title: Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection.
abstract: The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10-14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6 × 10(7) infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229-3039, USA. Han.vanderloo@cchmc.org
authors.forename: J C M
authors.initials: JC
authors.lastname: van der Loo
authors.forename: W P
authors.initials: WP
authors.lastname: Swaney
authors.forename: E
authors.initials: E
authors.lastname: Grassman
authors.forename: A
authors.initials: A
authors.lastname: Terwilliger
authors.forename: T
authors.initials: T
authors.lastname: Higashimoto
authors.forename: A
authors.initials: A
authors.lastname: Schambach
authors.forename: C
authors.initials: C
authors.lastname: Baum
authors.forename: A J
authors.initials: AJ
authors.lastname: Thrasher
authors.forename: D A
authors.initials: DA
authors.lastname: Williams
authors.forename: D L
authors.initials: DL
authors.lastname: Nordling
authors.forename: L
authors.initials: L
authors.lastname: Reeves
authors.forename: P
authors.initials: P
authors.lastname: Malik
mesh_terms: D000818:Animals; D061249:Batch Cell Culture Techniques / Q000379:methods / Q000592:standards; D019149:Bioreactors; D001709:Biotechnology; D002460:Cell Line; D017988:Gammaretrovirus / Q000235:genetics* / Q000302:isolation & purification; D005822:Genetic Vectors / Q000302:isolation & purification* / Q000592:standards*; D006801:Humans; D051379:Mice; D011786:Quality Control; D014162:Transfection / Q000379:methods*