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 comprising the steps of:
a) subjecting HSV particles to tangential flow filtration, wherein the tangential flow filtration is a hollow fiber system and the tangential flow filtration step has a transmembrane pressure of less than 4 psi; and
b) 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).
2. The method of claim 1 , wherein the hollow fiber system has a molecular weight cutoff of between 250-50 kDa.
3. The method of claim 1 , wherein 100-300 doses of purified HSV particles are recovered in step (b), wherein a dose of purified HSV is 1×10 7 PFU.
4. The method of claim 1 , wherein the HSV particles are obtained from a mammalian host cell culture that has been grown in a volume of at least 2 liters and a cell culture area of at least 6,320 cm 2 .
5. The method of claim 1 , wherein the HSV is herpes simplex virus type-2 (HSV-2) or herpes simplex virus type-1 (HSV-1).
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 4 , further comprising before step (a) the steps of:
(i) treating the mammalian host cell culture with a viral releasing agent to release HSV particles from the host cells; and
(ii) purifying the material of step (i) by a non-centrifugation-based method.
9. The method of claim 8 , wherein the product of step (ii) is treated with an endonuclease.
10. The method of claim 8 , wherein the material of step (i) is purified by anion exchange chromatography.
11. The method of claim 1 , wherein the purified HSV particles are recovered in a liquid stabilization buffer and wherein the liquid stabilization buffer comprises glutamate, 1-20 mM histidine and optionally glutamine, a salt, and a sugar, and wherein other than histidine and optionally glutamine, the liquid stabilization buffer does not contain any other amino acids.
12. The method of claim 11 , wherein the liquid stabilization buffer comprises 20-75 mM potassium glutamate, 1-20 mM histidine, 50-250 mM salt, and 5-20% sugar.
13. The method of claim 12 , wherein the liquid stabilization buffer comprises 50 mM potassium glutamate, 10 mM histidine, 160 mM salt, and 10% sugar.
14. The method of claim 13 , wherein the sugar is sucrose.
15. The method of claim 2 , wherein the hollow fiber system has a molecular weight cutoff of 100 kDa.
16. A method for the purification of Herpes Simplex Virus (HSV) particles comprising the steps of:
a) treating a mammalian host cell culture comprising host cells that comprise HSV particles with a viral releasing agent to release the HSV particles from the host cells;
b) purifying material of step (a) by a non-centrifugation-based method;
c) subjecting the HSV particles to tangential flow filtration, wherein the tangential flow filtration is a hollow fiber system and the tangential flow filtration step has a transmembrane pressure of less than 4 psi; and
d) 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).
17. A method for the purification of Herpes Simplex Virus (HSV) particles comprising the steps of:
a) subjecting HSV particles to tangential flow filtration, wherein the tangential flow filtration is a hollow fiber system and the tangential flow filtration step has a transmembrane pressure of less than 4 psi; and
b) 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), wherein the purified HSV particles are recovered in a liquid stabilization buffer that comprises glutamate, 1-20 mM histidine and optionally glutamine, a salt, and a sugar, and wherein other than histidine and optionally glutamine, the liquid stabilization buffer does not contain any other amino acids.