High-density eukaryotic cell transient transfection method for producing recombinant viral vectors

Abstract

The invention provides a method for preparing a recombinant virus vector in eukaryotic cells. In particular, the present invention relates to a method for preparing a recombinant viral vector by transient transfection of eukaryotic cells, comprising the step of transient transfection of cells in a cell culture wherein the cell culture has an unexpectedly high cell density of at least 25 MVC / mL. After the transfection step, an optional cell recovery step may be performed according to the host cell type, the exogenous nucleic acid, and the transient transfection method used. After the transfection step, or after an optional cell recovery step, the preparation step of the recombinant viral vector begins at an unexpectedly high cell density of at least 20 MVC / mL, a limited dilution or no dilution being performed after the transfection step.

Description

Technical Field

[0001] The present invention relates to the field of cell transient transfection and recombinant viral vector production. More specifically, the present invention relates to a high cell density transient transfection method for producing recombinant viral vectors. Background Art

[0002] Transient transfection is a gene transfer technique widely used to produce biologically active molecules or molecular assemblies, where exogenous nucleic acids are introduced into host cells for a limited period of time, rather than integrating into the host cell genome. After transient transfection, cells that have incorporated the target nucleic acid are able to express the transfected genetic material and proceed to the production phase of the biological product, which can be a protein, polymer, virus, virus-like particle, extracellular vesicle, etc. Compared to the use of stable production cell lines that take years to establish, transient transfection takes only a few days and is very versatile for host cell lines, transfection vectors, and target products. These advantages make it an excellent candidate for manufacturing large molecules such as recombinant proteins and viral vectors during drug discovery research and preclinical stages, or when the expressed form includes toxic moieties, where intensive screening and characterization of the target product and its manufacturing process parameters are performed. However, the biopharmaceutical industry has been reported to lack productivity of molecules such as viral vectors due to bottlenecks in the production process, such as low yield and high production costs.

[0003] In transient transfection, plasmid DNA is one of the common genetic materials delivered to host cells. Plasmid DNA is a small, circular, double-stranded DNA molecule. In some cases, co-transfection of multiple plasmids is required to produce certain products. For example, for the production of recombinant adeno-associated virus vectors (rAAV) in the field of gene and cell therapy, 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 vectors in the absence of helper adenovirus" Journal of Virology 72 (1998): 2224-2232; Grimm et al., "New tools for the production and purification of recombinant adeno-associated virus vectors" Hum Gene Ther 9 (1998): 2745-2760; Matsushita et al., "Enables efficient production of adeno-associated virus vectors in the absence of helper viruses" Gene Ther 5 (1998): 938-945]. Co-transfection is usually more challenging, since all genetic elements must be delivered simultaneously, but in some cases it remains the only or preferred option due to technical limitations. Moreover, the biosynthesis of recombinant viruses represents a very high level of complexity, in fact, it implies the biosynthesis and correct assembly of the capsid, composed of a total of 60 viral protein molecules, as well as the inclusion of the genome of interest in the capsid. It is known that the efficiency of this manufacturing process decreases when the cell density increases on a cell-specific basis, the so-called cell density effect [Maranga, L. et al., 2005 "PER.C6 during the growth and propagation of replication-defective adenoviral vectors"]. TM Characterization of cellular metabolic changes” Biotechnology and Bioengineering, 90(5), pp. 645-655; Henry, O. et al., 2005 “Metabolic flux analysis of HEK-293 cells in perfusion culture for adenoviral vector production” Metabolic Engineering, 7(5-6), pp. 467-476. Similar observations have been made for the production of rAAV in insect cells using a baculovirus-based expression system [Joshi et al., 2021 “Recombinant adeno-associated virus gene delivery using an insect cell baculovirus expression platform [Progress in molecular design and bioprocessing of vectors] Journal of Biotechnology, 16(4), pp. 2000021; Megarous et al., 2005 “Production of recombinant adeno-associated virus vectors using a baculovirus / insect cell suspension culture system: from shake flasks to 20 L bioreactors” Progress in Biotechnology, 21(1), pp. 154-160; Urabe et al., 2002 “Insect cells as factories for the production of adeno-associated virus type 2 vectors” Human Gene Therapy, 13(16), pp. 1935-1943].

[0004] The production process of recombinant viral vectors is usually divided into 3 steps: the first step is to obtain the required number of cells, that is, through cell expansion, transient transfection steps and production steps. First, the cells proliferated in the culture container are prepared for transient transfection, which is usually done by setting the density of the growing cells to 0.6 to 0.8 million viable cells per milliliter (MVC / mL) the day before transfection. On the day of transfection, the cell density reaches about 1 to 1.5 MVC / mL from cell growth, optionally exponentially. This operation provides a cell host with high cell viability for transfection and is carried out under culture conditions that are conducive to transfection efficiency.

[0005] In the case of transfection at high cell density, a high cell density cell suspension can be obtained by concentrating a low cell density cell culture or culturing cells at high density. After transient transfection, the cells incorporating the target nucleic acid are able to express the transfected genetic material and enter the production phase. In the case of high cell density transfection, a dilution step is usually performed after transfection to return the cells to a low cell density culture to achieve production. The time point for dilution and the cell density after dilution are highly dependent on the host cell type, the transient transfection method used, available laboratory instruments and control systems. After a period of production, usually 48 to 72 hours after transfection (hpT), the recombinant viral vector produced is harvested and the production titer is carefully checked before the next step of production (i.e., purification, etc.).

[0006] Various non-viral transient transfection methods are commercially available, including physical and chemical methods. Chemical transfection is a widely adopted method that utilizes transfection reagents to form a complex with the target nucleic acid, which is then delivered to 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 that condenses negatively charged DNA into PEI-DNA composite particles. The composite particles then bind to the cell surface and are transferred into the cell via endocytosis / phagocytosis. The PEI-mediated transfection process has become a popular technique due to its relatively low cost and ease of operation. According to the latest technology, chemical transfection for molecular production is usually performed at a cell density of ≤5 MVC / mL, and standardized protocols operate at 1-2 MVC / mL.

[0007] Another popular physical transfection technique is electroporation, where the transient transfection process can be performed at high cell density (up to 100 MVC / mL). This is achieved by concentrating the cell broth to a very high target cell density before electroporation, usually by centrifugation. After electroporation, the cell suspension is diluted back to a low cell density, such as ≤5 MVC / mL, such as 1-2 MVC / mL, to enter the production stage. However, the application of this technology is limited by the scale and cost of operations. Electroporation is used at laboratory scale, with batches below 6 mL and typically below 1 mL. Larger-scale solutions exist, such as flow electroporation technology commercially offered by Maxcyte, which can process up to 100 mL of cell suspension at a rate of 100 MVC / mL per batch, but needs to be diluted back to a low cell density, such as ≤5 MVC / mL, such as 1-2 MVC / mL, within 1 hour after electroporation for the production stage.

[0008] Therefore, the prior art methods have many disadvantages, such as low production yield, limited small-scale operation, and high production costs. The purpose of the present invention is to eliminate these disadvantages and provide a method for transiently transfecting cells and producing recombinant viral vectors, which has the potential for large-scale use to reduce costs. Summary of the invention

[0009] The present invention provides a method for producing a recombinant viral vector in a eukaryotic cell. Here, a process may also be referred to as a method, i.e., the terms are used interchangeably here.

[0010] In particular, the present invention relates to a method for transiently transfecting eukaryotic cells to produce a recombinant viral vector, comprising the step of transiently transfecting cells in a cell culture, wherein the cell culture has an unexpectedly high cell density of at least 25 MVC / mL.

[0011] After the transfection step, an optional cell recovery step or process may be performed depending on the host cell type, the exogenous nucleic acid or exogenous plasmid DNA, and the transient transfection method used. After the transfection step or after the optional cell recovery step or time period, the production step of the recombinant viral vector begins at an unexpectedly high cell density of at least 20 MVC / mL, with or without limiting dilution after the transfection step.

[0012] The method of the present invention comprises: a) obtaining a cell culture having a cell density of at least 25 million viable cells per milliliter; b) transiently transfecting cells in the cell suspension with exogenous plasmid DNA or several exogenous plasmid DNAs required for producing the recombinant viral vector, i.e., co-transfecting with multiple plasmid DNAs; c) Optionally, a period of cell recovery is allowed depending on the host cell type, exogenous plasmid DNA, and transient transfection method used. During cell recovery, the transfected cells are maintained in culture or solution with a large surface that facilitates gas exchange (such as oxygen or air exchange), and the transfected cells are generally kept stationary, or substantially stationary, and are generally kept stationary on a sufficiently large surface. This time period is typically greater than 1 minute and varies depending on the host cell type, the exogenous plasmid DNA and the transient transfection method used; d) after the transfection step b) and the optional cell recovery step c), starting the production step after limiting dilution or without dilution of the transfected cells to start the production phase from a cell density of at least 20 million viable cells per ml after the transfection step.

[0013] In other words, the present invention also discloses a method for producing a recombinant vector in a eukaryotic cell, the method comprising the following steps: a) obtaining a cell suspension having a cell density of at least 25 million viable cells per ml; b) transiently transfecting cells in the cell suspension with exogenous plasmid DNA or multiple different exogenous plasmid DNAs to obtain transfected cells; c) optionally, allowing a period of cell recovery, wherein the transfected cells remain substantially stationary on the surface during the cell recovery period; and d) after the transfection in step b) and / or the optional cell recovery step c), subjecting said transfected cells present in said cell suspension to a limiting dilution or to no dilution and thereafter starting production of said recombinant vector from a culture of said transfected cells at a cell density of at least 20 million viable cells per ml.

[0014] The surface of step c) above is typically the surface of a container or vessel or the like, as described elsewhere herein.

[0015] The target recombinant viral vector is then harvested some time after transfection, typically 24-240 hours post transfection (hpT), although longer production times are also envisioned, as described elsewhere herein.

[0016] By partially or completely eliminating the dilution step, returning it to a low cell density, such as ≤5 MVC / mL, such as down to 1-2 MVC / mL after transfection, the manufacturing process is greatly simplified. From the initial host cell inoculation to the final harvest of the target recombinant viral vector, the same container can be used for cell culture, transfection, and production. Therefore, the space occupied by the production equipment is largely minimized, making the entire manufacturing process scalable to large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The titers of rAAV capsids / mL produced during transient transfection of the present invention at 50 MVC / mL using different ratios of cell number to plasmid DNA, and different ratios of plasmid DNA to PEI (polyethyleneimine) are shown. Open bars: control group, cell density at transfection was 1 MVC / mL, in two different culture media; solid bars: groups 3 to 12, cell density at transfection was 50 MVC / mL and different ratios of cell number (million cells), plasmid DNA (μg) and PEI (μg). 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 1 MVC, and the ratios of DNA (μg): PEI (μg) were 1:1, 1:2, 1:4 and 1:8, respectively. In the 1:2:2, 1:2:4 and 1:2:8 groups, the plasmid DNA was 2 μg DNA per 1 million cells, and the ratios of DNA (μg): PEI (μg) were 1:1, 1:2 and 1:4, respectively. In the 1:4:4, 1:4:8 and 1:4:16 groups, the plasmid DNA was 4 μg DNA per 1 million cells, and the ratios of DNA (μg): PEI (μg) were 1:1, 1:2 and 1:4, respectively.

[0018] Figure 2 Shown are production titers of rAAV capsid / mL at 72 hpT during transient transfections performed at 50 MVC / mL, with different incubation times of cationic polymer and DNA, i.e., 5 min, 10 min, 15 min, or 0 min (“quick mix”), and with DNA added to the cell suspension and then the cationic polymer immediately added to the cell suspension without premixing the cationic polymer and DNA (“direct transfection”).

[0019] Figure 3 Shown are production titers of rAAV capsid / mL following transient transfection procedures of the invention at 100 MVC / mL, followed by dilution of AAV production harvested at 72 hpT at different cell densities using different media exchange frequencies; from left to right: 1) Dilute to 10 MVC / mL and perform medium exchange at 24 hpT and 48 hpT as a control for dilution of cell density outside the present invention; 2) dilute to 10 MVC / mL and perform a medium exchange at 48 hpT as a control for dilution cell density outside the present invention; 3) dilute to 30 MVC / mL and perform medium exchange at 18 hpT, 42 hpT, and 66 hpT; 4) Dilute to 30 MVC / mL and perform medium exchange at 24 hpT and 48 hpT.

[0020] Figure 4 The production of rAAV viral vectors at a cell density of 64 MVC / mL by the transient transfection process of the present invention in a perfusion bioreactor culture with a working volume of 220 mL is shown. Solid circles: viable cell density (MVC / mL); open circles: viability (%); open squares: transfection efficiency (GFP signal %).

[0021] Figure 5 The production of rAAV viral vectors by the transient transfection process of the present invention using flow electroporation at a cell density of >80 MVC / mL in a perfusion bioreactor culture with a working volume of 200 mL is shown. Solid circles: viable cell density (MVC / mL); open circles: viability (%); open squares: transfection efficiency (GFP signal %).

[0022] Figure 6 The production of rAAV viral vectors by the transient transfection process of the present invention at 50 MVC / mL in a perfusion bioreactor culture with a working volume of 200 mL is shown, and two control cultures at 2 MVC / mL viable cell density and 15 MVC / mL viable cell density are also shown, wherein the transfection process was carried out at 37°C CO 2 The experiments were performed in a pseudo-perfusion microbioreactor in an incubator, i.e., a 50 mL vessel with a vented cap.

[0023] Figure 6 (A) shows long-term production in a bioreactor, with viable cell densities up to 120 MVC / mL during the production phase; solid line with solid circles: viable cell density; open circles: cell viability; open squares with dashed lines: transfection efficiency for GFP expression; open diamonds: production titer viral genomes / mL; control cultures transfected at a cell density of 1 MVC / mL (left pattern - solid circles) with viral genome yield / mL (left solid diamonds) in a micro-bioreactor, and control cultures transfected at a cell density of 15 MVC / mL (right solid diamonds) in a micro-bioreactor with viral genome yield / mL (right pattern - solid circles) are represented in the same graph for convenience, but the time scale does not apply to these.

[0024] Figure 6 (B) shows the expression of 3, 6 and 7 days (3dpT, 6dpT and 7dpT) from Figure 6(A) The long-term consistent high infectious titer of the supernatant samples collected during the bioreactor run indicates the biological efficiency of the produced viral vector, as indicated by the GFP expression of infected cells in the transduction assay (solid bars). For comparison, the transfection in the reference (Ref) was performed at a low cell density of 1 MVC / mL in a shake flask, and the supernatant was collected 3 days after transfection, which is a common practice for rAAV production.

[0025] Figure 7 Several transient transfection production processes of rAAV viral vectors of the present invention at a cell density of 50 MVC / mL are shown. Two cell lines were used: HEK293F and virus production cell 2.0 (VPC2.0). Two different chemical transfection methods, cationic polymer PEI and cationic lipid-based transfection reagent (VPT), ​​were used, and three transfection media, virus production medium (VPM), Free Style 293 (FS293) and Balan CD HEK (BCD) were used. Figure 7 (A) shows the cell density and viability when 3dpT was harvested, and the transduction experiment using the supernatant of 3dpT harvested. Figure 7 (B) shows the relevant production titers. DETAILED DESCRIPTION

[0026] Definitions and Abbreviations

[0027] Terms not specifically defined herein shall have the meanings given to them by those skilled in the art based on the present disclosure and context. Unless otherwise specified, the following terms used in this specification have the meanings indicated.

[0028] The term "transient transfection" as used herein means that the introduced nucleic acid may only be present in the cell for a limited time and is not necessarily integrated into the cell genome. The transfected cells then express the transiently transfected gene for a limited time, usually limited to a few days. A common model of transfection is to express the green fluorescent protein GFP as a target gene. The production of GFP can be easily quantified based on its fluorescence in successfully transfected cells. "Transfection efficiency" gives the percentage of cells expressing GFP among all cells. In the present disclosure, the gene encoding GFP is used as a target gene or a cargo delivered by AAV used for gene therapy.

[0029] The term "cell suspension" as used herein refers to a solution of cells that are not adhered to a static support. Such cells are typically grown as a mixture in a stirred tank bioreactor or stirred culture vessel to ensure that nutrients are delivered to the cells. A cell suspension can be obtained from cells grown in culture, a concentration procedure, and / or a dilution procedure. Herein, the term may be used interchangeably with the term or expression "cell culture suspension", "suspension containing cells", etc.

[0030] The term "cell density" as used herein refers to the concentration of cells within a given volume, and biomass is quantified based on the cell concentration. Therefore, in this article, the term "cell density" is sometimes used interchangeably with the expression "cell concentration" and the like. A commonly used unit is million viable cells per milliliter (MVC / mL), which is widely used for eukaryotic cells. Quantification of cells can also be expressed as cell volume fraction; for example, in the present disclosure, in a cell suspension with a cell density of 25 MVC / mL and an average cell diameter of 17-22 μm, cells account for at least 6% of the culture volume (volume / volume).

[0031] Here, when "limiting dilution or no dilution" is performed as disclosed herein, it means that the dilution is limited, that is, the starting cell density for producing the recombinant vector is at least 20 million viable cells per milliliter, compared to conventional methods where dilution is usually performed to a low cell density ≤5 MVC / mL (usually 1-2 MVC / mL) to start the production of the recombinant vector.

[0032] As used herein, the term "perfusion" is a process in which cells are retained within a bioreactor or culture vessel using a dedicated device connected to the bioreactor or culture vessel while the culture medium in the reactor or vessel is continually exchanged at time points deemed appropriate for the particular process. The culture medium exchange provides fresh culture medium to the cells while removing spent culture medium containing cellular waste and byproducts. This method is known in the art, as are any variations of this method that can be applied to the present disclosure by those skilled in the art.

[0033] As used herein, the term "pseudo-perfusion" is a process that is equivalent in purpose to a perfusion process, i.e., the spent culture medium is completely or partially removed and replaced with fresh culture medium at a time point and ratio appropriate for the specific process, but this operation is performed by centrifugation or sedimentation of cells. This procedure can be performed manually or automatically using a robotic processor or variants thereof.

[0034] "Perfusion bioreactor culture" as used herein refers to cell culture in a bioreactor run as a perfusion process according to the present invention.

[0035] Riedl et al. previously reported a non-viral transfection of human T lymphocytes in which the transfection process was as high as 40 MVC / mL [Riedl et al. "Non-viral transfection of human T lymphocytes" Processes 2018, 6188]. This number should not be directly regarded as such, as naive T cells are approximately 5-7 μm in diameter. If human T lymphocytes were to occupy a 6% portion of the culture volume, the cell density of these cells would be 530 MVC / mL, while 6% of the culture volume of cells with a diameter of 17-22 μm corresponds to a density of 25 MVC / mL as described in the present disclosure. The term "cell recovery" as used herein refers to an optional period of time after transfection to allow cells to recover from the transfection process, thereby improving cell survival and further promoting the production of the target product. In non-viral transient transfection gene delivery, regardless of the method used, the exogenous DNA must overcome several obstacles to successfully express the target gene it carries. This process involves the host cell membrane being disrupted to a certain extent to allow the introduction of the exogenous DNA into the cell, followed by the transport of the DNA to the nuclear membrane within the cytoplasmic compartment and finally across the nuclear membrane. Gene expression will only occur when enough intact DNA enters the cell nucleus, which is why all transfection methods are considered to be harmful to the cell's part.

[0036] Post-transfection cell recovery is sometimes used in non-viral gene delivery methods, such as electroporation, because cell viability is greatly reduced during transfection. However, this is not commonly used in transfection methods using chemical reagents because the cell density used in chemical transfection is usually very low and ≤5 MVC / mL, usually 1 or 2 MVC / mL, so that the cells are not subjected to stress such as limited nutrient and oxygen access that usually occurs in cell suspensions with high cell density.

[0037] In the present disclosure, the transient transfection process is based on high cell density, so an optional cell recovery process is used in two embodiments. In both embodiments where a cell recovery process is applied after transfection, the transfected cell suspension at high cell density is transferred to a large container with a large surface area, in which the transfected cell suspension is well dispersed to obtain sterile air / oxygen. Cell recovery is not a necessary step in the present disclosure, and the cell recovery time can vary widely, depending on the transfection method used, cell type, plasmid DNA, etc.

[0038] Embodiment 4 herein is an example of AAV production without a cell recovery process.

[0039] In addition to the above definitions, the following abbreviations are used in the description and exemplary embodiments. If an abbreviation used herein is not defined, it has its generally accepted meaning. Detailed description

[0040] The present invention provides methods for transiently transfecting eukaryotic cells at high cell density to produce recombinant viral vectors in high yield. Such processes are used to produce recombinant vectors on a large scale to simplify and reduce the costs typically associated with such processes. Specific process steps or circumstances discussed in the context of any method or process herein are equally applicable to any other process or method disclosed herein.

[0041] The methods or processes disclosed herein are in vitro methods or processes, and the cells used in any such methods or processes have been isolated from their natural environment. Thus, the cells "obtained" or "provided" in the methods disclosed herein, etc., are not directly obtained from their natural environment.

[0042] Here, more specifically, a method for producing a recombinant vector in a eukaryotic cell is provided, the method comprising the following steps: a) obtaining a cell suspension having a cell density of at least 25 million viable cells per ml; b) transiently transfecting cells in the cell suspension with exogenous plasmid DNA or multiple different exogenous plasmid DNAs to obtain transfected cells; c) optionally, allowing a period of cell recovery, wherein the transfected cells remain substantially stationary on the surface during the cell recovery period; and d) after the transfection in step b) and / or the optional cell recovery step c), subjecting said transfected cells present in said cell suspension to a limiting dilution or to no dilution and thereafter starting production of said recombinant vector from a culture of said transfected cells at a cell density of at least 20 million viable cells per ml.

[0043] "Production" is sometimes also referred to herein as "production phase" and is related to the production of recombinant vectors. The surface may be a surface in a container or vessel, for example providing a large oxygen exchange.

[0044] The present invention also provides a method, wherein the cell density or concentration of the cell suspension of step a) is at least 30 million cells per milliliter. The present invention also provides a method, wherein the cell density or concentration of the cell suspension of step a) is at least 50 million cells per milliliter. The present invention also provides a method, wherein the cell density or concentration of the cell suspension of step a) is at least 80 million cells per milliliter. The present invention also provides a method, wherein the cell density or concentration of the cell suspension of step a) is at least 100 million cells per milliliter. Naturally, when the cell suspension of step a) has a higher cell density or concentration, such as 80 million cells per milliliter, the production of step d) started after the transfection of step b) or step c) can start from a cell density or concentration close to 80 million cells per milliliter, such as 75 million or 78 million cells.

[0045] Similar reasoning applies to other examples of cell density or cell concentration disclosed herein, ie the initial cell density or concentration of step a) compared to the density or concentration of step d).As previously mentioned, the terms cell density and cell concentration are used interchangeably herein.

[0046] The present invention also provides a method, wherein the method comprises: a medium exchange performed at one or more time points at the start of production in step d). This medium exchange can be performed at different time points, such as after 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and / or 48 hours, but is not limited to after the start of production.

[0047] The present invention also provides a method comprising perfusion.

[0048] The present invention also provides a method, wherein the method comprises: a medium exchange by perfusion at the beginning of the production or production phase of step d). Thus, the medium exchange is performed during the production process of step d). The perfusion can be performed in a perfusion bioreactor culture, as described herein.

[0049] Here, as described elsewhere herein, it may be advantageous to carry out no dilution or a limited or partial dilution before starting the production in step d).

[0050] The present invention also provides a method, wherein, before starting the production in step d), no dilution is performed which would result in a cell density below 20 MVC / mL.

[0051] The medium exchange and perfusion processes were shown to be particularly relevant for maintaining high cell densities after the transfection process and for obtaining high yields of the recombinant vector after the production phase.

[0052] As an alternative to perfusion bioreactor cultivation, a pseudo-perfusion process can be used.

[0053] The production of step d) of the methods described herein may be carried out for at least 24 hours, such as 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 may benefit from a longer production period.

[0054] The cell suspension of step a) may be such that the cells of the suspension occupy at least 6% of the culture volume, and wherein the cell suspension is selected from cell culture, microbial fermentation, cell tissue-derived cell suspension and blood-derived cell suspension.

[0055] Here, the eukaryotic cell can be selected from mammalian cells, human cells, avian cells, insect cells, and plant cells. More specifically, the cell is selected from 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, NSO, CRL7030, A group consisting of HsS78Bst cells, PER, C6, SP2, SPO, hybridoma cells, MRC-5, MDCK, Wl-98, CAP, EB66, HUVEC, AGE1, CR, CR, Trichomonas cells, Spodoptera frugiperda cells, SF9, SF21, Hi5, mesenchymal stem cells, endothelial cells, induced pluripotent stem cells, primary cells, tobacco cells, BY2, Nicotiana tabacum cells, rice cells, Arabidopsis cells, and carob cells.

[0056] The cells used in the present disclosure do not include cells involved in destroying human embryos. Therefore, such cells do not belong to the present disclosure and do not form a part of the present disclosure.

[0057] Here, the recombinant viral vector can be a viral vector, a virus, a virus-like particle (VLP) or any combination thereof, or they are derived from an assembly, or they are derived and combined with a small molecule of less than 1000 kDa. More specifically, recombinant adeno-associated virus (AAV) viral vector particles can be prepared herein, optionally, wherein the transfection of step b) is performed using a transient transfection (AAV) system including co-transfection of at least three plasmids. This will be illustrated in the experimental section, and the system is well known to those skilled in the art.

[0058] The cell density of step a) of the method is obtained by concentrating the cell content in the cell suspension and / or by adjusting the cell density of the culture suspension by culturing cells in the culture suspension. Alternatively, the cell density of step a) can be adjusted by concentrating the cells in the suspension, for example by centrifugation, filtration, microfiltration, precipitation, acoustic sedimentation or specific binding of cells, or by diluting the cells in the suspension. In addition, the cell density of step a) of the method is adjusted by cultivation, and the cultivation mode includes batch culture, fed batch culture, perfusion culture, constant culture, or a combination of at least two of these culture modes, wherein the cultivation can be performed in combination with cell concentration and / or cell dilution. These are all procedures well known in the art.

[0059] Here, the transient transfection includes chemical transfection using chemical reagents, and the chemical reagents are selected from cationic polymers, such as cationic polymers including DEAE dextran, polyisoprene, polyethyleneimine and its derivatives, dendritic macromolecules, calcium phosphate, polycations, and cationic lipids including liposomes and non-liposome transfection reagents and lipid nanoparticles. The polyethyleneimine is a synthetic cationic polymer with a linear or branched topological structure, with a molecular weight range of 1kDa to 1000kDa. In addition, in this article, the transfection includes a physical transfection method selected from the following: microinjection, optical transfection, biogun transfection (also known as particle bombardment), electroporation, iron oxide nanoparticle delivery (also known as magnetofection), sonoporation, laser irradiation, electric field induced molecular vibration.

[0060] The present invention also provides a method, more specifically, a method for producing AAV viral vectors by eukaryotic cells. In particular, the present invention relates to a method for transiently transfecting eukaryotic cells to produce AAV viral vectors, including the step of transiently transfecting cells in a cell suspension, wherein the cell density of the cell suspension is at least 25 MVC / mL. After transfection, an optional cell recovery process can be performed depending on the host cell type, plasmid DNA, and the transient transfection method used. During transfection or an optional cell recovery period, the production of AAV viral vectors begins at a cell density of at least 20 MVC / mL, and a limiting dilution step or a step without dilution is performed after transfection.

[0061] High cell density is one of the key factors in bioproduction processes to produce high expression levels of target products. Very high cell density can significantly increase the volumetric yield of the target product produced. However, to date, there have been no reports on very high cell density transfection and very high cell density production of viral vectors produced by transient transfection processes. This is due to the limitations of cell density and cell fragility during transfection. It is challenging to maintain the culture at high cell density while the cells are still producing, especially at above 50 MVC / mL, while maintaining an average good viability. This is why transient production of viral vectors is not performed in this cell density range. As mentioned before, all transfection methods are harmful to the cells.

[0062] The key factor to achieve high cell density culture is high average survival rate, which is difficult to maintain during transient transfection. Since the number of cells that need to be transfected in high cell density culture increases significantly, the amount of plasmid DNA and, in the case of chemical transfection, the chemical transfection reagent also increases significantly, which raises the issue of cell toxicity. Therefore, this will compromise cell survival rate and result in low viral vector yield.

[0063] It is important to note that the dilution step to a low density after high cell density transfection is not efficient and is considered a disadvantage from a process perspective. It creates an obstacle to simplified processing and increases the use of culture medium, which further leads to a larger footprint of production equipment for the entire manufacturing process and raises unnecessary storage and transportation issues. Prior to the present disclosure, Backliwal et al. reported in situ transfection of suspension-adapted HEK-293 cells with 25 kDa linear polyethyleneimine at densities up to 20 MVC / mL in complex culture medium, followed by production at a lower cell density (1 MVC / mL) [Backliwal et al. "High-density transfection of HEK-293 cells allows transient titer doubling and eliminates the 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 that can produce virus-like particles at higher transfection cell densities of 15-25 MVC / mL [Blackstock et al. "Comprehensive flow cytometric analysis of PEI-based transfection for virus-like particle production," Research, 2020, Article ID 1387402].

[0064] In their work, cells were centrifuged three hours after transfection and the cell density was set to 1 MVC / mL, which means that the transfected cell suspension was diluted to 1 MVC / mL at the beginning of the production phase. So far, the dilution step after transfection is necessary for high cell density transfection processes. Dilution is specifically mentioned when using physical transfection methods such as electroporation. In electroporation methods, high cell density has the advantage of close physical distance between host cells and plasmid DNA, but cells need to be diluted after electroporation to start the production phase. So far, the highest cell density reported after electroporation is 10 MVC / mL [Steger et al. Transient gene expression of CHO-S antibody titers >1 g / L using flow electroporation followed by rapid migration to high-yielding stable cell lines. J Biomol Sieve. 2015 Apr;20(4):545-51].

[0065] The present invention provides a method comprising: a step of transiently transfecting cells in a cell suspension, wherein the cell density of the cell suspension is at least 25 MVC / mL. After transfection, an optional cell recovery process can be performed if necessary. After transfection or an optional cell recovery period, the production of viral vectors is started at a cell density of at least 20 MVC / mL, with or without a limiting dilution step after transfection.

[0066] In other words, the present invention provides a method or process, wherein the process comprises: a) obtaining a cell suspension having a cell density of at least 25 million viable cells per ml; b) transiently transfecting cells in the cell suspension with exogenous plasmid DNA or several exogenous plasmid DNAs required for producing the recombinant viral vector, i.e., co-transfecting with multiple plasmid DNAs; c) Optionally, a period of cell recovery is performed depending on the host cell type, the exogenous plasmid DNA, and the transient transfection method used. During the cell recovery period, the transfected cells are maintained on a sufficiently large surface. This period of time may vary depending on the host cell type, the exogenous plasmid DNA, and the transient transfection method used; d) after the transfection step b and / or the optional cell recovery step c, subjecting the transfected cells to a limiting dilution or no dilution to start the production phase from a cell density of at least 20 million viable cells per ml.

[0067] In some embodiments of the disclosed methods, the cell culture suspension has a cell density of at least 30 million cells per milliliter.

[0068] In some embodiments of the disclosed methods, the cell culture suspension has a cell density of at least 50 million cells per milliliter.

[0069] In some embodiments of the disclosed methods, the cell culture suspension has a cell density of at least 80 million cells per milliliter.

[0070] In some embodiments of the disclosed methods, the cell culture suspension has a cell density of at least 100 million cells per milliliter.

[0071] In some embodiments of the methods of the present disclosure, the cell culture suspension is such that cells occupy at least 6% of the culture volume, and the cell culture suspension is selected from cell culture, microbial fermentation, cell tissue-derived cell suspension, and blood-derived cell suspension.

[0072] Cells useful in the disclosed methods are typically eukaryotic cells, such as mammalian cells, human cells, avian cells, insect cells, and plant cells.

[0073] Typically, the cell is selected from 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, Trichomonas cells, Spodoptera frugiperda cells, SF9, SF21, Hi5, primary T cells, leukemia cells, mesenchymal stem cells, endothelial cells, induced pluripotent stem cells, primary cells, tobacco cells, BY2, Nicotiana tabacum cells, rice cells, Arabidopsis cells, and carob cells.

[0074] The cells used in the present disclosure do not include cells involving the destruction of human embryos. Therefore, such cells do not belong to the present disclosure and do not form a part of the present disclosure.

[0075] Here, the recombinant viral vector can be a viral vector, a virus, a virus-like particle (VLP), or any combination thereof, or an assembly derived from them, or an assembly derived from them and bound to a small molecule of less than 1000 kDa. The cell density of the cell suspension in the disclosed method can be obtained by adjusting the cell density of the culture suspension by concentration and / or culture as described elsewhere herein.

[0076] 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 by diluting the cells.

[0077] In some embodiments, the cell density of the cell suspension is reached by culturing. Any suitable culturing method known in the art can be used, such as batch culture, fed-batch culture, perfusion culture, chemostat culture or a combination of at least two of these culture modes, and culturing can be combined with cell concentration or cell dilution.

[0078] Transfection is performed on the day when the cell density in culture reaches at least the target cell density. In case of cell density above the target, the total number of cells required for transfection is calculated and the cell suspension that produces the required total number of cells is removed and diluted with fresh growth medium to reach the target cell density and target cell suspension volume. Excess cells are then discarded from the system. After transfection, the transfected cell suspension is transferred to the same culture vessel used for cell expansion and proceeds to the production phase.

[0079] The transient transfection of the present invention is a non-viral gene delivery method, which can be a chemical transfection method or a physical transfection method. In the case of chemical transfection, it uses or may use a chemical agent selected from the following: cationic polymers, such as DEAE dextran, polyisoprene, polyethyleneimine and its derivatives, dendritic macromolecules, calcium phosphate, polycations; cationic lipids include liposomes and non-liposome transfection agents, lipid nanoparticles.

[0080] In the case of a physical transfection method, it refers or may refer to one or more methods selected from: microinjection, optical transfection, biolistic transfection (also known as particle bombardment), electroporation, iron oxide nanoparticle delivery (also known as magnetofection), sonoporation, laser irradiation, electric field induced molecular vibration, but is not limited thereto.

[0081] In one embodiment, the chemical agent is a cationic polymer. A representative cationic polymer is polyethyleneimine with a linear or branched topology. Typically, the molecular weight of polyethyleneimine ranges from 1 kDa to 1000 kDa.

[0082] Transient transfection according to the method of the present disclosure is or can be a physical transfection method selected from: direct microinjection into cells or cell nuclei, electroporation, biogun particle delivery (also known as particle bombardment), iron oxide nanoparticle delivery (also known as magnetofection), sonoporation, laser irradiation, electric field induced molecular vibration, but is not limited thereto.

[0083] Microinjection is a technique for delivering DNA directly to the nucleus, while electroporation uses an electrical potential to induce pores in the cell membrane to allow foreign DNA to enter the cell. Bioparticle delivery or microprojectile bombardment is a technique for delivering foreign genes to cells using heavy metal particles coated with foreign DNA. Sonoporation uses physical disturbances in fluids to induce pores in the cell membrane to deliver nucleic acids. Magnetofection is a transfection method that uses a magnetic field to focus particles containing foreign DNA into target cells. Laser irradiation penetrates individual cells by focusing a laser beam on a localized area of ​​the cell membrane to allow nucleic acids to enter.

[0084] In one embodiment, transient transfection is a physical transfection method selected from the group consisting of: microinjection, optical transfection, biolistic transfection (also known as particle bombardment), electroporation, iron oxide nanoparticle delivery (also known as magnetofection).

[0085] The invention will now be illustrated by the following experimental section describing specific embodiments. These embodiments are intended to illustrate the invention and are not intended to limit the scope of the invention to the embodiments described.

[0086] Experimental Section

[0087] Materials and methods

[0088] Cell lines and culture media

[0089] HEK293F cells were purchased from Thermo Fisher Scientific. The culture media were Balan CD HEK293(-) glucose medium (Fujifilm, USA), supplemented with glucose and L-glutamine for some transfection experiments specified in the text, FreeStyle 293 expression medium (Gibco), Free Style F17 expression medium (Gibco), and virus production medium (Gibco). When expanded in Erlenmeyer flasks and cultured in spin tubes (50 mL Corning) or tubular spin bioreactors (50 mL TPP), the cells were kept at 37°C, 5% CO 2 The stirring speeds were 120 rpm and 320 rpm in the incubator.

[0090] Transfection materials

[0091] Chemical transfection

[0092] Transient transfection in this study was performed by PEI-mediated chemical transfection, using PEI-MAX (mW40000, Polysciences) high-efficiency linear PEI. For the production of serotype AAV1, the plasmid was the rAAV helper-free packaging system containing 3 plasmids: pHelper, pRC1, and pGFP (Cell Biolabs). For the production of AAV9, pR2C9 (pAAV2 / 9nAddgene) was used with the same pHelper and pGFP used for AAV1 production. Transfection efficiency was monitored using a model gene for GFP, and the infectious titer of the produced rAAV was assessed in a transduction assay. HEK293F cells were cultured at 37°C and 5% CO 2 Grow in suspension in conical cell culture flasks in an incubator.

[0093] Physical transfection

[0094] Electroporation Maxcyte ExPERT STx is used with multiple CL2 units with a processing capacity of 100mL / unit of high-density cell suspension.

[0095] Experimental setup

[0096] For the first time, a small-scale system was established to screen and optimize key process parameters. Small-scale studies were performed in microbioreactors, i.e., 50 mL tubular containers with vented caps, called spin tubes, with a working volume of 5 mL, at 37 °C in a CO atmosphere. 2 The cells were shaken in an incubator. These operations were performed in pseudo-perfusion mode, with manual medium replacement by centrifugation and retention of all cells. Thereafter, the developed process was applied to perfusion cultures in a bioreactor system DASGIP (Eppendorf) with a working volume of 200 mL, using an alternating flow filter as a cell retention device with a microfilter hollow fiber cartridge (both Repligen). After transfection, the transfected cell suspension was kept in the same bioreactor until harvest. Unless otherwise specified in the text, culture harvesting was performed at 72 hours post-transfection (hpT) by centrifugation at 200 × g for 5 minutes, and both supernatants and cell pellets were stored at −80 °C for analysis. To determine the production titer, cell pellet samples were resuspended in a 50 mM Tris, 150 mM NaCl, and 2 mM Mg Cl 2 The cell suspension was then frozen and thawed for 3 rounds (10 min each) by alternating the tubes between an isopropanol bath at -80°C and a water bath at 37°C. The crude lysate was then incubated with 50 units / mL Denarase at 37°C for 45 min, then centrifuged at 3000 g for 10 min at 4°C, and the clear supernatant was collected for further analysis.

[0097] Analytical methods

[0098] Cell density was measured with Bioprofile FLEX (Nova Biomedical) or Norma XS (Iprasense). Concentrations of glucose, lactate, glutamine, and ammonium were measured by Cedex Bio (Roche). Transfection efficiency (GFP intensity) was measured by two flow cytometers, Gallios (Beckman Coulter) and Guava EasyCyte (Luminex Corporation). The produced viral vector capsids were quantified using AAV1 and AAV9 titration ELISA kits (Progen Biotechnik). The viral genome was analyzed by the AAVpro titration kit for qPCR (Takara Bio) and measured using the CFX96 real-time qPCR detection system (Bio-rad). Transduction assays were used to evaluate functional infectious viral vector production. For transduction assays, supernatants or cell lysates were added to healthy HEK293F cell suspensions and GFP expression levels were monitored 72 hours after transduction.

[0099] Example 1 (Implementation Method 1)

[0100] In Embodiment 1, the transient transfection process is a high cell density transfection process of 50 MVC / mL mediated by a cationic polymer PEI (polyethyleneimine) for the production of rAAV. The purpose of this experiment is to demonstrate that the high cell density transfection process is a universal process that can be applied to various transient transfection conditions with different ratios of plasmid DNA to cell number and different ratios of vector DNA to cationic polymer.

[0101] Transfection is accomplished using an AAV helper-free multi-plasmid transient transfection system that co-transfects 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 required for the production of infectious AAV. This function is required for the production of infectious AAV particles and is provided in vivo by a helper virus such as adenovirus, which is simply replaced by the pHelper gene during the manufacturing process. Plasmid pGFP contains the gene encoding GFP (green fluorescent protein) and is used as a model for the target gene or cargo of gene therapy. GFP is often used as a model gene for the target and is easily quantified due to fluorescence detection, demonstrating the efficiency of transfection.

[0102] HEK293 cells grown in suspension were maintained at 37°C in a CO atmosphere. 2Cells in the exponential growth phase were centrifuged to a viable cell density of 50 MVC / mL and continued to be cultured at high density in a microbioreactor, i.e., at 37°C with CO. 2 In an incubator, transfection was performed using a 50 mL centrifuge tube with a vented cap.

[0103] Several sets of transfection reagents were prepared with different ratios of plasmid DNA to PEI and different ratios of plasmid DNA to cell number. Two sets of transfections with 1 MVC / mL were performed at normal cell density as a reference according to standard transfection protocols commonly used in the field. During transfection, 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 steps were performed and the cultures were maintained by pseudo-perfusion in the same culture vessel in the incubator until harvesting 72 hours after transfection (hpT).

[0104] The production titer was then quantified by ELISA (enzyme-linked immunosorbent assay), which analyzes the assembled rAAV capsids in the harvest. Figure 1 In the figure, the two groups counted from the left are controls, where the cell density at the time of transfection was 1 MVC / mL. From the third group onwards, transfection was performed at 50 MVC / mL with varying ratios between cells, plasmid DNA and PEI. The production titer was then quantified by ELISA (enzyme-linked immunosorbent assay), which analyzes the assembled rAAV capsids in the harvest. Figure 1In the figure, the two groups counted from the left are controls, in which the cell density at the time of transfection was 1 MVC / mL. From the third group onwards, transfection was performed at 50 MVC / mL, and the ratios between cells, plasmid DNA, and PEI were different. The groups are named after the ratios between the number of cells (MVC): plasmid DNA (μg): PEI (μg): in the groups of 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 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 the groups of 1:2:2, 1:2:4, and 1:2:8, the plasmid DNA was 2 μg DNA per 1 million cells, and the ratios of DNA (μg): PEI (μg) were 1:1, 1:2, and 1:4, respectively. In the 1:4:4, 1:4:8, and 1:4:16 groups, the plasmid DNA was 4 μg DNA per 1 million cells, and the ratio of plasmid DNA (μg): PEI (μg) was 1:1, 1:2, and 1:4, respectively. As can be seen from the figure, except for the last group, 1 million cells: 4 μg plasmid DNA: 16 μg PEI, all other groups had production titers higher than the control group performed at 1 MVC / mL. Due to its cationic properties, PEI is well known for its cytotoxicity; therefore, it is reasonable that the productivity is reduced when the transfection reagent is overused.

[0105] Example 2 (Implementation Method 2)

[0106] In Example 2, the transient transfection process is a 50 MVC / mL high cell density transfection process mediated by a cationic polymer PEI for the production of rAAV. The purpose of this experiment is to demonstrate that the transfection reagent (a mixture of plasmid DNA and a cationic polymer) has a very high cell density transfection process at different incubation times. The transfection is completed by a multi-plasmid transient transfection system without an AAV adjuvant, which co-transfects three plasmids: pAAV-RC1, pHelper, and pGFP. Plasmid pAAV-RC1 provides viral rep (replication) and cap (capsid) genes. Plasmid pHelper carries the adenoviral gene products required for the production of infectious AAV. Plasmid pHelper carries the adenoviral gene products required for the production of infectious AAV. This function is necessary for the production of infectious AAV particles and is provided in vivo by helper viruses such as adenovirus, and is only replaced by the pHelper gene during the manufacturing process. Plasmid pGFP contains a gene encoding GFP (green fluorescent protein) and is used as a model for the target gene or cargo of gene therapy. GFP is often used as a model gene for transfection and is easily quantified due to fluorescence detection, demonstrating the efficiency of transfection.

[0107] HEK293 cells grown in suspension were maintained at 37°C in a CO atmosphere.2 The 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 microbioreactor in pseudo-perfusion mode, that is, at 37°C with CO 2 Transfection was performed in an incubator using a 50 mL container with a vented lid. Plasmid DNA was 1 μg per 1 million cells, and the ratio of DNA (μg): PEI (μg) was 1:2 for all groups. Using this ratio, the use of plasmid DNA and PEI was minimized, with equivalent production titers as described in Example 1, which significantly reduced the costs associated with plasmid production and PEI. After mixing DNA and PEI, different incubation times were used: 5 minutes "5min", 10 minutes "10min" or 15 minutes "15min" (see Figure 2 ). After incubation, the mixture of DNA and PEI was added to the cell suspension. In the "0 min quick mix" group, DNA was mixed with PEI, mixed quickly by inverting the mixture several times, and then immediately added to the cell suspension without any incubation. In the "0 min, direct transfection" group, DNA was added to the cell suspension and PEI was immediately added to the cell suspension, completely eliminating the pre-mixing of plasmid DNA and PEI. In all groups, no further dilution steps than 3:5 were performed after transfection, i.e., 2 mL of mixed DNA and PEI were added to 3 mL of cell suspension at 50 MVC / mL for transfection, and the culture was maintained in the incubator in the same culture vessel until harvest at 72 hpT. The production titer was then quantified by ELISA, which analyzed the assembled rAAV capsids in the harvest. Figure 2 It is shown that all used procedures ensured the production of AAV capsids in the final harvest, and the yields of all groups were comparable, except for "0 min, direct transfection" which produced a lower AAV yield. This indicates that transfection can be performed with or without the formation of cationic polymer / DNA complexes, and that different incubation times are applicable when the cationic polymer and DNA are premixed.

[0108] Example 3 (Implementation Method 3)

[0109] In Embodiment 3, the transient transfection process is a high cell density transfection process mediated by PEI at 100 MVC / mL for the production of rAAV. The purpose of this experiment is to demonstrate the process of extremely high cell density transfection of various high cell densities (10 MVC / mL as control, 30 MVC / mL) with different dilutions after transfection to the production stage. The transfection is completed by a multi-plasmid transient transfection system without AAV adjuvant, which co-transfects three plasmids: pAAV-RC1, pHelper, and pGFP. Plasmid pHelper carries the adenoviral gene products required for the production of infectious AAV. This function is necessary for the production of infectious AAV particles and is provided in vivo by helper viruses such as adenovirus and is only replaced by the pHelper gene during the manufacturing process. Plasmid pGFP contains a gene encoding GFP (green fluorescent protein) and is used as a model of the target gene or cargo for gene therapy. GFP is often used as a model gene for the target and is easily quantified due to fluorescence detection, proving the efficiency of transfection.

[0110] HEK293 cells grown in suspension were maintained at 37°C in a CO atmosphere. 2 conical cell culture flasks in an incubator. Prior to transfection, cells in the exponential growth phase were centrifuged to a viable cell density of 100 MVC / mL. The amount of plasmid DNA used was 1 μg per 1 million cells, and the ratio of plasmid DNA (μg): PEI (μg) in all groups was 1:2. Using such a ratio, the usage of plasmid DNA and PEI was minimal, with equivalent production titers as described in Example 1. The transfection process was exactly the same for all groups: plasmid DNA was mixed with PEI and incubated for 15 minutes, then added to the very high density cell suspension. After transfection, the transfected cell suspension was diluted to 4 different conditions for the production phase, two of which were 10 MVC / mL and two of which were 30 MVC / mL. Before the final harvest at 72 hpT, a different pseudo-perfusion medium exchange strategy was applied in each condition: 1) The first condition was to dilute to 10 MVC / mL and perform a medium exchange at 24 hours post-transfection (hpT) and once every 24 hours thereafter; 2) The second condition was dilution to 10 MVC / mL and a medium exchange at 48 hpT; 3) The third condition was dilution to 30 MVC / mL and medium exchange at 18 hpT and every 24 h thereafter; 4) The fourth condition was to dilute to 30 MVC / mL and perform medium exchange at 24 hpT and then every 24 hours.

[0111] After dilution, the culture was continued in a microbioreactor at 37°C in a CO atmosphere. 2The cells were incubated in a 50 mL tube container with a vented cap in an incubator until harvest at 72 hpT. Production titers were then quantified by ELISA, which analyzed the assembled rAAV capsids in the harvest. Figure 3 In a simple culture system, for example, during the production phase, at 37°C CO 2 Transfections at very high cell densities of 100 MVC / mL in shaking 50 mL tubes with vented caps in an incubator produced comparable titers after dilution to cell densities of 10 or 30 MVC / mL, as long as media exchanges were performed to replenish nutrients and remove byproducts to maintain the health of the cell culture, i.e., condition 3 compared to conditions 1 and 2. Under condition 4, media exchange was first performed at 24 hpT, which resulted in lower rAAV yields compared to condition 3 where the first media exchange was performed at 18 hours. This finding implies that if cell cultures can be maintained at very high densities, such as 100 MVC / mL, with adequate media exchanges, dilutions after transfection are not necessary.

[0112] Example 4 (Implementation Method 4)

[0113] In embodiment 4, the transient transfection process is a PEI-mediated very high cell density transfection process of 60 MVC / mL for the production of rAAV in a perfusion bioreactor culture with a working volume of 220 mL. The purpose of this experiment is to demonstrate a very high cell density transfection process in a perfusion bioreactor culture without any dilution after transfection to produce rAAV. The transfection is accomplished by a multi-plasmid transient transfection system without an AAV adjuvant, which co-transfects three plasmids: pAAV-RC1, pHelper, and pGFP. Plasmid pHelper carries the adenoviral gene products required for the production of infectious AAV. This function is required for the production of infectious AAV particles and is provided in vivo by a helper virus such as adenovirus, which is replaced only by the pHelper gene during the manufacturing process. Plasmid pGFP contains a gene encoding GFP (green fluorescent protein) and is used as a model of the target gene or cargo for gene therapy. GFP is often used as a model gene for the target and is easily quantified due to fluorescence detection, demonstrating the efficiency of transfection.

[0114] 7 days before transfection, HEK293 cells growing in suspension were inoculated into a bioreactor with a working volume of 220 mL, and perfusion was started shortly after inoculation using a hollow fiber cartridge operated in ATF (alternating tangential flow) mode. The target for transfection was a cell density between 60 and 100 MVC / mL. On the day when the viable cell density reached 61 MVC / mL, the transfection reagent was prepared according to the ratios proposed in Examples 2 and 3: 1 μg of plasmid DNA per 1 million cells, and a DNA (μg): PEI (μg) ratio of 1:2. Using such a ratio, the usage of plasmid DNA and PEI is minimal, with equivalent production titers as shown in Implementation Example 1, which significantly reduces production costs, especially in terms of bioreactor scale. Transfection efficiency was monitored daily using a flow cytometer that quantified GFP expression levels. Here, the culture was harvested when no further increase in GFP levels was observed. The results are shown in Figure 2. Figure 4 As shown. Due to the increase in the volume of transfection reagent, the viable cell density decreased from 61.4 MVC / mL to 58.7 MVC / mL after transfection. The viability decreased from 96% before transfection to 82% at 4 hpT (0 dpT), which is a normal decrease in viability for transfection reactions. Cell growth also decreased in the first 2 days after transfection. The slowly recovered cell growth and viability after 2 dpT indicated that the plasmid was gradually lost during cell division, which is a normal phenomenon for this transient transfection. The transfection efficiency increased every day after transfection, reaching a maximum of 56% at 5 dpT, and then decreased slightly at 6 dpT. The culture was harvested at 6 dpT. At this time, the cell density and viability recovered to 116 MVC / mL and 94%, respectively.

[0115] Example 5 (Implementation Method 5)

[0116] In Example 5, the transient transfection process is a flow electroporation process of >80 MVC / 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 an extremely high cell density transfection process in a perfusion bioreactor culture without any dilution after transfection to produce rAAV. The transfection is accomplished by a multi-plasmid transient transfection system without an AAV adjuvant, which co-transfects three plasmids: pAAV-RC1, pHelper, and pGFP. Plasmid pHelper carries the adenoviral gene products required for the production of infectious AAV. This function is necessary for the production of infectious AAV particles and is provided in vivo by a helper virus such as adenovirus, which is only replaced by the pHelper gene during the manufacturing process. Plasmid pGFP contains a gene encoding GFP (green fluorescent protein), which is used as a model of the target gene or cargo for gene therapy. GFP is often used as a model gene for the target and is easily quantified due to fluorescence detection, demonstrating the efficiency of transfection.

[0117] 13 days before transfection, HEK293 cells grown in suspension were inoculated into a bioreactor with a working volume of 200 mL and perfusion was started after the cell density reached 2 MVC / mL, using a hollow fiber cartridge operated in ATF mode. Here, the electroporator ExPERT STx from Maxcyte was used. According to the manufacturer and the normal function of this system currently used in the field, cells proliferating in culture medium at low cell density (about 1 to 2 MVC / mL) were concentrated by centrifugation to a concentration of 100 MVC / mL and transferred to the "sample bag" of the Maxcyte system. Electroporation was performed as flow electroporation and the resulting material was collected in a "collection bag".

[0118] In the present disclosure, before the experiment, the electroporator ExPERT STx from Maxcyte was modified, and the sample bag and the collection bag were removed. The electroporation unit on the STx was directly connected "in series" to the bioreactor A, the bioreactor served as the "sample bag", and the collection container B served as the "collection bag" of Maxcyte. The collection container B was also connected to the bioreactor A to transfer the collected cell suspension of the collection container B back to the bioreactor A. The collection container B is a 10L inflatable bag, i.e., a bag commonly used to store culture medium or buffer, and is pre-inflated with sterile air containing 5% carbon dioxide. The target cell density for transfection is 80 to 100 MVC / mL. On the day when the viable cell density reached 83 MVC / mL, 80 mg of plasmid DNA was electroporated, i.e., 400 μg of plasmid DNA per mL of culture. Two electroporation units CL2 were used to perform electroporation directly on the cell suspension from the bioreactor A at 80 MVC / mL. Each CL2 unit has a processing capacity of 100 mL of high-density cell suspension, so two CL2 units were used sequentially for the electroporation of 200 mL of cell suspension. The cell suspension from bioreactor A was pumped into the electroporation unit, electroporated, and then pumped into the collection container B at 3 mL per cycle until the entire 200 mL of cell suspension had been electroporated. 10 mL of DNase1 was added to the collection container B to neutralize the excess free DNA in the electroporated cell suspension. After electroporation, the cells were transferred to the collection container B, which was then placed in an incubator at 37 °C for 45 minutes. In this way, the transfected cells were well distributed on a large enough surface and could obtain oxygen for recovery. After a recovery time of 45 minutes, the transfected cell suspension was transferred back to the bioreactor A. From the moment the transfected cells were transferred back to the bioreactor A, the cell culture entered the production phase and the transfection efficiency was monitored daily using a flow cytometer that quantified the GFP expression level. Figure 5Results are shown. Immediately after transfection, the viable cell density dropped. This is due to the sudden cell death caused by the electroporation technique. The viability dropped from 97% to 91% after transfection, which is a normal transfection response for electroporation techniques. The GFP signal peaked at a maximum of 80% 3 days after transfection and then gradually decreased. The cultures were harvested when a severe drop in viable cell density, viability, and GFP signal was observed, which was in this case on day 8 after transfection. Please note that the decrease in cell density during the first two days of transfection was due to manual removal of part of the culture to keep the cell density within the target range in preparation for transfection.

[0119] Example 6 (Implementation Method 6)

[0120] In Example 6, the transient transfection process is a PEI-mediated 50 MVC / mL very high cell density transfection process for producing rAAV in a perfusion bioreactor culture with a working volume of 200 mL. The purpose of this experiment is to demonstrate a very high cell density transfection process in a perfusion bioreactor culture without any dilution after transfection to produce rAAV. The transfection is completed by a multi-plasmid transient transfection system without an AAV adjuvant, which co-transfects three plasmids: pAAV-RC9, pHelper, and pGFP. Plasmid pAAV-RC9 provides viral rep (replication) and cap (capsid) genes. Plasmid pHelper carries the adenoviral gene products required for the production of infectious AAV. This function is necessary for the production of infectious AAV particles and is provided in vivo by a helper virus such as adenovirus, which is only replaced by the pHelper gene during the manufacturing process. Plasmid pGFP contains a gene encoding GFP (green fluorescent protein) and is used as a model for the target gene or cargo of gene therapy. GFP is often used as a model gene for transfection and is easily quantified due to fluorescence detection, demonstrating the efficiency of transfection.

[0121] Eight days before transfection, HEK293 cells growing in suspension were inoculated into a bioreactor a with a working volume of 200 mL, and perfusion was started shortly after inoculation using a hollow fiber cassette operated in ATF (alternating tangential flow) mode. The target for transfection was a cell density between 50 and 80 MVC / mL. On the day when the viable cell density reached 70 MVC / mL, 85 mL of the suspension was mixed with 35 mL of fresh culture medium to prepare 120 mL of a 50 MVC / mL cell suspension. The preparation ratio of the transfection reagent was: 0.5 μg of plasmid DNA per 1 million cells, DNA (μg): PEI (μg) 1:2. Using such a ratio, the amount of plasmid DNA and PEI used was greatly reduced, which significantly reduced production costs, especially in terms of bioreactor scale. A total of 30 mg of plasmid DNA consisting of pRC9: pGFP: PElper in a ratio of 1:1:2 was diluted to a total of 40 mL with culture medium. 60 mg of PEI was diluted to a volume of 40 mL with culture medium. 40 mL of plasmid DNA and 40 mL of PEI solution were mixed to produce a transfection reagent mixture and incubated at room temperature for 15 minutes. The transfection reagent mixture was then added to 120 mL of cell suspension at 50 mVC / mL. After mixing the cell suspension and transfection reagent mixture, the transfected cell suspension was transferred to a 300 cm 2 The transfected cells were cultured in tissue culture flasks on a non-tissue culture treated surface for 30 minutes. After a 30-minute cell recovery time, the transfected cell suspension was transferred back to the bioreactor. From the moment the transfected cells were transferred back to the bioreactor, the cell culture entered the production phase and the transfection efficiency was monitored daily using a flow cytometer that quantified the GFP expression level. Here, the culture was harvested when no further increase in GFP levels was observed 10 days after transfection. Cell suspension samples were collected from bioreactor A every day during the production phase. The sample supernatant obtained by centrifugation was used to perform transduction assays. Transduction assays are used to evaluate the functionality of infectious viral vectors contained in the samples by placing the samples in cells susceptible to infection with these infectious viral vectors. To perform the transduction assays, the supernatant of the collected samples was added to a healthy HEK293F cell suspension and the GFP expression level was monitored 72 hours after transduction or within 3 days after transduction. Figure 6(A) shows viable cell density, survival rate, transfection efficiency, and production titer. It shows a long-term continuous production phase, which is very long and the cell density is very high, i.e., 120 MVC / mL is maintained for 4 days starting from 7 dpT. For comparison, for convenience, two control cultures are shown in the same figure, one is transfected at a cell density of 1 MVC / mL in a micro-bioreactor (solid circles in the left figure), viral genome yield / mL (solid diamond blocks on the left), and the other is transfected at a cell density of 15 MVC / mL (solid circles in the right figure), viral gene yield / mL, but the time scale is not applicable to these cultures. The cell density produced by the perfusion process is 33 times higher than that of the 1 MVC / mL control culture and 30 times higher vg / mL, and the control culture is harvested at 2 MVC / mL at 3 dpT. Compared with the small-scale process of 15 MVC / mL, the perfusion process produces 4.3 times the cell density and 8.4 times the vg / mL. It can be concluded that the perfusion process maintained the cell-specific productivity well compared to the 1 MVC / mL standard transfection process and almost doubled the cell-specific productivity compared to the 15 MVC / mL process in the spinning tube. This is likely due to the continuous medium renewal in the perfusion culture, which is not feasible in a small-scale system in batch mode. Figure 6 (B) shows the results of the transduction test of the same process. The 3dpT, 6dpT and 7dpT groups refer to the supernatants collected 3 days after transfection, 6 days after transfection and 7 days after transfection, respectively. The Ref group is a reference group, in which rAAV was produced by transfection in a shake flask at a low cell density of 1 MVC / mL, and the supernatant was collected 3 days after transfection, which is a common practice for producing rAAV. It demonstrates a long-term continuous production phase of a functional rAAV vector that is infectious to human cells, with an infection titer increased by about 50% compared to the control group transfected at 1 MVC / mL using the same cells and transfection reagent. This embodiment has demonstrated the possibility of achieving very high titers of functional rAAV vectors by extending the production phase.

[0122] Example 7

[0123] In Embodiment 7, a transient transfection process was performed at 50 MVC / mL using host cells, HEK293F, and virus production cells 2.0 (VPC2.0, a variant cell line of HEK293 cells purchased from ThermoFisher) to produce rAAV. Two different chemical transfection methods were used: cationic polymer PEI and cationic lipid-based transfection reagent (VPT), ​​and three transfection media were used: virus production medium (VPM), FreeStyle293 (FS293), and BalanCD HEK (BCD). The purpose of this experiment is to demonstrate that the present invention is applicable to various production cell lines, chemical transfection methods, and transfection media, which are commercially available and widely used in the field. Transfection is accomplished by a multi-plasmid transient transfection system without an AAV adjuvant, which co-transfects three plasmids: pAAV-RC9, pHelper, and pGFP. Plasmid pAAV-RC9 provides viral rep (replication) and cap (capsid) genes. Plasmid pHelper carries the adenoviral gene products required for the production of infectious AAV. Plasmid pHelper carries the adenoviral gene products required for the production of infectious AAV. This function is required for the production of infectious AAV particles and is provided in vivo by a helper virus such as adenovirus, which is simply replaced by the pHelper gene during the manufacturing process. Plasmid pGFP contains the gene encoding GFP (green fluorescent protein) and is used as a model for the target gene or cargo of gene therapy. GFP is often used as a model gene for the target and is easily quantified due to fluorescence detection, demonstrating the efficiency of transfection.

[0124] HEK293F cells and VPC2.0 cells grown in suspension were maintained at 37°C in a CO atmosphere. 2 Cells in the exponential growth phase were centrifuged to a viable cell density of 50 MVC / mL and incubated at 37°C in a CO 2 High-density cell culture was continued in a microbioreactor (i.e., a 50 mL volume container with a vented lid) in an incubator in preparation for transfection. Plasmid DNA was 1 μg per 1 million cells and the DNA (μg):PEI (μg") ratio was 1:2 for all groups. After incubation for 10-15 minutes, the mixture of DNA and PEI was added to the cell suspension. In all groups, no further dilution steps were performed after transfection and the cultures were kept in the same culture container in the incubator until harvesting at 72 hpT. Production titers were then obtained by ELISA to quantify assembled rAAV capsids and qPCR to quantify viral genomes. Figure 7Four different transfection processes are shown: 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 infected with PEI in BCD medium (from left to right). Figure 7 (A) shows the cell density, viability and transfection efficiency GFP% at harvest. It also includes the results of transduction experiments using the supernatant after centrifugation of the final harvested cell suspension. Figure 7 (B) shows the production titers of viral genomes / mL and capsids / mL in different groups, respectively. This indicates that the present invention can be applied to various transfection systems using different production cell lines and transfection media, or even different transfection methods.

[0125] References

[0126] 1. Xiao et al., "Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus," Journal of Virology 72 (1998): 2224-2232;

[0127] 2. Grimm et al., "New tools for production and purification of recombinant adeno-associated virus vectors," Hum Gene Ther 9 (1998): 2745-2760;

[0128] 3. Matsushita et al., "Efficient production of adeno-associated virus vectors in the absence of helper virus," Gene Ther 5 (1998): 938-945;

[0129] 4. Maranga, L. et al., 2005. PER.C6 during the growth and propagation of replication-defective adenoviral vectors. TM Characteristics of changes in cell metabolism”, Biotechnology and Bioengineering, 90(5), pp. 645-655;

[0130] 5. Henry, O. et al., 2005, "Metabolic flux analysis of HEK-293 cells in perfusion culture for adenoviral vector production," Metabolic Engineering, 7(5-6), pp. 467-476;

[0131] 6. Joshi et al., 2021. “Advances in the molecular design and bioprocessing of recombinant adeno-associated virus gene delivery vectors using an insect cell baculovirus expression platform,” Journal of Biotechnology, 16(4), p. 2000021;

[0132] 7. Meghrous et al., 2005. “Production of recombinant adeno-associated virus vectors using a baculovirus / insect cell suspension culture system: from shake flasks to 20-L bioreactors”, Progress in Biotechnology, 21(1), pp. 154-160.

[0133] 8. Urabe et al., 2002. “Insect cells as factories for the production of adeno-associated virus type 2 vectors.” Human Gene Therapy, 13(16), pp. 1935-1943;

[0134] 9. Riedl et al., “Nonviral transfection of human T lymphocytes,” Processes 2018, 6, 188;

[0135] 10. Backliwal et al. “High-density transfection of HEK-293 cells allows transient titer doubling and eliminates the need for a priori DNA complex formation with PEI.” Biotechnol Bioeng. 2008 Feb 15;99(3):721-7;

[0136] 11. Blackstock et al., “Comprehensive flow cytometric analysis of PEI-based transfection for virus-like particle production,” Research, 2020, Article ID 1387402;

[0137] 12. Steger et al. “CHO-S antibody titers >1 g / L using flow electroporation-mediated transient gene expression followed by rapid migration to a high-producing stable cell line.” J Biomol Screen. 2015 Apr;20(4):545-51.

Claims

1. A method for producing a recombinant vector in a eukaryotic cell, characterized in that: include: a) obtaining a cell suspension having a cell density of at least 25 million viable cells per ml; b) transiently transfecting the cells in the cell suspension with exogenous plasmid DNA or multiple different exogenous plasmid DNAs to obtain transfected cells; c) optionally allowing a period of cell recovery, wherein the transfected cells remain substantially stationary on the surface during the cell recovery period; and d) after the transfection in step b) and / or the optional cell recovery step c), subjecting said transfected cells present in said cell suspension to a limiting dilution or to no dilution and thereafter starting production of said recombinant vector from a culture of said transfected cells at a cell density of at least 20 million viable cells per ml.

2. The method according to claim 1, characterized in that The cell suspension of step a) has a cell density of at least 30 million cells per milliliter.

3. The method according to claim 1, characterized in that The cell suspension of step a) has a cell density of at least 50 million cells per milliliter.

4. The method according to claim 1, characterized in that The cell suspension of step a) has a cell density of at least 80 million cells per milliliter.

5. The method according to claim 1, characterized in that: The cell suspension of step a) has a cell density of at least 100 million cells per milliliter.

6. The method according to any one of the preceding claims, characterized in that The method comprises: performing a culture medium exchange at one or more time points after the start of production in step d).

7. The method according to any one of claims 1 to 5, characterized in that The method comprises: performing medium exchange by perfusion after the production in step d) starts.

8. The method according to claim 1, characterized in that: The perfusion is performed in a perfusion bioreactor.

9. The method according to any one of claims 6 to 8, characterized in that The production of step d) is performed for at least 24 hours, such as at least 48 hours or 72 hours, or 4, 5, 6, 7, 8, 9 or 10 days.

10. The method according to any one of claims 6 to 9, characterized in that No dilutions were performed that resulted in cell densities below 20 MVC / mL before starting the production in step d).

11. The method according to any one of the preceding claims, characterized in that The cell suspension of step a) is 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 culture, microbial fermentation, cell tissue-derived cell suspension, and blood-derived cell suspension.

12. The method according to any one of the preceding claims, characterized in that The eukaryotic cell is selected from the group consisting of a mammalian cell, a human cell, an avian cell, an insect cell, and a plant cell.

13. The method according to claim 13, characterized in that The cells are selected from 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, NSO, CRL7030, Hs A group consisting of S78Bst cells, PER, C6, SP2, SPO, hybridoma cells, MRC-5, MDCK, Wl-98, CAP, EB66, HUVEC, AGE1.CR, CR, Trichomonas cells, Spodoptera frugiperda cells, SF9, SF21, Hi5, mesenchymal stem cells, endothelial cells, induced pluripotent stem cells, primary cells, tobacco cells, BY2, Nicotiana tabacum cells, rice cells, Arabidopsis cells, and carob cells.

14. The method according to any one of the preceding claims, characterized in that The recombinant viral vector is a viral vector, a virus, a virus-like particle (VLPs), or any combination thereof, or an assembly derived from them, or an assembly derived from them and combined with a small molecule having a size less than 1000 kDa.

15. The method according to any one of the preceding claims, characterized in that Producing recombinant adeno-associated virus (AAV) viral vectors, optionally, the transfection of step b) is performed using a transient transfection (AAV) system comprising co-transfection of at least three plasmids.

16. The method according to any one of the preceding claims, characterized in that The cell density of step a) is obtained by concentrating the cell content in the cell suspension and / or by adjusting the cell density of the culture suspension by culturing the cells in the culture suspension.

17. A method according to any one of the preceding claims, characterised in that The cell density of step a) is adjusted by concentrating the cells in suspension, including centrifugation, filtration, microfiltration, sedimentation, acoustic sedimentation or specific binding of cells, or by diluting the cells in suspension.

18. The method according to any one of the preceding claims, characterized in that The cell density of step a) is adjusted by culturing, and the culturing mode includes batch culture, fed-batch culture, perfusion culture, constant temperature culture, or a combination of at least two of these culturing modes, wherein the culturing can be performed in combination with cell concentration and / or cell dilution.

19. The method according to any one of the preceding claims, characterized in that The transient transfection includes chemical transfection using a chemical agent selected from cationic polymers including DEAE dextran, polyisoprene, polyethyleneimine and its derivatives, dendrimers, calcium phosphate, polycations, and cationic lipids including liposomal and non-liposomal transfection agents and lipid nanoparticles.

20. The method according to claim 19, characterized in that The polyethyleneimines are synthetic cationic polymers with linear or branched topologies and molecular weights ranging from 1 kDa to 1000 kDa.

21. The method according to any one of the preceding claims, characterized in that The transfection includes a physical transfection method selected from the following: microinjection, optical transfection, biolistic transfection (also known as particle bombardment), electroporation, iron oxide nanoparticle delivery (also known as magnetofection), sonoporation, laser irradiation, electric field induced molecular vibration.

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