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 - 55 . (canceled)
56 . A method for manufacturing recombinant adeno-associated virus (rAAV) particles comprising:
a) producing a sample containing helper virus and rAAV particles by culturing mammalian or insect cells in a cell culture and providing the mammalian or insect cells with: (i) a plasmid that contains a rAAV genome, an AAV rep gene function and an AAV cap gene function; and (ii) helper virus; and
b) inactivating the helper virus in the sample by heating the sample to a temperature greater than or equal to 45° C. for between one minute and six hours in the presence of a buffer, wherein the buffer comprises a concentration of 10 mM or greater of divalent or trivalent cations, or a concentration of 10 mM or greater of kosmotropic salt.
57 . The method of claim 56 , wherein the inactivating step comprises heating the sample to a temperature between 45° C. and 65° C.
58 . The method of claim 56 , wherein the inactivating step comprises maintaining the temperature for a time period of between 10 and 180 minutes.
59 . The method of claim 56 , wherein the rAAV particle comprises a genome of 3.0 kb of DNA to 5.2 kb of DNA.
60 . The method of claim 56 , wherein the buffer further comprises:
a) a chaotropic salt; or
b) a polyol selected from the group comprising: glycerol, propylene glycol, and 1,6-hexanediol.
61 . The method of claim 56 , wherein the buffer maintains a pH between 3.0 and 10.0 at temperatures between 4° C. and 70° C.
62 . The method of claim 61 , wherein the buffer is a Tris buffer, a phosphate buffer, or a triazolamine buffer.
63 . The method of claim 56 , wherein the buffer further comprises: 40 mM bis-tris propane, 20 mM HEPES, 20 mM citrate, 200 mM NaCI, and 0.001% (w/v) Pluronic F68.
64 . The method of claim 56 , wherein the helper virus is an adenovirus.
65 . The method of claim 64 , wherein the adenovirus is Ad5.
66 . The method of claim 56 , wherein the concentration of divalent or trivalent cations is 25 mM to 500 mM.
67 . The method of claim 56 , wherein the divalent or trivalent cations are selected from the group consisting of: Mg, Ca, Mn, Ni, Zn, Co, Sr, Cu, Cr, Fe, and Sc.
68 . The method of claim 67 , 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+ .
69 . The method of claim 67 , wherein the trivalent cations are Sc 3+ .
70 . The method of claim 56 , wherein the kosmotropic salt is selected from the group consisting of ammonium sulfate, ammonium acetate, sodium citrate, sodium acetate, sodium sulfate, potassium phosphate, and cesium chloride.
71 . The method of claim 70 , wherein the concentration of the kosmotropic salt is 0.5 M to 0.6 M.
72 . A sample comprising rAAV particles and inactivated helper virus particles, wherein the sample is produced according to the method of claim 56 .
73 . A sample comprising rAAV particles purified from a sample containing rAAV particles and inactivated helper virus particles produced according to the method of claim 56 .
74 . A method for manufacturing rAAV particles comprising:
a) producing a sample containing helper virus and rAAV particles by culturing mammalian or insect cells in a cell culture and providing the mammalian or insect cells with: (i) a plasmid that contains a rAAV genome, an AAV rep gene function and an AAV cap gene function; and (ii) helper virus; and
b) inactivating the helper virus in the sample by heating the sample to a temperature greater than or equal to 45° C. for between one minute and six hours in the presence of a buffer, wherein the buffer comprises 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 salt.
75 . A plurality of rAAV particles manufactured according to the method of claim 74 .