A method for improving the production of rabies virus vlp protein expression in mammalian cells
By employing a fully suspended serum-free culture and batch harvesting method in a bioreactor, the problem of low expression levels of rabies virus VLP protein in mammalian cells was solved, achieving efficient and stable protein production and meeting the needs of high-efficiency vaccine formulations.
Patent Information
- Application Number
- CN202410900069.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-05
- Publication Date
- 2025-11-18
- Estimated Expiration
- 2044-07-05
AI Technical Summary
In existing technologies, the expression level of mammalian cell rabies virus VLP protein is low, and the production process of inactivated vaccines is complex and has many safety risks, making it difficult to meet the demand for efficient and safe vaccine formulations.
A method combining full suspension serum-free culture in a bioreactor with batch harvesting was adopted to improve cell density and protein expression levels by controlling the culture environment and optimizing the harvesting process. This included using HEK-293T-RABV VLP cells, a turbulent flow bioreactor, and a serum-free culture medium with a specific formulation for partial medium replacement harvesting.
It significantly increased the expression level of rabies virus VLP protein in mammalian cells, with stable culture conditions, high reproducibility, reduced production costs, improved vaccine cost-effectiveness, and reduced probability of contamination.
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Figure CN118812670B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biotechnology, and particularly relates to a production method for improving the expression amount of rabies virus VLP protein of mammalian cells. BACKGROUND
[0002] Rabies is a natural epidemic zoonosis caused by rabies virus (RV) infection, which is a serious and highly fatal disease, with a mortality rate of almost 100%. At present, there is no special effective treatment for rabies, and the currently effective treatment for rabies is still the main method for preventing infection of rabies, which is to inoculate rabies virus vaccine (hereinafter referred to as rabies vaccine) and use anti-rabies serum. Wild animals are the main reservoir hosts of RV, and the infection and incidence of human and domestic animals are mainly caused by contacting wild animals carrying rabies virus. In China, the main source of rabies transmission is dogs. Therefore, dogs are an important link in the control of rabies, and inoculating rabies vaccine for dogs is the most effective measure to control rabies. At present, the coverage rate of canine rabies vaccine is low, and the inactivated vaccine is mainly used. However, there are still many technical difficulties and safety hazards in the production process of inactivated vaccine, including complex preparation process, large-scale suspension culture of cells, expansion of virus, low virus titer and low effective antigen amount. Therefore, a more safe and effective, convenient to prepare, low-cost, and capable of rapidly inducing high immune response vaccine preparation is needed. The VLP (virus-like particle) vaccine can well meet these needs, because it has the advantages of high safety, high immunization efficacy, wide adaptability, diversity of expression system and can be used for differential diagnosis, and is considered as the best candidate vaccine form most likely to replace the traditional inactivated vaccine.
[0003] G protein is the main immunoprotective antigen of rabies virus, and is relatively conservative, which can induce the production of specific neutralizing antibodies, and can also stimulate the body to produce cellular immunity, and plays an important role in resisting the attack of rabies virus. Mammalian expression system is the most attractive platform for producing VLP. As a natural host, the post-translational modification mechanism provided by mammalian cells can ensure the correct folding of VLP. And it has the advantages of high-efficiency secretion expression, easy purification, etc., and is widely used in the research and development of gene therapy products, vaccines, antibodies, etc. SUMMARY
[0004] In view of the defects existing in the prior art, the application provides a production method for improving the expression amount of rabies virus VLP protein of mammalian cells. The production method provided by the application has high production capacity and stable culture conditions, can be harvested in multiple batches, and has high repeatability and stability, and has wide application prospect.
[0005] The method for improving the expression of rabies virus VLP protein of mammalian cells according to the present application comprises the following steps:
[0006] S1 Seed cell inoculation and culture: the seed cells of mammalian cells expressing rabies virus VLP protein are cultured in a shake flask using serum-free culture solution, then inoculated into a bioreactor, and continue to be cultured using serum-free culture solution;
[0007] S2 Protein harvesting and medium replacement: when the cell growth parameters and culture solution parameters reach the requirements, the culture solution supernatant is collected, and part of the culture solution is replaced, until the cell growth parameters cannot reach the required parameters, then all the culture solution is centrifuged to obtain the supernatant.
[0008] By using a bioreactor to control a good culture environment, and combining with the improvement of culture and harvesting process, i.e. partial medium replacement and batch harvesting, the cell culture efficiency can be greatly improved, and high protein expression can be obtained.
[0009] For mammalian-derived protein expression, 293 cells and CHO cells are undoubtedly the best choice, preferably, the seed cells are HEK-293T-RABV VLP cells expressing rabies virus VLP protein.
[0010] Preferably, the bioreactor is a jet flow bioreactor, which uses jet flow oxygen transfer mechanism for oxygen transfer, and the culture effect is better.
[0011] Preferably, the serum-free culture solution is HEK293 cell full-suspension serum-free culture solution added with 8 mmol / L L-glutamine, which further contains 4 mM glutamine, 6 g / L glucose and 1.5 g / L PF68.
[0012] Preferably, the seed cells are inoculated into the bioreactor after being cultured in a shake flask using serum-free culture solution to 1E+7 cells / ml.
[0013] Preferably, the inoculation density of the cells in the bioreactor is 1E+6 cells / ml.
[0014] Preferably, in the step S1, the culture conditions in the bioreactor are as follows: culture temperature: 37-37.5℃, cell culture pH: 7.0-7.5, cell culture DO: 40%-80%, rotation speed: 50-100 rpm; and glucose is added into the culture solution to a concentration of 4 g / L every day, and the flow feeding is performed every other day from the second day.
[0015] The requirements of cell parameters and culture solution parameters for batch harvesting can be determined according to the specific culture cells, such as cell activity, size, and culture solution composition, etc.
[0016] Preferably, the cell growth parameters require: cell density ≥ 1E+7 cells / ml, viability ≥ 80%, and diameter 16-18 μm.
[0017] Preferably, the culture fluid parameters require: at least one of the following ①-③, which can be one, two or three; ① glucose consumption is greater than 2 g / L per day, ② lactic acid concentration is higher than 20 mmol / L, and ③ L-glutamine content is less than 2 mmol / L.
[0018] Preferably, in the step S2, 1 / 2-3 / 4 volume (volume ratio of the whole culture fluid) of the culture supernatant is collected each time, and an equal amount of new serum-free culture fluid is replaced.
[0019] The interval days for each collection are determined according to the culture condition, and preferably, every 3-5 days.
[0020] Compared with the prior art, the production method provided by the present application has the following advantages:
[0021] (1) The method of the present application uses a bioreactor for full-suspension serum-free culture combined with batch harvesting, so that the cell density is high, the protein expression amount is high, a lot of manpower and material resources are saved, the vaccine has high cost performance, and has great application prospect.
[0022] (2) The method of the present application uses a bioreactor, and the liquid addition and collection in the whole process are controlled by the system, which reduces the operation steps, reduces the pollution probability, and further improves the stability of the production process.
[0023] (3) The present application further optimizes each parameter in the production process, and at most 4 batches of expressed proteins can be collected in one experiment, and the protein yield of each batch is stable, and the protein expression amount of each batch can reach more than 10 times the yield of a shake flask, which has high productivity and stable culture conditions, and has high repeatability and stability. BRIEF DESCRIPTION OF DRAWINGS
[0024] Figure 1 For the cell expression VLP of Example 1, 1-4 batches are respectively the proteins collected by the bioreactor, and 5-8 are standard proteins (200, 100, 50, 25 mg / L);
[0025] Figure 2 For the cell expression VLP of Comparative Example 1, 1-10 are 10 batches of proteins collected by Comparative Example 1, and 11-14 are protein standards (20, 40, 80, 160 mg / L). DETAILED DESCRIPTION
[0026] The application will be further explained below in connection with specific embodiments. It should be noted, however, that the following embodiments are only used to explain the application and cannot be used to limit the application. All technical solutions identical or similar to the application are within the protection scope of the application. In the present embodiments, the specific techniques or conditions not mentioned are operated according to the conventional technical methods and the instrument instruction book; the reagents or instruments not mentioned by the manufacturer are the conventional products available in the market.
[0027] Reagents and instruments:
[0028] Bioreactor: Jianshun Biotechnology Co., Ltd., CUR5-A;
[0029] Serum-free medium: Jianshun Biotechnology Co., Ltd., 293CD02;
[0030] Feed: Jianshun Biotechnology Co., Ltd., 293Feed A (CD 293FA) and 293Feed B (CD 293FB);
[0031] The remaining reagents are also purchased from domestic manufacturers.
[0032] Example 1
[0033] A production method for improving the expression amount of rabies virus VLP protein in mammalian cells, comprising the following steps:
[0034] S1 Seed cell inoculation and culture:
[0035] The stored seed cells HEK-293T-RABV VLP were taken out from the liquid nitrogen storage tank, placed in a constant temperature water bath at 37°C, rapidly thawed, centrifuged at 800 rpm for 5 min to remove the cell cryopreservation solution, diluted with serum-free medium, inoculated into a 125 ml cell culture flask, and when the cells grew to more than 1E+7 / ml, the cells were diluted and transferred to a 500 ml cell culture flask for continuous culture, and serum-free medium was added to dilute to 1E+6 / ml, and placed in a constant temperature oscillator for continuous culture. The cell culture conditions were set as follows: culture temperature: 37-37.5°C, cell culture pH: 7.0-7.2, rotation speed: 50-100 rpm; and glucose was added to the culture medium to a concentration of 4 g / L every day, and the feed was added every other day from the second day.
[0036] The serum-free medium is 293CD02 full-suspension serum-free medium with 8 mmol / L L-glutamine, which also contains 4 mM glutamine, 6 g / L glucose, and 1.5 g / L PF68. The feed is 293Feed A with an addition amount of 5% (volume ratio) and 293Feed B with an addition amount of 0.5% (volume ratio).
[0037] Harvesting and medium replacement of S2 protein:
[0038] When at least one of the cell growth parameters (cell density reaching 1E+7 cells / ml, viability greater than 80%, diameter 16-18 μm) and the culture medium parameters (① glucose consumption greater than 2 g / L per day, ② lactic acid concentration higher than 20 mmol / L, ③ L-glutamine content less than 2 mmol / L) reaches the required value, the culture supernatant accounting for 1 / 2-3 / 4 of the total culture medium volume is collected every 3-5 days, and an equal amount of new serum-free culture medium is replaced; when the cell viability and diameter do not meet the required parameters, all the culture medium is collected and centrifuged to obtain the supernatant.
[0039] Detection of protein expression: Western Blot was used to detect the expression of VLP protein. The specific method is as follows: purified rabies G protein was used as a standard, gradient dilution was used to prepare the standard, anti-rabies virus monoclonal antibody was used as the primary antibody, and goat anti-mouse IgG was used as the secondary antibody. The yield of each batch of VLP protein was calculated according to the standard curve by Western Blot method
[0040] The results of protein expression detection are shown in Table 1. Figure 1 As shown in Table 1, a total of 4 batches of protein were collected in this experiment, except for the last batch which was lower (80 mg / L), the other three batches were all higher than 100 mg / L, respectively 137, 155, 168 mg / L.
[0041] Comparative Example 1: shake flask culture comparison
[0042] A method for producing a mammalian cell rabies virus VLP protein, comprising the following steps:
[0043] S1 seed cell inoculation and culture:
[0044] The preserved seed cells HEK-293T-RABV VLP were taken out from the liquid nitrogen storage tank, placed in a constant temperature water bath at 37°C, and quickly thawed. The cells were centrifuged at 800 rpm for 5 min to remove the cell freezing solution, and then diluted with serum-free medium and inoculated into a 125 ml cell culture flask. When the cell density reached more than 1E+7 cells / ml, the cells were diluted and transferred to a 500 ml cell culture flask for further culture. Serum-free medium was added to dilute the cells to 1E+6 cells / ml, and the cells were cultured in a constant temperature shaker. The cell culture conditions were set as follows: culture temperature: 37-37.5°C, cell culture pH: 7.0-7.2, rotation speed: 50-100 rpm; and glucose was added to the culture medium to a concentration of 4 g / L every day, and the flow feeding was started from the second day.
[0045] The serum-free culture solution is 293CD02 full-suspension serum-free culture solution with 8 mmol / L L-glutamine, 4 mM glutamine, 6 g / L glucose, and 1.5 g / L PF68 added. The feed is 293FeedA added at 5% (volume ratio) and 293Feed B added at 0.5% (volume ratio).
[0046] Harvesting and replacing of S2 protein:
[0047] When at least one of the cell growth parameters (cell density reaching 1E+7 cells / ml, viability greater than 80%, and diameter 16-18 μm) and the culture solution parameters (① glucose consumption greater than 2 g / L per day, ② lactic acid concentration higher than 20 mmol / L, and ③ L-glutamine content less than 2 mmol / L) reaches the required value, the culture solution supernatant accounting for 1 / 2-3 / 4 of the total culture solution volume is collected every 3-5 days, and an equal amount of new serum-free culture solution is replaced. When the cell viability and diameter do not meet the required parameters, all the culture solution is centrifuged, and the supernatant is collected.
[0048] The HEK-293T cell culture flask expressing rabies virus VLP was cultured in a constant temperature shaker, and the other conditions were consistent with those in Example 1. Example 2 was cultured in a cell culture flask, and the cells grew well in the early stage, but the cell activity changed rapidly in the later stage. A batch was collected about 3 days later, and a total of 10 batches were harvested, with a culture time of about 30 days. The collected supernatant was detected for VLP protein expression using the detection method of Example 1, but the protein expression of each batch was low, with the highest batch being only 65 mg / L and the last batch being only 10 mg / L. The protein of most batches was only 1 / 10 of the yield of the bioreactor in Example 1.
[0049] Comparison of the harvesting method of Comparative Example 2
[0050] A method for producing rabies virus VLP protein in mammalian cells, comprising the following steps:
[0051] S1 Seed cell inoculation and culture:
[0052] The stored seed cells HEK-293T-RABV VLP were taken out from the liquid nitrogen storage tank, placed in a constant temperature water bath at 37°C, and quickly thawed. The cell cryopreservation solution was removed by centrifugation at 800 rpm for 5 min. The cells were diluted with serum-free culture solution and inoculated in a 125 ml cell culture flask. When the cell density reached more than 1E+7 / ml, the cells were diluted and transferred to a 500 ml cell culture flask for further culture. Serum-free culture solution was added to dilute the cells to 1E+6 / ml. The cell culture was continued in a constant temperature shaker, and the cell culture conditions were set as follows: culture temperature: 37-37.5°C, cell culture pH: 7.0-7.2, rotation speed: 50-100 rpm; and glucose was added to the culture solution to a concentration of 4 g / L every day. From the second day, the feed was added every other day.
[0053] The serum-free culture solution was 293CD02 full-suspension serum-free culture solution with the addition of 8 mmol / L L-glutamine, which also contained 4 mM glutamine, 6 g / L glucose, and 1.5 g / L PF68. The feed was 293Feed A added at a concentration of 5% (volume ratio) and 293Feed B added at a concentration of 0.5% (volume ratio).
[0054] Harvesting and medium replacement of S2 protein:
[0055] The cells were continuously cultured until the cell viability was less than 80% or the maximum culture volume was reached. The bioreactor was then removed, and the cell culture solution was collected and centrifuged to obtain the supernatant.
[0056] As the culture time continued, the cell density in the bioreactor continued to increase, almost doubling every day. However, when the cell number reached 0.8E+7 / ml, the cell proliferation rate gradually slowed down. When the cell number reached 1.5E+7 / ml, the cell density gradually decreased, and the viability also began to decrease. When the cell viability dropped below 80%, the reactor was removed, and all the culture solution was collected. After centrifugation at 1000 rpm for 10 min, the supernatant was collected. The collected supernatant was detected for VLP protein expression using the detection method of Example 1, and the results are shown in Table 1.
[0057] Table 1 Detection of the effect of different culture methods on the expression of rabies virus VLP by HEK-293T cell strain
[0058]
[0059] As can be seen, in Comparative Example 2, the HEK-293T cells expressing the rabies virus VLPs were continuously grown and expressed in the bioreactor without medium replacement and with partial medium replacement during the fed-batch. As shown in Table 1, the continuous culture did not significantly improve the VLP yield, and the culture production time was short, and the total amount of harvested protein was low. By partial medium replacement, the culture time can be extended, and more protein can be harvested.
[0060] Effect of different lactic acid contents on VLP protein yield in Comparative Example 3
[0061] In order to compare the effect of different lactic acid contents on VLP yield, Comparative Example 3 was set up, and a 4L bioreactor was used to collect the culture expression liquid with different lactic acid contents. The specific experimental process is shown in Example 1 (except that the lactic acid content in the culture liquid parameters in step S3 of Example 1 is set to be higher than 30 mmol / L, and the other conditions are the same as in Example 1). The specific results are shown in Table 2.
[0062] Table 2 Effect of different lactic acid contents on expression of rabies virus-like particles by HEK-293T cell strain
[0063]
[0064] As can be seen from Table 2, by comparison, when the lactic acid content is too high and the medium is replaced, the cell state and viability are significantly reduced, the VLP yield is significantly reduced, the highest expression batch and the total protein amount are significantly lower than those in Example 1; the growth maintenance time is also shortened, the harvestable batches are also reduced, and the total protein amount finally harvested is also reduced. It is proved that too high lactic acid content will lead to lactic acid accumulation in the cells, and the cell state and viability will decrease, even if the medium is replaced, the cell state and viability will not recover.
[0065] Effect of different concentrations of L-glutamine on protein yield in Comparative Example 4
[0066] In order to compare the effect of different concentrations of L-glutamine on protein expression, we used a 4L bioreactor to culture and express with different concentrations of L-glutamine. The specific experimental process is shown in Example 1 (except that the L-glutamine in the culture liquid parameters in step S3 of Example 1 is replaced by different concentrations of L-glutamine (less than 4 mmol / L for Comparative Example 4-1 and less than 1 mmol / L for Comparative Example 4-2), and the other conditions are the same. The specific results are shown in Table 3.
[0067] Table 3 Effect of different concentrations of L-glutamine on expression of rabies virus VLPs by HEK-293T cell strain
[0068]
[0069] As can be seen from Table 3, by comparison, in Comparative Example 4-1, when the L-glutamine concentration is less than 4 mmol / L, the cell growth expression time is short, the expression amount of protein per batch is not high, although the number of batches is more, but the total amount of protein is low. In Comparative Example 4-2, when the L-glutamine concentration is less than 1 mmol / L, the cell culture time per batch is longer, and the highest protein expression per batch is higher; but the nutrients in the cell culture medium are insufficient and the harmful substances accumulate in the later stage, which leads to the cell state getting worse, the number of expression batches decreases, and the VLP expression of the subsequent batches of cells is low, and the total amount of harvested protein is less than that of Example 1.
[0070] Finally, it should be noted that the above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.
Claims
1. A method for producing rabies virus VLP protein with increased expression in mammalian cells, characterized in that, Includes the following steps: S1: Seed cell inoculation and culture: Mammalian cells expressing rabies virus VLP protein were cultured in shake flasks using serum-free culture medium, and then inoculated into a bioreactor for continued culture in serum-free culture medium. The serum-free culture medium was a serum-free culture medium in which HEK293 cells were fully suspended and supplemented with 8 mmol / L L-glutamine, and also contained 4 mM glutamine, 6 g / L glucose, and 1.5 g / L PF68. The bioreactor is a turbulent flow bioreactor. The culture conditions in the bioreactor are as follows: culture temperature: 37-37.5℃, cell culture pH: 7.0-7.5, cell culture DO: 40%-80%, rotation speed: 50-100 rpm; and glucose is added daily until the concentration in the culture medium reaches 4 g / L, with feed added every other day starting from the second day. S2: Protein Harvesting and Medium Change: Once cell growth parameters and culture medium parameters meet the requirements, collect the culture medium supernatant and add an equal volume of fresh culture medium. Cell growth parameters are required as follows: cell density ≥ 1E+7 cells / ml, viability ≥ 80%, and diameter 16-18μm; culture medium parameters are required to meet the following three conditions: ① glucose consumption greater than 2g / L per day, ② lactate concentration greater than 20mmol / L, ③ L-glutamine content less than 2mmol / L; each time, collect 1 / 2-3 / 4 of the culture medium supernatant and replace it with an equal volume of fresh culture medium; Continue until the cell growth parameters fail to meet the required parameters, then collect all the culture medium, centrifuge, and take the supernatant.
2. The production method according to claim 1, characterized in that, The seed cells were HEK-293T-RABV VLP cells.
3. The production method according to claim 1, characterized in that, The seed cells were cultured in shake flasks with serum-free medium until they reached 1E+7 cells / ml, and then inoculated into the bioreactor.
4. The production method according to claim 1, characterized in that, The cell seeding density in the bioreactor was 1E+6 cells / ml.