Purification of herpes virus
The present disclosure provides a method to prepare purified enveloped viral particle preparations employing ion exchange chromatography and tangential flow filtration.
1. A method for the purification of herpes simplex virus (HSV) particles from a mammalian host cell culture comprising the steps of:
a) treating the mammalian host cell culture with a viral releasing agent to release the HSV particles from the host cells;
b) treating the material from step (a) with an endonuclease;
c) filtering the material from step (b) to remove any intact cells or cellular debris;
d) applying the solution obtained from step (c) to an anion exchange chromatography resin;
e) eluting the HSV particles from the anion exchange column;
f) subjecting the eluent from step (e) to tangential flow filtration; and
g) recovering the purified HSV particles.
2. The method of claim 1 , wherein the purified HSV particles contain greater than 1×10 7 PFU/mL.
3. The method of claim 1 , wherein the endonuclease is a genetically engineered endonuclease from Serratia marcescens produced and purified from E. coli strain W3110.
4. The method of claim 1 , wherein the viral releasing agent is dextran sulfate.
5. The method of claim 1 , wherein the purified HSV particles contain less than 10 ng host cell DNA per 1×10 7 plaque forming units (PFU).
6. The method of claim 1 , wherein the HSV is a replication defective HSV.
7. The method of claim 6 , wherein the replication defective HSV is HSV529.
8. The method of claim 1 , wherein the tangential flow filtration is a hollow fiber system.
9. The method of claim 8 , wherein the hollow fiber system has a molecular weight cutoff of 100 kDa.
10. The method of claim 1 , wherein the anion exchange chromatography comprises a membrane-based chromatography resin.
11. A method for the large scale purification of herpes simplex virus (HSV) particles from a mammalian host cell culture comprising the steps of:
a) treating the mammalian host cell culture with a viral releasing agent to release the HSV particles from the host cells;
b) treating the material from step (a) with an endonuclease;
c) filtering the material from step (b) to remove any intact cells or cellular debris;
d) applying the solution obtained from step (c) to an anion exchange chromatography resin, wherein the anion exchange chromatography comprises a membrane-based chromatography resin comprising quaternary amino groups or diethylaminoethane groups;
e) eluting the HSV particles from the anion exchange column;
f) subjecting the eluent from step (e) to tangential flow filtration, wherein the tangential flow filtration is a hollow fiber system and wherein the hollow fiber system has a molecular weight cutoff of between 250-50 kDa; and
g) recovering the purified HSV particles, wherein the purified HSV particles contain greater than 1×10 7 PFU/mL and less than 10 ng host cell DNA per 1×10 7 plaque forming units (PFU).
12. The method of claim 11 , wherein in step (c) the material from step (b) is filtered with a porous filter medium.
13. The method of claim 11 , wherein the HSV is a replication defective HSV529 and the mammalian host cell is a Vero cell line containing the UL29 and UL5 genes.
14. The method of claim 13 , wherein 100-300 doses of purified HSV particles are recovered in step (g), wherein a dose of purified HSV is 1×10 7 PFU.
15. The method of claim 11 , further comprising before step (a) a step of growing the mammalian host cell culture in a volume of at least 2 liters and a cell culture area of at least 6,320 cm 2 .
16. The method of claim 13 , wherein, the amount of Vero host cell protein following the elution step (e) is no more than 3 μg/mL.