Bioreactor culture of recombinant Drosophila melanogaster S2 cells: characterization of metabolic features related to cell growth and production of the rabies virus glycoprotein

Abstract

Although several reports have been published on recombinant protein expression using Drosophila cells, information on their metabolism and growth in vitro is relatively scarce. In the present study, we have analyzed the growth and metabolism of transfected S2 cells (S2AcRVGP) in bioreactor cultures with serum-free medium Sf900 II, to evaluate its potential for mass production of a rabies virus glycoprotein (RVGP). Cells were cultured in a 3 lstirred-tank bioreactor at 28°C with pH controlled at 6.2 and dissolved oxygen at 50% air saturation. The cells attained a specific growth rate and maximum cell density as high as 0.084 h -1 and 2.3 9 10 7 cell ml -1 , respectively. The main substrates consumed during this rapid growth phase were glucose, glutamine and proline. An atypical accumulation of ammonia and alanine was observed in the culture medium, up to 62 mM and 47 mM, respectively, but lactate was produced in low levels. After exhaustion of glutamine and proline as energy sources, alanine was consumed and production of ammonia increased. The production of recombinant RVGP reached concentrations as high as 178 lg l -1 . Premature exhaustion of glutamine, serine and cysteine could be related to degradation of the recombinant glycoprotein. In general, the results demonstrated that S2AcRVGP can be considered an effective vehicle for large-scale recombinant glycoprotein expression and that several critical factors of the bioprocess could be optimized to increase the quality and productivity of the RVGP.

Kinetic response of a Drosophila melanogaster cell line to different medium formulations and culture conditions

“…The low consumption of those amino acids by S2 cells as shown in this study (Fig. 5), as well as in Swiech et al (2008), is in accordance to the previous observation.…”

“…On the other hand, it was observed that the excess of GLN induced a less efficient metabolism, measured by the parameter Y X/GLN . Ammonia (NH 3 ) was not determined in our experiments, but data on a derived recombinant S2 cell line indicate that the amount of NH 3 synthesize, and its deleterious effects also depend on the culture medium employed (Swiech et al 2008;Galesi et al 2008). For SF900 II Ò (Swiech et al 2008), it could be observed concentrations up to 340 mg L -1 at the end of exponential phase, without any indication of cell growth inhibition.…”

“…Ammonia (NH 3 ) was not determined in our experiments, but data on a derived recombinant S2 cell line indicate that the amount of NH 3 synthesize, and its deleterious effects also depend on the culture medium employed (Swiech et al 2008;Galesi et al 2008). For SF900 II Ò (Swiech et al 2008), it could be observed concentrations up to 340 mg L -1 at the end of exponential phase, without any indication of cell growth inhibition. Nevertheless, for the TC-100-based formulation (Galesi et al 2008), a cell growth inhibition was attributed to the 50-60 mg L -1 of NH 4 + accumulated in culture.…”

Behavior of Wild-type and Transfected S2 Cells Cultured in Two Different Media

“…These include human plasminogen [14] and mouse endostatin [15]. Non-secreted and secreted recombinant proteins such as the G glycoprotein from rabies virus (RVGP) and the surface antigen of hepatitis B virus (HBsAg), respectively, can be expressed in Drosophila Expression System (DES), both showing to be appropriately processed and biologically active [13,[16][17][18][19][20][21][22][23]. Furthermore, S2AcRVGP2 cells cultivated in IPL-41 medium supplemented with 10 g L −1 glucose, 0.5 g L −1 fructose, 2 g L −1 lactose, 3.5 g L −1 glutamine, 0.6 g L −1 tyrosine, 1.48 g L −1 methionine, 6 g L −1 yeastolate ultrafiltrate, 1% chemically defined lipid concentrate, and 0.1% Pluronic F-68 are able to produce up to 31 ng RVGP L −1 in batch culture provided that dissolved oxygen and pH are controlled at 40% and 6.3, respectively [24].…”

Produção do fungo entomopatogênico Metarhizium rileyi (Farlow) por fermentação líquida e sólida

“…Levando em consideração a diversidade de fontes e a quantidade de nutrientes desse composto, é possível supor que, contrariamente ao exposto acima para os demais meios, a combinação SF-900 + Yeastolate Ultrafiltrado foi capaz de suprir as necessidades nutricionais do fungo no que diz respeito a produção de blastosporos. Todavia, há indicativos de esgotamento de nutrientes a partir do quarto dia, uma vez que houve uma queda brusca da contagem de blastosporos no sexto dia, fenômeno este já reportado quando houve utilização do SF-900 para cultura de células insetos, com indicações de esgotamento de alguns aminoácidos presentes no meio BOVO et al, 2008;SWIECH et al, 2008).…”