Adaptation of BTITN5B1-4 (High Five) Insect Cells for Large-Scale Production in a Stirred Bioreactor

Recombinant baculoviruses are used for the production of eukaryotic proteins in insect cell cultures. Because the system is capable of producing very high levels of recombinant proteins compared to mammalian cells, baculovirus technology has found its way to a number of industrial applications (i.e., vaccine production) and x-ray crystallographic structure/function studies. Commonly, the Spodoptera frugiperda SF-9 cell line is used because of its ability to grow in suspension and to express a high level of recombinant proteins. Recently, an attachment-dependent cell line, the Trichoplusia ni BTI-TN5B1-4 (High Five), has been shown to be superior to the SF9 cell for expression of both cytoplasmic and secreted glycosylated proteins (3,6,10). This cell line can optimally produce 26fold more human-secreted alkaline phosphatase and 28-fold more soluble tissue factor per cell than the SF9 in monolayer cultures (10,11). Previously believed to be attachment-dependent, High Five cells were not easy to grow in a bioreactor despite of the use of a microcarrier system (11,12). Several approaches have permitted these cells to adapt to single-cell suspension culture, either with a long adaptation period (6) or with the use of polyanions (1,2) or heparin (8). However, these methods can inhibit the baculovirus infection of High Five cells. Here, we propose an alternative method to adapt, in one step, High Five cells to single-cell suspension, based on the use of the serum-free medium (SFM) Xpress (BioWhittaker, Walkersville, MD, USA). High Five and SF9 cells were obtained from Invitrogen (Carlsbad, CA, USA) and ATCC (CRL-1711; Rockville, MD, USA), respectively. Xpress SFM was utilized for both cell lines. This medium is protein free and contains glucose as the only source of carbon. Its osmolarity and pH were estimated at 362 mOsm/kg and 6.2, respectively. No agents were added to the medium. A recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) was used to express the human class II histocompatibility glycoprotein molecules, HLA-DR1, under control of the polyhedrin promoter. Recombinant baculoviruses carrying truncated genes for the α and the β subunits of human HLA-DR1 were generated in the insect cell line SF9 (7). Viral stock was produced by infection of SF9 at low multiplicity. Viral titer was approximately 3 × 108 plaqueforming units (pfu)/mL. Cells were infected with the baculovirus at a multiplicity of infection (MOI) of 10, according to the procedure of O’Reilly et al. (4), which evaluated cells at a lesser density. In our study, High Five cells were progressively adapted, in 175-cm2 tissue culture flasks, to Xpress medium after 3 passages and were maintained at 27°C. This medium induced a morphological change: cells left their adherence and were able to grow in suspension with a doubling time of approximately of 16 h and a maximal density of 4 × 106 cells/mL. Once High Five cells were adapted to suspension culture, we determined fundamental parameters, such as the inoculum density and the time of infection (TOI) for a large-scale culture in a 15-L ADI 1030, Stirred-Bioreactor (Biocontroller, Applikon, The Neitherlands). Minimal starting cell density of 2 × 105 cells/mL seemed to be optimal to rapidly reach the exponential phase of High Five cells. The TOI was evaluated at 1 × 106 cells/mL with an optimal production of recombinant HLA-DR1 from 72–96 h post-infection. HLA-DR1 was measured with an enzyme-linked immunosorbent (ELISA) assay. The supernatent containing HLA-DR1 molecules was directly coated with 0.1 M sodium carbonate (pH 9.6). L243 biotinylated monoclonal antibodies were used to detect the bound HLA-DR1. The revelation system was the streptavidin-peroxydase. Finally, under identical conditions (medium and the density of cells), HLA-DR1 expression in the High Five cells was higher than in the SF9 cells (Figure 1). Because of the ability of High Five cells to grow easily in suspension and to Benchmarks