Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells.
Viral 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 Integrity(TM) iCELLis(TM) . 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.
Department of Gene Therapy, University of Ulm, Ulm, Baden-Württemberg, Germany. verena.emmerling@uni-ulm.de.|Development, Rentschler Biotechnologie GmbH, Laupheim, Baden-Württemberg, Germany. verena.emmerling@uni-ulm.de.
Verena V
VV
Emmerling
Development, Rentschler Biotechnologie GmbH, Laupheim, Baden-Württemberg, Germany.
Antje
A
Pegel
Human Health Therapeutics Portfolio, National Research Council of Canada, Montreal, QC, Canada.
Ernest G
EG
Milian
Human Health Therapeutics Portfolio, National Research Council of Canada, Montreal, QC, Canada.
Alina
A
Venereo-Sanchez
Development, Rentschler Biotechnologie GmbH, Laupheim, Baden-Württemberg, Germany.
Marion
M
Kunz
Development, Rentschler Biotechnologie GmbH, Laupheim, Baden-Württemberg, Germany.
Jessica
J
Wegele
Department of Bioengineering, McGill University, Montreal, Quebec, Canada.
Amine A
AA
Kamen
Department of Gene Therapy, University of Ulm, Ulm, Baden-Württemberg, Germany.
Stefan
S
Kochanek
Development, Rentschler Biotechnologie GmbH, Laupheim, Baden-Württemberg, Germany.
Markus
M
Hoerer
D057005:Bioengineering / Q000379:methods*; D000229:Dependovirus / Q000235:genetics / Q000502:physiology*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D014779:Virus Replication
Adeno-associated virus; HEK293T; Recombinant vector; Vector production; iCELLis