Title of the invention: Low serum medium composition for Vero cell culture and use thereof
technical field
[One]
Cross-Citation with Related Applications
[2]
This application claims the benefit of priority based on Korean Patent Application No. 10-2019-0174356 dated December 24, 2019, and all contents disclosed in the documents of the above application are incorporated herein by reference.
[3]
The present invention relates to a low-serum medium composition for culturing Vero cells, and a method for culturing Vero cells and a virus production method using the same.
background
[4]
Vero cells are a continuous cell line widely approved and used for the manufacture of viral vaccines. Vero cells were first isolated from African green monkey kidney cells and are used in the production of inactivated poliovirus vaccine (IPV), oral live polio vaccine, and rabies vaccine. Vero cells have broad susceptibility to various viruses, are reported to be safe from carcinogenesis, and do not pose a threat to human health by the World Health Organization.
[5]
In the conventional vaccine production using animal cells, it is common to use a medium to which an animal-derived material (eg, serum, trypsin, albumin, etc.) is added in the step of propagating the cells or infecting the cells with the virus to propagate the virus. In particular, serum contains substances that promote the survival and proliferation of cells, such as nutrients, hormones, growth factors, and cytokines. Since it facilitates adhesion and spreading, there is an advantage that the culture medium can be mimicked similarly to the in vivo environment. However, due to the complexity of the chemical composition of serum, it is difficult to produce a single product, providing various variables for cell growth, high unit price, and in the case of a vaccine produced using serum, there is a risk of infection by exogenous pathogenic factors, and complex purification process There are disadvantages such as requiring a downstream process.
[6]
Vero cells are adherent cells, and are generally cultured by attaching to a microcarrier in a normal medium containing 10% Fetal Bovine Serum (FBS). The main component contained in FBS is albumin, which is known as a protein that plays an important role in cell adhesion. However, due to the disadvantages of the serum medium as described above, the need for a serum-reduced or serum-free medium is emerging.
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[7]
Therefore, instead of reducing the amount of serum, it is required to develop a medium having a composition containing supplements that can help cell adhesion and promote cell growth.
means of solving the problem
[8]
An example of the present application, newborn calf serum (NCS) 1 ~ 7% (v / v); 1-6 g/L hydrolysate; And it provides a medium composition for culturing Vero cells, comprising 1 to 6 mg/L of insulin.
[9]
Another example of the present application, newborn calf serum (NCS) 1 ~ 7% (v / v); 1-6 g/L hydrolysate; And it provides a method of culturing Vero cells, comprising the step of culturing Vero cells in a medium composition comprising 1-6 mg/L of insulin.
[10]
Another example of the present application, newborn calf serum (NCS) 1 ~ 7% (v / v); 1-6 g/L hydrolysate; And it provides a virus production method comprising the step of culturing Vero cells in a medium composition comprising 1-6 mg/L of insulin.
Brief description of the drawing
[11]
1 shows the results of Vero cell culture in the medium condition according to an example (Experiment No. 1) of the present application.
[12]
Figure 2 shows the results of Vero cell culture in the medium condition according to an example (Experiment No. 2) of the present application.
[13]
Figure 3 shows the results of Vero cell culture in the medium conditions according to an example (Experiment No. 3) of the present application.
[14]
Figure 4 shows the results of Vero cell culture in the medium conditions according to an example (Experiment No. 4) of the present application.
[15]
5 shows the results of Vero cell culture in the medium condition according to an example (Experiment No. 5) of the present application.
[16]
Figure 6 shows the results of Vero cell culture in the medium conditions according to an example (Experiment No. 6) of the present application.
[17]
7 shows the results of Vero cell culture in the medium condition according to an example (Experiment No. 7) of the present application.
[18]
Figure 8 shows the results of Vero cell culture in the medium conditions according to an example (Experiment No. 7) of the present application.
Best mode for carrying out the invention
[19]
According to one aspect of the present invention, newborn calf serum (NCS) 1-7% (v/v); 1-6 g/L hydrolysate; And insulin comprising 1-6 mg/L, a medium composition for culturing Vero cells is provided.
[20]
In one embodiment, the medium composition may not include Fetal Bovine Serum (FBS).
[21]
In one embodiment, the hydrolyzate may be an animal component-free, protein-free hydrolyzate.
[22]
In one embodiment, the Vero cells may be cultured attached to a microcarrier (microcarrier).
[23]
In one embodiment, the Vero cells may include culturing attached to a microcarrier (microcarrier).
[24]
In one embodiment, the medium may include MEM medium.
[25]
According to another aspect of the present invention, there is provided a method for culturing Vero cells, comprising the step of culturing Vero cells in the medium composition.
[26]
In one embodiment, the culture may be performed on a scale of 10mL to 1000L.
[27]
According to another aspect of the present invention, the method comprising: infecting Vero cells cultured in the medium composition with a virus; and culturing the Vero cells infected with the virus, a virus production method is provided.
[28]
In one embodiment, the culture of the virus-infected Vero cells may be performed on a scale of 10mL to 2000L.
[29]
Hereinafter, the present invention will be described in more detail.
[30]
The term "Vero cell" refers to any Vero cell line or cell line cultured or passaged therefrom, including, but not limited to, genetically modified Vero cells. Non-limiting examples of known Vero cells include VERO (ATCC Number CCL-81), VERO C1008 (ATCC Number CRL-1586), VERO 76 (ATCC Number CRL-1587) and Vero-SF-ACF (ATCC Number CCL-) 81.5) and the like.
[31]
The term "Vero cell culture" includes any procedure in which Vero cells are grown or maintained in a viable state. For example, culturing of Vero cells includes not only a process in which the cells grow or proliferate, but also a process in which the number of cells is not substantially increased but a viable state is maintained.
[32]
As a preferred example, the Vero cells herein may be used as a vehicle for a viral vaccine, or a treatment regimen based on a virus or viral vector. For example, Vero cells may be infected with a virus to produce a virus. The virus that can be produced from Vero cells can be an attenuated virus, a recombinant virus, or an oncolytic virus. In addition, viruses that can be produced in Vero cells include poliovirus, enterovirus, IBDV (infectious cyst virus), rotavirus, measles virus, smallpox virus, influenza virus, Japanese encephalitis virus, rabies virus, Newcastle disease virus, respiratory syncytial virus. Virus (RSV), Sendai virus, simian virus 40 (SV40), chikungunia virus and dengue virus may be exemplified, but is not limited thereto.
[33]
The term, "culture medium" is used for culturing Vero cells and refers to a composition containing the components necessary for growth. As the medium for Vero cells herein, any medium generally known in the art, such as MEM, DMEM, DMEM/F12, MDSS2, CCM5, Medium 199, or RPMI, may be used as the basal medium. Such culture medium may contain a number of components including amino acids, vitamins, organic and inorganic salts and carbohydrate sources that support the culture of Vero cells.
[34]
As used herein, “comprising (comprising certain ingredients)” can mean comprising, or constituting essentially of, ingredients other than those described. .
[35]
As a preferred example, the medium for Vero cell culture herein is, in the basal medium, newborn calf serum (NCS) 1-7% (v/v), hydrolysate 1-6 g/L, and insulin 1 Medium supplemented with ~6 mg/L.
[36]
As another example, in the present application, the medium for Vero cell culture is 1 to 7% (v/v), for example, 1 to 7% (v/v), 1 to newborn calf serum (NCS) in the basal medium. 6% (v/v), 1-5% (v/v), 1-4% (v/v), 1-3% (v/v), 1% (v/v), 2% (v) /v), 3% (v/v), 4% (v/v), 5% (v/v), 6% (v/v) or 7% (v/v); Hydrolysate 1-6 g/L, such as 1-6 g/L, 1-5 g/L, 1-4 g/L, 1-3 g/L, 1-2 g/L, 2-4 g/L, 2-3 g/L or 2 g/L; and insulin 1-6 mg/L, such as 1-6 mg/L, 1-5 mg/L, 1-4 mg/L, 1-3 mg/L, 2-6 mg/L, 2-5 Medium supplemented with mg/L, 2-4 mg/L, 2-3 mg/L or 3 mg/L.
[37]
Neonatal calf serum (NCS) is serum from newborn calves, typically from calves of about 20 days or less, such as about 14 days or less, or about 10 days or less, or about 3 to 10 days old, It is sold at 1/10 the price of fetal bovine serum (FBS). It is essential to have price competitiveness in order for the developed vaccine to enter the bidding market, and the use of neonatal bovine serum (NCS) instead of fetal bovine serum (FBS) has a clear cost reduction effect. In addition, neonatal bovine serum (NCS) is more stable than fetal bovine serum (FBS) in terms of supply and has less lot variation, so consistent experimental results can be obtained, and it is advantageous from the viewpoint of animal ethics.
[38]
In the medium composition of the present application, neonatal bovine serum (NCS) is contained in 1 to 7% (v/v), and can be used at a lower concentration than 10% FBS, which is a condition generally used for Vero cell culture. According to the example of the present application, cell growth is promoted in 1-7% (v/v) NCS conditions, and virus productivity within the above range shows similar results, for example, 1-7% (v/v), 1~ 6% (v/v), 1-5% (v/v), 1-4% (v/v) or 1-3% (v/v), more specifically 1-3% (v/v) ) or 2% (v/v), etc. may be presented as preferred concentrations. However, the concentration range may be appropriately increased or decreased within the level of ordinary skill of those skilled in the art when other culture conditions affecting virus productivity are changed.
[39]
As used herein, "hydrolysate" refers to a substance obtained by breaking a peptide bond of a protein, which is derived from milk (eg, casein, whey protein), or animal-derived. It may be of protein (meat, collagen), or of plant origin. For example, the vegetable hydrolyzate may be, but is not limited to, soybean hydrolyzate, wheat hydrolyzate, potato hydrolyzate, cottonseed hydrolyzate, rice hydrolyzate, pea hydrolyzate, or corn hydrolyzate, and the like.
[40]
As a preferred example, in order to reduce the lot variation of the hydrolyzate and efficiently perform the downstream process, there is no animal component and protein produced by different methods such as digestion and processing (animal component-free, protein-free) hydrolyzate can be used. Commercially available animal component and protein free hydrolysates, specifically Hypep TM 1510, Hypep TM 1511, Hypep TM 5603, Sheff-CHO PF ACF, Sheff-CHO Plus PF ACF, Sheff-VAX PF ACF SheffVax Plus PF ACF, SheffVax Plus PF ACF VP and the like may be exemplified, but the scope of the present application is not limited thereto.
[41]
In the medium composition of the present application, the hydrolyzate may be included in an amount of 1 to 10 g/L, but in a high concentration condition, there may be a negative effect on cell growth. Therefore, in a preferred embodiment, considering the concentration that has a positive effect on cell growth, the hydrolyzate is 1-6 g/L, for example, 1-6 g/L, 1-5 g/L, 1-4 g /L, 1-3 g/L, 1-2 g/L, 2-4 g/L, 2-3 g/L or 2 g/L may be given as preferred concentrations. However, the concentration range may be appropriately increased or decreased within the level of ordinary skill of those skilled in the art when other culture conditions affecting virus productivity are changed.
[42]
The medium composition herein also includes insulin. For example, the insulin may be natural insulin or recombinant insulin, and more specifically, human recombinant insulin, but is not limited thereto.
[43]
Insulin in the medium composition of the present application is 1-6 mg/L, 1-5 mg/L, 1-4 mg/L, 1-3 mg/L, 2-6 mg/L, 2-5 mg/L, 2 It may contain ~4 mg/L, 2-3 mg/L or 3 mg/L. However, the concentration range may be appropriately increased or decreased within the level of ordinary skill of those skilled in the art when other culture conditions affecting virus productivity are changed.
[44]
In a preferred embodiment, Vero cells may be cultured by being attached to a carrier such as a microcarrier. A microcarrier has a micro-level diameter and refers to any solid support matrix to which Vero cells can be attached. For example, cells can be cultured in suspension in a liquid medium by attaching the cells to the surface. Examples of the material of the microcarrier include plastics such as polystyrene, polyethylene, polypropylene, polyester, polycarbonate, polyamide, polyacetal and polyurethane, and copolymers thereof; glass; ceramic; metal; acrylamide; silica; silicone rubber; polylysine; cellulose; dextran; collagen (gelatin); and glycosaminoglycans may be used, but is not limited thereto. A microcarrier is at least about 100 μm, at least about 200 μm, at least about 300 μm, at least about 400 μm, at least about 500 μm, at least about 600 μm, at least about 700 μm, at least about 800 μm, at least about 900 μm, or about It may have a diameter of 1 mm or more, and may be in the form of beads (spherical), disk, strip, sheet, fiber, filament, rod, disk, cube, tube, etc., but is not limited thereto. For example, the microcarrier may be porous.
[45]
In a preferred embodiment, the culture of Vero cells is 10mL to 1000L or more, for example, 10mL to 2000L or more scale. "Bioreactor" refers to any vessel that can be used to culture Vero cells, including a fixed bed bioreactor having a fixed bed containing microcarriers. In the present application, Vero cells may be cultured on a microcarrier in a bioreactor, and as another example, may be suspension cultured in a bioreactor in a batch process or fed-batch batch process.
[46]
In the process of culturing Vero cells, the medium may be exchanged as needed during the culture period. The pH of the medium is preferably about 6 to 8, and may be about 6.5 to 7.5, or about 7. Cultivation can be carried out for about 5 to 9 days at about 35° C. to 40° C., and aeration or agitation may be added as necessary, but is not limited thereto. The dissolved oxygen concentration (DO) during cell culture may be about 60 to 90%, about 70 to 80%, etc., but is not limited thereto.
[47]
The Vero cells cultured according to the present invention can be used to produce viruses as a vehicle for a viral vaccine or a treatment regimen based on a virus or viral vector. Accordingly, the present invention also comprises the steps of infecting the Vero cells cultured in the medium composition as described above with a virus; And it provides a virus production method comprising the step of culturing the virus-infected Vero cells.
[48]
Vero cells infected with the virus can be cultured under conditions optimized for propagating the virus. For example, Vero cells may be cultured at a first temperature prior to infection with the virus and cultured at a second temperature after infection with the virus, wherein the second temperature is lower than the first temperature. For example, Vero cells can be cultured at about 37° C. before infection with the virus, ie, before infection, and cultured at about 29° C. to about 37° C. after infection with the virus, ie, after infection. As another example, Vero cells may be cultured at about 33° C. after infection with the virus. As another example, Vero cells may be cultured at about 30° C. after infection with the virus. The time of infection may be 1 day, 2 days, or 3 or 4 days after cell inoculation.
[49]
After completion of the virus culture, the microcarrier can be removed and the virus solution containing the virus can be recovered. The recovered virus solution may be filtered through a filtration membrane or the like to remove cell debris, or may be concentrated and/or purified before or after inactivation. As the concentration method, an ultrafiltration method, ultracentrifugation method, dialysis method, etc. can be exemplified, and as a purification method, for example, a method using physical properties such as size, density, sedimentation coefficient, etc. of the object to be purified, chemical or physicochemical A method using a reaction (adsorption/desorption, etc.) may be exemplified, but the present invention is not limited thereto.
Modes for carrying out the invention
[50]
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
[51]
[52]
Confirmation of optimal medium composition for Vero cells
[53]
The following experiment was designed to find the optimal medium composition for Vero cells. Vero cells were attached to microcarriers and cultured in shake flasks or bioreactors.
[54]
[Table 1]
Experiment No. control experimental group Experiment scale
One 5% FBS - 2% FBS + 2.5 g/L Hydrolysate
- 1% FBS + 2.5 g/L Hydrolysate 40mL
(shake flask)
2 5% FBS - 2% FBS- 1% FBS
- 0% FBS
- 2% FBS + 2.5 g/L Hydrolysate + 3 mg/L Insulin
- 1% FBS + 2.5 g/L Hydrolysate + 3 mg/L Insulin
- 0% FBS + 2.5 g/ L L Hydrolysate + 3mg/L Insulin 30mL
(shake flask)
3 5% FBS - 1-2% FBS + 0-6 g/L Hydrolysate + 0-6 mg/L insulin 30mL (shake flask)
4 5% FBS - 1%, 2% FBS
- 1%, 2%, 5% NCS 30mL
(shake flask)
5 5% FBS - 2% FBS + 2 g/L Hydrolysate + 3 mg/L insulin
- 2% NCS + 2 g/L Hydrolysate + 3 mg/L insulin 2L
(BRX)
6 2% FBS + 2 g/L Hydrolysate + 3 mg/L Insulin - 2% NCS + 2 g/L Hydrolysate + 3 mg/L insulin
- 7% NCS + 2 g/L Hydrolysate + 3 mg/L insulin 2L
(BRX)
7 2L - 40L
- 1000L 40L
1000L
[55]
[56]
experimental material
[57]
Hydrolysate (Kerry, Ireland)
[58]
Insulin: Recombinant human insulin (Gibco, USA)
[59]
FBS: Fetal Bovine Serum (Hyclone, USA)
[60]
NCS: newborn calf serum (Hyclone, USA)
[61]
BRX: bioreactor (Model: Biostat B, Sartorius, Germany)
[62]
[63]
Experiment No.1.
[64]
When the concentration of FBS was reduced and hydrolysate was added, the growth effect of Vero cells was confirmed. For Vero cells, cells derived from ATCC Number CCL-81 were used. As a control group, MEM medium containing amino acids, vitamins, minerals, glucose, etc. supplemented with 5% FBS was used, and as an experimental group, 2% FBS + 2.5 g/L Hydrolysate, or 1% FBS + 2.5 g/L Hydrolysate was used. Each supplemented MEM medium was used. As a result of culturing the prepared Vero cells in 40 mL of the prepared medium for 5 days (incubated at 37° C., 5% CO 2 , 80±5% Humidity), 2% FBS + 2.5 g/L Hydrolysate was supplemented with similar to the control group. While it was possible to confirm the cell growth profile, it was confirmed that the cell growth was reduced in the medium supplemented with 1% FBS + 2.5 g/L Hydrolysate compared to the control group (FIG. 1).
[65]
[66]
Experiment No.2.
[67]
When the concentration of FBS was reduced and hydrolysate and insulin were added, the growth effect of Vero cells was confirmed. For Vero cells, ATCC Number CCL-81-derived cells were used. As a control group, MEM medium containing amino acids, vitamins, minerals, and glucose supplemented with 5% FBS was used, and as an experimental group, 2% FBS, 1% FBS, 0% FBS, 2% FBS + 2.5 g/L Hydrolysate was used. MEM medium supplemented with + 3 mg/L insulin, 1% FBS + 2.5 g/L Hydrolysate + 3 mg/L insulin, or 0% FBS + 2.5 g/L Hydrolysate + 3 mg/L insulin, respectively, was used. The result of subculture of the prepared Vero cells in 30mL of the prepared medium three times for 14 days (37°C, 5% CO 2 , cultured at 80±5% Humidity), 2% FBS + 2.5 g/L Hydrolysate + 3 mg/L insulin A cell growth profile higher than that of the control group was confirmed in this supplemented medium, whereas the cell growth was decreased in the medium supplemented with 1% FBS + 2.5 g/L Hydrolysate + 3 mg/L insulin compared to the control group, but higher than the 2% FBS group. It showed a growth profile, and it was confirmed that hydrolysate and insulin had a distinct growth-promoting effect. In the medium condition without FBS, cell growth did not occur even after adding hydrolysate and insulin, and the culture was terminated at 1st passage (FIG. 2).
[68]
[69]
Experiment No.3.
[70]
The optimal concentrations of hydrolysate and insulin were confirmed. For Vero cells, ATCC Number CCL-81-derived cells were used. As shown in the following table, MEM medium supplemented with 5% FBS was used as a control group, and MEM medium supplemented with 0-6 g/L Hydrolysate + 0-6 mg/L insulin was used as an experimental group, respectively.
[71]
[Table 2]
sample ID FBS (%) Hydrolysate (g/L) Insulin (mg/L)
M5 5 0 0
S1 One 0 0
S2 One 0 6
S3 One 6 0
S4 One 6 6
S5 1.5 3 3
S6 1.5 3 3
S7 1.5 3 3
S8 2 0 0
S9 2 0 6
S10 2 6 0
S11 2 6 6
S12 2 2 0
S13 2 2 3
S14 2 2 6
S15 2 4 0
S16 2 4 3
S17 2 4 6
[72]
As a result of culturing the prepared Vero cells in 30 mL of the prepared medium for 6 days (incubated at 37° C., 5% CO 2 , 80±5% Humidity), the final cell number was the highest in the case of 6 g/L Hydrolysate condition (S10). However, in the condition of 2 g/L Hydrolysate, the condition (S13) supplemented with 3 mg/L insulin was selected as the condition for culturing in the bioreactor. It was determined that the cell growth rate at the initial stage of the culture was fast, which was a more favorable condition for cell growth when fed-batch culture was performed in the bioreactor (FIG. 3).
[73]
[74]
Experiment No.4.
[75]
It was checked by concentration whether FBS could be replaced with NCS. For Vero cells, ATCC Number CCL-81-derived cells were used. MEM medium supplemented with 5% FBS was used as a control group, and MEM medium supplemented with 1% or 2% FBS, or 1%, 2% or 5% NCS, respectively, was used as an experimental group. As a result of culturing the prepared Vero cells in 30 mL of the prepared medium for 5 days (incubated at 37 °C, 5% CO 2 , 80±5% Humidity), FBS and NCS each showed a similar cell growth profile at the same concentration. was confirmed (Fig. 4), which suggests that FBS can be replaced with NCS.
[76]
[77]
Experiment No.5.
[78]
Conduct the experiment by expanding the conditions selected in Experiment No. 3 (2% FBS + 2 g/L Hydrolysate + 3 mg/L insulin condition) to a 2L incubator scale, and check whether FBS can be replaced with NCS 2% NCS + 2 g /L Hydrolysate + 3 mg/L insulin condition was added and confirmed. For Vero cells, ATCC Number CCL-81-derived cells were used. As a control group, seed culture and main culture were performed using MEM medium supplemented with 5% FBS (#R02), and as an experimental group, 2% NCS + 2 g/L Hydrolysate + 3 mg/L insulin (#R03), Or 2% FBS + 2 g / L Hydrolysate + 3 mg / L insulin was performed in MEM medium supplemented with seed culture and main culture (#R04) (temperature 37 ℃, pH 7.2 ±0.05, DO 50%, culture method: Fed-batch, culture days: seed culture and main culture, culture progressed for 18 days).
[79]
As a result, the condition using MEM medium supplemented with 5% FBS and the condition using the medium supplemented with 2% FBS + 2 g/L Hydrolysate + 3 mg/L insulin and 2% NCS + 2 g/L Hydrolysate + 3 mg/L It was confirmed that a similar cell growth profile was exhibited in the insulin condition, suggesting that the composition of 2% FBS or NCS supplemented with Hydrolysate and insulin can be substituted for the composition of 5% FBS, and that FBS can be substituted with NCS ( 5).
[80]
[81]
Experiment No.6.
[82]
In Experiment No.6, hydrolysate and insulin were tried to determine the effect on cell growth and virus productivity according to the concentration of NCS in the composition. For Vero cells, ATCC Number CCL-81-derived cells were used. As a control group, MEM medium supplemented with 2% FBS + 2 g/L Hydrolysate + 3 mg/L insulin was used (#R02), and as an experimental group, MEM supplemented with 2% NCS + 2 g/L Hydrolysate + 3 mg/L insulin was used. Medium (#R04) or MEM medium (#R03) supplemented with 7% NCS + 2 g/L Hydrolysate + 3 mg/L insulin was used. As a result of culturing the prepared Vero cells in a 2L bioreactor for 19 days using the prepared medium (temperature 37° C., pH 7.2 ±0.05, DO 50%, culture method: Fed-batch), culture days: species Culture and main culture 19 days), 2% NCS + 2 g / L Hydrolysate + 3 mg / L In MEM medium supplemented with insulin, a cell growth profile similar to that of the control was shown, confirming that FBS can be replaced with NCS, It was confirmed that cell growth was increased in the 7% NCS concentration condition than in the 2% NCS condition ( FIG. 6 ). Cells cultured under each condition were infected with virus and recovered after 4 days of incubation (temperature 32.5° C., pH 7.4±0.1, DO 25%, number of incubation days: 4 days incubation) to quantify the virus concentration and compare productivity. . The final cell number was the highest in the condition of 7% NCS + 2 g/L Hydrolysate + 3 mg/L insulin (#R03), but Virus productivity was confirmed to be at a similar level compared to the condition of 2% NCS + 2 g / L Hydrolysate + 3 mg / L insulin (#R04). Therefore, it was confirmed that FBS can be used instead of NCS based on the productivity of Vero cell culture growth and virus culture, and the culture scale expansion experiment was carried out by selecting the composition of 2% NCS + 2 g/L Hydrolysate + 3 mg/L insulin. (Fig. 6).
[83]
[84]
Experiment No.7.
[85]
Through the previous experiments, the culture scale was changed to 2L, 40L, and 1000L with a medium (2% NCS + 2 g/L Hydrolysate + 3 mg/L insulin) selected as a medium composition containing NCS, Hydrolysate, and insulin that can replace FBS. It was checked whether cell growth was maintained while expanding (temperature 37℃, pH 7.2 ±0.05, DO 50%, culture method: Fed-batch, number of culture days: seed culture and main culture 18 days) . Vero cells cultured under each condition were infected with the virus, cultured for 3 to 4 days, and recovered to compare virus productivity.
[86]
The results of comparing cell growth in the 2L and 40L culture scales are shown in the table below and FIG. 7, and it was possible to confirm the same or higher cell growth results even in the 40L scale.
[87]
[Table 3]
culture scale 2L (n=6) 40L (n=3)
Final cell density
(X10 5 cells/mL) 15.03 17.95
SGR, D1~4 (day -1 ) 0.68 0.71
SGR, D1~6 (day -1 ) 0.53 0.56
[88]
* SGR: specific growth rate
[89]
In addition, the results of comparing cell growth in a medium condition containing FBS and a medium condition containing NCS, Hydrolysate, and insulin at 1000L scale are shown in FIG. . The virus productivity before and after the change to NCS, Hydrolysate, and Insulin composition medium was compared by culture scale and shown in FIG. 8 . After changing the medium, the virus productivity was further increased, and even when it was expanded to a 2000L culture scale, it was possible to confirm the virus productivity of equal or higher.
[90]
From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.
Claims
[Claim 1]
A medium for culturing Vero cells, containing 1~7% (v/v) of newborn calf serum (NCS), 1~6 g/L of hydrolysate, and 1~6 mg/L of insulin composition.
[Claim 2]
The medium composition according to claim 1, characterized in that it does not contain Fetal Bovine Serum (FBS).
[Claim 3]
The medium composition according to claim 1, wherein the hydrolyzate is an animal component-free, protein-free hydrolyzate free from animal urea and protein.
[Claim 4]
The medium composition of claim 1, wherein the Vero cells are adhered to and cultured on a microcarrier.
[Claim 5]
The medium composition of claim 1 , wherein the medium comprises MEM medium.
[Claim 6]
A method of culturing Vero cells, comprising the step of culturing Vero cells in the medium composition of any one of claims 1 to 5.
[Claim 7]
The method according to claim 6, wherein the culturing is performed on a scale of 10 mL to 1000 L.
[Claim 8]
Infecting the Vero cells cultured in the medium composition of any one of claims 1 to 5 with a virus; and culturing the virus-infected Vero cells.
[Claim 9]
The method according to claim 8, wherein the culturing of virus-infected Vero cells is performed on a scale of 10 mL to 2000 L.