On-line monitoring and control of the cultivation of Spodoptera frugiperda Sf9 insect cells and β-galactosidase production by Autographa californica virus vector

Abstract

Spodoptera frugiperda Sf9 cells were cultivated in serum-containing (Grace and TC-100 + 10% FCS) and serum-free (Excell 401) medium in batch and continuous cultures. The cells, infected by recombinant Autographa californica, formed β-galactosidase which was only partly secreted. The total cell concentration was monitored on-line by measuring the optical density ( OD) and the viable cell concentration in situ by measuring the intensity of the background-corrected culture fluorescence ( FI). By plotting 1 FI vs. 1 OD , the phases of cell growth, virus infection, and expression of virus genome, and the lysis of the cells were identified. Extracellular lactate dehydrogenase (LDH) and intracellular β-glucosidase were monitored on-line. Serum-containing media yielded better productivity than the serum-free medium. In batch culture, cell concentrations up to 4 × 10 6 cells ml −1, a specific growth rate of μ = 0.0288 h −1, a doubling time of t D = 24 h, and with the baculovirus vector, specific β-glucosidase activities of 3.0–40 U 10 −6 cells which increased above 100 U 10 −6 cells by the end of the batch phase, were observed. During a 626 h continuous culture, steady-state cell concentrations of 10 6 cells ml −1 and specific β-galactosidase activities of 15–50 U 10 6 cells were obtained. DNA content, determined by the use of a laser flow cytometer, increased during the virus infection.