Influence of Baculovirus‐Host Cell Interactions on Complex N‐Linked Glycosylation of a Recombinant Human Protein

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Abstract
The conditions required for mammalian‐type complex N‐linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated. Marked alterations to N‐linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn‐4h) cells cultured in serum‐free medium, structural analyses indicated 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‐acetylglucosamine and/or galactose residues. In contrast, SEAP produced in Sf9 cells grown in serum‐containing medium with AcMNPV contained <1% hybrid oligosaccharides 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 alterations 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.