal stem cells, endothelial cells, induced pluripotent stem cells, primary cells, cells of tobacco, BY2, cells of Nicotiana benthamiana, cells of rice, cells of Arabidopsis thaliana, and cells of Solanum tuberosum. 【0065】 The cells used in the present disclosure do not contain cells involving the destruction of human embryos. Thus, such cells are excluded from the present disclosure and do not form part of the present disclosure. 【0066】 Here, the recombinant viral vector may be a viral vector, a virus, a virus-like particle (VLP), or any combination thereof, or an aggregate derived therefrom, or an aggregate that associates with a small molecule having a size of less than 1000 kDa derived therefrom. The cell density of the cell suspension in the process of the present disclosure can be obtained by adjusting the cell density of the culture suspension by concentration and / or culture as described elsewhere in this specification. 【0067】 In some embodiments, the cell density of the cell suspension is adjusted by cell concentration. Any suitable concentration method known in the art can be used, such as centrifugation, filtration, microfiltration, precipitation, acoustic sedimentation, or specific binding, or it can also be adjusted by cell dilution. 【0068】 In some embodiments, the cell density of the cell suspension is achieved by culture. Any suitable culture method known in the art can be used, such as batch culture, fed-batch culture, perfusion culture, chemostat, or a combination of at least two of these culture modes, and the culture can be carried out in combination with cell concentration or in combination with cell dilution. 【0069】 Transfection is performed on the day when the cell density in the culture reaches at least the target cell density. If it is higher than the target cell density, the total number of cells required for transfection is calculated, the cell suspension giving the required total number of cells is removed, and it is diluted with fresh growth medium to the target cell density and the target cell suspension volume. Then, the excess cells are discarded from the system. After transfection, the transfected cell suspension is transferred to the same culture vessel used for cell expansion, and the manufacturing stage is carried out. 【0070】 Transient transfection according to the present disclosure is a non-viral gene introduction method and is either a chemical transfection method or a physical transfection method. In the case of chemical transfection, chemical reagents selected from the group consisting of cationic polymers such as DEAE-dextran, polybrene, polyethyleneimine and their derivatives, dendrimers, calcium phosphate, polycations; cationic lipids, lipid nanoparticles including liposome and non-liposome transfection reagents can be utilized or used. 【0071】 In the case of physical transfection methods, it refers to or may refer to one or more methods selected from the group consisting of microinjection method, optical transfection method, biolistic transfection method (also known as particle bombardment method), electroporation method, iron oxide nanoparticle delivery method (also known as magnetofection method), sonoporation method, laser irradiation method, electric field-induced molecular vibration method, but is not limited thereto. 【0072】 In one embodiment, the chemical reagent is a cationic polymer. A representative cationic polymer is polyethyleneimine having a linear or branched topology. Typically, polyethyleneimine has a molecular weight in the range of 1 kDa to 1000 kDa. 【0073】 Transient transfection by the process of the present disclosure is a physical transfection method selected from the group consisting of direct microinjection method into cells or nuclei, electroporation method, biolistic particle delivery method (also known as particle bombardment method), iron oxide nanoparticle delivery method (also known as magnetofection method), sonoporation method, laser irradiation method, electric field-induced molecular vibration method, or may be, but is not limited thereto. 【0074】 Microinjection is a technique for directly introducing DNA into the cell nucleus, and electroporation is a technique that uses an electric potential to form pores in the cell membrane and allow foreign DNA to enter the cell. Biolistic particle delivery or microprojectile bombardment is a technique for delivering foreign genes to cells using heavy metal particles coated with exogenous DNA. Sonoporation utilizes physical disturbances in a liquid to create pores in the cell membrane for nucleic acid introduction. Magnetofection is a transfection method that uses a magnetic field to concentrate particles containing exogenous DNA on target cells. Laser irradiation perforates individual cells by focusing a laser beam on a confined area of the cell membrane to enable the entry of nucleic acids. 【0075】 In one embodiment, transient transfection is a physical transfection method selected from the group consisting of the microinjection method, the optical transfection method, the biolistic particle delivery method (also known as the particle bombardment method), the electroporation method, and the iron oxide nanoparticle delivery method (also known as the magnetofection method). 【0076】 Next, the present invention is illustrated by the following experimental items that describe specific embodiments. The embodiments are meant to be illustrative of the present invention and are not intended to limit the scope of the present invention to the described embodiments. 【Examples】 【0077】 Experimental items Materials and methods Cell lines and media HEK293F cells were obtained from Thermofisher Scientific. The media used were BalanCD HEK293(-) glucose medium (manufactured by Fujifilm, USA) supplemented with glucose and L-glutamine, FreeStyle293 expression medium (manufactured by Gibco), FreeStyleF17 expression medium (manufactured by Gibco), and the virus production medium (manufactured by Gibco) used in some of the transfection experiments specified in the text. When growing in Erlenmeyer flasks and culturing in spin tubes (50 mL, Corning) or TubeSpin bioreactors (50 mL, TPP), the cells were placed in an incubator at 37 °C and 5% CO2 and stirred at 120 rpm and 320 rpm, respectively. 【0078】 Transfection materials Chemical transfection In this study, highly active linear PEI of PEI MAX (mW 40,000, manufactured by Polysciences) was used to perform chemical transfection via PEI. For the production of serotype AAV1, the plasmid was an rAAV helper-free packaging system, which included three plasmids: pHelper, pRC1, and pGFP (manufactured by Cell Biolabs). For the production of AAV9, pR2C9 (pAAV2 / 9n, Addgene) was used together with the same pHelper and pGFP as those used for AAV1 production. The GFP, which is the target model gene, was used in the transduction assay to monitor the transfection efficiency and evaluate the infectivity titer of the produced rAAV. HEK293F cells were grown in suspension in Erlenmeyer flasks for cell culture in an incubator at 37 °C and 5% CO2. 【0079】 Physical transfection The electroporation device Maxcyte ExPERT STx was used together with multiple CL2 units having a high-density cell suspension processing capacity of 100 mL / unit. 【0080】 Experimental setup First, a small-scale system was constructed, and the main process parameters were screened and optimized there. The small-scale study was carried out in a mini bioreactor, namely, a 50 mL tube container with a vent cap called a well spin tube, with a working volume of 5 mL and shaking in a 37 °C CO2 incubator. These were operated in a pseudo-perfusion mode, and the medium was manually exchanged by centrifugation and retention of whole cells. Subsequently, the developed process was applied to perfusion culture in a bioreactor system DASGIP (manufactured by Eppendorf) with a working volume of 200 mL, using an alternating flow filter as a cell retention device with a microfilter hollow fiber cartridge (all manufactured by Repligen). After transfection, the transfected cell suspension was maintained in the same bioreactor until harvest. The harvest of the culture was performed by centrifugation at 200 xg for 5 minutes 72 hours post-transfection (hpT) unless otherwise specified in the text, and both the supernatant and the cell pellet were stored at -80 °C for analysis. To measure the production titer, the cell pellet sample was resuspended in a lysis buffer containing 50 mM Tris, 150 mM NaCl, and 2 mM MgCl2, and then the cell suspension was frozen / thawed 3 times (10 minutes each) by alternating the tube between an isopropanol bath at -80 °C and a water bath at 37 °C. Subsequently, the crude lysate was incubated with 50 units / mL of DNAse (Denarase) at 37 °C for 45 minutes, followed by centrifugation at 3000 g for 10 minutes at 4 °C to recover the clarified supernatant, which was then advanced to further analytical assays. 【0081】 Analysis method Cell density was measured by Bioprofile FLEX (manufactured by Nova Biomedical) or Norma XS (manufactured by Iprasense). The concentrations of glucose, lactate, glutamine, and ammonium were measured by Cedex Bio (manufactured by Roche). Transfection efficiency (GFP intensity) was measured with two flow cytometers, Gallios (manufactured by Beckman Coulter) and Guava EasyCyte (manufactured by Luminex Corporation). The produced viral vector capsids were quantified using the AAV1 and AAV9 titration ELISA kits (manufactured by Progen Biotechnik). The viral genome was analyzed using the AAVpro titration kit for qPCR (manufactured by Takara Bio) and measured using the CFX96 real-time qPCR detection system (manufactured by Bio-Rad). To evaluate the production of functional infectious viral vectors, a transduction assay was used. To perform the transduction assay, the supernatant or cell lysate was added to a healthy HEK293F cell suspension, and the GFP expression level 72 hours after transfection was monitored. 【0082】 (Example 1 (Embodiment 1)) In Embodiment 1, the transient transfection process is a cationic polymer PEI (polyethylene imine)-mediated high cell density transfection process at 50 MVC / mL for the production of rAAV. The purpose of this experiment is to demonstrate that this high cell density transfection process is a general-purpose process applicable to various transient transfection conditions with different ratios of plasmid DNA to cell number and plasmid DNA to cationic polymer. 【0083】 Transfection was performed via an AAV helper-free multi-plasmid transient transfection system that co-transfected three plasmids: plasmid pAAV-RC1 (serotype AAV1), plasmid pHelper, and plasmid pGFP. Plasmid pAAV-RC1 provides the viral rep (replication) and cap (capsid) genes. Plasmid pHelper carries the adenoviral gene products necessary for the production of infectious AAV. This function is required to generate infectious AAV particles and is provided in vivo by a helper virus such as adenovirus and replaced only by the pHelper gene in production. Plasmid pGFP contains a gene encoding GFP (green fluorescent protein), and GFP was used as a model for the therapeutic gene or cargo of gene therapy. GFP is generally used as a model for the therapeutic gene and can be easily quantified due to fluorescence detection, demonstrating the efficiency of transfection. 【0084】 HEK293 cells grown in suspension were maintained in a 37 °C CO2 incubator in a Erlenmeyer flask for cell culture. Cells in the exponential growth phase were centrifuged to a viable cell density of 50 MVC / mL, and high-density cell culture was continued in a mini bioreactor in a 37 °C CO2 incubator ready for transfection, namely a centrifuge tube with a vent cap, at a volume of 50 mL. 【0085】 Several groups of transfection reagents were prepared by varying the ratio of plasmid DNA to PEI and the ratio of plasmid DNA to cell number. Two groups transfected at 1 MVC / mL, run according to a standard transfection protocol commonly used in the art at normal cell density, were also included for reference. In the transfection process, plasmid DNA was mixed with PEI and incubated at room temperature for 15 minutes. After incubation, each transfection mixture was added individually to the cell suspension. No additional dilution step was performed and the cultures were maintained by pseudo-perfusion in the same culture vessel in the incubator until harvested 72 hours post-transfection (hpT). 【0086】 Thereafter, the production titer was quantified by ELISA (enzyme-linked immunosorbent assay) that analyzes the assembled rAAV capsids in the harvest. In Figure 1, the two groups counted from the left were experimental controls, and the cell density at the time of their transfection was 1MVC / mL. The groups after the third were transfected at 50MVC / mL while changing the ratios among cells, plasmid DNA, and PEI. Each group was named according to the ratio among the number of cells (MVC): plasmid DNA (μg): PEI (μg): In groups 1:1:1, 1:1:2, 1:1:4, and 1:1:8, the ratio of plasmid DNA to the number of cells was 1 μg of DNA per 1 million cells, and the ratios of DNA (μg): PEI (μg) were 1:1, 1:2, 1:4, and 1:8, respectively. In groups 1:2:2, 1:2:4, and 1:2:8, the plasmid DNA was 2 μg of DNA per 1 million cells, and the ratios of DNA (μg): PEI (μg) were 1:1, 1:2, and 1:4, respectively. In groups 1:4:4, 1:4:8, and 1:4:16, the plasmid DNA was 4 μg of DNA per 1 million cells, and the ratios of plasmid DNA (μg): PEI (μg) were 1:1, 1:2, and 1:4, respectively. As can be seen from the figure, all other groups except the last group, 1 million cells: 4 μg of plasmid DNA: 16 μg of PEI, showed a higher production titer compared to the experimental control group carried out at 1MVC / mL. Since the cytotoxicity due to the cationicity of PEI is well-known, it was reasonable that the productivity decreased when the transfection reagent was used in excess. 【0087】 (Example 2 (Embodiment 2)) In Embodiment 2, the transient transfection process is a cationic polymer PEI-mediated high cell density transfection process at 50MVC / mL for producing rAAV. The purpose of this experiment is to demonstrate the ultra-high cell density transfection process by varying the incubation time of the transfection reagent, which is a mixture of plasmid DNA and a cationic polymer. Transfection was performed using an AAV helper-free multi-plasmid transient transfection system that co-transfects three plasmids: pAAV-RC1, pHelper, and pGFP. Plasmid pAAV-RC1 provides the viral rep (replication) and cap (capsid) genes. Plasmid pHelper carries the adenoviral gene products necessary for the production of infectious AAV. This function is required to generate infectious AAV particles and is provided in vivo by a helper virus such as adenovirus and replaced only by the pHelper gene in production. Plasmid pGFP contains a gene encoding GFP (green fluorescent protein), which was used as a model for the gene of interest or cargo in gene therapy. GFP is commonly used as a model for the gene of interest and is easily quantifiable due to fluorescence detection, demonstrating the efficiency of transfection. 【0088】 HEK293 cells grown in suspension were maintained in a cell culture Erlenmeyer flask in a 37 °C CO2 incubator. Cells in the exponential growth phase were centrifuged to a viable cell density of 50 MVC / mL, and high-density cell culture was continued in a mini bioreactor in pseudo-perfusion mode, i.e., a 50 mL container with a vent cap, in a 37 °C CO2 incubator in a state ready for transfection. In all groups, the plasmid DNA was 1 μg per million cells, and the ratio of DNA (μg):PEI (μg) was 1:2. Using such a ratio, the usage amounts of both plasmid DNA and PEI were the lowest at a production titer equivalent to that described in Embodiment 1, clearly reducing the costs associated with plasmid production and PEI. After mixing DNA and PEI, different incubation times were used: 5 minutes ("5 min"), 10 minutes ("10 min") or 15 minutes ("15 min") were applied (see Figure 2). After incubation, the DNA and PEI mixture was added to the cell suspension. In the "0 min rapid mixing" group, DNA and PEI were mixed and the mixture was rapidly mixed by inverting it several times, and then immediately added to the cell suspension without incubation. In the "0 min, direct transfection" group, DNA was added to the cell suspension, and then PEI was immediately added to the cell suspension, completely eliminating the pre-mixing of plasmid DNA and PEI. In all groups, a dilution step exceeding 3:5 resulting from adding 2 mL of the mixed DNA and PEI to 3 mL of the 50 MVC / mL cell suspension for transfection was not performed after transfection, and the culture was maintained in the same culture container in the incubator until harvested at 72 hpT. Thereafter, the production titer was quantified by ELISA analyzing the assembled rAAV capsid in the harvest. Figure 2 shows that in the final harvest, all procedures used ensured the production of AAV capsids, and that all groups showed equivalent production except that "0 min, direct transfection" had low AAV production.This demonstrates that transfection can be carried out regardless of the formation of a complex between the cationic polymer and DNA, and that different incubation times are applicable in the case of pre-mixing the cationic polymer and DNA. 【0089】 (Example 3 (Embodiment 3)) In Embodiment 3, the transient transfection process is a PEI-mediated high cell density transfection process at 100MVC / mL for the production of rAAV. The purpose of this experiment is to demonstrate a transfection process at extremely high cell densities by varying the dilution to various high cell densities (10MVC / mL and 30MVC / mL as experimental controls) after transfection at the production stage. Transfection was performed by an AAV helper-free multi-plasmid transient transfection system that co-transfects three plasmids: pAAV-RC1, pHelper, and pGFP. Plasmid pHelper carries the adenoviral gene products necessary for the production of infectious AAV. This function is required to generate infectious AAV particles, which are provided in vivo by a helper virus such as adenovirus and replaced only by the pHelper gene in production. Plasmid pGFP contains a gene encoding GFP (green fluorescent protein), which was used as a model for the therapeutic gene or cargo of gene therapy. GFP is generally used as a model for the gene of interest and can be easily quantified due to fluorescence detection, demonstrating the efficiency of transfection. 【0090】 HEK293 cells grown in suspension were maintained in a 37 °C CO2 incubator in a Erlenmeyer flask for cell culture. Prior to transfection, cells in the exponential growth phase were centrifuged so that the viable cell density became 1,000,000 cells / mL (100MVC / mL). In all groups, the amount of plasmid DNA used was 1 μg per 1 million cells, and the ratio of plasmid DNA (μg):PEI (μg) was 1:2. Using such a ratio, the usage amounts of both plasmid DNA and PEI were the lowest at a production titer equivalent to that described in Embodiment 1. The transfection process was exactly the same in all groups: plasmid DNA was mixed with PEI, incubated for 15 minutes, and then added to a very high-density cell suspension. After transfection, the transfected cell suspension was diluted under four different conditions for the production stage, two of which were diluted to 10MVC / mL and two were diluted to 30MVC / mL. Prior to the final harvest at 72 hours post transfection (hpT), different medium exchange strategies of pseudo-perfusion were applied to each condition: 1) The first condition, diluted to 10MVC / mL, medium exchange was performed 24 hours after transfection (hpT), and medium exchange was performed once 24 hours later; 2) The second condition, diluted to 10MVC / mL, medium exchange was performed once at 48 hpT; 3) The third condition, diluted to 30MVC / mL, medium exchange was performed at 18 hpT, and medium exchange was performed once 24 hours later; 4) The fourth condition, diluted to 30MVC / mL, medium exchange was performed at 24 hpT, and medium exchange was performed once 24 hours later. 【0091】 After dilution, the culture was continued in a mini bioreactor, i.e., a 50 mL tube container with a vent cap, in a 37 °C CO2 incubator until harvesting at 72 hpT. Thereafter, the production titer was quantified by ELISA analyzing the assembled rAAV capsids in the harvest. Figure 3 shows that in a simple culture system such as a 50 mL tube container with a vent cap, even with transfection at a very high cell density of 100 MVC / mL by shaking in a 37 °C CO2 incubator during the production stage, as long as medium exchange is performed to replenish nutrients and remove by-products to maintain the integrity of the cell culture, titers equivalent to those after dilution to a cell density of 10 MVC / mL or 30 MVC / mL are obtained, which is condition 3 compared to conditions 1 and 2. In condition 4, medium exchange is performed for the first time at 24 hpT, which leads to a decrease in rAAV production compared to condition 3 where the first medium exchange is performed after 18 hours. This finding suggests that dilution after transfection is not a prerequisite when sufficient medium exchange is performed and cell culture can be maintained at a very high density such as 100 MVC / mL. 【0092】 (Example 4 (Embodiment 4)) In Embodiment 4, the transient transfection process is a PEI-mediated ultra-high cell density transfection process at 60 MVC / mL for the production of rAAV in perfusion bioreactor cultures with a working volume of 220 mL. The purpose of this experiment is to demonstrate a transfection process at ultra-high cell density without dilution after transfection in perfusion bioreactor cultures for rAAV production. Transfection was performed by an AAV helper-free multi-plasmid transient transfection system that co-transfects three plasmids: pAAV-RC1, pHelper, and pGFP. Plasmid pHelper carries the adenoviral gene products necessary for the production of infectious AAV. This function is required to generate infectious AAV particles, which are provided in vivo by a helper virus such as adenovirus and replaced only by the pHelper gene in production. Plasmid pGFP contains a gene encoding GFP (green fluorescent protein), which was used as a model for the therapeutic gene or payload of gene therapy. GFP is commonly used as a model for the gene of interest and can be easily quantified due to fluorescence detection, demonstrating the efficiency of transfection. 【0093】 HEK293 cells grown in suspension were inoculated into a bioreactor with a working volume of 220 mL seven days prior to transfection, and perfusion was initiated immediately after inoculation using a hollow fiber cartridge operating in the ATF (alternating tangential flow) mode. Transfection aimed for a cell density of 60 - 100 MVC / mL. On the day when the viable cell density reached 61 MVC / mL, the transfection reagent was prepared at the ratios shown in Embodiments 2 and 3: the plasmid DNA was 1 μg per million cells, and the ratio of DNA (μg):PEI (μg) was 1:2. Using such ratios, the usage amounts of both plasmid DNA and PEI were the lowest at a production titer equivalent to that described in Embodiment 1, thereby significantly reducing the manufacturing cost, especially at the bioreactor scale. The transfection efficiency was monitored daily using a flow cytometer that quantifies the GFP expression level. Here, the culture was harvested when no further increase in the GFP level was observed. The results are shown in Figure 4. The viable cell density after transfection decreased from 61.4 to 58.7 MVC / mL depending on the addition amount of the transfection reagent. The viability decreased from 96% immediately before transfection to 82% at 4 hpT (0 dpT) measurement, which is a normal decrease in viability as a reaction to transfection. A reduction in cell growth was also observed during the first two days after transfection. The gradual recovery of cell growth and viability after 2 dpT indicates that the plasmid is gradually lost during cell division, which is a normal phenomenon in such transient transfection. The transfection efficiency increased day by day after transfection, reaching a maximum of 56% at 5 dpT and then slightly decreasing at 6 dpT. The culture was harvested at 6 dpT. At this time, the cell density and viability had recovered to 116 MVC / mL and 94% respectively. 【0094】 (Example 5 (Embodiment 5)) In Embodiment 5, the transient transfection process is a flow electroporation process for the production of rAAV in perfusion bioreactor cultures with a working volume of 200 mL and at a level exceeding 80 MVC / mL. The purpose of this experiment is to demonstrate a transfection process at extremely high cell densities without dilution after transfection for rAAV production in perfusion bioreactor cultures. Transfection was performed using an AAV helper-free multi-plasmid transient transfection system that co-transfects three plasmids: pAAV-RC1, pHelper, and pGFP. Plasmid pHelper carries the adenoviral gene products necessary for the production of infectious AAV. This function is required to generate infectious AAV particles, which in the body are provided by a helper virus such as adenovirus and are replaced only by the pHelper gene in production. Plasmid pGFP contains a gene encoding GFP (green fluorescent protein), and GFP was used as a model for the gene of interest or cargo in gene therapy. GFP is commonly used as a model for the gene of interest and can be easily quantified thanks to fluorescence detection, demonstrating the efficiency of transfection. 【0095】 HEK293 cells grown in suspension were inoculated into a bioreactor with a working volume of 200 mL 13 days before transfection, and perfusion was started after the cell density reached 2 MVC / mL using a hollow fiber cartridge operating in ATF mode. Here, the electroporation device ExPERT STx from Maxcyte was used. According to the manufacturer, in the normal function of this system, for cells cultured and grown at a low cell density of about 1 - 2 MVC / mL, which are currently used on-site, they are concentrated to a concentration of 100 MVC / mL by centrifugation and transferred to the "sample bag" of the Maxcyte system. Electroporation was performed as flow electroporation, and the resulting material was collected in the "recovery bag". 【0096】 In this disclosure, before the experiment, the electroporation device ExPERT STx manufactured by Maxcyte was modified by removing both the sample bag and the recovery bag, and directly connecting the electroporation unit of the STx "in-line" to the functions of bioreactor A as the "sample bag" and recovery container B as the "recovery bag" of Maxcyte. The recovery container B was also connected to the bioreactor A, and the cell suspension recovered in the recovery container B was returned to the bioreactor A. The recovery container B was a 10L expandable bag, i.e., a bag typically used for storing culture medium or buffer, which had been pre-expanded with sterile air containing 5% carbon dioxide. Transfection was targeted at a cell density of 80 - 100MVC / mL. On the day when the viable cell density reached 83MVC / mL, electroporation was performed with 80mg of plasmid DNA, i.e., 400μg of plasmid DNA per 1mL of culture medium. This electroporation was directly performed on the 80MVC / mL cell suspension from the bioreactor A using two electroporation units CL2. Since each CL2 unit had a processing capacity of 100mL of high-density cell suspension, two CL2 units were continuously used for the electroporation of 200mL of cell suspension. The cell suspension from the bioreactor A was fed into the electroporation unit for electroporation, and 3mL was fed into the recovery container B per cycle until the entire 200mL of cell suspension was electroporated. To neutralize the excess free DNA in the electroporated cell suspension, 10mL of DNase1 was added to the recovery container B. After electroporation, the cells were transferred to the recovery container B and left standing in an incubator at 37°C for 45 minutes. In this way, the transfected cells were sufficiently dispersed on a surface of sufficient area to come into contact with oxygen for recovery. After a 45-minute recovery time, the transfected cell suspension was returned to the bioreactor A. From the moment the transfected cells were returned to the bioreactor A, the cell culture entered the production stage, and the transfection efficiency was monitored daily using a flow cytometer to quantify the GFP expression level. Figure 5 shows the results.A decrease in viable cell density occurred immediately after transfection. This was due to sudden cell death caused by the electroporation technique. After transfection, the survival rate decreased from 97% to 91%, which is a normal transfection reaction in the electroporation technique. The GFP signal reached a maximum of 80% three days after transfection and then gradually decreased. The culture was harvested when a significant decrease in viable cell density, survival rate, and GFP signal was observed, which was on the 8th day after transfection in this case. Note that the decrease in cell density two days before transfection was due to manually removing a part of the culture to adjust the cell density to the target range in preparation for transfection. 【0097】 (Example 6 (Embodiment 6)) In Embodiment 6, the transient transfection process is a PEI-mediated ultra-high cell density transfection process at 50MVC / mL for the production of rAAV in a perfusion bioreactor culture with a working volume of 200 mL. The purpose of this experiment is to demonstrate a transfection process at ultra-high cell density without dilution after transfection for rAAV production in perfusion bioreactor culture. Transfection was performed by an AAV helper-free multi-plasmid transient transfection system that co-transfects three plasmids: pAAV-RC9, pHelper, and pGFP. Plasmid pAAV-RC9 provides the viral rep (replication) gene and cap (capsid) gene. Plasmid pHelper carries the adenoviral gene products necessary for the production of infectious AAV. This function is required to generate infectious AAV particles, which are provided in vivo by a helper virus such as adenovirus and replaced only by the pHelper gene in production. Plasmid pGFP contains a gene encoding GFP (green fluorescent protein), which was used as a model for the therapeutic gene or cargo of gene therapy. GFP is commonly used as a model for the therapeutic gene and can be easily quantified due to fluorescence detection, demonstrating the efficiency of transfection. 【0098】 HEK293 cells grown in suspension were inoculated into bioreactor A with a working volume of 200 mL 8 days prior to transfection, and perfusion was initiated immediately after inoculation using a hollow fiber cartridge operating in the ATF (alternating tangential flow) mode. The transfection aimed for a target cell density of 50 - 80 MVC / mL. On the day when the viable cell density reached 70 MVC / mL, 120 mL of a 50 MVC / mL cell suspension was prepared by mixing 85 mL of the suspension and 35 mL of fresh medium. The transfection reagent was prepared at the following ratios: 0.5 μg of plasmid DNA per 1 million cells, and the ratio of DNA (μg):PEI (μg) was 1:2. Using such ratios significantly reduced the usage amounts of both plasmid DNA and PEI, particularly clearly reducing the manufacturing cost on the bioreactor scale. A total of 30 mg of plasmid DNA composed of pRC9:pGFP:pHelper in a ratio of 1:1:2 was diluted to a total volume of 40 mL in the medium. A total of 60 mg of PEI was also diluted to a volume of 40 mL in the medium. 40 mL of the plasmid DNA and 40 mL of the PEI solution were mixed to prepare a transfection reagent mixture, which was subjected to incubation at room temperature for 15 minutes. This transfection reagent mixture was added to 120 mL of a 50 MVC / mL cell suspension. After mixing this cell suspension and the transfection reagent mixture, the transfected cell suspension was placed on a surface area of 300 cm 2It was transferred onto the non-tissue culture-treated surface of the tissue culture flask for 30 minutes. After a 30-minute cell recovery period, the transfected cell suspension was returned to the bioreactor. From the moment the transfected cells were returned to the bioreactor, cell culture entered the production stage, and the transfection efficiency was monitored daily using a flow cytometer that quantifies the GFP expression level. Here, when no further increase in the GFP level was observed, on the 10th day after transfection, the culture was harvested. During the production stage, cell suspension samples were collected daily from Bioreactor A. The supernatant obtained from the samples by centrifugation was used to perform a transduction assay. The transduction assay was used to evaluate the functionality of the infectious viral vectors contained in the samples by placing the samples in the presence of cells susceptible to infection with the infectious viral vectors. To perform the transduction assay, the supernatant of the collected samples was added to a healthy HEK293F cell suspension, and the GFP expression level was monitored for 72 hours after transduction or up to 3 days after transduction. Figure 6A shows the viable cell density, viability, transfection efficiency, and production titer. This demonstrates a very long and very high cell density maintained for 4 days from 7 dpT, i.e., 120 MVC / mL, in a long-term continuous production stage. For comparison, experimental control cultures (circles filled with left-side patterns) in a mini-bioreactor transfected at a cell density of 1 MVC / mL and their viral genome production / mL (left-side black diamonds), and experimental control cultures (circles filled with right-side patterns) in a mini-bioreactor transfected at a cell density of 15 MVC / mL and their viral genome production / mL (right-side black diamonds) are represented in the same graph for convenience, but no time scale is applied to these. The perfusion process generated a 33-fold higher cell density and a 30-fold higher vg / mL compared to the experimental control culture at 1 MVC / mL harvested at 2 MVC / mL at 3 dpT. The perfusion process generated a 4.3-fold higher cell density and an 8.4-fold higher vg / mL compared to the small-scale process at 15 MVC / mL.The perfusion process maintained cell-specific productivity very well compared to the standard transfection process at 1MVC / mL, and it could be concluded that the cell-specific productivity was almost doubled compared to the 15MVC / mL process in spin tubes. This may be due to continuous medium renewal in perfusion culture, which is impossible in a small-scale batch-mode system. Figure 6B shows the results of the transduction assay in the same process. The 3dpT group, 6dpT group, and 7dpT group refer to the supernatants collected on the 3rd, 6th, and 7th days after transfection, respectively. The Ref group is a reference group in which the production of rAAV by transfection was performed in a shaking flask at a low cell density of 1MVC / mL, which is common in the production of rAAV, and the supernatant was collected on the 3rd day after transfection. This demonstrates a long-term continuous production stage of a functional rAAV vector infectious to human cells, with an approximately 50% increase in infectious titer compared to a reference group transfected at 1MVC / mL using the same cells and transfection reagents. This embodiment demonstrated the possibility of reaching a very high titer of functional rAAV vectors along with an extended production stage. 【0099】 (Example 7) In Embodiment 7, a transient transfection process for producing rAAV was performed at 50MVC / mL using both HEK293F, the host cell, and Virus Production Cell 2.0 (VPC2.0, a variant cell line of HEK293 cells obtained from ThermoFisher). Two different chemical transfection methods: the cationic polymer PEI and the cationic lipid-based transfection reagent (VPT) were used, and three types of transfection media: Virus Production Medium (VPM), FreeStyle293 (FS293), and BalanCD HEK (BCD) were used. The purpose of this experiment was to demonstrate that the present invention is applicable to various production cell lines, chemical transfection methods, and transfection media that are widely adopted and commercially available in the art. Transfection was performed by an AAV helper-free multi-plasmid transient transfection system that co-transfected three plasmids: pAAV-RC9, pHelper, and pGFP. Plasmid pAAV-RC9 provides the viral rep (replication) gene and cap (capsid) gene. Plasmid pHelper carries the adenoviral gene products necessary for the production of infectious AAV. This function is necessary to generate infectious AAV particles and is provided in vivo by a helper virus such as adenovirus and replaced only by the pHelper gene in production. Plasmid pGFP contains the gene encoding GFP (green fluorescent protein), and GFP was used as a model for the target gene or payload of gene therapy. GFP is generally used as a model for the target gene, is easily quantified thanks to fluorescence detection, and demonstrates the efficiency of transfection. 【0100】 HEK293F cells and VPC2.0 cells grown in suspension were maintained in a 500 mL Erlenmeyer flask in a 37 °C CO2 incubator. Cells in the exponential growth phase were centrifuged to a viable cell density of 50 MVC / mL, and the high-density cell culture was continued in a mini bioreactor, a 50 mL volume container with a vent cap, in a 37 °C CO2 incubator in a state ready for transfection. In all groups, the plasmid DNA was 1 μg per 1 million cells, and the ratio of DNA (μg):PEI (μg) was 1:2. After incubation for 10 - 15 minutes, the DNA and PEI mixture was added to the cell suspension. In all groups, no further dilution step was performed after transfection, and the culture was maintained in the same culture vessel in the incubator until harvest at 72 hpT. Next, the production titers were obtained by ELISA for quantifying the assembled rAAV capsid and qPCR for quantifying the viral genome. Figure 7 shows four different transfection processes: VPC2.0 cells transfected with VPT reagent in VPM, VPC2.0 cells transfected with PEI in FS293 medium, HEK293F cells transfected with PEI in FS293 medium, and HEK293F cells transfected with PEI in BCD medium (from left to right). Figure 7A shows the cell density, viability, and transfection efficiency GFP% at harvest. Also included are the results from the transduction assay performed using the supernatant after centrifuging the finally harvested cell suspension. Figure 7B shows the production titers of viral genome / mL and capsid / mL in different groups. This demonstrates that the present invention is applicable to various transfection systems using different production cell lines and transfection media, and even different transfection methods. 【0101】 (References) 1. Xiao, et al. “Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.” J Virol 72 (1998): 2224-2232; 2. Grimm, et al. “Novel tools for production and purification of recombinant adeno associated virus vectors.” Hum Gene Ther 9 (1998): 2745-2760; 3. Matsushita, et al. “Adeno-associated virus vectors can be efficiently produced without helper virus.” Gene Ther 5 (1998): 938-945; 4. Maranga, L., et al. 2005. “Characterization of changes in PER. C6 TM cellular metabolism during growth and propagation of a replication‐deficient adenovirus vector.” Biotechnology and bioengineering, 90(5), pp.645-655; 5. Henry, O. et al. 2005. “Metabolic flux analysis of HEK-293 cells in perfusion cultures for the production of adenoviral vectors.” Metabolic engineering, 7(5-6), pp.467-476; 6. Joshi et al., 2021. “Advancements in molecular design and bioprocessing of recombinant adeno‐associated virus gene delivery vectors using the insect‐cell baculovirus expression platform”. Biotechnology Journal, 16(4), p.2000021.; 7. Meghrous et al., 2005. “Production of recombinant adeno‐associated viral vectors using a baculovirus / insect cell suspension culture system: From shake flasks to a 20‐L bioreactor”. Biotechnology progress, 21(1), pp.154-160.; 8. Urabe et al., 2002. “Insect cells as a factory to produce adeno-associated virus type 2 vectors”. Human gene therapy, 13(16), pp.1935-1943; 9. Riedl, et al. “Non-Viral Transfection of Human T Lymphocytes.” Processes 2018, 6, 188; 10. Backliwal et al. “High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI.” Biotechnol Bioeng. 2008 Feb 15;99(3):721-7; 11. Blackstock et al. "Comprehensive Flow Cytometry Analysis of PEI-Based Transfections for Virus-Like Particle Production", Research, 2020, Article ID 1387402; 12. Steger et al. CHO-S antibody titers >1 gram / liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines. J Biomol Screen. 2015 Apr;20(4):545-51.

Claims

[Claim 1] A process for producing recombinant vectors in eukaryotic cells, wherein the process is: a) A step of obtaining a cell suspension having a cell density of at least 25 million viable cells per milliliter; b) Transient transfection of cells in a cell suspension with a single exogenous plasmid DNA or multiple different exogenous plasmid DNAs to obtain transfected cells; c) The step of optionally performing cell regeneration for a certain period of time, wherein during the cell regeneration period the transfected cells are kept substantially still on the surface; and d) After the transfection of step b) and / or the optional cell recovery step c), the dilution of the transfected cells present in the cell suspension is limited or not performed at all, and thereafter the preparation of the recombinant vector is started from a culture of transfected cells having a cell density of at least 20 million viable cells per milliliter. The above process, including. [Claim 2] The process according to claim 1, wherein the cell suspension in step a) has a cell density of at least 30 million cells per milliliter, for example, at least 50 million cells per milliliter. [Claim 3] The process according to claim 1, wherein the cell suspension in step a) has a cell density of at least 80 million cells per milliliter, for example, at least 100 million cells per milliliter. [Claim 4] The process according to any one of claims 1 to 3, comprising a medium exchange performed at one or more time points from the time when the manufacturing of step d) is initiated, wherein the medium exchange is performed by perfusion, for example, in a perfusion bioreactor culture. [Claim 5] The process according to any one of claims 1 to 3, wherein the manufacturing in step d) is carried out for at least 24 hours, for example, at least 48 hours or 72 hours, or for 4, 5, 6, 7, 8, 9, or 10 days. [Claim 6] The process according to any one of claims 1 to 3, wherein no dilution is performed before the commencement of step d) that results in a cell density lower than 20 MVC / mL. [Claim 7] The process according to any one of claims 1 to 3, wherein the cell suspension in step a) contains cells in the suspension in proportion to at least 6% of the culture volume, and the cell suspension is selected from the group consisting of cell cultures, microbial fermentations, cell suspensions derived from cell tissue, and cell suspensions derived from blood. [Claim 8] The process according to any one of claims 1 to 3, wherein the eukaryotic cells are selected from the group consisting of mammalian cells, human cells, avian cells, insect cells and plant cells. [Claim 9] The cells are CHO, CHO-DBX11, CHO-DG44, CHO-S, CHO-K1, Vero, BHK, HeLa, COS, MDCK, HEK-293, HEK-293T, HEK-2 93S, HEK-293F, L293, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0, CRL7030, HsS78Bst cells, PER. C6, SP2, SP0, hybridoma, MRC-5, MDCK, WI-98, CAP, EB66, HUVEC, AGE1. The process according to claim 8, comprising cells selected from the group consisting of CR, CR, cells of Trichoplusia ni, cells of Spodoptera Frugiperda, SF9, SF21, Hi5, mesenchymal stem cells, endothelial cells, induced pluripotent stem cells, primary cells, cells of Nicotiana tabacum, BY2, cells of Nicotiana benthamiana, cells of Oriza sativa, cells of Arabidopsis thaliana, and cells of Daucus carota. [Claim 10] The process according to any one of claims 1 to 3, wherein the recombinant viral vector is a viral vector, a virus, a virus-like particle (VLP), or any combination thereof, or aggregates derived therefrom, or aggregates derived therefrom that associate with small molecules less than 1000 kDa in size. [Claim 11] The process according to any one of claims 1 to 3, wherein a recombinant adeno-associated virus (AAV) viral vector is produced, and optionally, the transfection in step b) is carried out using a transient (AAV) transfection system comprising co-transfection of at least three plasmids. [Claim 12] The process according to any one of claims 1 to 3, wherein the cell density of step a) is obtained by adjusting the cell density of the culture suspension by concentrating the cell content of the cell suspension and / or culturing the cells in the culture suspension, wherein the concentration or culturing is optionally carried out by diluting the cells of the suspension by centrifugation, filtration, microfiltration, precipitation, acoustic sedimentation, or specific cell binding, by batch culture, by supply batch culture, by perfusion culture, by chemostat, or by at least two combinations of these culture modes, and the culturing can be carried out in combination with cell concentration and / or cell dilution. [Claim 13] The process according to any one of claims 1 to 3, wherein the transient transfection comprises a chemical transfection using a chemical reagent selected from the group consisting of cationic polymers such as DEAE-dextran, polybrene, polyethyleneimine and derivatives thereof, dendrimers, calcium phosphate, polycations; liposomes and non-liposome transfection reagents and cationic lipids including lipid nanoparticles. [Claim 14] The process according to claim 13, wherein the polyethyleneimine is a synthesized cationic polymer having a linear or branched topology and a molecular weight in the range of 1 kDa to 1,000 kDa. [Claim 15] The process according to any one of claims 1 to 3, wherein the transfection includes a physical transfection method from the group consisting of microinjection, optical transfection, biolithographic transfection (also known as particle impact), electroporation, iron oxide nanoparticle delivery (also known as magnetofection), sonoporation, laser irradiation, and electric field-induced molecular vibration.

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