Method for mass production of vaccinia virus using suspension cells

Abstract

The present invention relates to a method for mass-producing vaccinia virus using suspension cells. Conventional methods for producing vaccinia virus using adherent cells have been limited in their suitability for mass production of virus due to the characteristics of adherent cells. However, the present invention has established a technology that enables virus production in a bioreactor using suspension cells and a low appropriate cell number, MOI, culture FBS concentration, and medium, and has confirmed that the virus productivity is similar to that of adherent cells. Therefore, the vaccinia virus production technology using suspension cells according to the present invention enables mass production of vaccinia virus with high productivity, and the use of suspension cells can reduce production costs, time, and labor. Therefore, it is expected to be useful in clinical and commercial production fields where mass production of vaccinia virus is required.

Description

[Technical Field]

[0001] The present invention relates to a method for mass-producing vaccinia virus using suspension cells. [Background technology]

[0002] Vaccinia virus, also known as cowpox virus, is an enveloped DNA virus in the Poxvirus family with a linear, double-stranded DNA genome of approximately 180 kb encoding approximately 250 independent genes. It has a wide range of host species, including mammals and birds, and grows in a variety of cultured cell lines, forming white, forked viruses similar to but larger than the smallpox virus. Vaccinia virus infection is generally very mild, inducing a rash and fever but causing no symptoms in healthy individuals. The immune response induced by vaccinia virus infection protects the human body from fatal smallpox infection; for this reason, vaccinia virus has been used as a live virus vaccine against smallpox.

[0003] Vaccinia virus has been attracting attention as a vaccine carrier, not only for smallpox vaccines, but also for its relatively large linear DNA genome, which can carry various antigen genes. It also has a strong immune-inducing ability, making it suitable for use as a vaccine carrier for vaccines that are difficult to develop, such as therapeutic and preventive vaccines for infectious diseases and anti-cancer vaccines. Inserting foreign genes into attenuated vaccinia virus makes it possible to produce recombinant vaccines that are safer and have fewer side effects.

[0004] Recently, cancer treatment technologies using vaccinia virus as an oncolytic virus have been developed, and most of these are produced in adherent cells. U.S. Patent Publication US2014 / 0162342 relates to a method for producing vaccinia virus using adherent cells and roller bottles. HeLa cells, a type of uterine cancer cell line, were infected with vaccinia virus at an MOI of 0.01-0.05 pfu / cell and cultured, resulting in a productivity of 50 pfu. The study concluded that HeLa S3, a suspension cell line, was not suitable for producing vaccinia virus due to its low productivity. Another patent application describes a method for producing the NYCBOH strain of vaccinia virus in MRC-5 human fibroblast cells. Another paper reported a method for producing a temperature-dependent protein, rather than vaccinia virus, using HeLa S3 and the WR strain of vaccinia virus.

[0005] Thus, currently, most related companies and reported prior art in this field are known and using methods for producing vaccinia virus using adherent cells such as Vero, MRC-5, and HeLa. However, these conventional production methods have limitations that make them unsuitable for mass production of viruses, as they increase production labor, costs, and time due to limitations in scale-up caused by the characteristics of adherent cells. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, in order to establish a suitable production process for mass production of vaccinia virus, the inventors have explored and evaluated the conditions related to everything from initial culture using suspension cells to virus infection and production, and have secured a vaccinia virus production technology using suspension cells that shows high levels of virus productivity.

[0007] Therefore, an object of the present inventors is to provide a method for mass-producing vaccinia virus using suspension cells.

[0008] However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0009] In order to achieve the above-mentioned objects of the present invention, the present invention provides a method for mass-producing vaccinia virus, which comprises the following steps: (a) Initial culture of suspension cells, HeLa S3 or MDCK (Madin-Darby Canine Kidney); (b) The initially cultured cells are subcultured and dispensed at a density of 5.00E+04 to 1.00E+05 cells / mL, and then diluted to 0.01 to 0. 1 ofInfecting the cells with vaccinia virus at a multiplicity of infection (MOI) and culturing the cells; and (c) recovering the virus from the cell culture.

[0010] In one embodiment of the present invention, the initial culture in step (a) may be cultured up to the second to fourth passages of the cells.

[0011] In another embodiment of the present invention, the initial culture of step (a) may be cultured up to the second passage of the cells.

[0012] In yet another embodiment of the present invention, the cells of each passage in step (a) may be cultured for 3 to 5 days.

[0013] In yet another embodiment of the present invention, in the initial culture, the first passage cells may be dispensed at a density of 1.00E+05 to 3.00E+05 cells / mL, and the second passage cells may be dispensed at a density of 5.00E+04 to 1.00E+05 cells / mL.

[0014] In yet another embodiment of the present invention, the cells may be cultured in a medium supplemented with fetal bovine serum (FBS).

[0015] In yet another embodiment of the present invention, the medium may be Serum-like Modified Eagle's Medium (SMEM) or RPMI 1640 medium.

[0016] In yet another embodiment of the present invention, the fetal bovine serum may be added at a concentration of 5% to 10%.

[0017] In yet another embodiment of the present invention, the harvesting in step (c) may be performed 4 to 6 days after the viral infection.

[0018] In yet another embodiment of the present invention, the vaccinia virus may be any one selected from the group consisting of Western Reserve (WR), NYVAC (New York Vaccinia Virus), Wyeth (The New York City Board of Health), LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, IHD-J (International Health Division-J), IHD-W (International Health Division-White), variants thereof, and combinations thereof. [Effects of the Invention]

[0019] Previous methods for producing vaccinia virus using adherent cells had limitations, such as being unsuitable for mass production of virus due to the characteristics of adherent cells. However, the present inventors have developed a technology that enables virus production in a bioreactor using suspension cells and a low, appropriate cell number, MOI, culture FBS concentration, and medium, and have confirmed that the virus productivity is similar to that of adherent cells. Therefore, the vaccinia virus production technology using suspension cells according to the present invention enables mass production of vaccinia virus with high productivity, and the use of suspension cells reduces production costs, time, and labor, and is expected to be useful in clinical and commercial production fields where mass production of vaccinia virus is required. [Brief explanation of the drawings]

[0020] [Figure 1a] To select the initial (P+1) culture conditions for suspension cells HeLa S3, cells were dispensed at densities of 3.00E+05, 5.00E+05, and 1.00E+06 cells / mL, and the live cell number was measured daily during culture. [Figure 1b]To select the initial (P+1) culture conditions for suspension cells HeLa S3, cells were dispensed at densities of 3.00E+05, 5.00E+05, and 1.00E+06 cells / mL, and the cell viability was measured daily during culture. [Figure 1c] To select the initial (P+1) culture conditions for suspension cells HeLa S3, cells were dispensed at densities of 3.00E+05, 5.00E+05, and 1.00E+06 cells / mL, and the cell expansion fold was measured daily while culturing. The results are shown here. [Figure 2a] To select the initial (P+1) culture conditions for suspension cells HeLa S3, cells were dispensed at densities of 1.00E+05, 3.00E+05, and 5.00E+05 cells / mL, and the number of viable cells was measured daily during culture. [Figure 2b] To select the initial (P+1) culture conditions for suspension cells HeLa S3, cells were dispensed at densities of 1.00E+05, 3.00E+05, and 5.00E+05 cells / mL, and the cell viability was measured daily while culturing. [Figure 2c] To select the initial (P+1) culture conditions for suspension cells HeLa S3, cells were dispensed at densities of 1.00E+05, 3.00E+05, and 5.00E+05 cells / mL, and the cell proliferation rate was measured daily while culturing. [Figure 3a] To select the initial (P+2) culture conditions for suspension cells HeLa S3, cells were dispensed at the P+2 stage at densities of 5.00E+04, 1.00E+05, and 2.00E+05 cells / mL, and the number of viable cells was measured daily during culture. [Figure 3b] To select the initial (P+2) culture conditions for suspension cells HeLa S3, cells were dispensed at the P+2 stage at densities of 5.00E+04, 1.00E+05, and 2.00E+05 cells / mL, and the cell viability was measured daily during culture. [Figure 3c]To select the initial (P+2) culture conditions for suspension cells HeLa S3, cells were dispensed at the P+2 stage at densities of 5.00E+04, 1.00E+05, and 2.00E+05 cells / mL, and the cell proliferation rate was measured daily during culture. [Figure 4a] To select the initial (P+3) culture conditions for suspension cells HeLa S3, cells were dispensed at the P+3 stage at densities of 5.00E+04, 1.00E+05, and 2.00E+05 cells / mL, and the number of viable cells was measured daily during culture. [Figure 4b] To select the initial (P+3) culture conditions for suspension cells HeLa S3, cells were dispensed at the P+3 stage at densities of 5.00E+04, 1.00E+05, and 2.00E+05 cells / mL, and the cell viability was measured daily during culture. [Figure 4c] To select the initial (P+3) culture conditions for suspension cells HeLa S3, cells were dispensed at the P+3 stage at densities of 5.00E+04, 1.00E+05, and 2.00E+05 cells / mL, and the cell proliferation rate was measured daily during culture. [Figure 5] FIG. 1 shows a cell culture process illustrating cell dispensing density and FBS concentration conditions at P+3 to determine the FBS concentration during and after infection of suspension cells HeLa S3 with vaccinia virus. [Figure 6a] The results are shown below. HeLa S3 cells were cultured according to the process shown in Figure 5 under different cell dispensing densities and FBS concentrations at P+3, and the number of viable cells (Figures 6a to 6d) and cell viability (Figures 6e to 6h) were measured. [Figure 6b] The results are shown below. HeLa S3 cells were cultured according to the process shown in Figure 5 under different cell dispensing densities and FBS concentrations at P+3, and the number of viable cells (Figures 6a to 6d) and cell viability (Figures 6e to 6h) were measured. [Figure 6c]The results are shown below. HeLa S3 cells were cultured according to the process shown in Figure 5 under different cell dispensing densities and FBS concentrations at P+3, and the number of viable cells (Figures 6a to 6d) and cell viability (Figures 6e to 6h) were measured. [Figure 6d] The results are shown below. HeLa S3 cells were cultured according to the process shown in Figure 5 under different cell dispensing densities and FBS concentrations at P+3, and the number of viable cells (Figures 6a to 6d) and cell viability (Figures 6e to 6h) were measured. [Figure 6e] The results are shown below. HeLa S3 cells were cultured according to the process shown in Figure 5 under different cell dispensing densities and FBS concentrations at P+3, and the number of viable cells (Figures 6a to 6d) and cell viability (Figures 6e to 6h) were measured. [Figure 6f] The results are shown below. HeLa S3 cells were cultured according to the process shown in Figure 5 under different cell dispensing densities and FBS concentrations at P+3, and the number of viable cells (Figures 6a to 6d) and cell viability (Figures 6e to 6h) were measured. [Figure 6g] The results are shown below. HeLa S3 cells were cultured according to the process shown in Figure 5 under different cell dispensing densities and FBS concentrations at P+3, and the number of viable cells (Figures 6a to 6d) and cell viability (Figures 6e to 6h) were measured. [Figure 6h] The results are shown below. HeLa S3 cells were cultured according to the process shown in Figure 5 under different cell dispensing densities and FBS concentrations at P+3, and the number of viable cells (Figures 6a to 6d) and cell viability (Figures 6e to 6h) were measured. [Figure 7] FIG. 1 shows the cell culture and virus production process for analyzing virus productivity of HeLa S3 cells depending on cell seeding density at P+3 and FBS concentration conditions during and after vaccinia virus infection. [Figure 8a] The cell dispensing density and FBS concentration conditions were varied at P+3, and HeLa S3 cell culture and virus production were carried out according to the steps of FIG. 7, after which the number of viable cells was measured. [Figure 8b]The cell viability was measured after HeLa S3 cell culture and virus production were carried out according to the steps of FIG. 7 under different cell dispensing densities and FBS concentrations at P+3. [Figure 8c] The cell dispensing density and FBS concentration conditions at P+3 were varied, and HeLa S3 cell culture and virus production were carried out according to the steps of FIG. 7, after which virus productivity (TCID50 / cell) was measured. [Figure 9] FIG. 1 shows the cell culture and virus production process (primary experiment) for analyzing cell growth and virus productivity according to the MOI of infection of HeLa S3 cells with vaccinia virus, harvest date, and cell dispensing density conditions at P+3. [Figure 10a] The experiment was conducted according to the steps in Figure 9 with different infection MOIs when the cell dispensing density at P+3 was 1.00E+05 cells / mL, and then samples were collected on each collection day and the number of viable cells was measured. [Figure 10b] The experiment was conducted according to the steps in Figure 9, with the cell dispensing density at P+3 being 1.00E+05 cells / mL and the infection MOI varied. Samples were then collected on each collection day, and the cell viability was measured. [Figure 10c] The experiment was performed according to the steps in Figure 9, with different infection MOIs when the cell dispensing density at P+3 was 5.00E+05 cells / mL. After that, samples were collected on each collection day and the number of viable cells was measured. [Figure 10d] The experiment was performed according to the steps of Figure 9 with different infection MOIs when the cell dispensing density at P+3 was 5.00E+05 cells / mL, and then samples were collected on each collection day and cell viability was measured. [Figure 11a] 10a to 10d show the results of confirming the total virus productivity for each sample collected to analyze the virus productivity. [Figure 11b]10a to 10d show the results of confirming the virus production capacity per cell (TCID50 / cell) of each virus to analyze virus productivity for the collected samples. [Figure 12a] A secondary experiment was conducted to determine the MOI of infection of HeLa S3 cells with vaccinia virus, the harvest date, and the cell dispensing density conditions at P+3. The experiment was conducted using the same process as in Figure 9, but with a cell dispensing density of 5.00E+04 cells / mL and different harvest date conditions. Samples were collected on each harvest date and the number of viable cells was confirmed. [Figure 12b] A secondary experiment was conducted to determine the MOI of infection of HeLa S3 cells with vaccinia virus, the harvest date, and the cell dispensing density conditions at P+3. The experiment was conducted using the same process as in Figure 9, but with a cell dispensing density of 5.00E+04 cells / mL and different harvest date conditions. Samples were collected on each harvest date to confirm cell viability. [Figure 12c] A secondary experiment was conducted to determine the MOI of infection of HeLa S3 cells with vaccinia virus, the harvest date, and the cell dispensing density conditions at P+3. The experiment was conducted using the same process as in Figure 9, but with a cell dispensing density of 1.00E+05 cells / mL and different harvest date conditions. Samples were collected on each harvest date and the number of viable cells was confirmed. [Figure 12d] A secondary experiment was conducted to determine the MOI of infection of HeLa S3 cells with vaccinia virus, the harvest date, and the cell dispensing density conditions at P+3. The experiment was conducted using the same process as in Figure 9, but with a cell dispensing density of 1.00E+05 cells / mL and different harvest date conditions. Samples were collected on each harvest date to confirm cell viability. [Figure 13a] 12a to 12d show the results of confirming the total virus production amount to analyze the virus productivity for the collected samples. [Figure 13b] The results of confirming the virus production capacity per cell were used to analyze virus productivity for the samples collected in Figures 12a to 12d. [Figure 14a]To further select the date for harvesting cells and culture medium after virus infection, experiments were performed under fixed cell distribution density and infection MOI conditions, and all samples were harvested on days 3, 4, 5, 6, and 7, and the number of viable cells was analyzed. [Figure 14b] To further select the date for harvesting cells and culture medium after virus infection, experiments were performed under fixed cell distribution density and infection MOI conditions, and all samples were harvested on days 3, 4, 5, 6, and 7, respectively, and cell viability was analyzed. [Figure 15a] The total virus production was confirmed using the samples collected in Figures 14a and 14b. [Figure 15b] The results show that the virus production capacity per cell was confirmed using the samples collected in Figures 14a and 14b. [Figure 16] FIG. 1 shows a process for scaling up virus production, in which selected conditions for the initial culture conditions of HeLa S3 cells, cell seeding density for virus infection, MOI, and harvest date were applied in the examples. [Figure 17a] FIG. 17 shows the results of checking the cell count and cell viability before and after scale-up after conducting an experiment according to the steps of FIG. 16. [Figure 17b] FIG. 17 shows the results of confirming virus productivity before and after scale-up after conducting an experiment according to the steps of FIG. 16. [Figure 18] FIG. 1 shows the experimental steps for the selection of culture media for HeLa S3 cells. [Figure 19a] The experiment was carried out by changing the culture medium of HeLa S3 cells to SMEM, JMEM or RPMI 1640 according to the steps of FIG. 18, and the number of viable cells was then counted. [Figure 19b] The results are from experiments in which the culture medium for HeLa S3 cells was changed to SMEM, JMEM, or RPMI 1640 in steps P+1 to P+3 according to the process of FIG. 18, and then cell viability was confirmed. [Figure 20]The graph shows the results of comparative analysis of virus productivity after experiments were conducted using different culture media for HeLa S3 cells at P+1 to P+3 according to the process of FIG. DETAILED DESCRIPTION OF THE INVENTION

[0021] In order to establish a production process capable of mass-producing vaccinia virus, the present inventors have explored and evaluated conditions related to everything from initial culture using suspension cells to virus infection and production, and have thereby secured a vaccinia virus production technology using suspension cells that exhibits high levels of virus productivity.

[0022] The present invention will be described in detail below.

[0023] Thus, the present invention provides a method for producing a suspension cell line comprising: (a) initially culturing HeLa S3 or MDCK (Madin-Darby Canine Kidney) suspension cells; (b) subculturing the initially cultured cells and dispensing them at a density of 5.00E+04 to 1.00E+05 cells / mL, followed by subculturing at a density of 0.01 to 0. 1 of The present invention provides a method for mass-producing vaccinia virus, comprising the steps of (a) infecting cells with vaccinia virus at a multiplicity of infection (MOI) and culturing the cells; and (b) recovering the virus from the cell culture.

[0024] In the present invention, unless otherwise specified, the term "vaccinia virus" includes both vaccinia virus and a vaccinia virus solution containing the same. The vaccinia virus solution is not particularly limited as long as it contains vaccinia virus, and includes, for example, a culture supernatant obtained after culturing host cells infected with vaccinia virus and a virus suspension obtained after removing impurities from the culture supernatant.

[0025] The vaccinia virus may be any one selected from the group consisting of Western Reserve (WR), NYVAC (New York Vaccinia Virus), Wyeth (The New York City Board of Health), LC16m8, Lister, Copenhagen, Tian Tan, USSR, TashKent, Evans, IHD-J (International Health Division-J), IHD-W (International Health Division-White), variants thereof, and combinations thereof, preferably IHD-W, but not limited to this.

[0026] The term "suspension cells" used in the present invention refers to cells that grow in a suspended state in the medium without adhering to a culture plate during cell culture. When subculturing, the culture medium is not replaced, but dilution culture is performed. The suspension cells HeLa S3 and MDCK that can be used to produce vaccinia virus in the present invention have the properties of adherent cells but can be adjusted to grow in suspension. HeLa S3 is a clone derivative of the parent HeLa lineage, a human uterine cancer cell line, and is a suitable cell line for transfection. MDCK is a canine kidney-derived cell line known in the art as a cell line capable of propagating a variety of viruses.

[0027] In the present invention, an optimal production process that enables mass production of vaccinia virus using suspension cells was studied and established.

[0028] In the present invention, the step (a) is a step of initially culturing suspension cells HeLa S3 or MDCK.

[0029] The initial culture can be performed by thawing frozen floating cells, dispensing the cells, and culturing them up to the second to fourth passages, preferably up to the second passage (P+2). After dispensing the cells, the initial culture is preferably performed by culturing the cells of each passage for 3 to 5 days, and more preferably by carrying out subculture after 4 days of culture.

[0030] Furthermore, when initially culturing up to the second passage (P+2), it is preferable to dispense the cells of the first passage at a density of 1.00E+05 to 3.00E+05 cells / mL and the cells of the second passage at a density of 5.00E+04 to 1.00E+05 cells / mL, and more preferably, optimal cell growth can be induced when the cells of the first passage are dispensed at 1.00E+05 cells / mL and the cells of the second passage at 5.00E+04 cells / mL.

[0031] In one specific example of the present invention, in order to select the initial culture conditions before infecting suspension cells HeLa S3 with a virus, specifically the cell seeding density and culture period, the cells were cultured under various conditions, and the number of viable cells and cell viability were analyzed. It was confirmed that the above conditions were the optimal initial culture conditions for mass production of vaccinia virus (see Example 1).

[0032] The cells may be cultured in a medium supplemented with fetal bovine serum (FBS). Preferably, the cell culture medium is Serum-like Modified Eagle's Medium (SMEM) or RPMI 1640, with RPMI 1640 being more preferred from the viewpoints of cell culture efficiency and economy, but is not limited thereto. In this case, the fetal bovine serum may be added at a concentration of 5% to 10%, preferably at a concentration of 10% during the initial culture in step (a).

[0033] In addition to the conditions selected for the initial culture, the culture temperature, carbon dioxide concentration, and method for subculturing suspension cells are not particularly limited, and can be appropriately carried out by a person skilled in the art using ordinary conditions and methods conventionally used for cell culture.

[0034] In the present invention, step (b) is a step of infecting the cells dispensed after the initial culture with vaccinia virus. For example, if the initial culture is performed up to the second passage (P+2), the initially cultured cells are cultured to the third passage (P+3), dispensed, and then infected with vaccinia virus.

[0035] In step (b), the cell density during the virus infection is preferably 5.00E+04 to 1.00E+05 cells / mL, and more preferably 5.00E+04 cells / mL, which can induce optimal cell growth and virus production.

[0036] Furthermore, when infecting with the virus, the multiplicity of infection (MOI) is 0.01 to 0. 1 is Preferably, and more preferably, infection at an MOI of 0.01 can induce the highest cell growth and virus productivity.

[0037] The term "multiplicity of infection (MOI)" as used herein means the ratio of target (e.g., host cell) infections to an agent (e.g., a virus). It refers to the ratio of the number of virus particles to the number of target cells present in a limited space.

[0038] In addition, the medium used to culture the virus-infected suspension cells in step (b) may be SMEM or RPMI 1640 medium supplemented with 5% to 10% FBS, and more specifically, it is preferable to use a medium supplemented with 5% FBS.

[0039] Through experiments, the present inventors have confirmed that the above conditions in step (b) are optimal mass production conditions.

[0040] That is, in another specific example of the present invention, to determine the concentration of FBS to be added during virus infection and post-infection culture in suspension cells HeLa S3, cells were plated at different densities and cultured at FBS concentrations of 0, 2, 5, and 10%, respectively, and then cell growth and vaccinia virus productivity were compared and analyzed. The optimal cell plated density and FBS concentration for virus infection were selected as described above (see Example 2).

[0041] In yet another example of the present invention, experiments were conducted to determine the virus infection MOI, cell and culture medium recovery period, and cell dispensing density conditions for virus infection using suspension cells, HeLa S3. Specifically, cells were dispensed at different densities, and the MOI and recovery period were varied. Cell growth and vaccinia virus productivity were compared, and the optimal conditions were selected as described above (see Examples 3-1 to 3-3).

[0042] In the present invention, vaccinia virus productivity was evaluated by the total virus production and the virus production capacity per cell (TCID50 / cell). The virus production capacity per cell is a unit of measure for virus infectious titer, and is synonymous with the term "titer," which is frequently used in the art. Because viruses cannot be seen even with an optical microscope, their density (number / volume) cannot be measured microscopically like biological cells. Therefore, in the case of viruses, the infectious titer, which is measured using the ability to infect host cells, is used as a unit to surrogate for their quantity or concentration. For example, when an appropriately diluted virus suspension is added to a monolayer of host cells, the number of viruses is detected as plaques, and the infectious titer can be measured in plaque-forming units (pfu) / mL. Alternatively, the infectious titer can be measured by diluting the virus-containing liquid until 50% of the host cells become positive for infection, at a concentration of 50% (tissue culture infectious dose = TCID50) / mL. In this example, the productivity of vaccinia virus, that is, the infectious titer, was measured in terms of TCID50 / mL, but the present invention is not limited to this.

[0043] Other than the above-selected conditions, the method for culturing suspension cells and infecting the cells with a virus is not particularly limited, and can be carried out by a person skilled in the art by appropriately applying a method commonly used in the relevant technical field.

[0044] In the present invention, the step (c) is a step of recovering the virus from the cell culture to obtain the virus produced through the step (b).

[0045] To recover the virus, it is preferable to recover all of the cultured cells and the culture product. Recovery of the virus can be carried out 4 to 6 days after infection with the virus, and is preferably carried out 5 days later.

[0046] From the samples collected through the above steps, those skilled in the art can obtain the final produced virus by methods used in the art and analyze the productivity.

[0047] In yet another example of the present invention, to investigate whether the vaccinia virus production conditions of the present invention are applicable to actual virus mass production processes, a virus production scale-up experiment was conducted at a 1.8 L scale, and the viable cell count, cell viability, and virus productivity were analyzed. As a result, it was confirmed that the cell count and viability were similar at both scales (30 mL and 1800 mL), and that the virus productivity was also similar. This confirmed that the vaccinia virus production process and established conditions using suspension cells according to the present invention are applicable to mass production (see Examples 3 and 4).

[0048] In yet another example of the present invention, further experiments were conducted to select a culture medium for suspension cells, HeLa S3, in order to further increase virus productivity and ensure production cost savings in the selected virus production process. As a result, it was confirmed that RPMI 1640 also exhibited similar cell growth and virus production effects compared to SMEM used in the process. Based on these results, it was determined that RPMI 1640 medium, which is more readily available and allows for cost savings, is appropriate for use (see Example 4).

[0049] The vaccinia virus finally produced through the above production process of the present invention can be used in various ways in basic research and clinical fields, such as as a vaccine, an oncolytic virus for cancer treatment, or a viral vector as a carrier.

[0050] Preferred examples are presented below to aid in understanding the present invention. However, the following examples are provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0051] [Example] Example 1. Selection of culture conditions for suspension cells HeLa S3 In order to establish the culture conditions for suspension cells HeLa S3, a cell line for producing vaccinia virus, the present inventors conducted experiments to select the initial culture conditions from P+1 to P+3 after cell thawing.

[0052] 1-1. Selection of P+1 culture conditions First, to select the culture conditions for P+1, cells were thawed and seeded under the conditions shown in Table 1 below, and then cultured. Live cell number, cell viability, and cell expansion fold were measured every day.

[0053] [Table 1]

[0054] Cells were cultured under these conditions and experiments were performed. As shown in Figures 1a-1c, the maximum cell number that grew without medium change was approximately 2.00E+06 cells / mL, indicating that the higher the cell density during dispensing, the higher the cell number that could be obtained in a shorter time. However, by examining the cell proliferation fold, it was confirmed that the cell culture was most efficient when the cells were dispensed at the lowest density of 3.00E+05 cells / mL. Therefore, it was found that the culture efficiency was better when the cell density during dispensing was low than when it was high.

[0055] Based on the above results, in subsequent experiments, the cell dispensing density was changed to 1.00E+05, 3.00E+05, and 5.00E+05 as shown in Table 2 below, with 3.00E+05 cells / mL as the standard. cells / mL The cell growth was compared and analyzed.

[0056] [Table 2]

[0057] As a result of culturing cells under the conditions in Table 2, as can be seen from Figures 2a to 2c, the maximum cell numbers during the culture period were similar under all three cell density conditions, but cells / mL The lowest density of 1.00E+05 resulted in the highest cell viability. In addition, the graph peaked and then quickly declined under the other two density conditions, making it difficult to set the subculture period. cells / mL In this case, it was determined that subculture should be performed on the fourth day of culture, as the stationary phase lasts for 4 to 5 days.

[0058] Therefore, the conditions for P+1 culture were set as follows: cells were dispensed at 1.00E+05 cells / mL, and subcultured after 4 days.

[0059] 1-2. Selection of P+2 culture conditions Next, to select the culture conditions for P+2, cells were cultured under the conditions shown in Table 3 below, and the cell culture efficiency was compared and analyzed depending on the cell dispensing density conditions. During the process of setting the culture conditions for P+1, it was observed that an initial high cell density tended to lower the culture efficiency. Therefore, in this experiment, the dispensing density was further reduced to set the conditions and culture was performed.

[0060] [Table 3]

[0061] As shown in Figures 3a-3c, the time to reach the maximum cell number varied depending on the cell dispensing density. However, the final cell number was approximately 2.00E+06 cells / mL or higher in all cases. The lowest density of 5.00E+04 cells / mL demonstrated the highest cell viability and proliferation fold, confirming the highest culture efficiency. When selecting the P+2 culture conditions, considering that subculture was performed in the log phase, it was determined that dispensing cells at 5.00E+04 cells / mL and subculture on day 4, when viability was maintained and proliferation fold was high, was appropriate. These conditions have the advantages of high culture efficiency, allowing for later scaling up to approximately 750 mL in P+3, and the convenience of having the same culture cycle as P+1.

[0062] 1-3. Selection of P+3 culture conditions In addition, to select the conditions for P+3, cells were cultured under the P+1 and P+2 conditions selected in Examples 1-1 and 1-2, and then cultured for P+3 under the conditions in Table 4 below.

[0063] [Table 4]

[0064] As can be seen from Figures 4a-4c, a similar growth curve to P+2 was observed, and it was confirmed that the cell culture efficiency was highest at a low dispensing density of 5.00E+05 cells / mL. Furthermore, considering the culture efficiency and cell viability in the logarithmic phase, it was determined that subculture on day 4 after culture, similar to P+2, was appropriate.

[0065] Therefore, the initial culture conditions for P+1, P+2, and P+3 selected in the above example are summarized in Table 5 below, and HeLa S3 cells were cultured under these conditions in future experiments to select vaccinia virus production conditions.

[0066] [Table 5]

[0067] Example 2. Selection of FBS concentration when culturing suspension cells HeLa S3 The present inventors conducted the following experiment to determine the FBS concentration that can provide optimal cell growth and vaccinia virus productivity in the culture of suspension cells HeLa S3.

[0068] 2-1. Observation of cell growth depending on FBS concentration First, to observe the effect of FBS concentration in the culture medium on HeLa S3 cell growth, cells were cultured under the conditions selected in Example 1, as shown in FIG. 5, and then the cell dispensing densities at P+3 were set to 5.00E+04, 5.00E+05, and 1.00E+06 cells / mL, and the FBS concentration was varied to four conditions (0, 2, 5, and 10%), and the cell growth curve was analyzed.

[0069] The P+3 culture was analyzed for viable cell count and cell viability. As shown in Figures 6a–6h, under 0% FBS conditions, cells did not grow and viability remained low, as expected. Under 2% FBS conditions, a slight increase in cell number was observed, but growth efficiency was not significant. A decrease in cell viability was observed from the early stage of culture, except at the lowest cell density of 5.00E+04 cells / mL. Under 5% FBS, growth curves similar to those under the positive control (10% FBS) were observed at all cell densities up to day 2 of culture, and similar viability was observed at low cell densities. Based on these results, we predicted that virus productivity would be low due to poor cell health during virus infection and cell culture for pectin production under 0–2% FBS conditions. However, to confirm the actual effect of FBS concentration on virus productivity, we performed a virus productivity verification experiment under the same FBS conditions as above.

[0070] 2-2. Confirmation of vaccinia virus (VACV) productivity depending on FBS concentration and selection of conditions Next, to confirm the effect of FBS concentration on vaccinia virus productivity in suspension cells HeLa S3, an experiment was conducted according to the steps in Figure 7. In this experiment, the cell dispensing density was 5.00E+05 or 1.00E+06 cells / mL, the FBS concentration was 2, 5, or 10%, and the MOI was 0. 1 in The cells were infected with vaccinia virus, and after culturing for 4 days, the cells and culture medium were all collected and the virus productivity was evaluated under each condition.

[0071] We analyzed the viable cell count and cell viability as a function of FBS concentration under each of the two cell density conditions. As shown in Figures 8a-8c, we observed a decrease in cell count and viability as cell culture and virus production progressed. At 5.00E+05 cells / mL, cell count and viability increased slightly with increasing FBS concentration. In contrast, at 1.00E+06 cells / mL, cell count and viability remained similar as FBS concentration increased. These results suggest that at 5.00E+05 cells / mL, differences in virus productivity exist depending on the FBS concentration. Indeed, we evaluated vaccinia virus productivity using TCID50, which measures the virus production capacity per cell. At 5.00E+05 cells / mL, significant differences in virus productivity were observed between FBS conditions, with the 5% FBS condition demonstrating the highest productivity. Therefore, we ultimately selected a 5% FBS concentration during and after cell infection to produce high vaccinia virus titers.

[0072] Example 3. Selection of virus infection and recovery conditions for suspension cells HeLa S3 3-1. Primary experiment for selection of virus infection MOI, harvest day, and cell seeding density Using the FBS concentration conditions during the initial culture process of suspension cells HeLa S3 and infection and production of vaccinia virus selected through the experiments in Examples 1 and 2, an experiment was conducted to select the virus infection MOI (Multiplicity of infection), harvest date, and cell dispensing density conditions to further improve the virus productivity. The experiment was conducted according to the process and conditions shown in Figure 9, and specifically, the virus infection MOI was set at 0.01, 0.1, 1 of The cells and culture medium were collected on days 2, 3, 4, and 5 after virus infection, and virus productivity was evaluated. The cell densities for virus infection were 1.00E+05 cells / mL and 5.00E+05 cells / mL.

[0073] First, we analyzed the number of viable cells and cell viability according to the MOI and harvest date for each of the two cell density conditions. As shown in Figures 10a-10d, at the lowest MOI of 0.01 under both density conditions, the cell number increased and then either maintained or decreased. We also observed that cell viability decreased as the virus infection concentration and culture period increased. From these findings, we were able to predict the subsequent virus production through virus infection.

[0074] In addition, virus productivity was analyzed by TCID50 for samples harvested 2 to 5 days after the start of culture. As shown in Figure 11a, the total virus productivity was highest when cells were dispensed at 5.00E+05 cells / mL, infected with virus at an MOI of 0.01, and harvested 3, 4, and 5 days later. However, as shown in Figure 11b, the actual virus production per cell was highest when cells were dispensed at a density of 1.00E+05 cells / mL, infected with virus at an MOI of 0.01, and harvested 3, 4, and 5 days later. Furthermore, considering that vaccinia virus productivity using adherent HeLa cells is greater than 300 TCID50 / cell as an internal standard, virus production at greater than 300 TCID50 / cell was also confirmed when suspension HeLa S3 cells were used under the conditions described above. Although 5.00E+05 cells / mL uses five times more cells than 1.00E5 cells / mL, there was no significant difference in total production volume, indicating very low production efficiency. Therefore, we initially selected the following conditions: cells were dispensed at a density of 1.00E+05 cells / mL, infected with virus at an MOI of 0.01, and harvested 3-5 days later. Furthermore, referring to Figures 11a and 11b, we observed a tendency for virus productivity to increase with lower dispensed cell density and MOI. Therefore, in subsequent secondary experiments, we prioritized further reducing the cell density.

[0075] 3-2. Secondary experiment for selection of virus infection MOI, harvest date, and cell dispensing density Based on the results of Example 3-1, a secondary experiment was conducted to determine the time of vaccinia virus infection, MOI, harvest date, and cell injection density. The overall procedure was the same as that shown in Figure 9 in Example 3-1, except that the cell injection densities at P+3 were 5.00E+04 and 1.00E+05 cells / mL. Since low productivity was observed when samples were harvested two days after infection in Figures 11a and 11b, the harvest conditions excluded day 2, and all cells and culture medium were harvested three, four, and five days after infection, respectively.

[0076] First, we analyzed the number of viable cells and cell viability according to the MOI and harvest date under the two cell density conditions. As shown in Figures 12a to 12d, at an MOI of 0.01 and 5.00E+04 cells / mL, the cell number increased and viability was maintained until the fourth day of culture. At a higher concentration of 1.00E+05 cells / mL, the cell number increased and viability was maintained until the third day of culture.

[0077] In addition, cells were infected with the virus, and TCID50 was measured for samples harvested after 3 to 5 days of culture to analyze virus productivity. As shown in Figure 13a, as expected, virus productivity was higher at low cell densities (5.00E+04 cells / mL). As shown in Figure 13b, the virus productivity per cell was confirmed to be greater than 300 TCID50 / cell, which is the vaccinia virus productivity in adherent HeLa cells, the internal standard. In particular, under infection conditions with an MOI of 0.01, productivity did not decrease but instead increased as harvesting days progressed from 3 to 5. Based on these results, the cell dispensing density was selected to be 5.00E+04 cells / mL, lower than in the primary experiment in Example 3-1, and the virus was administered at an MOI of 0.0. 1 in The conditions selected were such that the cells and culture medium were all harvested 5 days after infection.

[0078] 3-3. Further collection date selection experiment Since Figure 13b shows that productivity increases with increasing harvest days, the inventors conducted an experiment to verify whether vaccinia virus productivity increases by further increasing the harvest days. The overall experimental procedure was the same as that shown in Figure 9 in Example 3-1, except that the SMEM medium was adjusted to 80 mL at P+2. Based on the experimental results, the cell dispensing density for virus infection was set to 5.00E+04 cells / mL, and the MOI was set to 0.0. 1 to The experiments were performed on days 3, 4, 5, 6 and 7 after fixation and recovery.

[0079] First, the cell number and viability were analyzed. As shown in Figures 14a and 14b, the number of cells increased 3 times after viral infection. ~4 It was shown that the cell number increased up to day 1 and then decreased, and it was confirmed that the viability decreased rapidly from day 4 onwards.

[0080] Furthermore, analysis of vaccinia virus productivity by harvest date showed that cell growth was maintained and virus production did not increase further after day 5, as shown in Figures 15a and 15b. Based on these results, we finally decided to infect the virus and harvest it after 5 days.

[0081] 3-4. Scaling up vaccinia virus production Based on the results of the above examples, the conditions for vaccinia virus production, i.e., initial cell culture conditions, virus injection density for infection, MOI, and harvest date, were selected. A virus production scale-up experiment was then conducted to produce vaccinia virus at a 1.8 L scale in a stirred tank reactor (STR) using the above conditions. Specifically, the experiment was conducted according to the steps in Figure 16, and the final analyzed virus productivity was compared with the results at a 30 mL EMF scale in the above examples.

[0082] Specifically, cells were infected with the virus, and after four days, all samples were collected and analyzed for viable cell count, cell viability, and virus productivity. As shown in Figure 17a, the cell count and viability were similar at both scales (30 mL and 1800 mL). As can be seen in Figure 17b, virus productivity was also similar. These results suggest that the process conditions selected at the small-scale 30 mL EMF scale are suitable for vaccinia virus production at the 1.8 L STR scale, and that the selected conditions can be used to produce vaccinia virus in the STR. Therefore, scaling up from EMF to STR is now possible.

[0083] Example 4. Selection of culture medium for suspension cells HeLa S3 The present inventors conducted an experiment to select a culture medium for HeLa S3 cells to further increase virus productivity or reduce production costs under the conditions selected in Examples 1 to 3. Initial cell culture and virus production were performed according to the conditions and process shown in Figure 18. The cells were cultured using different culture media, SMEM, JMEM, and RPMI 1640, and infected with vaccinia virus on P+3 to confirm virus productivity. To predict changes in virus productivity depending on the culture medium, harvesting was performed on days 3 to 5.

[0084] 4-1. Analysis of cell growth by medium type First, to examine how the change in medium type affects the growth of suspension cells HeLa S3, the number of viable cells and cell viability were analyzed.

[0085] As shown in Figures 19a and 19b, after two days of culture, there was no significant difference in cell number or viability when using the three media. Therefore, it was determined that changing the media would not pose a significant problem for HeLa S3 cell culture. Furthermore, it was determined that RPMI 1640 medium, which is readily available and inexpensive, is actually advantageous from the perspective of virus production, even when vaccinia virus productivity is excluded.

[0086] 4-2. Analysis of virus productivity by medium type The present inventors observed that HeLa S3 cells were cultured similarly regardless of the type of medium. Based on this result, we conducted an experiment to investigate the effect of the type of medium on actual virus productivity. To do this, we cultured the cells, infected them with viruses, and analyzed the virus productivity using TCID50.

[0087] As a result, as shown in Figure 20, except for JMEM medium, the previously used SMEM and RPMI 1640 showed excellent and similar levels of virus productivity, with an internal standard of 300 TCID50 / cell or more. Based on these results, it was concluded that RPMI 1640 can be used for vaccinia virus production. Overall, considering both the HeLa S3 cell culture efficiency and vaccinia virus productivity, it was determined that RPMI 1640 medium, which is readily available and cost-effective, would be appropriate. Therefore, RPMI 1640 was ultimately selected as the cell culture and virus production medium.

[0088] 4-3.Derivation of the final production process of vaccinia virus using suspension cells HeLa S3 Based on the results of the above examples, final process conditions were established that enable efficient production of vaccinia virus in suspension cells, HeLa S3. The final process conditions are shown in Table 6 below. When virus was produced under these conditions, it was found that by using suspension cells, virus could be produced at a rate of 300 TCID / cell or more, which is the vaccinia virus productivity in adherent cells, HeLa, used as an internal standard.

[0089] [Table 6]

[0090] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not limiting. [Industrial Applicability]

[0091] The present invention relates to a method for mass-producing vaccinia virus using suspension cells, and has established a mass-production technology for vaccinia virus by specifically establishing the appropriate cell number, MOI, culture FBS concentration, and medium conditions to mass-produce vaccinia virus, which was previously impossible. Therefore, the vaccinia virus production technology using suspension cells according to the present invention enables mass production of vaccinia virus with high productivity, and the use of suspension cells can reduce production costs, time, and labor, and is expected to be useful in clinical and commercial production fields where mass production of vaccinia virus is required.

Claims

1. The following steps: (a) initially culturing suspension cells HeLa S3 in a suspension state; (b) subculturing the initially cultured cells in a suspension state, distributing them at a density of 5.00E+04 to 1.00E+05 cells / mL, infecting them with vaccinia virus at a multiplicity of infection (MOI) of 0.01 to 0.1, and culturing them in a suspension state; and (c) recovering the virus from the cell culture. A method for mass production of vaccinia virus, comprising: The cells were cultured in a medium supplemented with fetal bovine serum (FBS), The method for mass-producing vaccinia virus is characterized in that the medium is SMEM (Serum-like Modified Eagle's Medium) or RPMI 1640 medium.

2. 2. The method for mass-producing vaccinia virus according to claim 1, wherein the initial culture in step (a) comprises culturing the cells for the second to fourth passages.

3. 3. The method for mass-producing vaccinia virus according to claim 2, wherein the initial culture of step (a) comprises culturing the cells up to the second passage.

4. 4. The method for mass-producing vaccinia virus according to claim 2 or 3, wherein the cells of each passage in step (a) are cultured for 3 to 5 days.

5. 4. The method for mass-producing vaccinia virus according to claim 3, wherein the first passage cells in the initial culture are dispensed at a density of 1.00E+05 to 3.00E+05 cells / mL, and the second passage cells are dispensed at a density of 5.00E+04 to 1.00E+05 cells / mL.

6. 2. The method for mass-producing vaccinia virus according to claim 1, wherein the fetal bovine serum is added at a concentration of 5% to 10%.

7. 2. The method for mass-producing vaccinia virus according to claim 1, wherein the harvesting in step (c) is carried out 4 to 6 days after the virus infection.

8. 2. The method for mass-producing a vaccinia virus according to claim 1, wherein the vaccinia virus is any one selected from the group consisting of Western Reserve (WR), NYVAC (New York Vaccinia Virus), Wyeth (The New York City Board of Health), LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, IHD-J (International Health Division-J), IHD-W (International Health Division-White), variants thereof, and combinations thereof.

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