title: Serum-free microcarrier based production of replication deficient influenza vaccine candidate virus lacking NS1 using Vero cells.
abstract: BACKGROUND
  Influenza virus is a major health concern that has huge impacts on the human society, and vaccination remains as one of the most effective ways to mitigate this disease. Comparing the two types of commercially available Influenza vaccine, the live attenuated virus vaccine is more cross-reactive and easier to administer than the traditional inactivated vaccines. One promising live attenuated Influenza vaccine that has completed Phase I clinical trial is deltaFLU, a deletion mutant lacking the viral Nonstructural Protein 1 (NS1) gene. As a consequence of this gene deletion, this mutant virus can only propagate effectively in cells with a deficient interferon-mediated antiviral response. To demonstrate the manufacturability of this vaccine candidate, a batch bioreactor production process using adherent Vero cells on microcarriers in commercially available animal-component free, serum-free media is described.
  
  
  RESULTS
  Five commercially available animal-component free, serum-free media (SFM) were evaluated for growth of Vero cells in agitated Cytodex 1 spinner flask microcarrier cultures. EX-CELL Vero SFM achieved the highest cell concentration of 2.6 × 10^6 cells/ml, whereas other SFM achieved about 1.2 × 10^6 cells/ml. Time points for infection between the late exponential and stationary phases of cell growth had no significant effect in the final virus titres. A virus yield of 7.6 Log10 TCID50/ml was achieved using trypsin concentration of 10 μg/ml and MOI of 0.001. The Influenza vaccine production process was scaled up to a 3 liter controlled stirred tank bioreactor to achieve a cell density of 2.7 × 10^6 cells/ml and virus titre of 8.3 Log10 TCID50/ml. Finally, the bioreactor system was tested for the production of the corresponding wild type H1N1 Influenza virus, which is conventionally used in the production of inactivated vaccine. High virus titres of up to 10 Log10 TCID50/ml were achieved.
  
  
  CONCLUSIONS
  We describe for the first time the production of Influenza viruses using Vero cells in commercially available animal-component free, serum-free medium. This work can be used as a basis for efficient production of attenuated as well as wild type Influenza virus for research and vaccine production.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, Centros, Singapore.
authors.forename: Allen
authors.initials: A
authors.lastname: Chen
authors.forename: Swan Li
authors.initials: SL
authors.lastname: Poh
authors.forename: Christian
authors.initials: C
authors.lastname: Dietzsch
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Roethl
authors.forename: Mylene L
authors.initials: ML
authors.lastname: Yan
authors.forename: Say Kong
authors.initials: SK
authors.lastname: Ng
mesh_terms: D000818:Animals; D019149:Bioreactors / Q000821:virology*; D002522:Chlorocebus aethiops; D016895:Culture Media, Serum-Free / Q000378:metabolism; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D007252:Influenza Vaccines / Q000096:biosynthesis* / Q000235:genetics; D008858:Microscopy, Phase-Contrast; D014613:Vaccines, Attenuated / Q000096:biosynthesis / Q000235:genetics; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014776:Virus Cultivation / Q000295:instrumentation* / Q000379:methods; D014779:Virus Replication
chemical_list: D016895:Culture Media, Serum-Free; C085820:INS1 protein, influenza virus; D007252:Influenza Vaccines; D014613:Vaccines, Attenuated; D017361:Viral Nonstructural Proteins