High yield suspension cell line, system and method for making same
Inventors
Denning, Gabriela D. C. • Gautney, Richard E
Assignees
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Publication Number
US-8986991-B2
Publication Date
2015-03-24
Expiration Date
Abstract
A system and method of adapting host cells to suspension cell culture and suspension cell lines ATCC PTA-12593 and ATCC PTA-12461 produced thereby are disclosed. The method includes the serial replating of substantially undiluted culture cells onto a surface area until cell clumps are visualized and then, upon cell clumping, moving the cells into a suspension culture system.
Core Innovation
The disclosure adapts previously adherent mammalian cells to suspension culture and provides high-yield clumped suspension cell lines, including BHK-Ms and derived clones. The adapted suspension cell lines are described as producing recombinant protein in serum-free/blood-protein-free media, and specific examples are identified as ATCC PTA-12593 and ATCC PTA-12461.
The disclosure addresses the need for improved recombinant protein production, including improved expression and manufacturability, for cells adapted to suspension culture while operating in serum-free/blood-protein-free media. It frames this as overcoming limitations associated with adherent cell formats by enabling suspension production and enhanced output.
As an example of the improved recombinant output, the disclosure reports enhanced expression and activity for fVIII constructs, including ET-801 and ET-3, in the adapted suspension cell lines. Performance improvements are stated as reported 5% to 1000% greater expression, and increased fVIII activity is described across spinner/flask and serum-free scale.
The disclosure further describes that suspension-adapted cells can produce functional virus, including third-generation lentivirus. It also includes characterization and in vivo hemophilia A mouse efficacy with reduced blood loss after ET-801 administration, tied to recombinant fVIII activity produced by the suspension-adapted cell lines.
Claims Coverage
The partial claims content provided contains one independent claim and two dependent claims. The independent claim is directed to a specific cell line, while the dependent claims add inventive limitations regarding quantitative expression improvement and the specific recombinant polypeptide expressed.
Specific cell line ATCC PTA-12461
Cell line ATCC PTA-12461.
Quantitative expression superiority of suspension cells
A suspension cell line exhibits about 5% to about 1000% greater expression of a polypeptide than the expression of the same polypeptide in a derived adherent cell line.
Suspension cell line expressing fVIII
A cell line is provided that expresses the polypeptide fVIII as recited.
Overall, the claim coverage centers on a specific suspension cell line identified as ATCC PTA-12461, with additional limitations requiring about a 5% to about 1000% greater polypeptide expression versus a derived adherent cell line and an fVIII-expressing suspension phenotype.
Stated Advantages
Enhanced recombinant protein production in serum-free/blood-protein-free media.
Improved polypeptide expression, reported as about 5% to about 1000% greater expression in a suspension cell line versus a derived adherent cell line.
Increased fVIII activity across spinner/flask and serum-free scale.
Ability of suspension-adapted cells to produce functional virus, including third-generation lentivirus.
Demonstrated in vivo hemophilia A mouse efficacy with reduced blood loss after ET-801 administration.
Documented Applications
Recombinant fVIII production using suspension-adapted clumped suspension cell lines, including fVIII constructs ET-801 and ET-3.
Virus production using suspension-adapted cells, including functional third-generation lentivirus.
In vivo hemophilia A mouse efficacy assessment, where reduced blood loss is reported after ET-801 administration.
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