Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells

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DOIResolve DOI: http://doi.org/10.1002/biot.201500176
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TypeArticle
Journal titleBiotechnology Journal
ISSN1860-6768
Volume11
Issue2
Pages290297; # of pages: 8
SubjectBioreactors; Body fluids; DNA; Gene therapy; Mammals; Manufacture; Productivity; Viruses; Adeno-associated virus; Bioprocess optimization; HEK293T; ICELLis; Manufacturing strategy; Recombinant adeno-associated virus; Stirred tank bioreactors; Volumetric productivity; Vectors
AbstractViral vectors used for gene and oncolytic therapy belong to the most promising biological products for future therapeutics. Clinical success of recombinant adeno-associated virus (rAAV) based therapies raises considerable demand for viral vectors, which cannot be met by current manufacturing strategies. Addressing existing bottlenecks, we improved a plasmid system termed rep/cap split packaging and designed a minimal plasmid encoding adenoviral helper function. Plasmid modifications led to a 12-fold increase in rAAV vector titers compared to the widely used pDG standard system. Evaluation of different production approaches revealed superiority of processes based on anchorage- and serum-dependent HEK293T cells, exhibiting about 15-fold higher specific and volumetric productivity compared to well-established suspension cells cultivated in serum-free medium. As for most other viral vectors, classical stirred-tank bioreactor production is thus still not capable of providing drug product of sufficient amount. We show that manufacturing strategies employing classical surface-providing culture systems can be successfully transferred to the new fully-controlled, single-use bioreactor system IntegrityTM iCELLisTM. In summary, we demonstrate substantial bioprocess optimizations leading to more efficient and scalable production processes suggesting a promising way for flexible large-scale rAAV manufacturing.
Publication date
PublisherWiley
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC)
Peer reviewedYes
NPARC number21277505
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Record identifier155978b4-5776-493c-9910-871683b2cd98
Record created2016-03-09
Record modified2016-05-09
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