Baculovirus production for gene therapy: the role of cell density, multiplicity of infection and medium exchange

Abstract

One of the major concerns regarding the use of insect cells and baculovirus expression vectors for the production of recombinant proteins is the drop in production observed when infecting cultures at high cell densities; this work attempts to understand this so-called cell density effect in the scope of baculovirus production for gene therapy purposes. A Spodoptera frugiperda insect cell line (Sf-9) was cultured and infected in serum-free medium, and the patterns of production of a recombinant baculovirus expressing the green fluorescent protein (GFP) were analyzed at different cell concentrations at infection (CCIs) and multiplicities of infection (MOIs). The results confirm that a cell density effect on productivity occurs which is dependent on the MOI used, with a high MOI "delaying" the drop in production to higher cell densities. Medium replacement at the time of infection using a high MOI considerably improved baculovirus production, with the different production indicators, namely the titer, specific yield, amplification factor, and time of harvesting, increasing with cell concentration for the CCI range tested. Virus titers as high as 2.6 x 10(10) IP x mL(-1) were obtained in cultures infected at 3.5 x 10(6) cells x mL(-1), while the amplification factor was roughly 19 times higher than the highest value obtained without medium exchange.

Optimization of the Strategy for Recombinant Baculovirus Infection of Suspended Insect Cells

“…Batch processes usually employ intermediate to high MOIs in the exponential phase of growth, which result in synchronous infection of the insect cells and optimal yields of recombinant products or of polyhedra as bioinsecticides (Kumar et al, 1995). A recent study regarding the production of budded viruses (BVs) using a high MOI demonstrated that medium replacement at the time of infection considerably improved BV production, which increased with the cell concentration and reached a virus titer of 2.6×10 10 infectious particles (IPs) per mL in cultures of 3.5×10 6 Sf9-infected cells per mL (Carinhas et al, 2009). However, the use of such complex mixtures would involve higher operation costs and lead to variability in the process performance (Carinhas et al, 2009).…”

“…A recent study regarding the production of budded viruses (BVs) using a high MOI demonstrated that medium replacement at the time of infection considerably improved BV production, which increased with the cell concentration and reached a virus titer of 2.6×10 10 infectious particles (IPs) per mL in cultures of 3.5×10 6 Sf9-infected cells per mL (Carinhas et al, 2009). However, the use of such complex mixtures would involve higher operation costs and lead to variability in the process performance (Carinhas et al, 2009). In the case of high MOIs and higher cell densities, the technology for BV or recombinant protein production becomes more complex.…”

“…Over the last two decades, much effort has been focused on identifying factors that affect the productivities of such expression systems, including the cell line (Wickham et al, 1995), medium (Caron et al, 1990), oxygen supply (Gotoh et al, 2002), reactor design (Rice et al, 1993;Zhang et al, 1993) and infection strategy, which involves the multiplicity of infection (MOI), time of infection (TOI), initial cell density (ICD) and medium replacement (Carinhas et al, 2009;Hu and Bentley, 2001;Radford et al, 1997;Rodas et al, 2005;Yamaji et al, 1999;Zhang et al, 2005). Batch processes usually employ intermediate to high MOIs in the exponential phase of growth, which result in synchronous infection of the insect cells and optimal yields of recombinant products or of polyhedra as bioinsecticides (Kumar et al, 1995).…”

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations

“…The flux of glucose was studied in BTI-TN-5B1-4 cells, resulting that the proportion oxidized in the tricarboxylic acids cycle was lower than in Sf9 cells (Benslimane et al, 2005). The influence of baculovirus infection on the metabolism of insect cells, and specifically on glucose metabolism, is a topic that has been scarcely addressed, and the information is contradictory (Bernal et al, 2009;Gioria et al, 2006). According to Mitsuhashi (1989), 15 amino acids are essential to insect cells.…”

“…However, these results are still controversial, because other studies have found that the percentage of glucose consumed that is derived to the tricarboxylic acid cycle is much lower (Benslimane et al, 2005). The production of other metabolites such as alanine, glycerol and ethanol, as well as fatty acid synthesis, could explain the fate of a significant fraction of carbon incorporated to Sf9 cells through glucose consumption (Drews et al, 2000;Bernal et al, 2009). BTI-TN-5B1-4 and BM5 cell lines have a different behavior: both cell lines produce lactate, displaying a behavior similar to that of mammalian cells (Rhiel et al, 1997;Stavroulakis et al, 1991).…”