title: Human amniocyte-derived cells are a promising cell host for adenoviral vector production under serum-free conditions.
abstract: Recombinant adenovirus vectors (AdVs) have been used for the development of vaccines, as gene therapy vectors and for protein production. Currently, the production of clinical grade batches of recombinant E1-deleted adenovirus type 5 vectors is performed using human-derived HEK293 or PER.C6(®) cell lines. In this work we describe the generation of a new human amniocyte-derived cell line named 1G3 and show that it can be used as a very promising cell host for AdV production in serum-free conditions, allowing for production in high cell density cultures and avoiding the typical cell density effect observed for HEK293. By design, this cell line makes the generation of replication-competent adenovirus during production of E1-deleted AdVs very unlikely. The impact of the culture system (static versus agitated) and AdV infection parameters such as multiplicity of infection, time of harvesting and cell concentration at infection were evaluated and compared with HEK293. Using stirred tanks bioreactors, it was possible to grow 1G3 cells to cell densities of up to 9 × 10(6) cells/mL using serum-free media. Moreover, without a medium exchange step at infection, a three-fold increase in AdV volumetric titers was obtained, as no cell density effect was observed at CCI 3. Overall, our results clearly demonstrate the potential of the human amniocyte-derived newly established cell line 1G3 for AdV production in a serum-free scalable process, paving the way for further process improvements based on fed-batch or perfusion strategies.
authors.affiliation: iBET - Instituto de Biologia Experimental e Tecnológica, Animal Cell Technology Unit, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Animal Cell Technology Unit, Oeiras, Portugal.
authors.forename: Ana Carina
authors.initials: AC
authors.lastname: Silva
authors.forename: Daniel
authors.initials: D
authors.lastname: Simão
authors.forename: Claudia
authors.initials: C
authors.lastname: Küppers
authors.forename: Tanja
authors.initials: T
authors.lastname: Lucas
authors.forename: Marcos F Q
authors.initials: MF
authors.lastname: Sousa
authors.forename: Pedro
authors.initials: P
authors.lastname: Cruz
authors.forename: Manuel J T
authors.initials: MJ
authors.lastname: Carrondo
authors.forename: Stefan
authors.initials: S
authors.lastname: Kochanek
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics / Q000254:growth & development*; D000653:Amniotic Fluid / Q000166:cytology*; D061249:Batch Cell Culture Techniques / Q000379:methods*; D019149:Bioreactors; D002452:Cell Count; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000378:metabolism*; D005260:Female; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D011247:Pregnancy; D019562:Viral Load; D014776:Virus Cultivation / Q000379:methods
chemical_list: D016895:Culture Media, Serum-Free
keywords: Adenovirus vectors; Human amniocyte cells; Replication-competent adenovirus; Serum-free medium; Suspension culture