title: Decline in baculovirus-expressed recombinant protein production with increasing cell density is strongly correlated to impairment of virus replication and mRNA expression.
abstract: The cell density effect is a well-established constraint in the baculovirus-insect cell expression platform, in which cell-specific productivity declines with increasing cell density, hence limiting the maximum achievable volumetric yield of protein product. A deeper elucidation of this phenomenon is sought in this study, by tracking the peak production of viral DNA (vDNA), recombinant LacZ mRNA, and β-galactosidase (β-gal) protein, over a wide range of cell densities. Sf9 suspension cell cultures were propagated in Sf-900 III serum-free medium and synchronously infected with rAcMNPV at multiple infection cell densities (ICDs) of between 0.5 and 8 × 10(6) cells/mL. There was a strong negative linear correlation between the specific β-gal yield and the peak cell density (PCD) post-infection, but contrary to previous reports, the yield decline started at a lower PCD of around 1 × 10(6) cells/mL. Most interestingly, there also was a corresponding strong negative linear correlation between the specific vDNA or LacZ mRNA yield, and the PCD. Comparing the infections at the highest and lowest PCDs tested, the yield decline was most dramatic for β-gal protein (95 %) and LacZ mRNA (90 %), while it was more moderate for vDNA (50 %). These declines were significantly reduced but not completely arrested, when spent medium was replaced with fresh at the ICD. These findings suggest that protein yield deterioration with increasing cell density originated from limitations during upstream events such as virus gene replication or transcription, rather than during the translational phase. Such limitations may be largely nutritional, but a more complex mechanism may be implicated.
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Qld 4072, Australia. hoai.huynh@uq.edu.au
authors.forename: Hoai T
authors.initials: HT
authors.lastname: Huynh
authors.forename: Trinh T B
authors.initials: TT
authors.lastname: Tran
authors.forename: Leslie C L
authors.initials: LC
authors.lastname: Chan
authors.forename: Lars K
authors.initials: LK
authors.lastname: Nielsen
authors.forename: Steven
authors.initials: S
authors.lastname: Reid
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000502:physiology*; D002452:Cell Count; D015870:Gene Expression; D005822:Genetic Vectors; D012333:RNA, Messenger / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D061987:Sf9 Cells; D018411:Spodoptera; D014779:Virus Replication
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins