Methods of heat inactivation of adenovirus
AssigneeUltragenyx Pharmaceutical Inc.
InventorsChristopher J. Morrison, James D. Maratt
Life of this patent
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Abstract
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.
Claims21
1-55. (canceled)
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.
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.
Dependencies are inferred from the claim text.