BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING Decline in baculovirus-expressed recombinant protein production with increasing cell density is strongly correlated to impairment of virus replication and mRNA expression Hoai T. Huynh & Trinh T. B. Tran & Leslie C. L. Chan & Lars K. Nielsen & Steven Reid Received: 20 January 2013 / Revised: 3 March 2013 / Accepted: 5 March 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract The cell density effect is a well-established con- straint in the baculovirus–insect cell expression platform, in which cell-specific productivity declines with increasing cell density, hence limiting the maximum achievable volu- metric 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 synchro- nously 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 corre- lation 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 signifi- cantly 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. Keywords Baculovirus . Sf9 cells . Cell density effect . β-Gal yield . mRNA expression . Viral DNA replication Introduction The baculovirus–insect cell expression vector system (BEVS) is a widely used and rapid method of producing heterologous proteins from a broad range of organisms including viruses, bacteria, invertebrates, mammalian cells, and plants, with the potential for high product titers (Drugmand et al. 2012; Kost et al. 2005; Lindsay and Betenbaugh 1992). In recent years, new applications for BEVS have been developed, such as human- ized N-glycoprotein and gene delivery vectors for mammalian cells, and industrial production of human and veterinary vac- cines (Aucoin et al. 2010; Kost et al. 2005; Mena and Kamen 2011). In addition, baculoviruses have been used as a biolog- ical control agent for insect pests, particularly as part of an integrated pest management program (Moscardi et al. 2011; Szewczyk et al. 2006, 2009). Yield improvement is a critical task to enhance the eco- nomic feasibility of a BEVS-based production process, par- ticularly for low margin products such as baculovirus biopesticides and animal vaccines. It is desirable to infect cells at a high cell density in order to maximize product yields (Bedard et al. 1994; Carinhas et al. 2009; Elias et al. 2000; Wong et al. 1996). However, it has been well established that the cell-specific yield of baculovirus-expressed recombinant proteins declines with increasing cell density, which corre- spond with a decline in the volumetric yield (Caron et al. 1990; Doverskog et al. 2000; Radford et al. 1997; Taticek and Shuler 1997; Wong et al. 1996). This bottleneck is com- mon to different biological systems, especially for animal cells (Ferreira et al. 2005; Henry et al. 2005). The loss of specific productivity with increasing cell density is referred to as the “cell density effect”, which is H. T. Huynh (*) : T. T. B. Tran : L. C. L. Chan : L. K. Nielsen : S. Reid Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Qld 4072, Australia e-mail: hoai.huynh@uq.edu.au Appl Microbiol Biotechnol DOI 10.1007/s00253-013-4835-8