Optimization of the production of virus-like particles in insect cells

Abstract

In this work the maximal operational hydrodynamic conditions (agitation and aeration rate) that cause no adverse effect in Sf‐9 cells growth in SF900II serum‐free medium were determined. Shear stresses higher than 1 N m−2 and aeration rates higher than 0.04 vvm affect cell growth and when these conditions increase to 1.5 N m−2 and 0.11 vvm, cell growth is completely inhibited with significant cell morphology changes and a strong decrease in viability. Although the pO2 did not show a significant effect upon cell growth in the range from 10 to 50%, cell infection and specific productivity were dramatically affected. The production was optimal at a pO2 of 25% with decreases higher than 50% being observed when the pO2 decreased to 10 or increased to 50%. The maximum product quality, i.e., the percentage of product in the form of high molecular weight particles, is not coincident with maximum product titer. Although the highest Pr55gag particle titer was obtained at 96 hours post infection (hpi) and at pO2 of 25%, the best product quality (defined by gel filtration chromatography and Western immunoblot) was obtained at 48 hpi, independently of the pO2 used. The effect of overcritical conditions upon productivity was also studied. As obtained for cell growth, cell infection is affected by shear stresses above 1 N m−2 and by aeration rates higher than 0.04 vvm, with decreases in Pr55gag particle titer higher than 70%, even when the overcritical values are still far from the limit at which cell death occurs. The results obtained and the optimization strategy used allowed the maximization of the oxygen supply without damaging the cells, with important consequences on the scale‐up of a production process involving this insect cell/baculovirus expression system. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 408–418, 1998.

Production of oncolytic adenovirus and human mesenchymal stem cells in a single‐use, Vertical‐Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier‐based cell culture processes

“…The estimation of shear stress ( τ ) (N/m 2 ) under stirred conditions as result of flow through Kolmogorov eddies was performed using Eq. , as described in the literature, and the Kolmogorov length scale (KLS) ( λ ), or eddy size (m) was estimated by Eq. : where (m 2 /s) is the kinematic viscosity, (m 2 /s 3 ) is the turbulent energy dissipation in the impeller zone, and μ (N.s/m 2 ) is the viscosity of the fluid.…”

On the Effect of Thermodynamic Equilibrium on the Assembly Efficiency of Complex Multi-Layered Virus-Like Particles (VLP): the Case of Rotavirus VLP

“…The pH was monitored but not controlled since it did not vary significantly throughout the culture time. The abovementioned conditions were previously optimized [49].…”

Adaptation of the Se301 insect cell line to suspension culture. Effect of turbulence on growth and on production of nucleopolyhedrovius (SeMNPV)

“…Maximum values of s assayed (see Table 2) were below 1.39 10 -3 N m -2 , much lower than those reported to be critical to other cell lines such as Sf21 (1-4 N m -2 ), Sf9 (0.59-1.5 N m -2 ), and T. ni TN-386 (0.1 N m -2 ) (Tramper et al 1986;Goldblum et al 1990;Cruz et al 1998). Assay 22 in Table 2 showed a growth rate similar to the static control at the maximum agitation speed tested.…”

“…It consisted in maintaining the cultures at a fixed shaking speed with the addition of additives until a constant growth rate during 4 generations was achieved. In this way, the shaking speed was increased step by step from 60 to 70, 90 and 120 rpm (Re = 2,190, 2,555, 3,285 and 4,381, respectively) as cells were adapted to the new shaking conditions until no cell damage was observed (Cruz et al 1998). Before subculturing, broths were allowed to settle for 15-20 min in order to remove clumps, and to collect the supernatant with the single cells to be later subcultured, as described by Rhiel et al (1997).…”

“…The baculovirus-insect cell system is considered as a powerful biotechnological tool to produce various valuable biotechnological products (Maranga et al 2004;Van Lier et al 1996;Cruz et al 1998;Negrete and Kotin 2007;Szewczyk et al 2006). However, considering that the technology for the in vitro protein expression is well-established (Agathos 1996), the in vitro production of baculovirus is an attractive cost-effective alternative (Enden et al 2005).…”