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 adenovirus in a sample containing adenovirus, adeno-associated virus particles, and a buffer comprising a concentration of 10 mM or greater of divalent or trivalent cations, or a concentration of 10 mM or greater of kosmotropic salts, wherein the method comprises:
heating the sample to a temperature greater than or equal to 45° C., thereby inactivating adenovirus.
2. The method of claim 1 , wherein the sample is heated to a temperature between 45° C. and 65° C.
3. The method of claim 1 , wherein the sample is maintained at the temperature for a time period of between 1 minute and 6 hours.
4. The method of claim 1 , wherein the method results in a log reduction of adenovirus of 3.7 or greater.
5. The method of claim 1 , wherein the adeno-associated virus particles comprise a genome of more than 4.7 kb of DNA.
6. The method of claim 1 , wherein the adeno-associated virus particles comprise a genome that is substantially self-complementary.
7. The method of claim 1 , wherein the buffer further comprises a chaotropic salt.
8. The method of claim 7 , wherein the chaotropic salt is a salt of urea or a salt of guanidine.
9. 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.
10. The method of claim 1 , wherein the buffer maintains pH between 3.0 and 10.0 at temperatures between 4° C. and 70° C.
11. 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.
12. The method of claim 10 , wherein the buffer is a Tris buffer; a phosphate buffer; or a triazolamine buffer.
13. The method of claim 1 , wherein the adenovirus is Ad5.
14. The method of claim 1 , wherein the concentration of divalent or trivalent cations is 10 mM to 500 mM.
15. The method of claim 1 , wherein the buffer comprises a concentration of 10 mM or greater of divalent or trivalent cations of metal selected from the group consisting of: Mg, Ca, Mn, Ni, Zn, Co, Sr, Cu, Cr, Fe, and Sc; wherein the divalent 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+ ; wherein the trivalent cations are Sc 3+ .
16. The method of claim 15 , wherein the cations are Mg 2+ .
17. The method of claim 1 , wherein the buffer comprises a concentration of 10 mM or greater of kosmotropic salts selected from the group consisting of ammonium sulfate, ammonium acetate, sodium citrate, sodium acetate, sodium sulfate, potassium phosphate, and cesium chloride.
18. The method of claim 17 , wherein the buffer comprises a concentration of 10 mM or greater of ammonium sulfate.
19. The method of claim 17 , wherein the concentration of kosmotropic salts is 0.1 M to 1 M.