Method for purifying enveloped viruses or viral vectors
The invention relates to a process for purifying enveloped viruses. The process of the invention is useful for recovering at a large scale enveloped viruses under conditions complying with good manufacturing practices and allowing viruses of a clinical grade to be obtained.
1. A process for purifying a pseudotyped lentivirus comprising an anion exchange chromatography step, the buffers used during said chromatography being:
of a pH below 6, or
of a pH greater than or equal to 6 and further comprising a polyol.
2. The process according to claim 1 , said anion exchange chromatography buffer(s) having a pH of less than 6 and also comprising a polyol.
3. The process according to claim 1 , the pH of the buffers being between 5.5 and 6.
4. The process according to claim 1 , the anion exchange chromatography step being preceded with an ultrafiltration/diafiltration step.
5. The process according to claim 4 , the ultrafiltration/diafiltration step comprising the use of one or several buffers with a pH between 5.5 and 7.5, said buffer(s) optionally comprising a polyol.
6. The process according to claim 1 , the process comprising:
(a) clarification of a cell culture medium containing cells producing said pseudotyped lentivirus thereby obtaining a clarified lentivirus;
(b) an ultrafiltration/diafiltration step for the clarified lentiviruses;
(c) an anion exchange chromatography; and
(d) an exclusion chromatography.
7. The process according to claim 6 , step (a) being carried out by filtration of the culture medium on a retention filter for which the retention threshold is between 0.2 and 0.45 μm.
8. The process according to claim 6 , step (b) being carried out by means of a tangential flow filtration.
9. The process according to claim 6 , step (d) comprising the use of an exclusion resin having an exclusion size between 300 and 1,000 kDa.
10. The process according to claim 6 , the resin used for the exclusion chromatography being a multimode resin, having a dual functionality of exclusion and adsorption.
11. The process according to claim 1 , the purified lentivirus being produced in a neutral medium or in a moderately acid medium.
12. The process according to claim 1 , the polyol being selected from sucrose, mannitol, sorbitol and trehalose.
13. The process according to claim 5 , the polyol being present in the buffer at a concentration between 1.5% and 15% by weight in the buffer.
14. The process according to claim 4 , the polyol being present in the buffers used during the ultrafiltration/diafiltration step and an anion exchange chromatography.
15. The process according to claim 6 , the polyol being present in the buffers in all the steps of the purification process.
16. The process according to claim 1 , the buffers used during said process also comprising a magnesium salt at a concentration between 0.1 mM and 5 mM.
17. The process according to claim 1 , wherein the anion exchange chromatography is a weak anion exchange chromatography and/or an anion exchange chromatography on a column.
18. A process for purifying a pseudotyped lentivirus, said process comprising an ultrafiltration/diafiltration step, said step being carried out using buffers containing a polyol.
19. The process according to claim 1 , wherein the lentivirus is pseudotyped with the GaLV-TR, VSV-g or MV envelope glycoprotein.
20. The process according to claim 1 , wherein the lentivirus is pseudotyped with the GaLV-TR envelope glycoprotein.
21. The process according to claim 1 , wherein the lentivirus is pseudotyped with the VSV-g envelope glycoprotein.
22. The process according to claim 13 , wherein the polyol in the buffer is sucrose at a concentration between 2% and 8% by weight.
23. The process according to claim 22 , where the polyol in the buffer is sucrose at a concentration of 5% by weight.