SO3 chromatography for use in a method for virus purification
The present invention refers to a process for purifying virus particles from cell culture, comprising the steps of subjecting the cell culture to centrifugation to get a supernatant fraction of virus particles, incubating said fraction with a nuclease, diluting the fraction with low conductivity buffer, subjecting said diluted fraction to a SO3 chromatography step, performing a washing step with low conductivity buffer, and eluting virus particles.
1. A process for purifying virus particles from cell culture, comprising the steps in the following order:
a) subjecting the cell culture to centrifugation to obtain a supernatant fraction of virus particles,
b) incubating the supernatant fraction with a nuclease,
c) diluting the supernatant fraction with a low conductivity dilution buffer to obtain a diluted fraction,
d) subjecting the diluted fraction to a SO3 chromatography step,
e) performing a washing step with low conductivity wash buffer, wherein the wash buffer has a conductivity of 12 mS/cm or less, and
f) eluting virus particles.
2. The process of claim 1 , wherein the virus is an influenza virus.
3. The process of claim 1 , wherein the virus particles are diluted after elution.
4. The process according to claim 1 , wherein an ultra and/or sterile filtration is performed after elution.
5. The process according to claim 1 , wherein the SO3 chromatography step is a column chromatography step.
6. The process according to claim 1 , wherein the dilution buffer has a conductivity of 5 mS/cm or less.
7. The process according to claim 1 , wherein the wash buffer further contains EDTA.
8. The process according to claim 1 , wherein the dilution buffer or the wash buffer comprises a component selected from the group consisting of Hepes, Tris, Tris base, potassium phosphate, sodium phosphate, Mops, Bis-Tris propane Bicine, Mes and Bis-Tris.
9. The process according to claim 1 , wherein the dilution buffer or the wash buffer further contains sucrose, sorbitol and/or mannitol.
10. The process according to claim 1 , wherein virus is eluted with a stepwise elution or linear gradient elution.
11. The process according to claim 2 , wherein the influenza virus is selected from the group consisting of influenza A, influenza B and influenza C virus.
12. The process according to claim 11 , wherein the influenza virus is selected from the group consisting of a wild type influenza virus and an influenza virus containing modifications, wherein the modifications comprise substitution mutations, insertions and/or deletions.
13. The process according to claim 1 , wherein centrifugation is performed at a speed of is least 2,000 g, at least 3,000 g, at least 4,000 g, or at least 4,100 g.
14. The process according to claim 1 , wherein cell culture is performed with Vero cells.
15. The process according to claim 1 , wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of cell protein is removed.
16. The process according to claim 1 , wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of cell DNA is removed.
17. The process according to claim 5 , wherein the column chromatography step is performed with a methacrylate monolith column.
18. The process according to claim 6 , wherein the dilution buffer has a conductivity of 1 mS/cm or less.
19. The process according to claim 10 , wherein the linear gradient elution is a sodium chloride gradient elution.