Methods of heat inactivation of adenovirus
Inventors
Morrison, Christopher J. • Maratt, James D.
Assignees
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Publication Number
US-11944658-B2
Publication Date
2024-04-02
Expiration Date
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.
Core Innovation
The invention relates to a method of inactivating helper virus in a sample containing helper virus, adeno-associated virus particles, and a buffer. The sample is heated to a temperature of 45 °C to 55 °C and maintained at that temperature for a time period of 1 minute to 6 hours, thereby inactivating helper virus. The buffer includes either 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.
The method protects AAV genomic integrity and biological activity during helper-virus inactivation by including kosmotropic salts and/or divalent or trivalent cations in the buffer. The approach addresses the background problem in which heat inactivation of helper virus can also degrade AAV genomic integrity and/or reduce AAV activity. The disclosed buffer compositions are used in combination with the defined heating conditions to achieve helper-virus inactivation while limiting adverse effects on the AAV particles.
The disclosure further characterizes the outcomes in terms of helper-virus log reductions and AAV genome degradation dependence on AAV serotype and vector genome size. Oversized AAV genomes are described as more heat-sensitive. It is reported that kosmotropic salts and cations improve post-heat AAV recovery, including recovery measured by DRP-qPCR, with reported helper-virus inactivation reaching log reductions greater than 5 and greater than 6.3 (limit of detection) in example helper-virus inactivation.
Claims Coverage
The independent claim is directed to heating a sample containing helper virus and adeno-associated virus particles in a defined buffer to inactivate helper virus, with inventive coverage anchored by the buffer-defined cation/kosmotrope ranges and the heat inactivation conditions. Dependent inventive features further refine buffer components and helper-virus identity and include quantitative constraints such as target helper-virus log reduction and sub-ranges for buffer concentration.
Heat inactivation with defined buffer cations or kosmotropic salts
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.
Helper-virus log reduction of 6.3 or greater
The method results in a log reduction of helper virus of 6.3 or greater.
Divalent or trivalent cations in 25 mM to 400 mM
The method uses divalent or trivalent cations at a concentration within the range of 25 mM to 400 mM.
Selected metal cations for divalent or trivalent cations
The buffer includes divalent or trivalent metal cations selected from Mg, Ca, Mn, Ni, Zn, Co, Sr, Cu, Cr, Fe, and Sc.
Kosmotropic salt selected from specified list
The buffer comprises a kosmotropic salt selected from ammonium sulfate, ammonium acetate, sodium citrate, sodium acetate, sodium sulfate, potassium phosphate, and cesium chloride.
Helper virus is an adenovirus
The helper virus is an adenovirus.
Across the independent claim and dependent refinements, the inventive concept is the combination of defined heating conditions with a buffer formulation defined by either divalent or trivalent cations or kosmotropic salts. Additional refinements include a quantitative helper-virus log reduction target of 6.3 or greater and specifying the helper virus as adenovirus.
Stated Advantages
Inactivates helper virus while protecting AAV genomic integrity and biological activity.
Provides helper-virus inactivation reaching log reductions greater than 5 and greater than 6.3 (limit of detection) in reported examples.
Improves post-heat AAV recovery, including recovery measured by DRP-qPCR.
Limits AAV genome degradation effects that depend on AAV serotype and vector genome size, with oversized genomes described as more heat-sensitive.
Documented Applications
Heat inactivation of helper virus in a sample containing adeno-associated virus particles while maintaining AAV recovery and integrity.
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