A culture medium and culture method suitable for large-scale suspension culture of Vero cells

Abstract

This invention discloses a culture medium and method suitable for large-scale full-suspension culture of Vero cells. The culture medium is a serum-free medium specifically designed and developed based on the growth and metabolic characteristics of Vero suspension cells and their different proliferation abilities when cultured in a reactor versus a shaker, combined with the differences in culture scale. It includes the following components: amino acids, proteins, vitamins, inorganic salts, serum replacement factors, and auxiliary components. Furthermore, this invention determines the control parameters of the full-suspension culture reactor based on cell growth characteristics, significantly increasing the VERO cell culture density. The cell doubling rate reaches 10-fold after 48 hours of culture, the cell production cycle is halved, and production efficiency is improved. The operation is simple, reducing the contamination rate in large-scale culture and solving the problem of long perfusion culture cycles. This is of great significance for the large-scale production of viral vaccines using VERO cells as a substrate.

Description

TECHNICAL FIELD

[0001] The present application relates to a culture medium suitable for full-suspension large-scale culture of Vero cells, and a method for full-suspension large-scale culture of Vero cells using the culture medium, and belongs to the technical field of cell culture. BACKGROUND

[0002] Vero cells (African green monkey kidney cells) were first isolated from the kidney of an African green monkey by Yasumura and Kawakita in 1962, and are the first aneuploid anchorage-dependent cells used for the production of human biological products. They are a cell line approved by the World Health Organization and the Chinese biological product regulations for vaccine production. Compared with primary cells, diploid cells and other some passage cell substrates used for vaccine production, Vero cells are convenient to obtain, have high biological safety, are sensitive to infection by a variety of viruses, have high virus proliferation titers, are ideal substrates for many viruses, and can be widely used for the production of human and animal vaccines. At present, the use of microcarrier culture technology has preliminarily established the culture of Vero cells by bioreactor-microcarrier culture technology. However, compared with full-suspension culture, the use of microcarriers not only increases the cost of cell culture, but also brings inconvenience to the scale-up of culture.

[0003] At present, the large-scale culture of Vero cells is mainly designed in a perfusion culture mode and a fed-batch culture mode. Although the final cell density is high, the production period generally lasts for 7-21 days, and the culture process needs to be supplemented and the culture parameters need to be adjusted, which is undoubtedly complicated and increases the risk of process contamination. In addition, commercial serum-free media for Vero cell culture have been successfully developed, but each cell strain has its unique nutritional requirements, so it is difficult to have a commercial serum-free medium that completely matches the nutritional requirements of a certain cell strain. Moreover, there is a great difference between the cell metabolic level and the shaker level during scale-up culture, so not only the reactor control parameters need to be adjusted, but also the medium components need to be adjusted to meet the needs of cell growth. However, the components of commercial media are secret, and it is impossible to solve the problems encountered in a targeted manner. Although Rourou S et al. obtained Vero cells that can grow in suspension in a shake flask by using a self-prepared medium, the cell density was about 2.0 x 10 6 cells / mL, and the cell productivity was low. Therefore, there is an urgent need to develop a full-suspension culture medium and a culture method for large-scale culture of Vero cells.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] One of the purposes of the present application is to provide a personalized culture medium suitable for large-scale bioreactor and full-suspension batch culture of Vero cells, which has relatively clear components and remarkable culture effect.

[0006] The second object of the present application is to provide a high-efficiency culture method suitable for full-suspension large-scale culture of Vero cells based on the above-mentioned culture medium.

[0007] In order to achieve the above-mentioned objects, the present application adopts the following technical means:

[0008] The culture medium suitable for full-suspension large-scale culture of Vero cells of the present application is a serum-free culture medium specially designed and developed by the laboratory according to the growth and metabolism characteristics of Vero suspension cells, according to the different proliferation abilities of Vero cells in a reactor and on a shaking table, and combining the differences in different scale cultures, and includes the following components: amino acids, proteins, vitamins, inorganic salts, serum replacement factors, auxiliary ingredients, etc. Specifically, the culture medium includes the following components:

[0009] ① Amino acid part

[0010] The amino acid part includes the following components:

[0011]

[0012]

[0013] ② Vitamin and balanced salt part

[0014] The vitamin and balanced salt part includes the following components:

[0015] ③ Other additives part

[0016] The other additives part includes the following components:

[0017]

[0018]

[0019] Preferably, the culture medium includes the following components:

[0020] ① Amino acid part

[0021] The amino acid part includes the following components:

[0022]

[0023] ② Vitamin and balanced salt part

[0024] The vitamin and balanced salt part includes the following components:

[0025]

[0026]

[0027] ③ Other additives

[0028] The other additives include the following components:

[0029]

[0030] Furthermore, the present invention also proposes the application of the aforementioned culture medium in large-scale suspension culture of Vero cells.

[0031] Furthermore, the present invention also proposes a method for large-scale culture of Vero cells in suspension, comprising the step of inoculating Vero cells adapted for suspension culture into the sterilized culture medium.

[0032] Preferably, the Vero cells adapted for suspension culture are named African green monkey kidney cells VERO-XW and are deposited at the China Center for Type Culture Collection with accession number CCTCC NO: C2022110.

[0033] Preferably, in the method, the cell seeding density is 0.60 × 10⁶. 6 -0.80×10 7 The cells / mL culture was controlled under the following conditions: pH 6.8–7.4, dissolved oxygen 40%–80%, stirring speed 70–110 r / min, and temperature 36.5℃–37.2℃.

[0034] Preferably, the method includes the following steps:

[0035] (1) Cell resuscitation and shaker culture

[0036] One cell line was resuscitated from a Vero cell working cell bank in suspension culture and thawed in pure water at 37°C for 1 minute. An appropriate amount of fresh, sterile culture medium preheated to 37°C as described above was placed in a centrifuge tube. The cells were added to the centrifuge tube, mixed well, and centrifuged at 600 rpm for 5 minutes to remove the cryoprotectant. The supernatant was discarded, and an appropriate amount of fresh, sterile culture medium as described in claim 1 or 2 was added. The cells were then inoculated into culture flasks at a density of 0.7 × 10⁻⁶. 6 Cells / mL, shaken on a CO2 shaker at 37℃ and 100 rpm for 48 h, then sampled and counted; once the cell culture volume reached a certain quantity, the cells were collected to obtain Vero suspension cell seed.

[0037] (2) Bioreactor cultivation, carried out in four stages:

[0038] 10L reactor culture: Take well-grown Vero suspension cells and culture them at a density of 0.6 × 10⁶ cells / year. 6 Cells / mL were inoculated into fresh, sterile culture medium as described above and cultured in a 10L reactor in a fully suspended batch for 48 hours. The reactor control parameters were: pH 7.0–7.2, dissolved oxygen 40%–60%, stirring speed 100–120 r / min, and temperature 36.5℃–37.2℃.

[0039] 100L reactor culture: Take well-grown Vero suspension cells from a 10L reactor and culture them at a density of 0.6 × 10⁶ cells / year. 6 Cells / mL were inoculated into fresh, sterile culture medium as described above and cultured in a 100L reactor in a fully suspended batch culture for 48 hours. The reactor control parameters were: pH 6.9–7.1, dissolved oxygen 40%–60%, stirring speed 100–120 r / min, and temperature 36.5℃–37.2℃.

[0040] Cultured in a 500L reactor: Seed cells of well-grown Vero suspension cells were taken from a 100L reactor and cultured at a density of 0.6 × 10⁶ cells / year. 6 Cells / mL were inoculated into fresh, sterile culture medium as described above and cultured in a 500L reactor in a fully suspended batch culture for 48 hours. The reactor control parameters were: pH 6.9–7.1, dissolved oxygen 40%–60%, stirring speed 80–100 r / min, and temperature 36.5℃–37.2℃.

[0041] Cultured in a 3000L reactor: Seed cells of well-grown Vero suspension cells from a 500L reactor were cultured at a density of 0.8 × 10⁶ cells / year. 6 Cells / mL were inoculated into fresh, sterile culture medium as described above and cultured in a 3000L reactor in a fully suspended batch for 48 hours. The reactor control parameters were: pH 6.8–7.0, dissolved oxygen 60%–80%, stirring speed 80–100 r / min, and temperature 36.5℃–37.2℃.

[0042] Preferably, the control parameters for the 10L reactor culture are: pH 7.1, dissolved oxygen 50%, stirring speed 110 r / min and temperature 36.8℃;

[0043] The control parameters for the 100L reactor culture were set as follows: pH 7.0, dissolved oxygen 50%, stirring speed 110 r / min, and temperature 36.8℃.

[0044] The control parameters for the 500L reactor culture were set as follows: pH 7.0, dissolved oxygen 50%, stirring speed 90 r / min, and temperature 36.9℃.

[0045] The 3000L reactor was set with the following control parameters: pH 6.9, dissolved oxygen 70%, stirring speed 90 r / min, and temperature 36.9℃. Compared with the prior art, the advantages of this invention are:

[0046] This invention utilizes a mechanically stirred bioreactor and a fully suspended batch culture process. By developing a personalized culture medium for specific cell lines and methods, and further defining upper and lower limits for concentration during the culture process, reactor control parameters were established based on the high efficiency of the fully suspended batch culture. This culture process significantly increases VERO cell culture density, achieving a 10-fold cell doubling rate after 48 hours of culture, and halving the cell production cycle. While increasing cell yield, production efficiency is also improved. Furthermore, the culture process is simple to operate, highly controllable, with clearly defined process control indicators, stable culture process, and low production costs. It achieves a 3000L culture scale for the first time in China, reducing contamination rates in large-scale culture and solving the problem of long perfusion culture cycles. This is of great significance for the large-scale production of viral vaccines using VERO cells as a substrate. Attached Figure Description

[0047] Figure 1 A graph showing the analysis of the content of various amino acids in the culture medium;

[0048] Figure 2 The cell yield after 48 hours at different glucose concentrations;

[0049] Figure 3 The effect of different lactate concentrations on specific cell growth rate;

[0050] Figure 4 The effect of different ammonia concentrations on the specific growth rate of VERO cells;

[0051] Figure 5 This shows the utilization of amino acids in Vero cells after formula adjustment.

[0052] Figure 6 Response surface methodology for the interaction of pH, dissolved oxygen, stirring speed, and temperature on cell growth density;

[0053] Figure 7 A graph showing the trend of cell distribution;

[0054] Figure 8 This is a diagram showing cell stability passaging at different scales.

[0055] Information on strain preservation:

[0056] Bacterial strain name: African green monkey kidney cells VERO-XW

[0057] Classification and nomenclature: African green monkey kidney cells VERO-XW

[0058] Accession number: CCTCC NO: C2022110

[0059] Depository Institution: China Center for Type Culture Collection

[0060] Location of collection: Wuhan University, Wuhan, China

[0061] Deposit date: May 18, 2022 Detailed Implementation

[0062] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0063] Example 1: Study of Vero Cell Nutritional Metabolic Flux and Determination of Culture Medium Formulation

[0064] 1. Materials

[0065] 1.1 Cell line: Vero suspension cells were introduced by Nongweite Biotechnology Co., Ltd. in February 2015 from the American Type Culture Collection (ATCC), with the accession number CCL-81 adherent Vero cells. The Vero suspension cells obtained through autonomous suspension domestication were named African green monkey kidney cells VERO-XW, with the accession number CCTCC NO: C2022110, and were provided by the seed bank.

[0066] 1.2 Culture medium: MEM medium, purchased from MERCK.

[0067] 1.3 Reagents and Equipment

[0068] 1.3.1 Main reagents: Amino acid analysis column (ACCQ-TAG TRAC18 1.7μm, 2.1*100mm); 20 amino acid standards; amino acid derivatization kit; acetonitrile (chromatographic grade); NOVAL biochemical reagent kit; NOVAL enzyme membrane; CASYton liquid; PBS; 70% ethanol; DAPI; Triton X-100; deionized water, etc.

[0069] 1.3.2 Main equipment: CO2 shaker (Thermo Fisher Scientific); cell analyzer (INNOVATIS, Germany); ultra-high performance liquid chromatograph (Waters, USA); biochemical analyzer (NOVA PLUS 100); microscope (Olympus, Japan); clean bench (Suzhou Jingantai Co., Ltd.); 10L, 100L, 500L, and 3000L bioreactors.

[0070] 2 methods

[0071] 2.1 Main determination methods

[0072] 2.1.1 Cell Counting and Analysis

[0073] Follow the instructions in the cell analyzer's user manual. Based on the results measured by the cell analyzer, calculate the cell growth rate and determine the cell's proliferative capacity.

[0074] 2.1.2 Amino acid detection and analysis: U-HPLC was used for detection.

[0075] 2.2 Cell resuscitation and culture

[0076] 2.2.1 One VERO-XW cell line was resuscitated from the working cell bank of suspended Vero cells and thawed in pure water at 37°C for 1 minute;

[0077] 2.2.2 Centrifuge the cells obtained in 2.2.1 at 600 r / min for 5 min, remove the cryoprotectant, discard the supernatant, and resuspend them in an appropriate amount of fresh culture medium;

[0078] 2.2.3 The cell suspension obtained in 2.2.2 was seeded into culture flasks at a seeding density of 0.7 × 10⁻⁶ cells / year. 6 The cells were counted at approximately 100 cells / mL. The cells were then incubated on a CO2 shaker (37°C, 100 rpm) for approximately 48 hours, and samples were taken for counting.

[0079] 2.2.4 Once the cell culture volume reaches a certain quantity, collect the cells and scale up the culture step by step.

[0080] 2.3 Study on the requirements of Vero cells for amino acids, glucose and metabolic byproducts

[0081] 2.3.1 The cells obtained in 2.2.4 were batch cultured in a 10L bioreactor. Cell density and cell viability were used as evaluation indicators. Cell samples were taken at 0h, 24h, 48h, 72h and 96h of culture. The cells were centrifuged at 1000r / min for 5min at below 4℃ and the cell supernatant was collected.

[0082] 2.3.2 The cell supernatant obtained in 2.3.1 was analyzed for glucose and glutamine, the main nutrients. The concentrations of glucose, glutamine, ammonia and lactate were determined using a NOVA PLUS 100 biochemical analyzer.

[0083] 2.3.3 The cell supernatant obtained in 2.3.1 was analyzed for the main nutrient amino acids. The amino acid concentration was determined by pre-column derivatization in ultra-high performance liquid chromatography. Finally, the data were processed to calculate the amino acid content.

[0084] 2.4 Optimization and Application of Culture Medium Formulation

[0085] Based on the cell growth and metabolism parameters detected in step 2.3 above, and combined with the cell metabolic flux monitoring results, the content of each nutrient that mainly affects cell growth and metabolism was determined, the tolerance concentration limit of byproducts during cell metabolism was examined, and the main nutrients were supplemented or reduced, thereby adjusting the basal culture medium MEM.

[0086] 3 Results

[0087] 3.1 Effects of amino acids, glucose, and metabolic byproducts on cell proliferation

[0088] The contents of 20 amino acids in the culture medium at 0 h and 48 h after cell inoculation were determined by HPLC, as shown in Table 1. Combined with cell growth and metabolic levels, the amount of each amino acid required for the growth of one cell was determined. Figure 1 As shown, this allows for the monitoring of amino acid metabolism during cell growth. The changes in the amount of amino acids required for cell proliferation, as well as the concentrations of glucose and metabolic byproducts, are shown in the figures below. Figures 2-5 As shown.

[0089] Table 1. Consumption of various amino acids (mM) during Vero cell batch culture.

[0090]

[0091] from Figure 3 It can be seen that when the lactate concentration is greater than 30 mM, the growth of Vero cells is strongly inhibited, with a cell specific growth rate of almost 0 and no logarithmic growth phase observed. However, when the lactate concentration is below 25 mM, the maximum viable cell density is comparable to that of the control, and there is no significant inhibitory effect on the growth of Vero cells. The cell specific growth rate is significantly higher than that of other experimental groups, indicating that the tolerance concentration of the metabolic byproduct lactate for Vero cells is 25 mmol / L.

[0092] from Figure 4It is evident that when the ammonia concentration reaches 5.5 mM, it inhibits cell growth at the beginning of batch culture; when the ammonia concentration reaches 12 mM, cell growth is completely inhibited, the cell growth rate is negative, and the viable cell density remains around the initial cell density, indicating that Vero cells tolerate ammonia, a metabolic byproduct, at a concentration of 5.5 mmol / L.

[0093] 3.2 Determination of glucose, glutamine, ammonia and lactic acid concentrations

[0094] The concentrations of glutamine, glucose, ammonia, and lactate in the cell supernatant after 0h, 24h, 48h, 72h, and 96h of culture were measured using a NOVAPLUS 100 biochemical analyzer. The results are shown in Table 2.

[0095] Table 2. Changes in the concentration of major metabolites during batch culture.

[0096]

[0097] 3.3 Determination and Validation of Culture Medium Formulation

[0098] Based on the above cell metabolic flux monitoring results, the contents of various nutrients that mainly affect cell growth and metabolism, as well as the tolerance concentration of byproducts during cell metabolism, were determined to design a culture medium formulation, named V-P1611. The culture medium includes an amino acid component, a balanced salt component, a vitamin component, other additives, serum, and distilled water, with a pH of 7.0–7.2. The final concentrations of other raw materials in the culture medium are shown in Table 3 below.

[0099] Table 3 Final concentrations of each raw material in the culture medium

[0100]

[0101]

[0102] Example 2: Determination and Optimization of Key Parameters of Bioreactor

[0103] 1. Materials

[0104] 1.1 Cell lines

[0105] Vero suspension cells (African green monkey kidney cells VERO-XW, CCTCC NO: C2022110) were used after passing the scale-up culture test.

[0106] 1.2 Culture medium

[0107] The serum-free culture medium for Vero cells prepared in Example 1 was named V-P1611.

[0108] 2 methods

[0109] 2.1 Cell resuscitation and culture

[0110] 2.1.1 One branch of African green monkey kidney cells (VERO-XW) was resuscitated from the Vero cell working cell bank in suspension culture and rehydrated in pure water at 37°C for 1 min;

[0111] 2.1.2 Centrifuge the cells obtained in 2.1.1 at 600 r / min for 5 min, remove the cryoprotectant, discard the supernatant, and resuspend them in an appropriate amount of fresh V-P1611 culture medium.

[0112] 2.1.3 The cell suspension obtained in 2.1.2 was inoculated into a culture flask and cultured at 37°C with shaking at a speed of 90 r / min. After the cell culture volume reached the specified number, the cells were collected for subsequent use as cell seed.

[0113] 2.2 Optimization of Key Parameters of Bioreactor

[0114] 2.2.1 Response Surface Design

[0115] To find the optimal experimental conditions and interactions between factors, a Box-Behnken response surface methodology was used. Cell density was used as the response value, and temperature, pH, dissolved oxygen, stirring speed and temperature were used as influencing factors. A response surface methodology with 4 factors and 3 levels was designed for a total of 29 experiments. The optimal culture parameters were optimized and screened. The factors and levels are shown in Table 4.

[0116] Table 4 Factors and Levels in Box-Behnken Design

[0117]

[0118] 2.2.2 Passaging of Vero cells in a reactor

[0119] The cells collected in 2.1 were seeded into a 3000L bioreactor and cultured at a seeding density of 0.60 × 10⁶ cells / year. 6 The cells / mL culture parameters were set according to the parameters in Table 4 of 2.2.1. After culturing for 48 hours, the cells were passaged, cell samples were taken, cell density and viability were measured, cell passage density was recorded, and the experimental results were analyzed to determine the optimal culture parameters.

[0120] 2.2.3 Validation of optimization results

[0121] Vero cells were cultured using the optimal culture parameters determined in section 2.2.2. The cell density under these conditions was compared with the extreme values ​​determined by multivariate function analysis and the variable values ​​corresponding to the extreme values ​​to verify the reliability of the model and determine the final optimization result.

[0122] 3 Results

[0123] 3.1 Results of Response Surface Design Experiments

[0124] The response surface methodology diagram showing the effect of the interaction between pH, dissolved oxygen, stirring speed, and temperature on cell growth density is shown below. Figure 6 As shown in Table 5, the central composite experimental design and results are presented.

[0125] Table 5. Centralized Composite Experimental Design and Results

[0126]

[0127]

[0128] 3.2 Analysis of Response Surface Methodology Results and Determination of Optimal Culture Parameters

[0129] The response surface plot is saddle-shaped, indicating the existence of extreme values. The coefficient of determination (R²) is 0.9528, meaning that 95.28% of the variation in Y values ​​can be explained by this model, indicating a good model fit. Based on the changes in the response surface, at an inoculation density of 0.60 × 10⁻⁶... 6 Under conditions of cells / mL, pH 6.8–7.4, dissolved oxygen 40%–80%, stirring speed 70–110 r / min, and temperature 36.5℃–37.2℃, the cell density ranged from 3.22 to 6.42 × 10⁻⁶ cells / mL. 6 The optimal values ​​for temperature, pH, dissolved oxygen, and stirring speed were 36.82℃, 7.07%, 61.81%, and 91.9 r / min, respectively. Under these conditions, the cell density reached 6.35 × 10⁻⁶ cells / mL. 6 cells / mL.

[0130] 3.3 Validation of Optimization Results

[0131] The optimal reaction parameters determined in section 3.2 are: inoculum density of 0.60 × 10⁻⁶. 6 Vero cells were cultured at 36.82℃, pH 7.07, dissolved oxygen 61.81%, and stirring speed 91.9 r / min, with a maximum cell concentration of 6.35 × 10⁻⁶ cells / mL. 6 The cells / mL figure was 99.3% of the predicted value, indicating a high degree of agreement.

[0132] Example 3 is applicable to the Vero cell suspension culture process at different scales.

[0133] 1. Materials

[0134] 1.1 Cell lines

[0135] Vero suspension cells (African green monkey kidney cells VERO-XW, CCTCC NO: C2022110) were used after passing the scale-up culture test.

[0136] 1.2 Culture medium

[0137] The serum-free culture medium for Vero cells prepared in Example 1 was named V-P1611.

[0138] 2 methods

[0139] 2.1 Cell Counting and Analysis

[0140] Follow the instructions in the cell analyzer's user manual. Based on the results measured by the cell analyzer, calculate the cell growth rate and determine the cell's proliferative capacity.

[0141] 2.2 Cell resuscitation and culture

[0142] 2.2.1 One branch of African green monkey kidney cells (VERO-XW) was resuscitated from the Vero cell working cell bank in suspension culture and rehydrated in pure water at 37°C for 1 min;

[0143] 2.2.2 Centrifuge the cells obtained in 2.2.1 at 600 r / min for 5 min, remove the cryoprotectant, discard the supernatant, and resuspend in an appropriate amount of fresh culture medium V-P1611;

[0144] 2.2.3 The cell suspension obtained in 2.2.1 was inoculated into a culture flask and cultured at 37°C with shaking at a speed of 90 r / min. After the cell culture volume reached the specified number, the cells were collected for subsequent use as cell seed.

[0145] 2.3 Passage culture of Vero cells in a 500L bioreactor

[0146] The cell seeds collected in section 2.2 were cultured on a 500L bioreactor at an inoculation density of 0.65 × 10⁶ cells / year. 6 The reactor parameters were as follows: temperature 36.79℃, DO value 59.31%, pH value 7.04, and rotation speed 79.81 r / min, all automatically controlled. The culture period was 48 h, followed by subculturing for 40 days (20 generations).

[0147] 2.4 Passage culture of Vero cells in a 3000L bioreactor

[0148] The cell seeds collected in section 2.2 were cultured on a 3000L bioreactor at an inoculation density of 0.7 × 10⁻⁶. 6The reactor parameters were as follows: temperature 36.79℃, DO value 59.31%, pH value 7.04, and rotation speed 79.81 r / min, all automatically controlled. The culture period was 48 h, followed by subculturing for 40 days (20 generations).

[0149] 3. Results

[0150] The key control parameters for cultivation determined above were used to verify the application of cultivation at scales of 500L and 3000L.

[0151] 3.1 Analysis of Cell Proliferation Status During Large-Scale VERO Cell Culture

[0152] Table 6 Vero cell growth status

[0153]

[0154]

[0155] As shown in Table 6 and Figure 7 As shown, Vero cells exhibited similar growth characteristics in 500L and 3000L reactors. Under suspension culture conditions at each scale, cell growth rates were essentially similar, with the fastest growth occurring between 24 and 48 hours, followed by a significant decrease in growth rate between 48 and 72 hours. The lack of significant difference in the suspension culture effect of Vero cells between the 500L and 3000L bioreactors demonstrates that the culture process determined in this study can be linearly scaled up.

[0156] The application of V-P1611 culture medium was validated at different scales of cell suspension culture (500L and 3000L). The stability and passage of cells at different scales were as follows: Figure 8 As shown, Vero cells were continuously cultured for 40 days (20 passages) using the V-P1611 culture medium determined in this invention, and the cell number reached 5.0 × 10⁻⁶. 6 With a cell / mL ratio of over 93% and a viability of over 93%, this efficient culture process can be applied to large-scale Vero cell culture.

[0157] Example 4: Establishment of a method suitable for large-scale culture of Vero cells in full suspension

[0158] 1. Materials

[0159] 1.1 Cell lines

[0160] Vero suspension cells (African green monkey kidney cells VERO-XW, CCTCC NO: C2022110) were used after passing the scale-up culture test.

[0161] 1.2 Culture medium

[0162] The serum-free culture medium for Vero cells prepared in Example 1 was named V-P1611.

[0163] 2 methods

[0164] (1) Cell resuscitation and shaker culture

[0165] One VERO-XW cell line was resuscitated from the Vero cell working cell bank in suspension culture and thawed in pure water at 37°C for 1 min. An appropriate amount of fresh culture medium preheated to 37°C was placed in a centrifuge tube, and the cells were added. After mixing, the cells were centrifuged at 600 rpm for 5 min to remove the cryoprotectant. The supernatant was discarded, and an appropriate amount of fresh V-P1611 culture medium was added. The cells were then seeded into culture flasks at a density of 0.7 × 10⁻⁶ cells / mL. 6 Cells / mL, cultured on a CO2 shaker (37℃, approximately 100 rpm) for 48 h, then sampled and counted. Once the cell culture volume reached a certain quantity, the cells were collected.

[0166] (2) Bioreactor cultivation, carried out in four stages:

[0167] 10L reactor culture: Take well-grown Vero suspension cells and culture them at a density of 0.6 × 10⁶ cells / year. 6 Cells / mL were seeded in serum-free V-P1611 medium and cultured in a batch suspension for 48 h. Reactor control parameters were: pH 7.0–7.2, dissolved oxygen 40%–60%, stirring speed 100–120 rpm, and temperature 36.5–37.2 °C. Preferably, the control parameters were: pH 7.1, dissolved oxygen 50%, stirring speed 110 rpm, and temperature 36.8 °C. During the culture, key parameters for evaluating cell culture performance, such as cell viability, density, and specific growth rate, were analyzed.

[0168] 100L reactor culture: Take well-grown Vero suspension cells from a 10L reactor and culture them at a density of 0.6 × 10⁶ cells / year. 6 Cells / mL were seeded in serum-free V-P1611 medium and cultured in a batch suspension for 48 h. Reactor parameters were controlled as follows: pH 6.9–7.1, dissolved oxygen 40%–60%, stirring speed 100–120 rpm, and temperature 36.5–37.2 °C. Preferably, the control parameters were: pH 7.0, dissolved oxygen 50%, stirring speed 110 rpm, and temperature 36.8 °C. During the culture, key parameters for evaluating cell culture performance, such as cell viability, density, and specific growth rate, were analyzed.

[0169] Cultured in a 500L reactor: Seed cells of well-grown Vero suspension cells were taken from a 100L reactor and cultured at a density of 0.6 × 10⁶ cells / year. 6Cells / mL were seeded in serum-free V-P1611 medium and cultured in a batch suspension for 48 h. Reactor control parameters were: pH 6.9–7.1, dissolved oxygen 40%–60%, stirring speed 80–100 rpm, and temperature 36.5–37.2 °C. Preferably, the control parameters were: pH 7.0, dissolved oxygen 50%, stirring speed 90 rpm, and temperature 36.9 °C. During the culture, key parameters for evaluating cell culture performance, such as cell viability, density, and specific growth rate, were analyzed.

[0170] Cultured in a 3000L reactor: Seed cells of well-grown Vero suspension cells from a 500L reactor were cultured at a density of 0.8 × 10⁶ cells / year. 6 Cells / mL were seeded in serum-free V-P1611 medium and cultured in a batch suspension for 48 h. Reactor control parameters were: pH 6.8–7.0, dissolved oxygen 60%–80%, stirring speed 80–100 rpm, and temperature 36.5–37.2 °C. Preferably, the control parameters were: pH 6.9, dissolved oxygen 70%, stirring speed 90 rpm, and temperature 36.9 °C. During the culture, key parameters for evaluating cell culture performance, such as cell viability, density, and specific growth rate, were analyzed.

Claims

1. A culture medium suitable for large-scale suspension culture of Vero cells, characterized in that, The culture medium consists of the following components: ① Amino acid portion The amino acid portion is composed of the following components: L-Aspartic acid 6.50 mg / L L-glutamic acid 5.23 mg / L L-Asparagine 68.34 mg / L L-serine 197.45 mg / L L-Lysine 107.16 mg / L L-glycine 26.64 mg / L L-histidine 48.38 mg / L L-arginine 319.28 mg / L L-Threonine 68.28 mg / L L-alanine 3.11 mg / L L-proline 21.68 mg / L L-Tyrosine 218.38 mg / L L-valine 63.73 mg / L L-methionine 73.97 mg / L L-cysteine ​​58.47 mg / L L-Isoleucine 59.56 mg / L L-Leucine 63.67 mg / L L-Phenylalanine 43.28 mg / L L-Tryptophan 11.45 mg / L ② Vitamins and Balanced Salts Section The vitamin and balanced salt portion consists of the following ingredients: Nicotinamide 36mg / L Pyridoxal 36 mg / L Thiamine 36 mg / L Pantothenic acid 36 mg / L choline chloride 36 mg / L Inositol 80mg / L Riboflavin 5.0 mg / L Folic acid 7.0 mg / L NaCl 6.4 g / L KCl 4.0g / L Magnesium sulfate 0.09 g / L Sodium dihydrogen phosphate 0.14 g / L ③ Other additives The other additives consist of the following components: Glucose 1.98 g / L Glutamine 0.47 g / L Hypoxanthine 0.106g / L Thymidine 0.050 g / L Fe(NO3)3·9H2O 0.800mg / L Yeast powder 0.150g / L Ferric citrate 35.0 mg / L Transferrin 11.0 mg / L Zinc sulfate 8.6 mg / L Sodium selenite 28.00 mg / L Vitamin E 80.00 mg / L Soy protein hydrolysate 3.00 g / L Phenol red 5.7 mg / L Pluronic F-68 1.0g / L Sodium bicarbonate 2.0 g / L Insulin 3.5 mg / L.

2. The application of the culture medium according to claim 1 in the large-scale suspension culture of Vero cells.

3. A method suitable for large-scale culture of Vero cells in full suspension, characterized in that, The step includes inoculating Vero cells adapted for suspension culture into the sterilized culture medium of claim 1.

4. The method as described in claim 3, characterized in that, The Vero cells adapted for suspension culture were named African green monkey kidney cells VERO-XW and deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2022110.

5. The method as described in claim 3 or 4, characterized in that, The cell seeding density was 0.60 × 10⁶. 6 -0.80×10 7 The cells / mL culture conditions were controlled as follows: pH 6.8-7.4, dissolved oxygen 40%-80%, stirring speed 70-110 r / min, and temperature 36.5℃-37.2℃.

6. The method as described in claim 3 or 4, characterized in that, The method includes the following steps: (1) Cell resuscitation and shaker culture One cell line was resuscitated from a Vero cell working cell bank in suspension culture and thawed in pure water at 37 °C for 1 min. An appropriate amount of fresh, sterile culture medium (preheated to 37 °C) as described in claim 1 was placed in a centrifuge tube. The cells were added to the centrifuge tube, mixed well, and centrifuged at 600 rpm for 5 min to remove the cryoprotectant. The supernatant was discarded, and an appropriate amount of fresh, sterile culture medium (as described in claim 1) was added. The cells were then inoculated into culture flasks at a density of 0.7 × 10⁻⁶ cells / mL. 6 Cells / mL, shaken on a CO2 shaker at 37℃ and 100 rpm for 48 h, then sampled and counted; once the cell culture volume reached a certain quantity, the cells were collected to obtain Vero suspension cell seed. (2) Bioreactor cultivation is carried out in four stages: 10L reactor culture: Take well-grown Vero suspension cells and culture them at a density of 0.6 × 10⁶ cells / year. 6 Cells / mL were inoculated into fresh, sterile culture medium as described in claim 1 and cultured in a 10L reactor in a fully suspended batch for 48 hours. The reactor control parameters were: pH 7.0~7.2, dissolved oxygen 40%~60%, stirring speed 100~120 r / min and temperature 36.5℃~37.2℃. 100 L reactor culture: Take well-grown Vero suspension cells from a 10 L reactor and culture the cells at a density of 0.6 × 10⁶ cells / year. 6 Cells / mL were inoculated into fresh, sterile culture medium as described in claim 1 and cultured in a 100 L reactor in a fully suspended batch for 48 h. The reactor control parameters were: pH 6.9-7.1, dissolved oxygen 40%-60%, stirring speed 100-120 r / min, and temperature 36.5℃-37.2℃. 500 L reactor culture: Take well-grown Vero suspension cells from a 100 L reactor and culture the cells at a density of 0.6 × 10⁶ cells / year. 6 Cells / mL were inoculated into fresh, sterile culture medium as described in claim 1 and cultured in a 500 L reactor in a fully suspended batch for 48 h. The reactor control parameters were: pH 6.9-7.1, dissolved oxygen 40%-60%, stirring speed 80-100 r / min, and temperature 36.5℃-37.2℃. Cultured in a 3000 L reactor: Take well-grown Vero suspension cells from a 500 L reactor and culture the cells at a density of 0.8 × 10⁶ cells / year. 6 Cells / mL were inoculated into fresh, sterile culture medium as described in claim 1 and cultured in a 3000 L reactor in a fully suspended batch for 48 h. The reactor control parameters were: pH 6.8-7.0, dissolved oxygen 60%-80%, stirring speed 80-100 r / min, and temperature 36.5℃-37.2℃.

7. The method as described in claim 6, characterized in that, The control parameters for the 10L reactor culture were set as follows: pH 7.1, dissolved oxygen 50%, stirring speed 110 r / min and temperature 36.8℃; The control parameters for the 100 L reactor culture were set as follows: pH 7.0, dissolved oxygen 50%, stirring speed 110 r / min and temperature 36.8℃; The control parameters for the 500 L reactor culture were set as follows: pH 7.0, dissolved oxygen 50%, stirring speed 90 r / min, and temperature 36.9℃. The control parameters for the 3000 L reactor culture were set as follows: pH 6.9, dissolved oxygen 70%, stirring speed 90 r / min, and temperature 36.9℃.

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