Use of tonicifying agents to enhance recombinant adeno-associated virus yield
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Publication Number
US-11795473-B2
Publication Date
2023-10-24
Expiration Date
Abstract
The invention provides methods for the production of recombinant adeno-associated virus vectors (rAAV), comprising culturing producer cells in media with increased osmolality. Also provided are methods for decreasing the production of helper virus by a rAAV producer cell, comprising culturing the producer cell in media with increased osmolality.
Core Innovation
The invention relates to an rAAV production system that increases total rAAV production compared to total rAAV production from a system comprising a cell culture medium having an osmolality of 266 mOsm/kg. The system comprises HeLa host cells capable of producing rAAV and a helper virus, where a cell culture medium having an osmolality of 360 mOsm/kg or higher is used when measured immediately after the HeLa host cells are introduced into the cell culture medium and throughout rAAV production. The medium includes one or more non-ionic tonicifying agents selected from sucrose, fructose, glucose, galactose, mannose, maltose, and trehalose.
The disclosed approach addresses the problem that helper virus production can accompany rAAV production, and the invention targets conditions that increase rAAV output while reducing helper virus output. The described comparisons use a baseline system with an osmolality of 266 mOsm/kg, and higher osmolality conditions are associated with changes in total helper virus production and total rAAV production. The document further specifies that the medium osmolality can be chosen to achieve specified percentage decreases in total helper virus production and/or specified percentage increases in total rAAV production versus the 266 mOsm/kg baseline.
In addition to non-ionic tonicifying agents, the document discloses embodiments using ionic tonicifying agents for increasing cell culture osmolality, and it includes example comparisons regarding extracellular rAAV and helper virus levels. The document describes tonicifying-agent selections, osmolality thresholds, and helper-virus-related outcomes measured using qPCR, including differentiation between extracellular and total rAAV and extracellular and total helper virus.
Claims Coverage
The independent claim covers an rAAV production system that increases total rAAV production versus a baseline system using a cell culture medium at 266 mOsm/kg, using HeLa host cells and a helper virus in a medium at 360 mOsm/kg or higher that includes one or more non-ionic tonicifying agents. The claim further requires that the osmolality be maintained immediately after introduction of the HeLa host cells and throughout rAAV production.
HeLa-based rAAV production with helper virus under maintained high osmolality
An rAAV production system comprising HeLa host cells capable of producing rAAV, a helper virus, and a cell culture medium having an osmolality of 360 mOsm/kg or higher when measured immediately after the HeLa host cells are introduced into the cell culture medium and throughout rAAV production.
Non-ionic tonicifying agents in high-osmolality medium
The cell culture medium comprises one or more non-ionic tonicifying agents selected from the group consisting of sucrose, fructose, glucose, galactose, mannose, maltose, and trehalose.
Overall, claim coverage centers on increasing total rAAV production by running a HeLa-plus-helper-virus rAAV production system in a cell culture medium maintained at 360 mOsm/kg or higher, with the medium including selected non-ionic tonicifying agents, and with comparative performance assessed against a 266 mOsm/kg baseline.
Stated Advantages
Increases total rAAV production compared to total rAAV production from a system using a cell culture medium having an osmolality of 266 mOsm/kg.
Can reduce helper virus production while improving rAAV production (helper virus reduction and rAAV increase are described comparatively versus the 266 mOsm/kg baseline).
Documented Applications
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