Method for mass production of vaccinia virus using suspension cells

Abstract

The present invention relates to a method for mass production of vaccinia virus using suspension cells. Although a method for producing vaccinia virus using adherent cells in the related art has limitations in being unsuitable for mass production of virus due to the characteristics of adherent cells, the present inventors have developed a technology capable of producing virus even with low appropriate cell number, MOI, culture FBS concentration, and culture medium when using suspension cells in a bioreactor, and also confirmed that the present invention has a high virus production capacity similar to that of using adherent cells. Accordingly, the technology for producing vaccinia virus using suspension cells according to the present invention can mass-produce vaccinia virus at a high yield. Since production costs and time, manpower, etc. using suspension cells can be reduced, it is expected that the technology will be effectively used in the clinical and commercial production fields requiring mass production of vaccinia virus.

Description

Technical Field

[0001] This invention relates to a method for mass production of vaccinia virus using suspended cells. Background Technology

[0002] Vaccinia virus, also known as vaccinia virus, belongs to the family Poxviridae and is an enveloped DNA virus with a double-stranded linear DNA genome of approximately 180 kb, encoding about 250 independent genes. Vaccinia virus has a wide range of hosts, including mammals and birds, and replicates in various cultured cells to form a white fork, which is similar to but larger than the smallpox virus. Vaccinia virus infection is usually very mild and can induce a rash and fever, but does not cause symptoms in healthy individuals. Because the immune response caused by vaccinia virus infection protects the body from fatal smallpox infection, vaccinia virus has been used as a live virus vaccine against smallpox.

[0003] Vaccine viruses have attracted attention not only as smallpox vaccines but also as vaccine delivery systems. They possess relatively large linear DNA genomes, allowing them to carry a variety of antigen genes. Furthermore, vaccinia viruses have a strong ability to induce immunity, making them a potential vaccine delivery system for difficult-to-develop vaccines used to treat and prevent infectious diseases, as well as anti-cancer vaccines. By inserting exogenous genes into attenuated vaccinia viruses, safer recombinant vaccines with fewer side effects can be produced.

[0004] Recently, cancer therapy techniques utilizing vaccinia virus as an oncolytic virus have been developed, and most of these are produced in adherent cells. US Patent Publication US2014 / 0162342 discloses a method for producing vaccinia virus using adherent cells and a rolling flask. HeLa cells, uterine cancer cells, were infected with vaccinia virus at an MOI of 0.01-0.05 pfu / cell and cultured to confirm a yield of 50 pfu. The conclusion was that when using HeLaS3 cells for vaccinia virus production, HeLaS3, being a suspension cell, was unsuitable as a cell line for producing the aforementioned virus due to its low vaccinia virus yield. In another patent application, a method for producing the vaccinia virus NYCBOH strain in MRC-5 cells (human fibroblasts) has been filed, and another article reports a method for producing a temperature-dependent protein instead of vaccinia virus using HeLaS3 and the vaccinia virus WR strain.

[0005] As mentioned above, in most companies involved in the relevant technologies and reported related technologies, methods for producing vaccinia virus using adherent cells such as Vero, MRC-5, and HeLa are known and used. However, due to the characteristics of adherent cells, this existing production method increases labor, cost, and time due to limitations in scale-up, making it unsuitable for large-scale virus production.

[0006] Invention Disclosure

[0007] Technical issues

[0008] Therefore, the inventors of this application have established a suitable production method capable of producing vaccinia virus in large quantities by exploring and evaluating the relevant conditions from the initial culture of suspension cells to virus infection and production, thus ensuring the technology for producing vaccinia virus using suspension cells that exhibit high levels of virus production capacity.

[0009] Therefore, one objective of the inventors of this application is to provide a method for mass production of vaccinia virus using suspended cells.

[0010] However, the technical problems to be solved by the present invention are not limited to those described above, and those skilled in the art will clearly understand from the following description other problems not mentioned.

[0011] Technical solution

[0012] To achieve the objectives of the present invention as described above, the present invention provides a method for large-scale production of vaccinia virus, the method comprising the following steps:

[0013] (a) Initial culture of suspension HeLa S3 or Madin-Darby canine kidney (MDCK) cells;

[0014] (b) Passage culture of initially cultured cells, seeding passaged cells at a density of 5.00E+04 to 1.00E+05 cells / mL, then infecting cells with vaccinia virus at a multiple of infection (MOI) of 0.01 to 0.1 and culturing the infected cells; and

[0015] (c) Harvesting viruses from cell cultures.

[0016] As an exemplary embodiment of the present invention, the initial culture in step (a) may be to culture cells until passage 2 to 4 times.

[0017] As another exemplary embodiment of the present invention, the initial culture in step (a) can be culturing cells until passage 2.

[0018] As another exemplary embodiment of the present invention, in step (a), the cells in each generation can be cultured for 3 to 5 days.

[0019] As another exemplary embodiment of the present invention, in the initial culture, cells from passage 1 can be seeded at a density of 1.00E+05 to 3.00E+05 cells / mL, and cells from passage 2 can be seeded at a density of 5.00E+04 to 1.00E+05 cells / mL.

[0020] As another exemplary embodiment of the present invention, the cells can be cultured in a medium supplemented with fetal bovine serum (FBS).

[0021] As another exemplary embodiment of the present invention, the culture medium may be serum-modified Eagle's medium (SMEM) or RPMI 1640 medium.

[0022] As another exemplary embodiment of the present invention, fetal bovine serum can be added at a concentration of 5% to 10%.

[0023] As another exemplary embodiment of the present invention, the harvest in step (c) can be carried out 4 to 6 days after the cells are infected with the virus.

[0024] As another exemplary embodiment of the present invention, the vaccinia virus may be selected from any of the following strains: Western Reserve (WR), New York Vaccine Virus (NYVAC), New York City Council of Health (Wyeth), LC16m8, Lister, Copenhagen, Tiantan, USSR, Tashkent, Evans, International Health Division-J (IHD-J), International Health Division-White (IHD-W), variants thereof, and combinations thereof.

[0025] Beneficial effects

[0026] Methods for producing vaccinia virus using adherent cells in related technologies have limitations due to the characteristics of adherent cells, making them unsuitable for large-scale virus production. However, the inventors of this application have developed a technique for producing virus using suspension cells, even with low appropriate cell numbers, MOI, culture FBS concentration, and culture medium in a bioreactor, and have demonstrated that the present invention achieves a similar high virus yield as in the case of using adherent cells. Therefore, the technique for producing vaccinia virus using suspension cells according to the present invention enables high-yield, large-scale production of vaccinia virus. Since it can reduce production costs, time, and manpower associated with using suspension cells, this technology is expected to be effectively used in clinical and commercial production fields requiring large-scale production of vaccinia virus. Attached Figure Description

[0027] Figure 1A illustrates the results obtained by measuring the number of viable cells daily when cells were seeded and cultured at densities of 3.00E+05, 5.00E+05, and 1.00E+06 cells / mL, respectively, to select the initial (P+1) culture conditions for suspension HeLa S3 cells.

[0028] Figure 1B illustrates the results obtained by measuring cell viability daily when cells are seeded and cultured at densities of 3.00E+05, 5.00E+05, and 1.00E+06 cells / mL, respectively, to select the initial (P+1) culture conditions for suspension HeLa S3 cells.

[0029] Figure 1C illustrates the results obtained by measuring the cell expansion fold daily when cells were seeded and cultured at densities of 3.00E+05, 5.00E+05, and 1.00E+06 cells / mL, respectively, to select the initial (P+1) culture conditions for suspension HeLa S3 cells.

[0030] Figure 2A illustrates the results obtained by measuring the number of viable cells daily when seeding and culturing cells at densities of 1.00E+05, 3.00E+05, and 5.00E+05 cells / mL, respectively, to select the initial (P+1) culture conditions for suspension HeLa S3 cells.

[0031] Figure 2B illustrates the results obtained by measuring cell viability daily when cells are seeded and cultured at densities of 1.00E+05, 3.00E+05, and 5.00E+05 cells / mL, respectively, to select the initial (P+1) culture conditions for suspension HeLa S3 cells.

[0032] Figure 2C illustrates the results obtained by measuring the cell expansion fold daily when cells are seeded and cultured at densities of 1.00E+05, 3.00E+05, and 5.00E+05 cells / mL, respectively, to select the initial (P+1) culture conditions for suspension HeLa S3 cells.

[0033] Figure 3A illustrates the results obtained by measuring the number of viable cells daily when seeding and culturing cells at densities of 5.00E+04, 1.00E+05, and 2.00E+05 cells / mL in step P+2, to select the initial (P+2) culture conditions for suspended HeLa S3 cells.

[0034] Figure 3B illustrates the results obtained by measuring cell viability daily during seeding and culturing of cells at densities of 5.00E+04, 1.00E+05, and 2.00E+05 cells / mL in step P+2, in order to select the initial (P+2) culture conditions for suspension HeLa S3 cells.

[0035] Figure 3C illustrates the results obtained by measuring the cell expansion fold daily during seeding and culturing of cells at densities of 5.00E+04, 1.00E+05, and 2.00E+05 cells / mL in step P+2, in order to select the initial (P+2) culture conditions for suspension HeLa S3 cells.

[0036] Figure 4A illustrates the results obtained by measuring the number of viable cells daily when cells are seeded and cultured in P+3 at densities of 5.00E+04, 1.00E+05, and 2.00E+05 cells / mL, respectively, in order to select the initial (P+3) culture conditions for suspension HeLa S3 cells.

[0037] Figure 4B illustrates the results obtained by measuring cell viability daily when cells are seeded and cultured in P+3 at densities of 5.00E+04, 1.00E+05, and 2.00E+05 cells / mL, respectively, in order to select the initial (P+3) culture conditions for suspended HeLa S3 cells.

[0038] Figure 4C illustrates the results obtained by measuring the cell expansion fold daily when cells are seeded and cultured in P+3 at densities of 5.00E+04, 1.00E+05, and 2.00E+05 cells / mL, respectively, in order to select the initial (P+3) culture conditions for suspension HeLa S3 cells.

[0039] Figure 5 Examples of cell culture methods at P+3 with cell seeding density and FBS concentration conditions are provided to determine FBS concentration during and after infection of suspension HeLa S3 cells with vaccinia virus.

[0040] Figures 6A-6H illustrate how changing the cell seeding density and FBS concentration at P+3 can lead to the following results: Figure 5 The results were obtained by culturing HeLa S3 cells and measuring the number of viable cells (Fig. 6A-6D) and cell viability (Fig. 6E-6H).

[0041] Figure 7 An example is given of the cell culture and virus production process of HeLa S3 cells so that cell seeding density and virus yield at P+3 can be analyzed during and after infection with vaccinia virus, based on FBS concentration conditions.

[0042] Figure 8A illustrates how changing the cell seeding density and FBS concentration in P+3 can lead to the following results: Figure 7 The results were obtained by measuring the number of viable cells after HeLa S3 cell culture and virus production using the method described above.

[0043] Figure 8B illustrates how changing the cell seeding density and FBS concentration at P+3 can lead to the following results: Figure 7 The results were obtained by measuring the cell viability of HeLa S3 cells after culture and virus production using a method described above.

[0044] Figure 8C illustrates how changing the cell seeding density and FBS concentration at P+3 can lead to the following results: Figure 7 The results were obtained by measuring viral yield (TCID50 / cell) after HeLa S3 cell culture and virus production.

[0045] Figure 9 An example illustrates the cell culture and virus production process (preliminary experiment) for analyzing cell growth and virus production based on the MOI of vaccinia virus infection, the harvest date, and the cell seeding density of HeLa S3 cells at P+3.

[0046] Figure 10A illustrates, for example, that at a cell seeding density of 1.00E+05 at P+3, the infection MOI was adjusted according to... Figure 9 After conducting the experiment using the method, samples were harvested and the number of live cells was measured on each harvest day.

[0047] Figure 10B illustrates, for example, how changing the infection MOI at a cell seeding density of 1.00E+05 cells / mL at P+3, based on... Figure 9 The method involves harvesting samples and measuring cell viability on each harvest day after the experiment.

[0048] Figure 10C illustrates, for example, that at a cell seeding density of 5.00E+05 cells / mL at P+3, the infection MOI was adjusted based on... Figure 9 After conducting the experiment using the method, samples were harvested and the number of live cells was measured on each harvest day.

[0049] Figure 10D illustrates, for example, that at a cell seeding density of 5.00E+05 cells / mL at P+3, the infection MOI was adjusted based on... Figure 9 After conducting the experiment using the method, samples were harvested on each harvest day and the results of cell viability were measured.

[0050] Figure 11A illustrates the results of confirming the total virus yield of each sample harvested in Figures 10A-10D, in order to analyze the virus yield.

[0051] Figure 11B illustrates the results of confirming the ability of each sample collected in Figures 10A-10D to produce virus per cell (TCID50 / cell) to analyze viral yield.

[0052] Figure 12A illustrates the results of determining the MOI of vaccinia virus infection in HeLa S3 cells, the harvest day, and the cell inoculation density conditions at P+3 through a second experiment, in which the inoculation density was set to 5.00E+04 cells / mL and the harvest day conditions were varied, with samples harvested on each harvest day.

[0053] Figure 12B illustrates the results of confirming cell viability by conducting a two-stage experiment to determine the MOI of vaccinia virus infection in HeLa S3 cells, the harvest date, and the cell seeding density conditions at P+3. The experiment was conducted by setting the seeding density to 5.00E+04 cells / mL and varying the harvest date conditions, and harvesting samples on each harvest date.

[0054] Figure 12C illustrates the results of determining the MOI of vaccinia virus infection in HeLa S3 cells by conducting a second experiment, confirming the number of viable cells on the harvest day and at cell inoculation density conditions at P+3, where the inoculation density was set to 1.00E+05 cells / mL and the harvest day conditions were varied, with samples harvested on each harvest day.

[0055] Figure 12D illustrates the results of confirming cell viability by conducting a two-stage experiment to determine the MOI of vaccinia virus infection in HeLa S3 cells, the harvest date, and the cell seeding density conditions at P+3. The experiment was conducted by setting the seeding density to 1.00E+05 cells / mL and changing the harvest date conditions, and harvesting samples on each harvest date.

[0056] Figure 13A illustrates the results of confirming the total virus yield of the samples harvested in Figures 12A to 12D, in order to analyze the virus yield.

[0057] Figure 13B illustrates the results confirming the ability of the samples harvested in Figures 12A to 12D to produce virus per cell (TCID50 / cell) for viral yield analysis.

[0058] Figure 14A illustrates the results of analyzing viable cell counts by conducting experiments under conditions of fixed cell seeding density and infection MOI, and then harvesting all samples on days 3, 4, 5, 6 and 7, respectively, to alternatively select the date for harvesting virus-infected cells and culture medium.

[0059] Figure 14B illustrates an example of conducting experiments under conditions of fixed cell seeding density and infection MOI, and then harvesting all samples on days 3, 4, 5, 6 and 7, respectively, to further select the date for harvesting virus-infected cells and culture medium to analyze cell viability results.

[0060] Figure 15A illustrates the results of confirming the total viral load using samples harvested in Figures 14A and 14B.

[0061] Figure 15B illustrates the results of confirming the ability of each cell to produce virus using samples harvested in Figures 14A and 14B.

[0062] Figure 16The method for scaling up virus production is illustrated with examples, including the initial culture conditions of HeLa S3 cells, the cell seeding density for virus infection, the MOI, and the selection of the harvest day.

[0063] Figure 17A illustrates an example based on... Figure 16 The results of the experiment, before and after scale-up, were used to confirm the cell number and viability.

[0064] Figure 17B illustrates an example based on... Figure 16 The method was used to confirm the viral yield results before and after the scale-up experiment.

[0065] Figure 18 An example is given illustrating the experimental method used to select the culture medium for HeLa S3 cells.

[0066] Figure 19A illustrates an example of how, after conducting the experiment, the results were obtained by... Figure 18 The method involves changing the culture medium of HeLa S3 cells to SMEM, JMEM, or RPMI 1640 at P+1 to P+3 to confirm the viable cell count.

[0067] Figure 19B illustrates an example of how, after conducting the experiment, one can... Figure 18 The method involves changing the culture medium of HeLa S3 cells from P+1 to P+3 using SMEM, JMEM, or RPMI 1640 to confirm cell viability.

[0068] Figure 20 Examples illustrate the situation based on Figure 18 The method involved conducting experiments by altering the culture medium of HeLa S3 cells at points P+1 to P+3, followed by comparative analysis of the viral yield results.

[0069] Invention Model

[0070] The inventors of this application have established a production method capable of mass-producing vaccinia virus by exploring and evaluating the relevant conditions from the initial culture of suspension cells to virus infection and production, thereby ensuring the technology for producing vaccinia virus using suspension cells that exhibit high levels of virus production capacity.

[0071] The present invention will be described in detail below.

[0072] Therefore, the present invention provides a method for large-scale production of vaccinia virus, the method comprising: (a) initially culturing suspended HeLa S3 or Madin-Darby canine kidney (MDCK) cells; (b) passage culturing the initially cultured cells, seeding the passaged cells at a density of 5.00E+04 to 1.00E+05 cells / mL, then infecting the cells with vaccinia virus at a multiplicity of infection (MOI) of 0.01 to 0.1 and culturing the infected cells; and (c) harvesting the virus from the cell culture.

[0073] In this invention, "vaccinia virus" includes vaccinia virus and vaccinia virus solution containing the virus, unless otherwise stated. Vaccinia virus solution is not particularly limited as long as it contains vaccinia virus and includes, for example, culture supernatant after culturing host cells infected with vaccinia virus and virus suspension after removing impurities from the culture supernatant.

[0074] The vaccinia virus can be selected from any of the following strains: Western Reserve (WR), New York Vaccine Virus (NYVAC), New York City Council of Health (Wyeth), LC16m8, Lister, Copenhagen, Tiantan, USSR, Tashkent, Evans, International Health Division-J (IHD-J), International Health Division-White (IHD-W), variants thereof, and combinations thereof, and may preferably be IHD-W, but is not limited thereto.

[0075] As used herein, the term "suspension cell" refers to cells that proliferate in suspension in a culture medium without adhering to a culture plate during cell culture, and are diluted during passage culture without changing the culture medium. The suspension HeLa S3 and MDCK cells used in this invention to generate vaccinia virus are cells with adherent cell characteristics but modifiable for growth in suspension. HeLa S3 is a clonal derivative of the parental HeLa lineage, which is a human uterine cancer cell line and is suitable for transfection; MDCK is a canine kidney-derived cell line and is known in the art as a cell line capable of propagating various viruses.

[0076] In this invention, an optimal production method for producing vaccinia virus in large quantities using suspended cells was studied and established.

[0077] In this invention, step (a) is the initial culture of suspended HeLa S3 or MDCK cells.

[0078] Initial culture can be performed by thawing frozen suspension cells, inoculating the thawed suspension cells, and culturing the inoculated suspension cells until passage 2 to 4, preferably until passage 2 (P+2). For initial culture, it is preferable to culture each generation of cells for 3-5 days after cell inoculation, more preferably to perform passage culture after 4 days of culture.

[0079] Furthermore, during the initial culture up to passage 2 (P+2), it is preferable to seed passage 1 cells at a density of 1.00E+05 to 3.00E+05 cells / mL and passage 2 cells at a density of 5.00E+04 to 1.00E+05 cells / mL. More preferably, optimal cell growth can be induced when passage 1 cells and passage 2 cells are seeded at 1.00E+05 cells / mL and 5.00E+04 cells / mL, respectively.

[0080] In a specific exemplary embodiment of the present invention, it was demonstrated that initial culture conditions, particularly cell seeding density and culture period, were selected before infecting suspended HeLa S3 cells with the virus by culturing cells under various conditions and analyzing the number of viable cells and cell viability. These conditions were the optimal initial culture conditions for the mass production of vaccinia virus (see Example 1).

[0081] Cells can be cultured in a medium supplemented with fetal bovine serum (FBS), and preferably, serum-modified Eagle's medium (SMEM) or RPMI 1640 medium can be used as the cell culture medium, and more preferably, RPMI 1640 is used in terms of cell culture efficiency and economic feasibility, but the medium is not limited to these. In this case, fetal bovine serum can be added at a concentration of 5% to 10%, and preferably at a concentration of 10% during the initial culture in step (a).

[0082] Apart from the selection of conditions for initial culture, there are no particular restrictions on culture temperature, carbon dioxide concentration, and the method of passage culture of suspension cells, and those skilled in the art can appropriately perform initial culture according to typical conditions and methods used for cell culture in the relevant field.

[0083] In this invention, step (b) is the step of inoculating the cells with vaccinia virus after initial culture. For example, in the case of initial culture up to passage 2 (P+2), step (b) is the step of culturing the initially cultured cells to passage 3 (P+3) and inoculating them with passaged cells, and then inoculating the inoculated cells with vaccinia virus.

[0084] In step (b), the preferred cell seeding density during viral infection is 5.00E+04 to 1.00E+05 cells / mL. More preferably, optimal cell growth and viral yield can be induced when cells are seeded at 5.00E+04 cells / mL.

[0085] Furthermore, during viral infection, the multiple of infection (MOI) is preferably 0.01-0.1, and more preferably, the highest cell growth and viral yield can be induced when cells are infected with an MOI of 0.01.

[0086] As used herein, the term "multiple of infection (MOI)" refers to the ratio of an agent (e.g., a virus) to an infection target (e.g., a host cell). It is the ratio of the number of viral particles to the number of target cells present in a confined space.

[0087] Furthermore, in step (b), as the culture medium for culturing suspension cells infected with the virus, SMEM or RPMI 1640 medium supplemented with 5% to 10% FBS can be used, and more specifically, a medium supplemented with 5% FBS is preferred.

[0088] The inventors of this application have demonstrated through examples that the conditions in step (b) above are optimal conditions for mass production.

[0089] That is, in another specific exemplary embodiment of the present invention, in order to determine the concentration of FBS added during the infection of suspended HeLa S3 cells and cultures with the virus after infection, the cells were seeded at different densities and cultured with concentrations of 0, 2, 5 and 10% FBS, respectively. The cell growth and yield of vaccinia virus were then compared and analyzed, and the optimal cell seeding density and FBS concentration for virus infection were selected as described above (see Example 2).

[0090] In another exemplary embodiment of the invention, experiments were conducted to select the MOI for virus infection, the cell and culture medium harvest period, and the cell seeding density conditions for virus infection in suspended HeLa S3 cells. Specifically, cell growth and vaccinia virus yield were compared when cells were seeded at different densities and MOIs and the harvest period was varied to select the optimal conditions as described above (see Examples 3-1 to 3-3).

[0091] In this invention, the yield of vaccinia virus is evaluated by the total amount of virus produced and the ability of each cell to produce virus (TCID50 / cell). The ability of each cell to produce virus is synonymous with "titer," which is commonly used in the art as a unit to indicate the viral infection titer. Since viruses cannot be seen using an optical microscope, their density (number / volume) cannot be measured under a microscope as it would with biological cells. Therefore, in the case of viruses, the infectious titer against the host cells is used as the unit, instead of their amount or concentration. For example, when a viral suspension diluted to an appropriate proportion is added to a monolayer of host cells, the number of viruses is detected as plaques, and the infection titer can be measured as plaque-forming units (pfu) / mL. Alternatively, the infection titer can be measured by diluting the virus-containing liquid and setting the concentration at which 50% of the host cells produce a positive infection as a 50% tissue culture infection dose (TCID50) / mL. In this embodiment, the yield of vaccinia virus, i.e., the infection titer, is measured as TCID50 / mL, but the invention is not limited thereto.

[0092] Apart from the conditions selected above, there are no particular limitations on the methods for culturing suspension cells and infecting cells with viruses, and those skilled in the art can appropriately apply and implement methods commonly used in the art.

[0093] In this invention, step (c) is the step of harvesting the virus from the cell culture to obtain the virus produced by step (b).

[0094] To harvest the virus, it is preferable to harvest cultured cells and cultures. The virus can be harvested 4-6 days after infection, preferably 5 days after infection.

[0095] From the samples collected through this step, those skilled in the art can obtain the final generated virus and analyze the yield using methods used in the art.

[0096] In another exemplary embodiment of the invention, a virus production scale-up experiment was conducted at a scale of 1.8L to examine whether the production conditions of the vaccinia virus of the present invention could be applied to a real-world large-scale virus production process. The number of viable cells, cell viability, and virus yield were then analyzed. The results confirmed that the cell number and viability were similar at both scales (30 mL and 1800 mL), and the virus yield was also similar. This demonstrates that the method for producing vaccinia virus using suspension cells according to the present invention and the established conditions can be applied to large-scale production (see Examples 3 and 4).

[0097] In another exemplary embodiment of the invention, further experiments were conducted to select a culture medium for the suspension of HeLa S3 cells in order to further improve viral yield or ensure cost reduction during the selected viral production process. The results confirmed that RPMI 1640 exhibited similar cell growth and viral production effects compared to SMEM used in the steps, and based on these results, it was determined that the use of RPMI 1640 medium was suitable, as it is more readily available and can reduce costs (see Example 4).

[0098] The vaccinia virus ultimately produced by the production method of the present invention can be used in various fields, such as basic research and clinical applications, including vaccines, oncolytic viruses for cancer treatment, and viral vectors as delivery systems.

[0099] Preferred embodiments of the invention will be presented below to aid in understanding the invention. However, these embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited to these embodiments. Example

[0100] Example 1. Suspended HeLa Selection of S3 cell culture conditions

[0101] In order to establish culture conditions for suspended HeLa S3 cells (a cell line that produces vaccinia virus), the inventors of this application conducted experiments and selected initial culture conditions from P+1 to P+3 after the cells were thawed.

[0102] 1-1. Selection of P+1 culture conditions

[0103] First, in order to select the culture conditions for P+1, the cells were thawed and seeded under the conditions shown in Table 1 below, and then the number of viable cells, cell viability and cell expansion were measured daily while the cells were being cultured.

[0104] [Table 1]

[0105]

[0106] As shown in Figures 1A to 1C, the results of culturing cells and conducting experiments under the above conditions revealed that the maximum cell number that could be achieved without changing the culture medium was approximately 2.00E+06 cells / mL, and higher cell density resulted in higher cell numbers within a faster timeframe. However, to confirm the cell expansion fold, it was confirmed that cells were cultured at the lowest density of 3.00E+05 cells / mL. Therefore, it can be seen that during cell seeding, lower cell density results in better culture efficiency than higher cell density.

[0107] Based on the above results, cell growth was compared and analyzed by changing the cell seeding density to 1.00E+05, 3.00E+05, and 5.00E+05 cells / mL, as shown in Table 2 below. In subsequent experiments, 3.00E+05 cells / mL was used as the standard.

[0108] [Table 2]

[0109]

[0110]

[0111] As the results of cell culture under the conditions shown in Table 2, as can be seen from Figures 2A to 2C, it was confirmed that the maximum number of cells during the culture period was similar for all three cell density conditions, but viability was highest when cells were seeded at the lowest density of 1.00E+05 cells / mL. Furthermore, under the other two density conditions, the graph peaked and immediately declined, making it difficult to set up passage culture cycles. However, at 1.00E+05 cells / mL, since the period between day 4 and day 5 showed a stationary phase, day 4 of culture was determined to be suitable for cell passage.

[0112] Therefore, during P+1 culture, the conditions were set to seed cells at 1.00E+05 cells / mL and passage the cells after 4 days.

[0113] 1-2. Selection of P+2 culture conditions

[0114] Next, to select the culture conditions for P+2, cells were cultured according to the conditions shown in Table 3 below, and the cell culture efficiency was compared and analyzed based on the cell seeding density conditions. In this case, it was observed that the method of setting the culture conditions for P+1 with an initial high cell density tended to lead to low culture efficiency, and in this experiment, the cell seeding density was further reduced to set the conditions and culture the cells.

[0115] [Table 3]

[0116] culture medium SMEM (10% FBS, 1% AA) Training scale 30 mL in 125 mL EMF RPM 110rpm Incubator conditions 37℃ 5% CO2 Cell seeding density 5.00E+04, 1.00E+05, 2.00E+05 cells / mL

[0117] As the experimental results, as shown in Figures 3A to 3C, confirmed that the time to reach the maximum cell number varied depending on the cell seeding density, but the final cell number in all cases was approximately 2.00E+06 cells / mL or higher. Furthermore, based on observations showing the highest cell viability and fold expansion at the lowest density, cell efficiency was optimal at a minimum density of 5.00E+04 cells / mL. When considering cell passage in the logarithmic phase under the selected P+2 culture conditions, it was determined that a seeding density of 5.00E+04 cells / mL and passage on day 4 were suitable for both high fold expansion and maintained viability. The advantage of these conditions is that the culture efficiency is so high that the culture scale can be expanded to approximately 750 mL in P+3, and because the culture cycle is the same as in P+1, it offers high convenience.

[0118] 1-3. Selection of P+3 culture conditions

[0119] In addition, in order to select the P+3 conditions, cells were cultured under the P+1 and P+2 conditions selected by Examples 1-1 and 1-2, and then cultured under the P+3 conditions in Table 4 below.

[0120] [Table 4]

[0121] culture medium SMEM (10% FBS, 1% AA) Training scale 30 mL in 125 mL EMF RPM 110rpm Incubator conditions 37℃ 5% CO2 Cell seeding density 5.00E+04, 1.00E+05, 2.00E+05 cells / mL

[0122] As shown in Figures 4A-4C, the culture results confirmed a similar growth curve to that observed in P+2, with the highest cell culture efficiency at 5.00E+05 cells / mL, which represents a low seeding density. Furthermore, considering the culture efficiency and cell viability in the logarithmic growth phase, it was determined that passage culture on day 4 after culturing the cells in the same manner as in P+2 was appropriate.

[0123] Therefore, the initial culture conditions for P+1, P+2 and P+3 selected in the examples are summarized in Table 5 below, and HeLa S3 cells were cultured under the above conditions during the experiments used to select conditions for vaccinia virus production in the future.

[0124] [Table 5]

[0125] culture medium SMEM (10% FBS, 1% AA) Training scale 30 mL in 125 mL EMF RPM 110rpm Incubator conditions <![CDATA[37℃ 5%CO2]]> P+1 cell seeding density and passage cycle 1.00E+05 cells / mL, Day 4 P+2 cell seeding density and passage cycle 5.00E+04 cells / mL, Day 4 P+3 cell seeding density and passage cycle 5.00E+04 cells / mL, Day 4

[0126] Example 2. Suspended HeLa Selection of FBS concentration during S3 cell culture

[0127] The inventors of this application conducted the following experiments to select the FBS concentration that could exhibit optimal cell growth and vaccinia virus yield in the culture of suspended HeLa S3 cells.

[0128] 2-1. Observe cell growth based on FBS concentration.

[0129] First, in order to observe the cell growth of HeLa S3 based on the FBS concentration in the culture medium, the following conditions were met: Figure 5 Cells were cultured under the conditions selected in Example 1, and then the cell seeding density at P+3 was set to 5.00E+04, 5.00E+05, and 1.00E+06 cells / mL. Cell growth curves were analyzed while culturing under four conditions (0, 2%, 5%, and 10%) in each case.

[0130] As a result of analyzing viable cell count and cell viability during P+3 culture, it was found that, as expected, cells did not grow and remained at low viability under 0% FBS conditions, as shown in Figures 6A to 6H. Although a slight increase in cell count was observed under 2% FBS conditions, no significant growth efficiency was observed, and a decrease in cell viability was observed from the initial stages of culture, except for the case where 5.00E+04 cells / mL was the lowest cell density. Under 5% FBS conditions, growth curves similar to those of the 10% FBS (as a positive control) were observed at all cell densities up to day 2 of culture, and similar cell viability was observed at low densities. Based on these results, low viral yield was expected, as the cell state under 0–2% FBS conditions during viral infection and cell culture is unfavorable for vaccinia virus production. However, to confirm substantial viral yield dependent on FBS concentration, viral yield validation experiments were performed under the same FBS conditions as described above.

[0131] 2-2. Confirm the yield of vaccinia virus (VACV) based on the selection of FBS concentration and conditions.

[0132] Next, to confirm the yield of vaccinia virus in suspended HeLa S3 cells based on FBS concentration, according to Figure 7 The experiment was conducted using the following method. In this case, cells were infected with vaccinia virus at a seeding density of 5.00E+05 or 1.00E+06 cells / mL, a FBS concentration of 2, 5, or 10%, and an MOI of 0.1. Subsequently, the cells were cultured for 4 days, and the cells and culture medium were collected, and viral yield was evaluated under each condition.

[0133] As shown in Figures 8A to 8C, the results of analyzing the number of viable cells and cell viability based on FBS concentration under each of the two cell density conditions revealed a decrease in cell number and viability with increasing cell culture and virus production. At 5.00E+05 cells / mL, cell number and viability increased slightly with increasing FBS concentration. Conversely, at 1.00E+06 cells / mL, cell number and viability showed a similar trend to increasing FBS concentration. These results suggest that viral yield at 5.00E+05 cells / mL will vary depending on FBS concentration. Indeed, as a result of assessing vaccinia virus yield using TCID50, which confirms the ability of each cell to produce virus, a significant difference in viral yield was found depending on each FBS condition at 5.00E+05 cells / mL, with the highest yield confirmed at 5% FBS. Therefore, a 5% FBS concentration was ultimately chosen for high-titer vaccinia virus production during and after cell infection.

[0134] Example 3. Suspended HeLa Selection of Viral Infection and Harvesting Conditions in S3 Cells

[0135] 3-1. Preliminary experiment on MOI of virus infection, harvest date and cell seeding density

[0136] Experiments were conducted to select the multiple of infection (MOI), harvest date, and cell seeding density conditions to further improve viral yield, using the initial culture method for suspension HeLa S3 cells selected through experiments in Examples 1 and 2, and the FBS concentration conditions during vaccinia virus infection and production. Figure 9 The methods and conditions described herein were used in experiments. Specifically, the MOI for viral infection was set to 0.01, 0.1, and 1, and viral yield was evaluated by harvesting cells and culture medium on days 2, 3, 4, and 5 post-infection. Furthermore, the seeding density of virus-infected cells was set to two conditions: 1.00E+05 cells / mL and 5.00E+05 cells / mL.

[0137] First, as the results of analyzing viable cell count and cell viability based on MOI and harvest date, respectively, under two cell density conditions, as shown in Figures 10A to 10D, at the lowest MOI of 0.01, an increase in cell count was observed, followed by a period of stagnation or decrease. Cell viability was also observed to decrease with increasing viral infection concentration and incubation period. This pattern suggests that subsequent viral production via viral infection can be predicted.

[0138] Furthermore, viral yield was analyzed by TCID50 analysis of samples harvested on days 2–5 after the start of culture. The results, as shown in Figure 11A, showed the highest total viral yield when cells were seeded at a density of 5.00E+05 cells / mL, infected with virus at an MOI of 0.01, and harvested after days 3, 4, and 5. However, as shown in Figure 11B, it was confirmed that the actual viral production capacity per cell was highest when cells were seeded at a density of 1.00E+05 cells / mL, then infected with virus at an MOI of 0.01, and harvested after days 3, 4, and 5. Moreover, considering that the vaccinia virus yield was 300 TCID50 / cell or higher when using adherent HeLa cells as an internal standard, it can be confirmed that even under the conditions described above in this invention, using suspension HeLa S3 cells can produce 300 TCID50 / cell or higher of virus. Using 5 times more cells at 5.00E+05 cells / mL than at 1.00E5 cells / mL did not result in a significant difference in total production, indicating very low production efficiency. Therefore, cells were primarily seeded at a density of 1.00E+05 cells / mL, infected with the virus at an MOI of 0.01, and harvested on days 3-5 for selection. Furthermore, referring to Figures 11A and 11B, it was observed that lower cell density and MOI correlated with higher virus yield. Therefore, in subsequent secondary experiments, an attempt was made to further reduce the cell density.

[0139] 3-2. Secondary experiments used to select the MOI for virus infection, harvest date, and cell seeding density.

[0140] Based on the results of Example 3-1, a second experiment was conducted to select the MOI for vaccinia virus infection, the harvest date, and the cell inoculation density. The entire process was the same as that performed in Example 3-1. Figure 9 The conditions were the same, with cell seeding densities adjusted to 5.00E+04 and 1.00E+05 cells / mL at P+3, and in Figures 11A and 11B, cells and culture medium solutions were harvested on days 3, 4, and 5 post-infection, respectively, when samples were harvested on day 2 post-infection, except for day 2 under harvest conditions, as productivity was low in all cases.

[0141] First, as shown in Figures 12A to 12D, the results of analyzing the number of viable cells and cell viability under the two cell density conditions described above, based on MOI and harvest date, indicate that an increase in cell number was observed, and viability was maintained until day 4 of culture. This confirms that at the higher concentration of 1.00E+05 cells / mL, the cell number increased and viability was maintained until day 3 of culture.

[0142] Furthermore, as shown in Figure 13A, the viral yield was analyzed by infecting cells with the virus and measuring the TCID50 of each sample harvested after 3–5 days of culture. This confirmed that the viral yield was as expected at low cell densities (5.00E+04 cells / mL), and Figure 13B confirmed that the virus was produced at 300 TCID50 / cell or higher, which is the yield of vaccinia virus in adherent HeLa cells, used as an internal standard. Specifically, the infection status at an MOI of 0.01 showed a pattern where productivity continued to increase without decreasing as the harvest day increased from day 3 to day 5. Based on these results, a cell seeding density of 5.00E+04 cells / mL was selected, lower than the density of the preliminary experiment in Example 3-1, and conditions were chosen under which cells were infected with the virus at an MOI of 0.01, and cells and culture medium were collected after 5 days.

[0143] 3-3. Experiments on alternative harvest days

[0144] Since the yield increases with the extension of the harvest day as shown in Figure 13B, the inventors of this application conducted an experiment to verify whether the yield of vaccinia virus increases with the extension of the harvest day. The experiment was conducted in the same manner as in Examples 3-1. Figure 9 A similar overall experimental procedure was performed under the conditions of P+2, with the SMEM medium adjusted to 80 mL. Based on the above experimental results, the virus-infected cell seeding density was fixed at 5.00E+04 cells / mL and the MOI was fixed at 0.01. The experiment was conducted only on days 3, 4, 5, 6, and 7.

[0145] First, as shown in Figures 14A and 14B, as a result of the analysis of cell number and viability, it was found that the cell number increased 3-4 days after viral infection, then decreased, and viability was confirmed to decrease sharply from day 4.

[0146] Furthermore, as shown in Figures 15A and 15B, the results of analyzing vaccinia virus yield based on the harvest date revealed that, starting from day 5, virus production ceased to increase while maintaining cell growth. Based on these results, cells were ultimately selected for virus infection, and the virus was harvested after 5 days.

[0147] 3-4. Scale up vaccinia virus production

[0148] Since the conditions for producing vaccinia virus, namely the initial cell culture conditions, the virus inoculation density for infection, the MOI, and the harvest day conditions, were selected based on the results of the above embodiments, a virus production scale-up experiment was conducted to produce vaccinia virus on a scale of 1.8 L in a stirred tank reactor (STR). Specifically, according to Figure 16The method described in the experiment was used, and the final viral productivity was compared with the results of the 30 mL EMF scale in the above examples.

[0149] Specifically, as shown in Figure 17A, the results of analyzing each of the following—viable cell count, cell viability, and viral yield—by infecting cells with the virus and collecting all samples after 4 days confirmed that similar cell counts and viability were observed at both scales (30 mL, 1800 mL), and as shown in Figure 17B, similar viral yields were also confirmed. These results indicate that the conditions selected for vaccinia virus production in STRs can be used below, as the aforementioned process conditions selected on a small scale of 30 mL EMF are suitable for vaccinia virus production on a 1.8 L STR scale. Therefore, scale-up from EMF to STR is possible.

[0150] Example 4. Suspended HeLa Selection of S3 cell culture medium

[0151] The inventors of this application conducted experiments on the culture medium for selecting HeLa S3 cells to further increase virus yield or ensure the effect of reducing production costs under the conditions selected in Examples 1 to 3. According to Figure 18 Initial cell culture and virus production were performed under the conditions and procedures shown. Cells were cultured by changing the culture medium to SMEM, JMEM, and RPMI 1640, respectively, and virus production was confirmed by infecting cells with vaccinia virus at P+3. Furthermore, experiments were conducted to assess the expected changes in virus yield induced by the culture medium by setting harvest days 3–5.

[0152] 4-1. Analyze cell growth by culture medium type

[0153] First, we analyzed the number of live cells and cell viability to investigate how changes in culture medium type affect the growth of suspended HeLaS3 cells.

[0154] As shown in Figures 19A and 19B, there were no significant differences in cell number and viability when using the three different media after 2 days of culture. Therefore, it was determined that changing the media did not cause any major problems in culturing HeLa S3 cells, and that the more readily available and cheaper RPMI 1640 medium was advantageous for actual virus production, except for the yield of vaccinia virus.

[0155] 4-2. Analyze virus yield by culture medium type

[0156] The inventors of this application observed that HeLa S3 cells were cultured similarly regardless of the type of culture medium, and based on these results, experiments were conducted to study the effect of culture medium type on actual viral yield. For this purpose, after culturing the cells, they were infected with the virus, and viral yield was analyzed by comparison using TCID50.

[0157] As a result, Figure 20 As shown, the results confirm that, in addition to JMEM medium, when using SMEM and RPMI 1640, which are already used in related technologies, the virus yield is superior to internal standards of 300 TCID50 / cell or higher, and shows similar levels. Based on these results, it can be inferred that RPMI 1640 can be used in the production of vaccinia virus. In summary, considering the culture efficiency of HeLaS3 cells and the production capacity of vaccinia virus, the use of RPMI 1640 medium was determined to be suitable. This medium is readily available and can reduce costs; therefore, RPMI 1640 was ultimately selected as the medium for cell culture and virus production.

[0158] 4-3. Final Production Process of Vaccine Virus Derived from Suspended HeLa S3 Cells

[0159] Based on the results of the examples, the final process conditions for efficient production of vaccinia virus in suspended HeLa S3 cells were established. The final processing conditions are shown in Table 6 below. It can be seen that when the virus is produced under these conditions, a level of virus equal to or higher than 300 TCID50 / cell can be produced. 300 TCID50 / cell is the yield of vaccinia virus in adherent HeLa cells, using suspended cells as an internal standard.

[0160] [Table 6]

[0161]

[0162]

[0163] The above description of the present invention is provided for illustrative purposes, and those skilled in the art will understand that the invention can be readily modified into other specific forms without altering its technical spirit or essential characteristics. Therefore, it should be understood that the above embodiments are exemplary in all respects only and not restrictive.

[0164] Industrial applicability

[0165] This invention relates to a method for mass production of vaccinia virus using suspended cells, and to ensure mass production of vaccinia virus that is impossible to achieve with existing technologies, a technique is developed to specifically establish suitable cell numbers, MOI, culture FBS concentration, and culture medium conditions. Therefore, the technique for producing vaccinia virus using suspended cells according to this invention enables high-yield mass production of vaccinia virus. Since it can reduce production costs, time, and manpower associated with using suspended cells, this technology is expected to be effectively used in clinical and commercial production fields requiring large-scale production of vaccinia virus.

Claims

1. A method for mass production of vaccinia virus, the method comprising the following steps: (a) Initial culture of HeLa S3 cells in suspension; (b) Passage the initially cultured cells in suspension, seed the passaged cells at a density of 5.00E+04 to 1.00E+05 cells / mL, then infect the cells with vaccinia virus at a multiplicity of infection (MOI) of 0.01 to 0.1, and culture the infected cells in suspension; and (c) Harvesting vaccinia virus from cell cultures. The cells were cultured in a medium supplemented with fetal bovine serum (FBS). The culture medium mentioned therein is serum-modified Eagle's medium (SMEM) or RPMI 1640 medium, and Fetal bovine serum is added at a concentration of 5% to 10%.

2. The method according to claim 1, wherein the initial culture in step (a) is to culture the cells for 2 to 4 generations.

3. The method according to claim 2, wherein the initial culture in step (a) is to culture the cells up to the second generation.

4. The method according to claim 2 or 3, wherein in step (a), the cells in each generation are cultured for 3 to 5 days.

5. The method of claim 3, wherein in the initial culture, cells in the first generation are seeded at a density of 1.00E+05 to 3.00E+05 cells / mL, and cells in the second generation are seeded at a density of 5.00E+04 to 1.00E+05 cells / mL.

6. The method of claim 1, wherein the harvest in step (c) is carried out 4 to 6 days after the cells are infected with the virus.

7. The method of claim 1, wherein the vaccinia virus is selected from any of the following strains: Western Reserve (WR), New York Vaccine Virus (NYVAC), New York City Council of Health (Wyeth), LC16m8, Lister, Copenhagen, Tiantan, USSR, Tashkent, Evans, International Health Division-J (IHD-J), International Health Division-White (IHD-W), variants thereof, and combinations thereof.

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