New virus-free insect cell lines derived from BTI-TN-5B1-4 exhibit high performance in recombinant protein production

Abstract The Trichoplusia ni -derived BTI-TN-5B1-4 (Tn5) cell line, commonly referred to as High Five ™ , is widely used for recombinant protein production because of its high productivity and strong secretion capacity in the baculovirus-insect cell expression system. However, adventitious viral contamination remains a significant concern, particularly in the manufacture of vaccines and therapeutic proteins, for which ensuring viral safety is paramount. In 2007, a latent infection with Tn5 Cell Line Virus (TnCLV), a member of the genus Alphanodavirus, was identified in Tn5 cells. Here, we report the discovery of a second adventitious virus, Bombyx mori macula-like virus (BmMLV), in the Tn5 cell line, and demonstrate that both viruses can be successfully eliminated using a co-culture protocol with Sf9 cells across a permeable membrane. Clonal cell lines were established from the resulting virus-free Tn5 cells and comprehensively characterized in serum-free suspension culture. Selected lines exhibited growth kinetics comparable to those of commercial High Five cells, supported productive baculovirus infection, and achieved recombinant protein yields comparable to or higher than those of High Five cells. These products included secreted alkaline phosphatase (SEAP), β-galactosidase (β-gal), the adenovirus penton-based virus-like particle ADDomer, and the malaria vaccine candidate PfRipr5. These new, fully traceable, virus-free cell lines provide an enhanced biosafety profile, potentially increasing their suitability for regulated biomanufacturing, and represent robust and reliable platforms for high-level recombinant protein production in both research and industrial applications.