Optimized insect cell culture for the production of recombinant heterologous proteins and baculovirus particles.

Insect cells are natural hosts for baculoviruses. Following infection, nuclear polyhedral inclusion bodies (PIBs) and budded virus (BV) are produced. Virion-rich PIBs are ingested by insect larvae as food contaminants. Upon PIB digestion in the insect midgut, virions replicate in midgut cells and spread to other tissues, leading larvae to become a fluid-filled sac containing large amounts of new PIBs. PIBs derived from the Anticarsia gemmatalis multicapsid nuclear polyhedrosis virus (AgMNPV) have already been applied as a biopesticide against the soybean worm Anticarsia gemmatalis, and cell culture-derived PIB production on an industrial scale can be envisaged (1,4,15). One of the most widely used tools for the production of large amounts of recombinant proteins is the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV), employed as an expression vector for foreign genes. The baculovirus expression system has been developed and utilized to produce foreign proteins in insect cells (3,5,8, 14,17,19). Using recombinant baculovirus vectors based on AcMNPV and Spodoptera frugiperda (Sf9) insect cells, it has been possible to produce secreted proteins, transmembrane proteins, and nuclear proteins. The protein yield was greatly dependent not only on the protein nature but also on the virus construct and on the cells serving as virus hosts for heterologous protein production (17,19). Another factor that decisively affects the protein yield is the cell culture system (6,16,20). For several studies, these systems are based on technologies currently used in research laboratories, such as tissue culture flasks and spinner or shaker bottles. The main difficulties of these procedures are (i) the low protein yield in relation to the amount required for most research studies and (ii) the lack of scale-up possibilities. Shaker bottles are very convenient for insect cell cultures, but only a small fraction of the whole bottle volume can be utilized for cell cultivation because of the limitations of oxygen transfer through the medium surface (9,13,16). Total culture volumes beyond 1 L already represent cumbersome procedures. Bioreactor cultures, in volumes ranging from 1 to 4000 L, may fulfill the requirements of a large number of users, provided a simple and practical culture procedure is available (6). Here, we describe a scaleable standardized procedure for the cultivation and infection of the widely used Sf9 insect cells in a 1-L bioreactor that mimics the characteristics encountered with the operation of larger volumes. Several laboratories have studied the optimization of oxygen uptake rate, temperature, and hydrodynamic stress in insect cell cultures (2,7,18,21–23). The experimental conditions described below were based on these findings and on our previous studies of virus replication in bioreactor cell cultures and then applied to the production of proteins derived from the baculovirus expression system (10–12). Cells adapted to grow in serum-free medium were cultured in Sf900II medium (Invitrogen, Carlsbad, CA, USA), and the cell viability was determined by the Trypan blue exclusion method. Cells were first grown in a New Brunswick 4330 shaker in 1-L “Schott” shaker bottles containing 100 mL as the working volume. Cell cultures, which were inoculated at a density of 5 × 105 cells/mL and attained 5.5 × 106 cells/ mL after 4 days at 28°C under agitation (100 rpm), were then transferred to a 1-L working volume Inceltech/SGI bioreactor. This bioreactor culture was started with 5 × 105 cells/mL, 98% of dissolved oxygen, a temperature of 28oC, and agitated at 40 rpm (Figure 1). The dissolved oxygen level in the culture medium decreased constantly and attained levels below 20% after 4 days of cultivation, indicating an active cell metabolism and oxygen consumption. From this time on, the bioreactor culture was constantly supplied with air, and the dissolved oxygen was controlled at 20%. Agitation was increased to 60 rpm after culturing for 24 h and, Benchmarks