Method for efficiently preparing recombinant oncolytic adenovirus by using 293 suspension culture cells
By adjusting the stirred culture conditions of 293 cells, the cell viability is reduced, and the ventilation and nutrients are restored after toxicity is restored, the problem of low efficiency of virus amplification of suspended cells is solved, and the efficient preparation of high-titer recombinant oncolytic adenovirus is achieved.
Patent Information
- Application Number
- CN202311257138.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-26
- Publication Date
- 2025-03-28
AI Technical Summary
In the prior art, the efficiency of suspended cell amplification virus is low, resulting in low preparation efficiency of oncolytic adenovirus and cannot meet the needs of high titer viruses.
By controlling the stir culture conditions of 293 cells, including adjusting the stirring speed and ventilation state, reducing cell viability to improve the efficiency of viral infection, and restoring ventilation and adding nutrients after toxicity is carried out to promote virus amplification.
The recombinant oncolytic adenovirus was effectively amplified in a stirred bioreactor, and the infection titer per unit volume of virus harvesting solution was increased by 1.5 to 2 times, meeting the preparation needs of high titer viruses.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for efficiently preparing a recombinant oncolytic adenovirus. Background Art
[0002] Oncolytic viruses can kill tumor cells in a targeted manner without killing normal tissue cells. At the same time, oncolytic viruses can kill tumor cells that resist apoptosis and will not antagonize existing anti-tumor treatment regimens, making them suitable for combined drug use.
[0003] Adenovirus has the following advantages when used as a vector in the development of oncolytic viruses: compared with other viral vectors, adenovirus has higher gene transduction efficiency, broad tissue affinity, low pathogenicity, no integration into the host genome, clear genetic background, and high expression level of exogenous genes. Adenovirus vectors can accommodate about 8-10kb of exogenous genes, can express multiple genes at the same time, have a fast proliferation rate, are easy to culture, can produce higher viral titers, can be used in gene vaccines and anti-cancer treatments, and are suitable for multiple pathways of immunization. Currently, many clinical trials have confirmed the safety and effectiveness of adenovirus vectors in the above-mentioned treatment trials. Based on the above advantages, the use of adenovirus as a vector to develop oncolytic viruses will have broad application prospects.
[0004] However, oncolytic adenoviruses often require a higher infection titer to play a role in anti-tumor therapy. Therefore, it is particularly important to improve the preparation efficiency of oncolytic adenoviruses and achieve efficient amplification of adenoviruses, so as to obtain a sufficient number of high-titer oncolytic adenoviruses in a single batch.
[0005] The growth characteristics of cells that have been successfully suspended and domesticated into completely serum-free suspension culture have changed, or the ability to amplify viruses has been significantly reduced. Studies have shown that after HeLa cells were suspended and domesticated, the ability to amplify JX594 (Wyeth strain) viruses was significantly reduced, and the single-cell virus production dropped from 72pfu / cell (adherent culture) to 0.4pfu / cell (suspension culture), which has lost its commercial application value.
[0006] In summary, it is particularly important to improve the efficiency of virus amplification in suspension cells. Summary of the invention
[0007] In order to solve the above-mentioned technical problems existing in the prior art, the object of the present invention is to provide a method for efficiently preparing a recombinant oncolytic adenovirus, so as to improve the toxin-producing ability of Expi293 suspension culture cells in preparing a recombinant oncolytic adenovirus.
[0008] The technical solution of the present invention is as follows:
[0009] A method for efficiently preparing a recombinant oncolytic adenovirus using 293 suspension culture cells, wherein the oncolytic adenovirus is an unedited oncolytic adenovirus or a recombinant oncolytic adenovirus or a genetically modified chimeric adenovirus, the method comprising the following steps:
[0010] (1) Cell culture: During the culture process, the culture system was ventilated. The culture conditions were as follows: temperature: 36-38°C, pH: 7.2-7.4, and stirring speed: 90-110 rpm. The suspension cultured 293 cells were cultured to a cell density of not less than 6×10 6 cells / ml.
[0011] (2) Virus inoculation: Stop aerating the culture system and adjust the stirring speed to 30-50 rpm for 1-2 h until the cell viability drops to 90%-95%. Adjust the 293 cell density to (2-4)×10 6 cells / ml, and oncolytic adenovirus inoculation was completed at MOI=5-10pfu / cell.
[0012] (3) Virus acquisition: After virus inoculation, glucose and L-glutamine were added at 1 g / L culture medium, respectively. The stirring speed was adjusted to 90-110 rpm, the temperature was maintained at 34-36°C, and the pH was 7.2-7.4. Aeration of the culture system was resumed to a dissolved oxygen (DO) of 40%-60%. The virus was harvested after culturing for 48 h-72 h.
[0013] Furthermore, the culture conditions of step (1) are: 36°C to 38°C, pH: 7.2 to 7.4, and dissolved oxygen (DO): 40% to 60%.
[0014] Furthermore, step (1) is to culture under stirring conditions, and the stirring speed is 90 to 110 rpm.
[0015] Furthermore, in step (1), the 293 cells in suspension culture are cultured to a cell density of (6-9)×10 6 cells / ml.
[0016] In a particular embodiment, the step (1) is to ensure the dissolved oxygen demand in the cell fluid by oscillation, the culture temperature is set to 36°C to 38°C, the culture system is ventilated during the culture process of the reactor to ensure the dissolved oxygen demand, the stirring speed is set to 90 to 110 rpm, and the suspension cultured 293 cells are cultured to a cell density of not less than 6×10 6 cells / ml.
[0017] Furthermore, the culture temperature in step (2) is set to 34°C to 36°C.
[0018] Furthermore, the operation of harvesting the virus in step (3) is to harvest the cells by repeated freezing and thawing, lysing the cells, and harvesting the virus.
[0019] The present invention provides a good environment for the growth of 293 cells and ensures that the cells grow to the cell density required for infection by controlling the rotation speed of 293 cell stirring culture, including setting the stirring speed to 90-110 rpm and the DO value to 40%-60% when 293 cells are inoculated into a stirring bioreactor for amplification.
[0020] In order to improve the efficiency of recombinant oncolytic adenovirus infection of 293 cells before inoculation, it is necessary to make the growth state of the cells worse, and reduce the viability of 293 cells to make the cells more susceptible to virus infection; To achieve this goal, the specific measures are to adjust the speed of the stirred bioreactor to 30-50 rpm before inoculation, stop ventilation, maintain for 1-2 hours, reduce the dissolved oxygen (DO) in the stirred bioreactor, and thus slightly reduce the cell viability (target value 90%-95%);
[0021] Then complete the inoculation operation to allow more viruses to infect and enter the cells for amplification. At this time, in order to achieve efficient cell amplification of the virus, sufficient dissolved oxygen and nutrients (carbon source and nitrogen source) must be guaranteed. The specific measures are to adjust the speed of the stirred bioreactor to 90-110rpm, restore ventilation, set the dissolved oxygen (DO) to 40%-60%, and add glucose and L-glutamine at 1g / L culture medium respectively.
[0022] The above method can effectively improve the ability of amplifying recombinant oncolytic adenovirus per unit volume of cell culture fluid in a stirred bioreactor. Compared with the conventional virus amplification method (the stirring speed is always maintained at 90-110 rpm, ventilation is not interrupted, and no feed is added before infection), the virus infection titer per unit volume of virus harvest fluid can be increased by 1.5 to 2 times.
[0023] The Expi293F or 293 cells described in the present invention refer to human kidney epithelial cell lines. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Cell growth curve in bioreactor;
[0025] Figure 2 Changes in cell growth and viability in bioreactors;
[0026] Figure 3 Comparative study on the efficiency of preparing recombinant oncolytic adenovirus in bioreactors. DETAILED DESCRIPTION
[0027] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.
[0028] Example 1 Suspension culture of 293 host cells and preparation of recombinant adenovirus
[0029] (1) Cell source: purchased from Gibco, revived, amplified, aliquoted, cryopreserved and stored.
[0030] (2) Culture medium: Dynamis medium refers to Dynamis medium containing 8 mM L-Glutamine; Dynamis medium and L-Glutamine were purchased from Gibco.
[0031] (3) Recovery: Take out one frozen 293 cell from the liquid nitrogen biological container, place in a water bath at 36-38°C for 90s±30s, transfer aseptically into a 125mL shake flask after thawing, and culture with Dynamis medium (containing 8mM-Glutamine) in a culture volume of 30mL in a CO2 shaking incubator. The culture conditions are set as follows: 36°C-38°C, 5%±0.5% CO2.
[0032] (4) Continue culturing for 3 days and take samples to test cell density and viability. The growth density of 293 cells in a 125 mL shake flask should be (2-3) × 10 6 cells / ml, use Dynamis medium (containing 8mM-Glutamine) to dilute the cell solution to (0.5±0.1)×10 6 cells / ml were subcultured to 500 mL shaking flasks with a culture volume of 120 mL and placed in a CO2 shaking incubator for culture at 36-38°C and 5%±0.5% CO2.
[0033] (5) Continue culturing for 3 days and take samples to test cell density and viability. The growth density of 293 cells in a 500 mL shake flask is (3-5)×10 6 cells / ml, use Dynamis medium (containing 8mM-Glutamine) to dilute the cell solution to (0.5±0.1)×10 6 cells / ml were subcultured to 2L shaking flasks with a culture volume of 500mL and placed in a CO2 shaking incubator for culture at 36-38°C and 5%±0.5% CO2.
[0034] (6) Continue culturing for 3 days and take samples to test cell density and viability. The growth density of 293 cells in a 2L shake flask is (3-5)×10 6 cells / ml, use Dynamis medium (containing 8mM-Glutamine) to dilute the cell solution to (1.0±0.2)×106 cells / ml were subcultured to a 5 L bioreactor for further culture, with a culture volume of 1 L. Culture conditions: stirring speed: 110 rpm, temperature: 36-38°C, pH: 7.2-7.4, DO: 40%-60%.
[0035] (7) Continue culturing for 3 days (72 hours after tank loading), and take samples every 24 hours to test cell density and viability. The growth density of 293 cells in the 5L bioreactor is (6-9)×10 6 When the cell viability drops to 90% to 95%, the cell solution is diluted with Dynamis medium (containing 8 mM-Glutamine). The culture volume after dilution is 2 L and the cell density is (2 to 4) × 10 6 cells / ml, MOI = 5-10pfu / cell to complete the inoculation, glucose and L-glutamine were added at 1g / L culture medium. The culture conditions were set as follows: temperature: 90rpm, 34-36℃, pH: 7.2-7.4, ventilation was restored, DO: 40%-60%.
[0036] (8) After inoculation for 1 day (96 hours after tank loading), samples were taken to test cell density and viability. The growth density of 293 cells in a 5L bioreactor should be (4-6)×10 6 cells / ml.
[0037] (9) After culturing for 2 days (120 h after tank loading), samples were taken to test cell density and viability. The growth density of 293 cells in a 5L bioreactor was (4-7)×10 6 cells / ml, sampled to test cell density and viability, sampled 100mL, repeatedly frozen and thawed 3 times (freeze in liquid nitrogen, thaw in a 37°C water bath) to lyse the cells and harvest the virus. Samples were sent for testing of infection titer, and the test results are shown below Figure 1 , 2 , as shown in Figure 3.
[0038] (10) After culturing for 3 days (144 h after tank loading), samples were taken to test cell density and viability. The growth density of 293 cells in a 5L bioreactor should be (3-6)×10 6 cells / ml, sample 100 mL, freeze and thaw repeatedly 3 times (freeze in liquid nitrogen, thaw in 37°C water bath) to lyse the cells and harvest the virus.
[0039] Take samples and send them for infection titer testing. The results are shown in Figure 3 ; The growth curve of cells in the bioreactor is shown in Figure 1 , the changes in cell viability are shown in Figure 2 .
[0040] Comparative Example 1 Suspension culture of 293 host cells and preparation of recombinant adenovirus (common method for preparing recombinant adenovirus: the stirring speed is always maintained at 90-110 rpm, ventilation is not interrupted, and no feed is added before inoculation)
[0041] (1) Cell source: purchased from Gibco, revived, amplified, aliquoted, cryopreserved and stored.
[0042] (2) Culture medium: Dynamis medium refers to Dynamis medium containing 8 mM L-Glutamine; Dynamis medium and L-Glutamine were purchased from Gibco.
[0043] (3) Cell recovery: Take out one frozen 293 cell from the liquid nitrogen biological container, place it in a water bath at 36-38°C for 90s±30s, and after thawing, transfer it aseptically into a 125mL shaking flask, culture it with Dynamis medium (containing 8mM-Glutamine), and place the culture volume of 30mL in a CO2 shaking incubator. The culture conditions are set as follows: 36°C-38°C, 5%±0.5% CO2.
[0044] (4) After 3 days of continuous culture, samples were taken to test cell density and viability. The growth density of 293 cells in a 125 mL shake flask was (2-3) × 10 6 cells / ml, use Dynamis medium (containing 8mM-Glutamine) to dilute the cell solution to (0.5±0.1)×10 6 cells / ml were subcultured to 500 mL shaking flasks for further culture, and the culture volume was 120 mL and placed in a CO2 shaking incubator for culture at 36-38°C and 5%±0.5% CO2.
[0045] (5) After 3 days of continuous culture, samples were taken to test cell density and viability. The growth density of 293 cells in a 500 mL shake flask was (3-5)×10 6 cells / ml, use Dynamis medium (containing 8mM-Glutamine) to dilute the cell solution to (0.5±0.1)×10 6 cells / ml were subcultured to 2L shaking flasks with a culture volume of 500mL and placed in a CO2 shaking incubator for culture at 36-38°C and 5%±0.5% CO2.
[0046] (6) After 3 days of continuous culture, samples were taken to test cell density and viability. The growth density of 293 cells in a 2L shake flask was (3-5)×10 6cells / ml, use Dynamis medium (containing 8mM-Glutamine) to dilute the cell solution to (1.0±0.2)×10 6 cells / ml were subcultured to a 5 L bioreactor for further culture, with a culture volume of 1 L. Culture conditions: stirring speed: 110 rpm, temperature: 36-38°C, pH: 7.2-7.4, DO: 40%-60%.
[0047] (7) After 3 days of continuous culture (72 hours after tank loading), samples were taken every 24 hours to test cell density and viability. The growth density of 293 cells in the 5L bioreactor was (6-9)×10 6 cells / ml, the cell viability was ≥95%, and the cell solution was diluted with Dynamis medium (containing 8 mM-Glutamine). The culture volume after dilution was 2 L, and the cell density was (3-4)×10 6 cells / ml, cell viability ≥95%, MOI = 5-10pfu / cell to complete the virus inoculation. Culture conditions: stirring speed: 90rpm, 34℃~36℃, pH: 7.2~7.4, DO: 40%~60%.
[0048] (8) After inoculation for 1 day (96 hours after tank loading), samples were taken to test cell density and viability. The growth density of 293 cells in a 5L bioreactor should be (4-6)×10 6 cells / ml.
[0049] (9) After culturing for 2 days (120 h after tank loading), samples were taken to test cell density and viability. The growth density of 293 cells in a 5L bioreactor was (4-7)×10 6 cells / ml, sampled to test cell density and viability, sampled 100mL, repeatedly frozen and thawed 3 times (freeze in liquid nitrogen, thaw in 37℃ water bath) to lyse cells and harvest viruses. Submitted for infection titer testing, test results are as follows Figure 1 , 2 , as shown in Figure 3.
[0050] (10) After 3 days of inoculation (144 hours after tank loading), the growth density of 293 cells in the 5L bioreactor was (3-6)
[0051] ×10 6 cells / ml, sampled to detect cell density and viability, sampled 100 mL, and repeated freezing and thawing for 3 times (freezing in liquid nitrogen and thawing in a 37°C water bath) to lyse the cells and harvest the virus.
[0052] Submit for infection titer testing, see Figure 3 ; The growth curve of cells in the bioreactor is shown in Figure 1 , the changes in cell viability are shown in Figure 2 .
[0053] The results show:
[0054] Figure 1 The cell growth curve shows that: according to the path shown in the embodiment and comparative example 1, the cell amplification is carried out, and the 0-96h cell density change curve is basically the same, and the trend is that the cell density gradually increases from 0 to 72h, and the cell density decreases when the 74h dilution is completed; the cell density increases slowly from 74 to 96h. The cell density of the path shown in comparative example 1 first rises and then decreases at 96 to 144h, while the cell density of the path shown in the embodiment gradually decreases at 96 to 144h. In summary, the cell growth curve is consistent with expectations, that is, after changing the stirring speed of the reactor in the path shown in the embodiment, ventilation is stopped at the same time, and the cell viability decreases, which is more conducive to virus-infected cells. At this time, glucose and L-glutamine are supplemented, which can realize a large number of virus amplification in cells, thereby causing a short-term increase in cell density after infection (74 to 96h), and then the virus amplifies in cells in large quantities and lyses some cells, and the cell density decreases rapidly (96 to 144h).
[0055] The cell viability of the path shown in the embodiment decreased, while the cell viability of the path shown in comparative example 1 remained at the same level; 74 to 144 hours after infection, the cell viability of the path shown in comparative example 1 decreased slowly, while the cell viability of the path shown in the embodiment decreased rapidly. In summary, the cell viability change curve is consistent with expectations, that is, after changing the reactor stirring speed in the path shown in the embodiment, the ventilation is stopped at the same time, the cell viability decreases, which is more conducive to virus infection of cells and amplification in cells, thereby causing the cell viability to decrease rapidly after infection.
[0056] Figure 2 The cell growth viability change curve shows that: when the cell amplification is carried out according to the path shown in the embodiment and the comparative example 1, the cell viability is maintained at the same level from 0 to 72 hours; the cell viability of the path shown in the embodiment decreases from 72 to 74 hours, and the cell viability of the path shown in the comparative example 1 is maintained at the same level; the cell viability of the path shown in the comparative example 1 decreases slowly from 74 to 144 hours after infection, and the cell viability of the path shown in the embodiment decreases rapidly. The viability change curve is consistent with expectations, that is, after changing the stirring speed of the reactor in the path shown in the embodiment, the ventilation is stopped at the same time, and the cell viability decreases, which is more conducive to the virus infecting the cells and amplifying them in large quantities in the cells, thereby causing the cell viability to decrease rapidly after infection.
[0057] Figure 3The results of virus infection titer show that: the infection titer of the harvested liquid 48 hours after infection is higher than that of the harvested liquid 48 hours after infection according to the path shown in Comparative Example 1 for cell amplification, virus inoculation and amplification; the 72h harvested liquid obtained by the path shown in the embodiment is higher than the 72h harvested liquid titer after infection according to the path shown in Comparative Example 1. The infection titer of the 48h harvested liquid and the 72h harvested liquid obtained by the path shown in the embodiment and Comparative Example 1 are at the same level. Therefore, the path shown in the preferred embodiment, 48h harvested virus, can prepare a recombinant oncolytic adenovirus with a higher titer.
[0058] In summary, according to the path shown in the embodiment: the cells grow in a stirred bioreactor for 72 hours, the speed is adjusted to 30-50 rpm, the ventilation is stopped, and it is maintained for 1-2 hours to reduce the dissolved oxygen (DO) in the stirred bioreactor, thereby slightly reducing the cell viability (target value 90%-95%); the inoculation operation is completed, and glucose is added at 1g / L culture medium, and L-glutamine is added at 1g / L culture medium. Then the speed of the stirred bioreactor is adjusted to 90-110 rpm, ventilation is restored, the dissolved oxygen (DO) is set to 40%-60%, and the culture is continued to 120 hours (ie 48h±6h after inoculation), and the virus is harvested to obtain a recombinant oncolytic adenovirus with a higher titer.
[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for efficiently preparing a recombinant oncolytic adenovirus using 293 suspension culture cells, characterized in that: The oncolytic adenovirus is an unedited oncolytic adenovirus or a recombinant oncolytic adenovirus or a genetically modified chimeric adenovirus, and the method comprises the following steps: (1) Cell culture: During the culture process, the culture system was ventilated. The culture conditions were as follows: temperature: 36-38°C, pH: 7.2-7.4, stirring speed: 90-110 rpm, and the suspension cultured 293 cells were cultured to a cell density of not less than 6×10 6 cells / ml; (2) Virus inoculation: Stop aerating the culture system and adjust the stirring speed of the bioreactor to 30-50 rpm for 1-2 h until the cell viability drops to 90%-95%. Adjust the 293 cell density to (2-4)×10 6 cells / ml, and oncolytic adenovirus inoculation was completed at MOI = 5-10 pfu / cell; (3) Virus acquisition: After virus inoculation, glucose and L-glutamine were added to the culture medium at 1 g / L, respectively. The stirring speed of the bioreactor was adjusted to 90-110 rpm, the temperature to 34-36°C, and the pH to 7.2-7.
4. Aeration of the culture system was resumed to a dissolved oxygen (DO) of 40%-60%. The virus was harvested after culturing for 48 h-72 h.
2. The efficient preparation method of oncolytic adenovirus according to claim 1, characterized in that: The culture conditions of step (1) are as follows: culture temperature in the bioreactor: 34° C. to 38° C., pH: 7.2 to 7.4, and dissolved oxygen (DO): 40% to 60%.
3. The efficient preparation method of oncolytic adenovirus according to claim 1, characterized in that: Step (1) is to culture under stirring conditions in a bioreactor, and the stirring speed is 90 to 110 rpm.
4. The efficient preparation method of oncolytic adenovirus according to claim 1, characterized in that: Step (1) Cultivate the 293 cells in suspension culture to a cell density of (6-9)×10 6 cells / ml (in bioreactor).
5. The efficient preparation method of oncolytic adenovirus according to claim 1, characterized in that: Step (2) The culture temperature is set at 34°C to 36°C.
6. The efficient preparation method of oncolytic adenovirus according to claim 1, characterized in that: The operation of harvesting the virus in step (3) is to harvest the cells by repeated freezing and thawing, lyse the cells, and harvest the virus.