SweetBac: A New Approach for the Production of Mammalianised Glycoproteins in Insect Cells

Abstract

Recombinant production of therapeutically active proteins has become a central focus of contemporary life science research. These proteins are often produced in mammalian cells, in order to obtain products with post-translational modifications similar to their natural counterparts. However, in cases where a fast and flexible system for recombinant production of proteins is needed, the use of mammalian cells is limited. The baculoviral insect cell system has proven to be a powerful alternative for the expression of a wide range of recombinant proteins in short time frames. The major drawback of baculoviral systems lies in the inability to perform mammalian-like glycosylation required for the production of therapeutic glycoproteins. In this study we integrated sequences encoding Caenorhabditis elegans N-acetylglucosaminyltransferase II and bovine β1,4-galactosyltransferase I into the backbone of a baculovirus genome. The thereby generated SweetBac virus was subsequently used for the production of the human HIV anti-gp41 antibody 3D6 by integrating heavy and light chain open reading frames into the SweetBac genome. The parallel expression of target genes and glycosyltransferases reduced the yield of secreted antibody. However, the overall expression rate, especially in the recently established Tnao38 cell line, was comparable to that of transient expression in mammalian cells. In order to evaluate the ability of SweetBac to generate mammalian-like N-glycan structures on 3D6 antibody, we performed SDS-PAGE and tested for the presence of terminal galactose using Riccinus communis agglutinin I. The mammalianised variants of 3D6 showed highly specific binding to the lectin, indicating proper functionality. To confirm these results, PNGase A released N-glycans were analyzed by MALDI-TOF-MS and shown to contain structures with mainly one or two terminal galactose residues. Since the presence of specific N-glycans has an impact on antibodies ability to exert different effector functions, we tested the binding to human Fc gamma receptor I present on U937 cells.

MultiBac turns sweet

“…Especially the results for the recently established Tnao38 cell line must be highlighted, because yields of 30 μg/ml cell culture supernatant were reported. 35 The presence of terminal galactose residues on SweetBac expressed antibodies was confirmed by analyzing PNGase A released N-glycans with MALDI-TOF-MS. The more complex type N-glycan structures had no influence on antibodies target binding ability, but caused a significantly enhanced binding to human Fc gamma receptor I (CD64).…”

“…33 However, all these approaches are based on Sf 9 cells, but for the expression of secreted proteins, cell lines derived from Trichoplusia ni have been demonstrated to produce significantly higher amounts in many cases. 9,34,35 The recently developed SweetBac system, 35 a platform for the expression of mammalianised proteins in insect cells that is based on MultiBac, might serve to solve the problems stated above (a schematic representation of SweetBac is shown in Fig. 3).…”

Adding Functions to Biomaterial Surfaces through Protein Incorporation

“…Baculovirus-based systems provide routine expression of heterologous proteins in a robust, safe and scaleable manner [96] characterized by extremely high yields while glycosylation patterns similar to those found in human proteins can be achieved for in vivo applications. [97] Mammalian cell expression is the most reliable technology for the production of complex human proteins. [98,99] The major advantages of mammalian platforms remain their advanced folding, secretion and glycosylation capacities, while alternative cell lines have been engineered for improved expression of complex human glycostructures.…”

The MultiBac Baculovirus/Insect Cell Expression Vector System for Producing Complex Protein Biologics

“…Successful Tn7 and also Cre integration is imposed by antibiotic selection against the resistance markers encoded by the DNA plasmid modules integrated into the MultiBac genome. The integration sites can be used to integrate genes encoding for one or several multiprotein complexes of choice, but also for additional functionalities that may be required to activate or inactivate the complex (kinases, phosphatases, acetylases, deacetylases, others), support its folding (chaperones) or posttranslational processing such as glycosylation [19,23,[44][45][46].…”