Efeito da temperatura na otimização da produção de glicoproteína do vírus da raiva em cultivos em suspensão de células recombinantes de Drosophila melanogaster S2
Abstract
The increment of the expression of complex correctly processed recombinant proteins,
like the ones produced by animal cells, is essential for the development of an efficient large
scale bioprocess. The manipulation of culture conditions plays a very important role in the
controlled and optimized expression of these proteins in bioreactors. The response of
mammalian and insect cells to temperature effect has been studied by several researchers as a
potential strategy to enhance production of recombinant proteins. The present study evaluated
the influence of the temperature in the culture of recombinant Drosophila melanogaster S2
cells in Sf-900 II medium supplemented with aminoacids with the aim of enhancing the
production of the rabies virus glycoprotein (RVGP). The cultures were carried out in Schott
flasks (in shaker) as well as in a bubble-free stirred tank bioreactor. Five temperatures were
tested in Schott flasks cultures: 16, 20, 24, 28 and 34ºC. The results obtained in cultures at
those temperatures in terms of maximum specific growth rate (µmax) and maximum RVGP
concentration (CGPV
max) were, respectively: 0.009, 0.020, 0.039, 0.036 and 0.022 h-1, and
0.075, 2.973, 0.480, 1.404 and 0.013 mg.mL-1. The best temperature for RVGP production
(20oC) was chosen to be evaluated in the bioreactor operated in batch and feed-batch mode,
keeping 28oC as control. The results of µmax and CGPV
max in batch mode were: 0,061 h-1 and
0,149 mg.mL-1 at 28oC and 0,027 h-1 and 0.354 mg.mL-1 at 20oC. A batch experiment with
stepwise temperature decrease from 20ºC to 16ºC presented a µmax ranging from 0.024 to
0.012 h-1 and a final CGPV
max of 0.567 mg.mL-1. On the other hand, at 20ºC in feed-batch
mode, µmax was 0.024 h-1 and CGPV
max 1.155 mg.mL-1. The larger production of RVGP
observed in Schott flasks can be a consequence of a more favorable condition for the
expression of the protein at low values of dissolved oxygen concentrations that are seen in
flasks but not in controlled bioreactor cultures at 50% DO. Overall, these results show an
increase of recombinant protein expression at lower temperatures, especially when µmax is
kept fairly low. The largest production of RVGP was obtained in the culture at 20°C in Schott
flask when CGPV
max was 2.973 mg.mL-1, while the production obtained at the control
temperature of 28ºC was only of 1.404 mg.mL-1. Thus, the use of reduced culture
temperatures, around 20°C, seems to be the best strategy for the optimization of RVGP
production.