ELSEVIER Journal of Biotechnology33 (1994) 249-258 journal of blotechnology Optimization of the physiochemical parameters for the culture of Bombyx mori insect cells used in recombinant protein production Junli Zhang, Nicolas Kalogerakis, Leo A. Behie * Pharmaceutical Production Research Facility (PPRF), Faculty of Engineering, The Uniuersityof Calgary, Calgary, Alberta, Canada T2N 1N4 (Received 27 April 1993; revision accepted 15 October 1993) Abstract Silkworm (Bombyx mori, Bm5) cell growth at different environmental conditions was systematically studied in controlled bioreactors. The physiochemical parameters, which affect the growth of Bm5 cells, were defined and optimized. The growth rate was maximal at a medium osmolality of about 370 mosm kg-1; and more than 90% of the maximum growth rate was achieved with osmolality between 350 and 385 mosm kg- 1. The optimum pH for Bm5 cell growth was from 6.10 to 6.30. Moreover, the effect of dissolved oxygen concentrations (higher than 20% air saturation) on growth was minimal. However, the maximum cell density decreased markedly with dissolved oxygen tensions less than 20%. The shear protection additive Pluronic F-68 had a very important effect on the growth of Bm5 cells in bioreactors with an optimum concentration of 0.2% (w/v). Compared to the original culture condition, the optimized conditions resulted in a twofold increase in cell yield and a reduced population doubling time from 48 to 36 h. Furthermore, the yield of a recombinant protein (CAT) was increased substantially in the optimized cultures. Key words: Bioreactor; Insect cell culture; Osmolality; pH; Dissolved oxygen; Pluronic F-68 I. Introduction The baculovirus/insect cell system has shown great promise recently for the production of re- combinant proteins (Summers, 1991). The rising popularity of using genetically engineered bac- ulovirus/insect cell systems is due to a number of facts. First of all, the native baculovirus follows an unique biphasic replication in insect ceils, which consists of an earlier essential viral produc- * Corresponding author. tion and a late nonessential polyhedra expression by the polyhedrin gene within the virus (O'Reilly et al., 1992). This nonessential polyhedrin gene can be replaced with a foreign gene of choice. The inserted foreign gene within the double stranded, circular genome (100-150 kb) of the baculovirus can express abundantly its protein in insect cells under the control of the strong poly- hedrin gene promoter. Secondly, expressed pro- teins in the baculovirus/insect cell system are biologically active in most cases (Summers, 1991). Thirdly, the baculovirus/insect cell system is not pathogenic to animals. Consequently, the thera- 0168-1656/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1656(93)E0107-9