Influence of baculovirus-host cell interactions on complex N-linked glycosylation of a recombinant human protein

The conditions required for mammalian-type complex N-linked glycosylation o f human proteins produced in insect cells with the baculovirus expression v ector system were investigated. Marked alterations to N-linked glycosylatio n of human placental secreted alkaline phosphatase (SEAP) were observed wit h different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) wa s used to produce SEAP in Trichoplusia ni (Tn-4h) cells cultured in serum-f ree medium, structural analyses indicated <1% hybrid and no complex oligosa ccharides attached to SEAP, a typical result with the baculovirus expressio n vector system. However,when fetal bovine serum was added to the culture m edium, 48 +/- 4% of the oligosaccharides were hybrid or complex (but asialy lated) glycans. When a recombinant T. ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn-4h cells cultured in serum-containing medium, only 24 +/- 3% of the glycans contained terminal N-acetylglucosamin e and/or galactose residues. In contrast, SEAP produced in Sf9 cells grown in serum-containing medium with AcMNPV contained <1% hybrid oligosaccharide s and no complex oligosaccharides. The results illustrate that baculovirus type, host cell type, and the growth medium all have a strong influence on the glycosylation pathway in insect cells, resulting in significant alterat ions in structures and relative abundance of N-linked glycoforms. Although the addition of sialic acid residues to the SEAP glycans was not detected, possible approaches to obtain sialylated glycans are discussed.