Method for culturing MDBK suspension cells and method for producing virus

Abstract

The invention relates to an MDBK suspension cell culture method and a virus production method, and the culture method comprises the following steps: inoculating MDBK suspension cells, and carrying out cell culture; and after the cells are cultured until the cell density reaches 1 * 10 < 6 > / mL to 5 * 10 < 6 > / mL, perfusion is started, the perfusion rate is 0.5 VVD to 4VVD, and the method can be used for maintaining high-density full-suspension growth of bovine kidney cell MDBK cells and maintaining a relatively high motility rate.

Description

Technical Field

[0001] The present application relates to the technical field of biological products, and in particular to a method for culturing MDBK suspension cells and a method for producing viruses. Background Art

[0002] MDBK cells are an adherent cell line isolated and cultured from the kidney tissue of a healthy adult bull in the United States in 1953. This cell line is susceptible to a variety of pathogens (bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), pseudorabies virus (PRV), etc.), and is widely used in the fields of virus infection, detection and culture, especially for the proliferation of infectious bovine rhinotracheitis virus. It has high virus infection efficiency, rapid proliferation and is not easy to mutate. It is recognized as a cell matrix suitable for the production of infectious bovine rhinotracheitis vaccine. At present, the adherent process is mostly used for the production of related vaccines, but due to the limitations of its adherent growth characteristics, it is mostly produced by roller bottle culture and microcarrier culture. The production cycle is long, the downstream process is relatively complex, and industrial large-scale production is difficult. With the increasing maturity of suspension culture technology, suspension cells can be amplified and cultured in serum-free medium. The domesticated fully suspended MDBK cells minimize or avoid the addition of animal-derived serum, giving them better stability and safety in passage. They also have the advantage of linearly scalable culture, enabling large-scale and efficient production in bioreactors.

[0003] Fed-batch culture is an effective way to scale up cell culture. It mainly refers to first loading a certain amount of culture fluid into the reactor, inoculating cells under appropriate conditions, and culturing to allow the cells to grow continuously and the products to be formed continuously. In this process, as nutrients are continuously consumed, new nutrients are continuously added to the system to allow the cells to further grow and metabolize until the products are taken out after the entire culture is completed. Fed-batch culture not only avoids affecting the growth and metabolism of cells and the formation of products when the initial concentration of a certain nutrient is too high, but also prevents certain limiting nutrients from being exhausted during the culture process and affecting cell growth and product formation. It is an effective means to ensure that cells maintain proliferation.

[0004] In view of the wide application of MDBK cells in bovine virus preparation, it is necessary to scale up the culture of MDBK cells. However, batch feeding culture or perfusion culture has not been successfully applied to MDBK cells. Summary of the invention

[0005] Based on this, it is necessary for one embodiment of the present application to provide a method for culturing MDBK suspension cells with improved cell density and cell viability, as well as a method for producing bovine viruses with greatly improved virus titers.

[0006] The technical solutions include the following:

[0007] A method for culturing MDBK suspension cells comprises the following steps:

[0008] Inoculating MDBK suspension cells for cell culture; and

[0009] Culture until the cell density reaches 1×10 6 / mL~5×10 6 After perfusion, the perfusion rate was 0.5VVD~4VVD.

[0010] In one embodiment, the cells were cultured until the cell density reached 3×10 6 / mL~5×10 6 After the perfusion reaches 1VVD / mL, the perfusion is started at a rate of 1VVD~2VVD.

[0011] In one embodiment, the perfusion rate is maintained constant.

[0012] In one embodiment, the culture medium used is MDBK serum-free chemically defined medium.

[0013] In one embodiment, the cell culture satisfies one or more of the following conditions:

[0014] The culture temperature is 31℃~38℃;

[0015] The pH value of the culture system is 6.5-7.8;

[0016] Dissolved oxygen concentration 30%~80%; and

[0017] Stirring speed 30rpm~200rpm.

[0018] A method for producing a virus comprises the following steps:

[0019] Cultivating MDBK suspension cells using the culture method; and

[0020] Inoculate virus and culture;

[0021] Wherein, the virus is a bovine virus or a pseudorabies virus.

[0022] In one embodiment, the bovine virus includes bovine viral diarrhea virus and / or infectious bovine rhinotracheitis virus.

[0023] In one embodiment, the cell density of the MDBK suspension cells reaches 10×10 6 / mL~40×10 6 After the concentration of 1g / mL was increased, the virus was inoculated.

[0024] In one embodiment, the cell density of the MDBK suspension cells reaches 20×10 6 / mL~30×10 6 After the concentration of 1g / mL was increased, the virus was inoculated.

[0025] In one embodiment, the culture after inoculation of the virus includes one or more of perfusion culture, batch culture and fed batch culture.

[0026] In one embodiment, the culture after virus inoculation is perfusion culture, and the perfusion rate is 0.5VVD~4VVD.

[0027] In one embodiment, the perfusion rate is 1VVD~2VVD.

[0028] In one embodiment, perfusion culture is started 0.5h to 5h after virus inoculation.

[0029] A method for preparing a vaccine, comprising the step of producing a virus by the production method.

[0030] Compared with the traditional technology, this application has the following beneficial effects:

[0031] The present application provides a method for high-density culture of bovine virus host cells (MDBK suspension cells) by perfusion culture process. The selected culture conditions and perfusion conditions are suitable for the growth of MDBK suspension cells. The method can obtain higher cell density, cell viability and virus infection than batch and conventional perfusion processes. Through the perfusion culture method of the present application, high-density cell culture can be achieved, and the virus titer is greatly improved after inoculation of the corresponding bovine virus.

[0032] The method provided in the present application can culture cells to a high density and obtain a high-titer virus harvest solution. Those skilled in the art can combine the purification method to obtain a high-purity stock solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0034] Figure 1This is a comparison chart of the highest density of MDBK cells in perfusion culture and batch culture;

[0035] Figure 2 This is a comparison chart of the titer of infectious bovine rhinotracheitis virus stock solution cultured by perfusion process and batch process. DETAILED DESCRIPTION

[0036] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0038] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0039] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0040] In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0041] In this article, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel options of "yes" or "no". If there are multiple "options" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "option" is independent.

[0042] Herein, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0043] In this article, "VCD" stands for Viable Cell Density, which means viable cell density.

[0044] In this article, "batch culture" means that the cells and culture medium are first loaded into the reactor at one time for culture. The cells continue to grow. After a period of culture, the desired product is obtained and the culture is terminated. No feed, including glucose, is added during the process.

[0045] In this article, "fed-batch culture" is a culture method between batch culture and continuous culture. A certain amount of culture medium is added to the initial culture system, and cells are inoculated under appropriate conditions and cultured to allow cells to grow continuously and products to be formed continuously. During the culture process, fresh culture medium is regularly added according to the growth status and metabolic needs of the cells to meet the needs of continuous cell growth and product synthesis.

[0046] In this article, “VVD” represents the medium replacement rate, which usually refers to the ratio of the daily working volume to the volume of perfusion medium during perfusion culture.

[0047] In this article, "perfusion culture" is a cell culture technique in which fresh culture medium is continuously introduced into the culture system, while culture fluid containing cell metabolites and dead cells is continuously discharged to maintain a good environment for cell growth.

[0048] In one embodiment of the present application, a method for culturing MDBK suspension cells comprises the following steps (1) to (2):

[0049] Step (1), inoculating host cells and performing cell culture.

[0050] In one specific example, the host cell is an MDBK suspension cell.

[0051] In a specific example, MDBK suspension cells are suspension cells acclimated by adhesion or purchased through commercial channels, are stable in state, have good dispersibility, are fully adapted to suspension culture in serum-free suspension growth medium, and can be produced on a large scale.

[0052] In a specific example, the host cell is a cell capable of packaging bovine viruses, in particular, a cell capable of packaging bovine viral diarrhea virus and infectious bovine rhinotracheitis virus. MDBK suspension cell host cells can be grown in adherent culture or suspension culture, in particular, adapted to suspension culture.

[0053] In a specific example, the host cell is an autonomously domesticated MDBK suspension cell, such as, but not limited to, an MDBK clone cell line.

[0054] In a specific example, in step (1), the cell culture process includes but is not limited to batch culture, fed-batch culture or perfusion culture. Optionally, in perfusion culture, the time strategy for starting perfusion can be optimized, which can be the first day, the second day, or a specified interval.

[0055] In a specific example, in step (1), the cell culture is carried out in a bioreactor (eg, a stirred reactor or a turbulent flow reactor or other types of reactors).

[0056] In a specific example, the medium used for cell culture is any medium known to be suitable for the growth of host cells. In a specific example, the medium is MDBK serum-free chemically defined medium, a commercial medium such as CD MDBK211, but not limited thereto.

[0057] MDBK serum-free chemically defined medium refers to a medium capable of culturing MDBK cells, and the medium is a serum-free chemically defined medium.

[0058] In a specific example, before step (1), a cell recovery and cell culture step may be included.

[0059] Specifically, the frozen host cells are revived using commercial culture medium, the cell seeding density is adjusted, cultured under appropriate conditions, and subcultured and amplified as needed.

[0060] In a specific example, step 1 of the above method may also include a step of amplifying and culturing the host cells.

[0061] In one specific example, the subculture density of the expanded culture is 1×10 6 / mL~5×10 6 / mL, optional 1×10 6 / mL, 2×10 6 / mL, 3×10 6 / mL, 4×10 6 / mL, 5×10 6 Pieces / mL, or the range consisting of any two of the above values.

[0062] In a specific example, the subculture time for amplification culture is 48 h to 90 h, which can be selected as 48 h, 60 h, 72 h, 84 h, 90 h, or a range consisting of any two of the above values.

[0063] In a specific example, the temperature of the amplified culture can be 32°C to 38°C, and can be selected as 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, or a range consisting of any two of the above values.

[0064] In a specific example, the CO2 concentration for amplifying cell culture can be 4% to 8%, and can be optionally 4%, 5%, 6%, 7%, 8%, or a range consisting of any two of the above values.

[0065] In a specific example, the stirring speed of the above-mentioned cell culture can be 30rpm~200rpm, which can be 30rpm, 40rpm, 50rpm, 60rpm, 80rpm, 100rpm, 120rpm, 140rpm, 160rpm, 180rpm, 200rpm, or a range consisting of any two of the above values.

[0066] Step (2), culture until the cell density reaches 1×10 6 / mL~5×10 6 After perfusion, the perfusion rate was 0.5VVD~4VVD (container volume / day).

[0067] In a specific example, the cell density is cultured until it reaches the following optional value or range and then perfused: 1×10 6 / mL, 2×10 6 / mL, 3×10 6 / mL, 4×10 6 / mL, 5×10 6 Pieces / mL, or the range consisting of any two of the above values.

[0068] In one specific example, the cells were cultured until the cell density reached 3×10 6 / mL~5×10 6 After the cell count reaches 100 cells / mL, perfusion is started to obtain a higher cell density and cell viability.

[0069] In a specific example, the perfusion rate may be selected as 0.5 VVD, 1 VVD, 1.5 VVD, 2 VVD, 2.5 VVD, 3 VVD, 3.5 VVD, 4 VVD, or a range consisting of any two of the above values.

[0070] In a specific example, the perfusion rate is 1VVD~2VVD, which can achieve a higher cell density in culture.

[0071] In a specific example, during the perfusion process, the perfusion rate is maintained constant. This operation can simplify the operation steps in perfusion culture, reduce the deviation of the operation process, and obtain higher cell density and cell viability. At the same time, the volume of the perfusion culture medium can be controlled throughout the process, reducing the extra-budgetary liquid preparation cost and culture medium cost.

[0072] In a specific example, perfusion is performed by using a continuous perfusion device; more specifically, an alternating tangential flow cell retention system is used, wherein the retention pore size of the hollow fiber column is 0.1 μm~0.8 μm, which can be 0.1 μm, 0.2 μm, 0.4 μm, 0.6 μm or 0.8 μm.

[0073] In a specific example, the perfusion fluid replacement rate can be 1 L / h to 12 L / h, which can be 1 L / h, 2 L / h, 3 L / h, 4 L / h, 5 L / h, 6 L / h, 7 L / h, 8 L / h, 9 L / h, 10 L / h, 11 L / h, 12 L / h, or a range consisting of any two of the above values.

[0074] In a specific example, the cell culture temperature can be 31°C to 38°C, and can be 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, or a range consisting of any two of the above values.

[0075] In a specific example, the pH value of the cell culture is 6.5-7.8, which can be 6.5, 6.6, 6.8, 7.0, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, or a range consisting of any two of the above values.

[0076] In a specific example, the dissolved oxygen concentration of the cell culture is 30% to 80%, which can be 30%, 40%, 50%, 60%, 70%, 80%, or a range consisting of any two of the above values.

[0077] In a specific example, the CO2 concentration of the cell culture may be 4% to 8%, and may be 4%, 5%, 6%, 7%, 8%, or a range consisting of any two of the above values.

[0078] In a specific example, the stirring speed of the cell culture can be 30 rpm to 200 rpm, which can be 30 rpm, 40 rpm, 50 rpm, 60 rpm, 80 rpm, 100 rpm, 120 rpm, 140 rpm, 160 rpm, 180 rpm, 200 rpm, or a range consisting of any two of the above values.

[0079] An embodiment of the present application also provides a method for producing a virus, the method comprising the following steps a to b:

[0080] Step a, culturing host cells, wherein the host cells are MDBK suspension cells;

[0081] Step b, inoculating the virus and culturing.

[0082] In a specific example, in step a, the MDBK suspension cell culture method described above is used for culture.

[0083] In a specific example, the virus can be a virus that can infect a host cell, including but not limited to a bovine virus or a pseudorabies virus, a bovine virus, in particular a bovine viral diarrhea virus and an infectious bovine rhinotracheitis virus. Optionally, the above-mentioned virus can be any subtype (viral gene subtype) thereof. In a specific example, the above-mentioned bovine virus is an infectious bovine rhinotracheitis virus.

[0084] In one specific example, in step b, the cell density of the MDBK suspension cells reached 10 × 10 6 / mL~40×10 6 After inoculation of virus, the cell density can reach 10×10 6 / mL, 20×10 6 / mL, 30×10 6 / mL, 40×10 6 / mL, or a range consisting of any two of the above values, and then inoculate the virus.

[0085] In one specific example, in step b, the cell density of the MDBK suspension cells reached 20 × 10 6 / mL~40×10 6 After inoculating the virus with a concentration of 100 μg / mL, a higher virus titer can be obtained.

[0086] In a specific example, the culture method after virus inoculation includes perfusion culture, batch culture or fed-batch culture. Preferably, perfusion culture.

[0087] In a specific example, the perfusion rate is 0.5VVD~4VVD, which can be selected as 0.5VVD, 1 VVD, 2 VVD, 3VVD, 4VVD, or a range consisting of any two of the above values.

[0088] In a specific example, the perfusion rate is 1VVD~2VVD, which can be 0.5VVD, 1 VVD, 2 VVD, 3 VVD, 4VVD, or a range consisting of any two of the above values.

[0089] In a specific example, perfusion is started 0.5h to 5h after inoculation of the virus to obtain a higher virus titer. Optionally, perfusion is started 0.5h, 1h, 1.5h, 2h, 2.5, 3h, 3.5h, 4h, 4.5h or 5h after inoculation of the virus.

[0090] In a specific example, in step b, the culture medium used for culturing can be any known culture medium suitable for amplification and production of infectious bovine rhinotracheitis virus, such as CD MDBK 211 culture medium.

[0091] In a specific example, the other culture conditions in step b have the definitions of the corresponding conditions in the above-mentioned culture method of the viral host cells (MDBK suspension cells).

[0092] In a specific example, the virus production method may further include step c: harvesting the virus.

[0093] One embodiment of the present application provides a method for preparing a vaccine, comprising the step of producing a virus using the above-mentioned virus production method.

[0094] The bovine kidney cell suspension culture method and bovine virus production method provided in the present application, especially the method for preparing infectious bovine rhinotracheitis virus vaccine through perfusion culture process, can be used to maintain high-density full-suspension growth of bovine kidney cell MDBK cell suspension and maintain a high viability, thereby improving the production capacity of bovine kidney cell MDBK cell-related products.

[0095] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0096] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0097] The MDBK cells involved in the following examples are obtained by adherent domesticated suspension cells or suspension cells purchased from commercial channels (Gansu Jianshun Biotechnology Co., Ltd., self-domesticated suspension cells).

[0098] Example 1 Screening of MDBK perfusion culture medium

[0099] MDBK seed cells were revived and expanded, and the seed cell concentration and viability were measured using a cell viability analyzer. 6 The cell volume was calculated based on the initial cell density of 10 / mL and the working volume of 30 mL. The calculated cell solution was transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cell pellet was gently patted to disperse. The cells were resuspended in 5 different MDBK culture media. The cell density was adjusted to 1×10 6 / mL. Using TPP ® TubeSpin culture, 30mL working volume, temperature 37℃, stirring speed 200rpm (50mm orbital diameter). When the cells grow to the third day, TPP ® The cells in the TubeSpin were centrifuged and the medium of the corresponding conditions was used, i.e., the perfusion rate was 1VVD per day until the culture system reached the highest cell density. The density and viability of MDBK cells cultured under different conditions were tested, and the results are shown in Table 1.

[0100] Table 1 Density and viability of MDBK cells in different perfusion media

[0101]

[0102] Several culture media in the experimental design can support the growth of MDBK cells. After perfusion culture, it was found that CD MDBK211 (Jian Shun Bio, catalog number 10501-211) perfusion culture had the highest cell density, while TPP ® TubeSpin culture can obtain 29.47×10 6 The cell density was 100 / mL. Therefore, CD MDBK 211 medium was selected for subsequent perfusion process parameter exploration. This method was used for preliminary screening. If the perfusion was continuous and the rate was stable in the reactor, a higher cell density could be obtained.

[0103] Example 2 Optimization of VCD parameters for perfusion culture of MDBK cells

[0104] After the MDBK cells were revived, they were expanded and inoculated into the reactor. The culture temperature was 37°C, the pH was set to 6.6-7.8, the dissolved oxygen concentration was 40%, and the stirring speed was 200 rpm. When the cell density reached the experimental design density in Table 2, perfusion culture was started. Initially, the perfusion rate was fixed at 1VVD until the culture system reached the highest cell density. The density and viability of MDBK cells cultured under different conditions were tested, and the results are shown in Table 2.

[0105] Table 2 Density and viability of MDBK cells perfused cultured under different initial VCD conditions

[0106]

[0107] The results showed that the cell density obtained by perfusion culture was 1-2 times higher than that of batch culture. 6 The highest cell density and viability can be obtained when the perfusion rate is 1VVD at a density of 1×10 6 Cells perfusion time too early affects the cell growth rate. 6 The cell growth density was also higher under the condition of 3×10 / mL, but its viability and density were slightly lower than 3×10 6 / mL density condition, therefore, the experiment selected 3×10 6 The density condition of cells / mL was taken as the actual perfusion density.

[0108] Example 3 Optimization of perfusion rate parameters for culturing MDBK cells using the perfusion method

[0109] MDBK cells were expanded and inoculated into the reactor. When the cell density reached 3×10 6 / mL, and then the perfusion was started. The perfusion rates were selected as 0.5 VVD, 1 VVD, 2 VVD, 3VVD, and 4VVD, respectively. The perfusion rate was kept constant during the entire culture process. The culture temperature was 37°C, the pH was set to 6.6-7.8, the dissolved oxygen concentration was 40%, and the stirring speed was 200rpm. The density and viability of MDBK cells cultured with different perfusion processes were tested, and the results are shown in Table 3.

[0110] Table 3 Density and viability of cultured MDBK cells at different perfusion rates

[0111]

[0112] The results showed that a higher cell density could be obtained by using perfusion rates of -2 and -3, that is, a perfusion rate of 1-2 VVD. The nutrients provided by a perfusion rate of 0.5 VVD were insufficient to support the growth of cells at a higher density, while the density and viability of perfusion rates of 3 VVD and above were slightly lower than those of 1-2 VVD. At the same time, too high a perfusion rate would cause a waste of culture medium, leading to increased production costs.

[0113] Example 4 Comparison of cell density and viability between perfusion culture and batch culture

[0114] Experiment 1: The culture was carried out using a conventional batch culture process in the art.

[0115] Experiment 2: Using the perfusion rate of Example 3-3, MDBK cells were expanded and inoculated into the bioreactor. When the cell density reached 3×10 6 After the concentration of 100 μg / mL was reached, perfusion was started at a rate of 2VVD.

[0116] In experiments 1 and 2, other parameters (such as culture temperature, pH, dissolved oxygen concentration, stirring speed, etc.) during the culture process remained the same. The cell density and viability after the culture was completed were tested. The results are shown in Table 4.

[0117] Table 4 Viability and density of 293 cells cultured by batch and perfusion processes

[0118]

[0119] The results showed that the perfusion process can achieve higher cell density, and the cell density and viability are higher than those of the batch process. The highest culture density of batch culture is 13×10 6 Perfusion culture can continuously add fresh culture medium to the reactor through a continuous perfusion device to replace the cell metabolic waste in the reactor and maintain sufficient nutrition for the cells. The cell density of perfusion culture can reach up to 63×10 6 / mL, which is about 5 times that of batch culture and has obvious advantages.

[0120] Example 5 Comparison of virus culture under different density conditions in perfusion culture process

[0121] Experiment 1: Cultivate cells according to the perfusion process in Example 4 until the cell density reaches 20×10 6 Infectious bovine rhinotracheitis virus seed (prepared according to known prior art, refer to CN201110137299.1 and CN201711098901.9) was inoculated at a perfusion rate of about 2VVD. When the cell density reached 20×10 6 When the virus culture was completed, the virus harvest solution was taken and the TCID50 Titer determination method.

[0122] Experiment 2: Cultivate cells according to the perfusion process in Example 4 until the cell density reaches 30×10 6 The perfusion rate was selected as 2VVD, and when the cell density reached 30×10 6 When the virus culture was completed, the virus harvest solution was taken and the TCID 50 Titer determination method.

[0123] Experiment 3: Cultivate cells according to the perfusion process in Example 4 until the cell density reaches 40×10 6 The perfusion rate was selected as 2VVD, and when the cell density reached 40×10 6 When the virus culture was completed, the virus harvest solution was taken and the TCID 50 Titer determination method.

[0124] The other parameters in the culture process of experiments 1-3 were basically the same, including the inoculation amount and the harvest time. The results are shown in Table 5.

[0125] Table 5 Comparison of toxin production under different densities of perfusion culture

[0126]

[0127] The results showed that the cells were cultured by perfusion technology until the cell density reached 20×10 6 When the virus is inoculated at about 30×10 / m, a higher virus titer can be obtained. After inoculation, due to virus adsorption and expression, the perfusion is not turned on. The high initial cell density may cause nutrient deficiency during the later viral expression, resulting in a decrease in virus titer. Therefore, according to the highest cell growth density, the cell density at the time of inoculation is 30×10 6 The concentration of each cell was about 100 μg / mL, and perfusion was attempted after inoculation to evaluate the virus culture effect of the final perfusion culture process.

[0128] Example 6 Optimization of process parameters for perfusion culture of viruses

[0129] The cells were cultured according to the perfusion process in Example 4 until the cell density reached 30×10 6 Inoculate the infectious bovine rhinotracheitis virus at a rate of about 10 / mL. When the cells grow, the perfusion rate is 2VVD. When the cell density reaches 30×10 6 When the concentration of 50 μg / mL was about 100 μg / mL, the perfusion was stopped and the poison was administered.

[0130] In experiment 1, the perfusion culture was not started after the virus was inoculated. After the virus culture was completed, the virus harvest liquid was taken and TCID50 Titer determination method.

[0131] In experiment 2, perfusion culture was started within half an hour after virus inoculation, with a perfusion rate of 2VVD until the virus culture was completed. The virus harvest fluid was taken and TCID 50 Titer determination method.

[0132] Experiment 3: Perfusion culture was started 2 hours after virus inoculation, with a perfusion rate of 2VVD until the virus culture was completed. The virus harvest solution was collected and TCID 50 Titer determination method.

[0133] Experiment 4: Perfusion culture was started 5 hours after virus inoculation at a perfusion rate of 2VVD until the virus culture was completed. The virus harvest solution was collected and TCID 50 Titer determination method.

[0134] The other parameters in the culture process of experiments 1-4 were basically the same, including the inoculation amount and the harvest time. The results are shown in Table 6.

[0135] Table 6 Comparison of virus production in perfusion culture

[0136]

[0137] The results showed that the cells were cultured by perfusion technology until the cell density reached 30×10 6 Inoculate the virus at about 100 cells / m2, stop perfusion when inoculating, and start perfusion 2-5 hours after inoculation to obtain a higher virus titer. Start perfusion immediately after inoculation, and the virus adsorption is not sufficient, resulting in some viruses flowing out of the tank during the perfusion of the culture medium. Therefore, the actual inoculation amount is lower than the theoretical inoculation amount.

[0138] Example 7 Comparison of virus yields between perfusion culture and batch culture

[0139] Perfusion process: Culture the cells according to the perfusion process in Example 4 until the cell density reaches 30×10 6 Inoculate the infectious bovine rhinotracheitis virus at a rate of about 10 / mL. When the cells grow, the perfusion rate is 2VVD. When the cell density reaches 30×10 6 When the virus culture was about 100 / mL, the perfusion was stopped and the virus was inoculated. The perfusion culture was started 2 hours after the virus inoculation, with a perfusion rate of 2VVD until the virus culture was completed. The virus harvest solution was taken and TCID 50 Titer determination method.

[0140] Batch process: Batch culture up to 8×10 6 When the number of cells / mL is about 4×10 6 After the virus culture is completed, the virus harvest solution is taken and TCID 50Titer determination method.

[0141] The other parameters in the culture process were basically the same. The results are shown in Table 7.

[0142] Table 7 Comparison of toxin production of MDBK cells in perfusion and batch culture

[0143]

[0144] Comparing the virus yield per unit volume of perfusion culture and batch culture, perfusion culture can obtain a higher virus titer, which is about 50 times higher than batch culture.

[0145] In addition, the expression effect of pseudorabies virus using this embodiment was also evaluated, and the perfusion culture method increased the virus by about 30 times compared with batch culture.

[0146] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0147] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A method for culturing MDBK suspension cells, characterized in that: The following steps are included: Inoculating MDBK suspension cells for cell culture; and Culture until the cell density reaches 1×10 6 / mL~5×10 6 After perfusion, the perfusion rate was 0.5VVD~4VVD.

2. The culture method according to claim 1, characterized in that Culture until the cell density reaches 3×10 6 / mL~5×10 6 / mL and then perfused, the perfusion rate is 1VVD~2VVD; Optionally, the perfusion rate is maintained constant.

3. The culture method according to claim 1, characterized in that The culture medium used was MDBK serum-free chemically defined medium.

4. The culture method according to claim 1 or 2, characterized in that The cell culture meets one or more of the following conditions: The culture temperature is 31℃~38℃; The pH value of the culture system is 6.5-7.8; Dissolved oxygen concentration 30%~80%; and Stirring speed 30rpm~200rpm.

5. A method for producing a virus, characterized in that: The steps include: Cultivating MDBK suspension cells using the culturing method according to any one of claims 1 to 4; and Inoculate virus and culture; Wherein, the virus is a bovine virus or a pseudorabies virus; Optionally, the bovine virus comprises bovine viral diarrhea virus and / or infectious bovine rhinotracheitis virus.

6. The production method according to claim 5, characterized in that The cell density of the MDBK suspension cells reached 10 × 10 6 / mL~40×10 6 After 1000 cells / mL, the virus is inoculated; Optionally, the cell density of the MDBK suspension cells reaches 20×10 6 / mL~30×10 6 After the concentration of 1g / mL was increased, the virus was inoculated.

7. The production method according to claim 5, characterized in that: The culture after inoculation of the virus includes one or more of perfusion culture, batch culture and fed-batch culture.

8. The production method according to claim 7, characterized in that: The culture after virus inoculation is perfusion culture, and the perfusion rate is 0.5VVD~4VVD; Optionally, the perfusion rate is 1VVD~2VVD.

9. The production method according to claim 8, characterized in that: Perfusion culture began 0.5h~5h after virus inoculation.

10. A method for preparing a vaccine, characterized in that: The method comprises the step of producing the virus according to any one of claims 5 to 9.

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