Paucimannose or oligomannose structures are usually attached to glycoproteins produced by insect cells, while mammalian glycoproteins usually have complex glycans. The lack of complex glycosylation has limited the use of the insect cell baculovirus expression vector system (BEVS), despite its high productivity and versatility. The availability of cell lines capable of complex glycosylation can overcome such a problem and potentially increase the utility of BEVS. In this work the capability of two novel cell lines, one from Pseudaletia unipuncta (A7S) and one from Danaus plexippus (DpN1), to produce and glycosylate a recombinant protein (secreted human placental alkaline phosphatase, SeAP) was assessed. SeAP produced by Tn5B1-4 cells at a low passage number (<200) was utilized for comparison. The optimal conditions for the production of SeAP by DpN1 cells were defined, and the glycosylation profiles of SeAP produced by the cell lines were quantitatively determined. Both the A7S and the DpN1 cells produced lower concentrations of SeAP than the Tn5B1-4 cells. Less than 5% of the glycans attached to SeAP produced by the Tn5B1-4 cells had complex forms. Glycans attached to SeAP from A7S cells contained 4% hybrid and 8% complex forms. Galactosylated biantennary structures were identified. Glycans attached to SeAP produced by the DpN1 cell line had 6% hybrid and 26% complex forms. Of the complex forms in SeAP from DpN1, 13% were identified as sialylated glycans. The galactosyltransferase activity of the three cell lines was measured and correlated to their ability to produce complex forms. Even though neither novel cell line produced as much recombinant protein as the Tn5B1-4 cells, the glycosylation of SeAP expressed by both cell lines was more complete. These novel cell lines represent interesting alternatives for the production of complex glycosylated proteins utilizing the BEVS.

Secreted human placental alkaline phosphatase (SEAP) was produced in a nonengineered Trichoplusia ni insect cell line, Tn-4s, using a recombinant Autographa californica baculovirus expression vector. The effect of culture conditions on SEAP specific yield and glycosylation was studied. When cultured in the high aspect ratio vessel (HARV) or in tissue culture flasks (T-flasks), baculovirus-infected Tn-4s cells produced high levels of SEAP (13 and 23 U/10(6) cells, respectively; 4 days postinfection), but in those conditions SEAP possessed only high mannose, paucimannosidic, and hybrid structures. In spinner flasks, lower SEAP yields were obtained (<4 U/10(6) cells, 3 days postinfection), but in such cultures, sialylation of SEAP could be achieved. Several spinner-flask culture conditions were tested and resulted in different SEAP specific yields and levels of sialylation. The highest level of sialylation (9%) was obtained in the culture with the lowest agitation rate and lowest yield (1.2 U/10(6) cells), suggesting a limiting capacity of the Tn-4s cells to process glycoproteins to sialylation. High specific yield, low passage number Tn5B1-4 cells did not produce SEAP with complex glycosylation when cultured in a low agitation rate spinner-flask. On the basis of these results, we propose that the Golgi apparatus has a limited capacity for processing proteins to complex glycosylation and sialylation and that this capacity is easily overwhelmed by high levels of foreign protein productivity. Selected media additives such as Pluronic F-68, dextran sulfate (MW 12 500) and a lipids premix did not allow improvement of the specific yield of sialylated SEAP when supplemented to spinner-flask cultures.

Using a nonengineered Trichoplusia ni insect cell line, Tn-4s, infected with an Autographa californica recombinant baculovirus, 20% sialylation of human secreted placental alkaline phosphatase (SEAP) was observed. In contrast to this level of sialylation, intermediate complex forms with terminal galactose or N-acetylglucosamine were found in low proportions (<3% and <1%, respectively). We tested whether time of harvest or degradation of intermediate complex forms is responsible for this distribution of glycoforms. Spinner-flask cultures were infected with the SEAP baculovirus expression vector, and the cultures were harvested 48, 72, and 96 h post-infection. Structural analysis revealed that the glycoform distribution of SEAP was very similar at the different times of harvest, indicating that the cellular machinery was not significantly affected by the progress of infection and that the glycoforms obtained were stable. High levels of beta-galactosidase and N-acetylglucosaminidase activity were detected throughout infection. In contrast, sialidase activity was below detection level both in cell extracts and in supernatants. These levels of glycosidases activities raise the possibility that intermediate complex glycoforms may be degraded while sialylated forms should not experience significant degradation in this cell line. However, culture in the presence of extracellular beta-galactosidase and N-acetylglucosaminidase inhibitors did not significantly improve glycosylation, suggesting that extracellular degradation processes are not taking place. Instead, results suggest that the intracellular machinery of the Tn-4s cells tends to either shunt the glycans to paucimannosidic forms or drive them completely to sialylation.

A recombinant N-linked glycoprotein, secreted human placental alkaline phosphatase (SEAP), was produced in two Trichoplusia ni insect cell lines using the baculovirus expression vector. Silkworm hemolymph (SH) was added to TNMFH + 10% fetal bovine serum (FBS) medium to a concentration of 2.5% or 5%, and SEAP production and glycosylation in the presence of SH were compared with controls devoid of hemolymph. Growing Tn-4s cells in 5% SH-supplemented medium required progressive adaptation of the cells to SH, and adapted cells had a SEAP specific yield decreased by 2.5-fold compared with control cells not exposed to SH. Although SEAP produced in the control possessed little complex glycosylation (<1%), SEAP produced by SH-adapted cells in the presence of 5% SH possessed 8.7% sialylated structures, as well as unusual, asialylated, agalactosylated structures with a high degree of polymerization (DP). On the basis of enzymatic and mass-spectrometric analyses, we propose that these structures are glucosylated, high-mannose oligosaccharides. SEAP was also produced by Tn-4s cells without adaptation to SH when SH was added just prior to baculovirus infection, but SEAP specific yield was adversely affected (approximately fourfold reduction compared with control devoid of hemolymph), and glycosylation of SEAP produced under these conditions was characterized by large amounts of high-mannose and high-DP structures and an absence of complex structures. Similarly, Tn5B1-4 cells that were not adapted to SH had a SEAP specific yield reduced by approximately fivefold in SH-containing medium; however, these cells were able to produce 13.5% sialylated SEAP in the presence of 2.5% SH, whereas complex structures were not produced in the absence of SH. We propose that SH improves glycosylation either directly or indirectly by decreasing SEAP specific yield.