Effect of hypothermic temperatures on production of rabies virus glycoprotein by recombinant Drosophila melanogaster S2 cells cultured in suspension

Abstract

Aiming at maximizing the production of transmembrane rabies virus glycoprotein (rRVGP), the influence of hypothermic temperature on a recombinant Drosophila melanogaster S2 cell culture in Sf-900II medium was investigated. Cell growth and rRVGP production were assessed at 4 culture temperatures in Schott flasks: 16, 20, 24 and 28 °C. The maximum specific growth rates μ(max) were, respectively: 0.009, 0.019, 0.038 and 0.035 h(-1), while the maximum rRVGP levels C(max)(rRVGP) were: 0.075, 2.973, 0.480 and 1.404 mg L(-1). The best production temperature (20 °C) was then tested in a bioreactor with control of pH and dissolved oxygen in batch and fed-batch modes. In the batch culture, μ(max) and C(max)(rRVGP) were 0.060 h(-1) and 0.149 mg L(-1) at 28 °C and 0.026 h(-1) and 0.354 mg L(-1) at 20 °C, respectively. One batch-culture experiment was carried out with adaptation of the cells by the temperature falling in steps from 20 °C to 16 °C, so that μ(max) fell from 0.023 to 0.013 h(-1), while C(max)(rRVGP) was improved to 0.567 mg L(-1). In the fed-batch mode at 20 °C, μ(max) was 0.025 h(-1) and C(max)(rRVGP) was 1.155 mg L(-1). Taken together, these results indicate that the best strategy for optimized rRVGP production is the culture at hypothermic temperature of 20 °C, when μ(max) is kept low and with feeding of limitant aminoacids.

Transient expression of rabies virus G-glycoprotein using BHK-21 cells cultured in suspension

“…The maximum volumetric yield for RVGP (cell cultures plates with agitation) observed in the present work was lower than those reported for the same protein using stably transfected Drosophila melanogaster cells (1.5-3 mg L) with optimized culture conditions in bioreactors (Ventini et al 2010;Rossi et al 2012). Homogenization and aeration improvements could increase the RVGP expression.…”

“…At present, volumetric yield in spinner flask for RVGP are inferior to values of this parameter for the same heterologous protein and other proteins expressed with SFV vector in bioreactors (Blasey et al 2000;Ventini et al 2010;Rossi et al 2012). The diminution of time for maximum protein expression peak is another evidence of improved conditions for infection in spinner flask when compared with cell culture plate.…”

“…For experiment performed in cell culture plate at 33°C, the RVGP increase in supernatant over the course of post-viral adsorption time could be associated to membrane debris caused by cell lysis after prolonged infection time with SFV vector (Benmaamar et al 2009). This is a consequence of ''shut-off'' effect of the protein synthesis in host cell induced by SFV replication (Rossi et al 2012). However, as a rule, higher percentages of RVGP were located into the cell with respect to supernatant, for all assessed experimental conditions.…”

The use ofDrosophilaS2 cells in R&D and bioprocessing

“…Insights gained through detailed studies of S2 processes have led to a 9.3-fold increase in RVGP yield by lowering the production temperature to 22°C compared with the initial process conducted at 28°C [90]. Following this study, a detailed ana lysis of the effect of culture temperature on RVGP production between 16 and 28°C demonstrated that the best production was attained at 20°C while feeding limiting amino acids, as suggested by the study of Swiech et al [91]. An interesting 60% increase in RVGP production was seen for the addition of Lonomia oblique hemolymph [92]; it was shown that the positive effect was mainly early in the culture and was related to an increased maximum specific productivity of 60 ng/cell/day ver- Taken from [97].…”

Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives

“…, 2013; Rossi et al . , 2012). Historically, CHO cells allowed gene amplification, resulting in a higher recombinant protein yield (up to the gram per liter range for some proteins) and specific productivity, which was previously an issue in other mammalian cell lines (Carlage et al .…”