METHOD FOR PREPARING ADENOVIRUS VECTOR VACCINE BY MEANS OF PERFUSION CULTURE PROCESS

Abstract

Provided is a method for preparing an adenovirus vector vaccine by means of a perfusion culture process. The method comprises a step of culturing adenovirus host cells, and in particular a step of adjusting the perfusion rate by means of at least two stages according to cell density. The method increases the single cell yield of a virus after infection and the specific activity of a virus harvest liquid while achieving high-density growth of adenovirus host cells.

Claims

1. A method for culturing an adenovirus host cell, comprising the following steps: (1) inoculating host cells for cell culture; (2) starting perfusion at a perfusion rate of 1-3 VVD after a cell density reaches 1×10.sup.6-5×10.sup.6 cells/mL; (3) adjusting the perfusion rate to 2-4 VVD after the cell density grows to 5×10.sup.6-10×10.sup.6 cells/mL.

2. The method according to claim 1, wherein the perfusion is performed by using a continuous perfusion device; the continuous perfusion device uses an alternating tangential flow filtration system, wherein a filtration pore size of a hollow fiber column is 0.1-0.8 μm.

3. The method according to claim 1, wherein a concentration of glutamine is maintained at 2 mM or more in the cell culture.

4. The method according to claim 1, wherein the host cell is a 293 cell.

5. The method according to claim 4, wherein the host cell is an HEK293 cell or an HEK293.CS cell.

6. A method for producing an adenovirus, comprising the following steps: (1) inoculating host cells for cell culture; (2) starting perfusion at a perfusion rate of 1-3 VVD after a cell density reaches 1×10.sup.6-5×10.sup.6 cells/mL; (3) adjusting the perfusion rate to 2-4 VVD after the cell density grows to 5×10.sup.6-10×10.sup.6 cells/mL; (4) inoculating virus for culture.

7. The method according to claim 6, wherein in the step (4), the culture is a perfusion culture at a perfusion rate of 1-3 VVD.

8. The method according to claim 6, wherein the perfusion is performed by using a continuous perfusion device; the continuous perfusion device uses an alternating tangential flow filtration system, wherein a filtration pore size of a hollow fiber column is 0.1-0.8 μm.

9. The method according to claim 6, wherein a concentration of glutamine is maintained at 2 mM or more in the culture.

10. The method according to claim 6, wherein the host cell is a 293 cell.

11. The method according to claim 10, wherein the host cell is an HEK293 cell or an HEK293.CS cell.

12. The method according to claim 6, wherein the adenovirus is a human adenovirus or a chimpanzee adenovirus.

13. The method according to claim 6, wherein the adenovirus is selected from: AdHu5, AdHu4, AdHu7, AdHu11, AdHu26, AdHu55, AdC68, AdC3.

14. The method according to claim 6, wherein the adenovirus is a recombinant adenovirus comprising a coding exogenous gene.

15. The method according to claim 14, wherein the adenovirus comprises a gene of a structural protein of SARS-CoV-2, the structural protein is selected from: one or more of the followings: S protein, M protein, E protein, N protein.

16. A method for preparing an adenovirus vector vaccine, comprising the following steps: (1) inoculating host cells for cell culture; (2) starting perfusion at a perfusion rate of 1-3 VVD after a cell density reaches 1×10.sup.6-5×10.sup.6 cells/mL; (3) adjusting the perfusion rate to 2-4 VVD after the cell density grows to 5×10.sup.6-10×10.sup.6 cells/mL.

17. The method according to claim 16, wherein a concentration of glutamine is maintained at 2 mM or more in the cell culture.

18. The method according to claim 16, wherein the host cell is a 293 cell.

19. The method according to claim 18, wherein the host cell is an HEK293 cell or an HEK293.CS cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG. 1 is a graph showing the comparison between the highest density of 293 cells in perfusion culture and that in batch culture.

[0068] FIG. 2 is a graph showing the comparison between a specific activity of adenovirus stock solution in perfusion culture process and that in batch culture process.

DETAILED DESCRIPTION

[0069] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention relates.

[0070] Genes of SARS-CoV-2 and its structural proteins can be searched by techniques known in the art, for example, the genes of SARS-CoV-2 can be shown in GenBank: MT419849.1, and its structural proteins: the genes of S protein, E protein, M protein can be shown in 21387-25208, 26069-26296, 26347-27015 of GenBank: MT419849.1.

[0071] The disclosures of the various publications, patents, and published patent specifications cited herein are hereby incorporated by reference in their entirety.

[0072] The technical schemes of the present invention will be clearly and completely described below with reference to the examples of the present invention, and it is obvious that the described examples are only a part of the examples of the present invention but not all of them. Based on the examples of the present invention, all other examples obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

Example 1: Determination of Process Parameters for 293 Cells Cultured by Perfusion Method

[0073] Perfusion process 1: 293 cells were resuscitated, amplified and then inoculated into a fermenter, and the perfusion was started at a perfusion rate of 2 VVD after the cell density reached 1×10.sup.6 cells/mL. The cells were continuously cultured, and the perfusion rate was adjusted to 3 VVD after the cell density grew to 5×10.sup.6 cells/mL.

[0074] Perfusion process 2: 293 cells were resuscitated, amplified and then inoculated into a fermenter, and the perfusion was started after the cell density reached 1×10.sup.6 cells/mL or 5×10.sup.6 cell/mL. The perfusion rate was maintained constant at 1 VVD or 3 VVD throughout the culture.

[0075] Perfusion process 3: 293 cells were resuscitated, amplified and then inoculated into a fermenter, and the perfusion was started after the cell density reached 1×10.sup.6 cells/mL or 5×10.sup.6 cell/mL. The perfusion rate was maintained constant at 2 VVD or 4 VVD throughout the culture.

[0076] In the above 3 processes, the reactor parameters were as follows: the culture temperature was 37° C., the pH value was adjustable from 6.5 to 7.5, the concentration of dissolved oxygen was 30%-80%, and the stirring velocity was adjustable from 30 rpm to 40 rpm.

[0077] The density and viability of 293 cells cultured by different perfusion processes were measured. The results are shown in Tables 1 to 3.

TABLE-US-00001 TABLE 1 The density and viability of 293 cells cultured by perfusion process (perfusion process 1) Density of inoculation 1 × 10.sup.6 cells/mL Notes Perfusion velocity in the 2VVD first stage Perfusion velocity in the 3VVD Started when the cells grew second stage to 5 × 10.sup.6 cells/mL Density 11 × 10.sup.7 cells/mL Viability 98%

[0078] The results showed that the perfusion was started at the perfusion rate of 2 VVD after the cell density reached 1×10.sup.6 cells/mL. The cells were continuously cultured, and the perfusion rate was adjusted to 3 VVD after the cell density grew to 5×10.sup.6 cells/mL. The highest growth density of the cells could reach 11×10.sup.7 cells/mL.

TABLE-US-00002 TABLE 2 The density and viability of 293 cells cultured by perfusion process (perfusion process 2) 1 × 10.sup.6 cells/mL 5 × 10.sup.6 cells/mL 1VVD 3VVD 1VVD 3VVD Density 65 × 10.sup.6 70 × 10.sup.6 50 × 10.sup.6 60 × 10.sup.6 cells/mL cells/mL cells/mL cells/mL Viability 94% 96% 92% 93%

[0079] The results showed that the perfusion was started after the cell density reached 1×10.sup.6 cells/mL or 5×10.sup.6 cells/mL, and the perfusion rate was maintained constant at 1 VVD or 3 VVD throughout the culture. The highest growth density of the cells could reach 7×10.sup.7 cells/mL.

TABLE-US-00003 TABLE 3 The density and viability of 293 cells cultured by perfusion process (perfusion process 3) 1 × 10.sup.6 cells/mL 5 × 10.sup.6 cells/mL 2VVD 4VVD 2VVD 4VVD Density 85 × 10.sup.6 80 × 10.sup.6 70 × 10.sup.6 80 × 10.sup.6 cells/mL cells/mL cells/mL cells/mL Viability 96% 96% 93% 94%

[0080] The results showed that the perfusion was started after the cell density reached 1×10.sup.6 cells/mL or 5×10.sup.6 cells/mL, and the perfusion rate was maintained constant at 2 VVD or 4 VVD throughout the culture. The highest growth density of the cells could reach 70×10.sup.6 cells/mL.

[0081] Based on the results above, higher cell density could be obtained by using the perfusion process 1, and the cell viability was also higher than that in the processes 2 and 3.

Example 2: Comparisons Between Cell Density and Viability in Perfusion Culture Process and that in Batch Culture Process

[0082] Test 1: the culture was performed by using conventional batch culture process in the art.

[0083] Test 2: the perfusion process 1 in the Example 1 was used, i.e., 293 cells were resuscitated, amplified and then inoculated into a fermenter, and the perfusion was started at a perfusion rate of 2 VVD after the cell density reached 1×10.sup.6 cells/mL. The cells were continuously cultured, and the perfusion rate was adjusted to 3 VVD after the cell density grew to 5×10.sup.6 cells/mL.

[0084] The other parameters (such as culture temperature, pH, concentration of dissolved oxygen and stirring velocity) in the culture process of Tests 1 and 2 were substantially the same, and the cell density and viability after the culture were measured. The results are shown in Table 4.

TABLE-US-00004 TABLE 4 The viability and density of 293 cells cultured by batch process and perfusion process Batch process-Test 1 Perfusion process-Test 2 Density 3.8 × 10.sup.6 cells/mL 13 × 10.sup.7 cells/mL Viability 96% 98%

[0085] The results showed that higher cell density could be obtained by using the perfusion process, and the cell density and viability were higher than those in the batch process.

Example 3: Effect of Glutamine on Cell Production in Perfusion Process

[0086] Test 1: 293 cells were resuscitated, amplified and then inoculated into a fermenter, and the perfusion was started at a perfusion rate of 2 VVD after the cell density reached 1×10.sup.6 cells/mL. The cells were continuously cultured, and the perfusion rate was adjusted to 3 VVD after the cell density grew to 5×10.sup.6 cells/mL. The concentration of glutamine was not supplemented in the perfusion process.

[0087] Test 2: 293 cells were resuscitated, amplified and then inoculated into a fermenter, and the perfusion was started at a perfusion rate of 2 VVD after the cell density reached 1×10.sup.6 cells/mL. The cells were continuously cultured, and the perfusion rate was adjusted to 3 VVD after the cell density grew to 5×10.sup.6 cells/mL. The concentration of glutamine in the perfusion process was monitored, and glutamine was supplemented to maintain the concentration of glutamine at 2 mM.

[0088] Test 3: 293 cells were resuscitated, amplified and then inoculated into a fermenter, and the perfusion was started at a perfusion rate of 2 VVD after the cell density reached 1×10.sup.6 cells/mL. The cells were continuously cultured, and the perfusion rate was adjusted to 3 VVD after the cell density grew to 5×10.sup.6 cells/mL. The concentration of glutamine in the perfusion process was monitored, and glutamine was supplemented to maintain the concentration of glutamine at 10 mM.

[0089] Test 4: 293 cells were resuscitated, amplified and then inoculated into a fermenter, and the perfusion was started at a perfusion rate of 2 VVD after the cell density reached 1×10.sup.6 cells/mL. The cells were continuously cultured, and the perfusion rate was adjusted to 3 VVD after the cell density grew to 5×10.sup.6 cells/mL. The concentration of glutamine in the perfusion process was monitored, and glutamine was supplemented to maintain the concentration of glutamine at 20 mM.

[0090] In the above Tests 1 to 4, the reactor parameters were as follows: the culture temperature was 37° C., the pH value was adjustable from 6.5 to 7.5, the concentration of dissolved oxygen was adjustable from 30% to 80%, and the stirring velocity was adjustable from 30 rpm to 40 rpm.

[0091] The highest cell density and viability after the end of the culture were measured. The results are shown in Table 5.

TABLE-US-00005 TABLE 5 The density and viability of 293 cells cultured by different perfusion processes Test 1 Test 2 Test 3 Test 4 Concentration 0 2 10 20 of glutamine mM mM mM Density 11 × 10.sup.7 29 × 10.sup.7 30 × 10.sup.7 30 × 10.sup.7 cells/mL cells/mL cells/mL cells/mL Viability 98% 99% 99% 99%

[0092] The results showed that higher cell density could be obtained by using the perfusion process with the supplementation of glutamine concentration, and the cell density and viability were higher than those in the process without the supplementation of glutamine.

Example 4: Comparison Between the Highest Density of 293 Cells in Perfusion Culture and that in Batch Culture

[0093] Test 1: the culture was performed by using conventional batch culture process in the art.

[0094] Test 2: the perfusion culture process of the perfusion process test 2 in Example 3 was used, i.e., 293 cells were resuscitated, amplified and then inoculated into a fermenter, and the perfusion was started at a perfusion rate of 2 VVD after the cell density reached 1×10.sup.6 cells/mL. The cells were continuously cultured, and the perfusion rate was adjusted to 3 VVD after the cell density grew to 5×10.sup.6 cells/mL. The concentration of glutamine in the perfusion process was monitored, and glutamine was supplemented to maintain the concentration of glutamine at 2 mM.

[0095] The highest cell density and viability after the end of the culture were measured. The results are shown in FIG. 1.

[0096] The results in FIG. 1 showed that the highest culture density in the batch culture reached 3.8×10.sup.6 cells/mL. In contrast, in the perfusion culture, a fresh medium could be continuously supplemented into the reactor through the continuous perfusion device to replace cell metabolic waste within the reactor and maintain sufficient nutrients for the cells. The cell density cultured by the perfusion process could reach up to 33×10.sup.7 cells/mL, which was about 100 times higher than that of the cells cultured by the batch process. Therefore, the perfusion culture has significant advantages.

Example 5: Virus Yield in Perfusion Culture Process

[0097] Test 1: a perfusion process: perfusion culturing was performed until the cell density reached 10×10.sup.6 cells/mL, recombinant novel coronavirus (adenovirus vector) strains (prepared according to the known prior art) were inoculated, the glutamine concentration in the medium was monitored and then supplemented to 3 mM or more, and the perfusion rate of 1 VVD or 3 VVD was selected. After the completion of culture, the titer of the virus harvest liquid was determined by enzyme-linked immunosorbent assay.

[0098] Test 2: a batch process: viruses were inoculated for batch culture, glutamine was not supplemented, and the perfusion was not performed after the inoculation of viruses.

[0099] Other parameters were substantially the same during the culture in Tests 1 to 2. The results are shown in Table 4.

TABLE-US-00006 TABLE 6 Comparison between virus production of 293 cells in perfusion culture and that in batch culture Perfusion culture Batch culture 1VVD 3VVD N/A Cell 11.2 × 10.sup.6 15.2 × 10.sup.6 0.9 × 10.sup.6 density/viability cells/mL cells/mL cells/mL when inoculating 94% 97% 94% viruses Titer of virus 1.364 × 10.sup.10 1.7×10.sup.10 1.1 × 10.sup.8 harvest liquid IFU/mL IFU/mL IFU/mL Total virus amount 8.6 × 10.sup.14 7.6×10.sup.14 5.5 × 10.sup.12 IFU IFU IFU Single cell yield 1218 IFU/cell 1118 IFU/cell 122 IFU/cell

[0100] Comparing the virus production of single cell in the perfusion culture and that in the batch culture, higher yield of the single cell could be obtained by using the perfusion culture method, which was increased by about 10 times, and the production capacity in single tank was increased by nearly 100 times.

Example 6: Study on Perfusion Culture Process with Higher Virus Specific Activity

[0101] The adenovirus harvest liquids in perfusion culture and in batch culture were purified, respectively. The titer and the number of virus particles were measured, and the specific activities were calculated. The results are shown in FIG. 2.

[0102] The results showed that the specific activity of the adenovirus stock solution obtained by batch culture was 3.8%, and the specific activity of the adenovirus stock solution obtained by perfusion culture was 7.1%. Therefore, more live virus particles could be obtained by perfusion culture of adenovirus, which was superior to that obtained by batch culture.

[0103] The above description is only for the purpose of illustrating the preferred example of the present invention, and is not intended to limit the scope of the present invention.

[0104] Any modifications, equivalents and the like made without departing from the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0105] The foregoing examples and methods described herein may vary based on the abilities, experience, and preferences of those skilled in the art.

[0106] The certain order in which the steps of the method are listed in the present invention does not constitute any limitation on the order of the steps of the method.