Methods of heat inactivation of adenovirus
The present disclosure generally relates to methods of protecting the genomic integrity and/or biological activity of AAV viral particles in a sample containing both AAV particles and helper virus particles during heat inactivation. The methods include heating, to a temperature greater than or equal to 45′C, a sample containing helper virus particles, AAV particles, and a buffer. The buffer includes a concentration of 10 mM or greater kosmotropic salts and/or a concentration of 10 mM or greater of divalent or trivalent cations.
1. A method of inactivating helper virus in a sample containing helper virus, adeno-associated virus particles, and a buffer comprising a concentration of 25 mM to 500 mM of divalent or trivalent cations, or a concentration of 0.5 M to 0.6 M of kosmotropic salts, wherein the method comprises:
heating the sample to a temperature of 45° C. to 55° C.,
and maintaining the sample at the temperature for a time period of 1 minute to 6 hours, thereby inactivating helper virus.
2. The method of claim 1 , wherein the sample is maintained at the temperature for a time period of 10 to 180 minutes.
3. The method of claim 1 , wherein the method results in a log reduction of helper virus of 6.3 or greater.
4. The method of claim 1 , wherein the adeno-associated virus particle comprises a genome of approximately or more than 4.7 kb of DNA.
5. The method of claim 1 , wherein the buffer further comprises a chaotropic salt.
6. The method of claim 1 , wherein the buffer further comprises a polyol selected from the group consisting of: glycerol, propylene glycol, and 1,6-Hexanediol.
7. The method of claim 1 , wherein the buffer maintains a pH of 3.0 to 10.0 at temperatures of 4° C. to 70° C.
8. The method of claim 1 , wherein the buffer further comprises: 40 mM bis-tris propane, 20 mM HEPES, 20 mM citrate, 200 mM NaCl, and 0.001% (w/v) Pluronic F68.
9. The method of claim 7 , wherein the buffer is a Tris buffer, a phosphate buffer, or a triazolamine buffer.
10. The method of claim 1 , wherein the helper virus is an adenovirus.
11. The method of claim 10 , where the adenovirus is Ad5.
12. The method of claim 1 , wherein the concentration of divalent or trivalent cations is 25 mM to 400 mM.
13. The method of claim 1 , wherein the buffer-comprises divalent or trivalent cations of a metal selected from the group consisting of: Mg, Ca, Mn, Ni, Zn, Co, Sr, Cu, Cr, Fe, and Sc.
14. The method of claim 13 , wherein the cations are selected from the group consisting of: Mg 2+ , Ca 2+ , Mn 2+ , Ni 2+ , Zn 2+ , Co 2+ , Sr 2+ , Cu 2+ and Cr 2+ .
15. The method of claim 14 wherein the cations are Ca 2+ .
16. The method of claim 14 wherein the cations are Mg 2+ .
17. The method of claim 13 , wherein the cations are Sc 3+ .
18. The method of claim 1 , wherein the buffer comprises a kosmotropic salt selected from the group consisting of ammonium sulfate, ammonium acetate, sodium citrate, sodium acetate, sodium sulfate, potassium phosphate, and cesium chloride.
19. The method of claim 18 , wherein the buffer comprises ammonium sulfate.
20. The method of claim 18 , wherein the concentration of kosmotropic salts is 0.5 M.