Formulation of sugar solutions for continuous ultracentrifugation for virus purification
View Patent ↗The present invention provides a method for purification of a virus or virus antigen comprising providing a virus preparation and centrifugation of said virus preparation in a gradient of a sugar established by the addition of two or more buffered sugar layers of different concentration. The method leads to higher yields and reduces unwanted aggregation of the virus or virus antigen by increasing the volume of the peak pool.
1. A method for purifying a virus, comprising
providing
(i) a buffered sugar solution having a density of 1.15 kg/l to 1.23 kg/l and comprising a first physiological buffer and
(ii) a buffered sugar solution having a density of 1.23 kg/l to 1.32 kg/l and which is higher than the buffered sugar solution (i), and comprising a physiological buffer which is the same or different from the physiological buffer of (i),
wherein the volume ratio of buffered sugar solution (i) to buffered sugar solution (ii) is greater than or equal to 3:1;
providing a virus preparation;
centrifuging the virus preparation and buffered sugar solutions (i) and (ii) in a ultracentrifuge comprising an ultracentrifuge rotor to obtain a peak pool; and
extracting the peak pool to obtain the virus.
2. The method of claim 1 , wherein the volume of buffered sugar solutions (i) and (ii) is between 5% to 100% of the volume of the ultracentrifugation rotor.
3. The method of claim 1 , wherein the peak pool has a density between 1.20 kg/l to 1.24 kg/l.
4. The method of claim 1 , wherein one of said buffers has a concentration of between 5 mM to 50 mM.
5. The method of claim 4 , wherein said buffer has a concentration of between 15 mM to 30 mM.
6. The method of claim 5 , wherein said buffer has a concentration of between 18 mM to 25 mM.
7. The method of claim 1 , wherein the step of centrifuging the virus preparation and buffered sugar solutions is performed with a relative centrifugation of at least 20,000 g.
8. The method of claim 7 , wherein said relative centrifugation force is at least 30,000 g.
9. The method of claim 8 , wherein said relative centrifugation force is at least 90,000 g.
10. The method of claim 1 , wherein said volume ratio of said buffered sugar solution (i) to said buffered sugar solution (ii) is less than 20:1.
11. The method of claim 10 , wherein said volume ratio is less than 10:1.
12. The method of claim 11 , wherein said volume ratio is less than 8:1.
13. The method of claim 12 , wherein said volume ratio is between 6:1 to 3:1.
14. The method of claim 1 , wherein the virus preparation is obtained by centrifugation without a preclarifier.
15. The method of claim 1 , wherein said virus preparation comprises cells inoculated with the virus.
16. The method of claim 15 , wherein said cells are of an animal cell culture or cell line.
17. The method of claim 15 , wherein said cells are epithelial cells.
18. The method of claim 17 , wherein said cells are kidney epithelial cells.
19. The method of claim 18 , wherein said cells are Vero cells.
20. The method of claim 1 , wherein at least one of the physiological buffers is an amine buffer.
21. The method of claim 20 , wherein at least one of the physiological buffers is a trishydroxymethylaminomethane (TRIS) buffer.
22. The method of claim 21 , wherein the physiological buffer is TRIS-buffered saline.
23. The method of claim 1 , wherein the volume of buffered sugar solutions (i) and (ii) is less than 100% of the volume of the ultracentrifuge rotor.
24. The method of claim 23 , wherein the volume of buffered sugar solutions (i) and (ii) is less than 90% of the volume of the ultracentrifuge rotor.
25. The method of claim 1 , wherein the volume of buffered sugar solutions (i) and (ii) is greater than or equal to 50% of the volume of the ultracentrifuge rotor.
26. The method of claim 24 , wherein the volume of buffered sugar solutions (i) and (ii) is less than 90% of the volume of the ultracentrifuge rotor.
27. The method of claim 1 , wherein the virus is a parvovirus.