Method for the production of adenoviral vectors
The invention provides methods for large-scale production of recombinant adenovirus 35, using perfusion systems and infection at very high-cell densities.
1. A method for producing at least 1×10 12 recombinant adenovirus serotype 35 (rAd35) virus particles (VP)/mL, the method comprising:
culturing producer cells in a first bioreactor in suspension with a perfusion system;
transferring the cultured producer cells from the first bioreactor into a second bioreactor with a perfusion system;
infecting the producer cells in the second bioreactor at a density of between 10×10 6 and 16×10 6 viable cells/mL with rAd35; and
further culturing the infected producer cells in the second bioreactor to produce rAd35,
wherein, after between about 1.5 and about 5 days, the produced rAd35 particles in the infected cell culture of the second bioreactor reach a concentration of at least 1×10 12 VP/mL, and the virus particle to infectious particle (VP/IU) ratio of the produced rAd35 in the infected cell culture of the second bioreactor is less than 30:1.
2. The method according to claim 1 , wherein the producer cells are infected with rAd35 at a density of between about 10×10 6 and 14×10 6 viable cells/mL.
3. The method according to claim 1 , wherein the producer cells are cultured with an alternating tangential flow (ATF) perfusion system after infection with rAd35.
4. The method according to claim 1 , wherein the rAd35 lacks at least a portion of the E1 region of the adenovirus genome, and wherein the rAd35 comprises a heterologous nucleic acid.
5. The method according to claim 1 , wherein the producer cells are cultured with an alternating tangential flow (ATF) perfusion system prior to infection with rAd35.
6. The method according to claim 1 , wherein the particle to infectious particle (VP/IU) ratio of the produced rAd35 in the infected cell culture of the second bioreactor is less than 20:1.
7. A bioreactor comprising:
culture medium,
producer cells in a suspension thereof, and
at least 1×10 12 recombinant adenovirus serotype 35 (rAd35) virus particles/mL (VP/mL), wherein the virus particles in the infected cell culture have an infectious particle (VP/IU) ratio of less than 30:1, and wherein the bioreactor has a working volume of between 2 L and 1000 L.
8. The bioreactor of claim 7 , wherein the bioreactor comprises an alternating tangential flow (ATF) perfusion system.
9. The method according to claim 1 , further comprising harvesting the rAd35 virus particles from the infected producer cell culture after-between about 1.5 and about 5 days.
10. The bioreactor of claim 7 , wherein the bioreactor has a working volume of between 50 L and 500 L.
11. The bioreactor of claim 10 , wherein the bioreactor is connected to an alternating tangential flow (ATF) perfusion system.
12. The bioreactor of claim 7 , wherein the rAd35 virus particles in the infected cell culture of the second bioreactor have a VP/IU ratio of less than 20:1.
13. The method according to claim 2 , wherein the producer cells are cultured with an alternating tangential flow (ATF) perfusion system after infection with rAd35.
14. The method according to claim 2 , further comprising harvesting rAd35 virus particles from the infected producer cell culture after culturing in the second bioreactor for between about 1.5 and about 5 days.
15. The method according to claim 3 , further comprising harvesting rAd35 virus particles from the infected producer cell culture after culturing in the second bioreactor for between about 1.5 and about 5 days.
16. The method according to claim 9 , further comprising purifying the harvested rAd35 virus particles.
17. The method according to claim 16 , further comprising producing a pharmaceutical composition containing the purified rAd35 virus particles.
18. The method according to claim 14 , further comprising purifying the harvested rAd35 virus particles.
19. The method according to claim 18 , further comprising producing a pharmaceutical composition containing the purified rAd35 virus particles.
20. The method according to claim 15 , further comprising purifying the harvested rAd35 virus particles.
21. The method according to claim 20 , further comprising producing a pharmaceutical composition containing the purified rAd35 virus particles.
22. A method for producing at least 1×10 12 recombinant adenovirus serotype 35 (rAd35) virus particles (VP)/mL, wherein the rAd35 is replication deficient because of a deletion in the E1 region of the adenovirus genome, and wherein the producer cells complement the deletion in the E1 region, the method comprising:
culturing producer cells in suspension in a first bioreactor having an alternating tangential flow (ATF) perfusion system;
transferring producer cells from the first bioreactor to a second bioreactor having an ATF perfusion system;
infecting the producer cells at a density of between 10×10 6 and 16×10 6 viable cells/mL with rAd35; and
further culturing the infected producer cells in suspension with the perfusion system in the second bioreactor to produce rAd35 virus particles
wherein the produced rAd35 particles in the infected cell culture reach a concentration of at least 1×10 12 VP/mL after between about 2 and about 3 days,
wherein the producer cells produce the rAd35 virus particles with a unit productivity of between about 0.5×10 5 and 1.5×10 5 VP/cell, and
wherein the virus particle to infectious particle (VP/IU) ratio of the produced rAd35 virus particles in the infected cell culture is less than 20:1 after between about 2 and about 3 days.
23. The method according to claim 22 , wherein the rAd35 comprises a heterologous nucleic acid.
24. The method according to claim 22 , wherein the producer cells produce the rAd35 virus particles with a unit productivity of between 4.7×10 4 VP/cell and 1.2×10 5 VP/cell.
25. The method according to claim 1 , wherein the producer cells are mammalian cells.
26. The bioreactor of claim 7 , wherein the producer cells are mammalian cells.
27. The method according to claim 22 , wherein the producer cells are mammalian cells.