Optimization of Growth Methods and Recombinant Protein Production in BTI‐Tn‐5B1‐4 Insect Cells Using the Baculovirus Expression System

Abstract

A novel insect cell line from Trichoplusia ni, BTI‐Tn 5B1‐4 (Tn 5), was compared to Spodoptera frugiperda, Sf 9, cells for production of two recombinant secreted proteins: truncated Epstein‐Barr viral attachment protein (EBV gp¹⁰⁵) and truncated, soluble tissue factor (sTF). Under optimum conditions for both cell lines, Tn 5 cells produced 28‐fold more secreted sTF than Sf 9 cells, respectively, on a per cell basis. The total production of gp¹⁰⁵ was similar for the two cell lines. However, Tn5 cells secreted gp¹⁰⁵ much more efficiently, resulting in 5‐fold higher levels in the extracellular medium. Despite these increases, Tn 5 cells are attachment‐dependent, and protein production is sensitive to the cell density (cells/cm²), unlike the Sf9 cell line which can be easily grown and scaled up in cell suspension cultures without significantly affecting its per cell production. Thus, protein production from Tn 5 cells above 0.1L scales was optimized with respect to cell density using standard techniques for the growth of attachment‐dependent cells. Roller bottles precoated with DEAE‐based microcarriers and suspension cultures employing collagen‐coated microcarriers were found to be effective ways of culturing Tn 5 cells. Predetermined optimal cell densities were used to produce EBV gp¹⁰⁵ in microcarrier‐coated roller bottles or in suspension cultures using collagen‐coated microcarriers at concentrations close to those observed in tissue culture flasks.