Characterization of the Glycan Structures of GlycoproteinGA733‐Fc Expressed in a Baculovirus‐Insect Cell System

Abstract

Baculovirus-insect cell systems have been used to express functional recombinant biopharmaceutical proteins. Two pFastBac Dual vectors carrying the gene encoding antigen GA733, a cell-surface glycoprotein, fused to the IgG Fc (GA733-Fc) or KDEL (endoplasmic reticulum [ER] retention sequence) (GA733-FcK) genes were constructed to generate baculoviruses expressing the corresponding recombinant proteins in insect cells. The expression of the recombinant GA733-Fc and GA733-FcK proteins and their glycan structure profiles were assessed under various conditions by analyzing the cell line (Sf9 and High Five), the postinfection (PI) time (48, 72, and 96 h), and the harvested sample (cell culture media [CM] or cell lysate [CL]). Immunoblotting showed nonidentical expression of both GA733-Fc and GA733-FcK under various conditions. Glycosylation analysis revealed that varying conditions affected the glycan structure profiles. These results suggest that the PI time, subcellular localization, and cell type affect recombinant protein expression as well as glycosylation in the baculovirus-insect cell system. ExperimentalBacmid Generation. The GA733-Fc and GA733-FcK genes were cloned under the control of the polyhedrin promoter

Baculovirus titration method based on MOI values for optimizing recombinant protein expression of the anti‐cancer vaccine candidate GA733‐Fc using Sf9 insect cells

“…The baculovirus expression vector system (BEVS) has been proven useful for the production of recombinant proteins in eukaryotic insect cells (Deparis et al 2003;Song et al 2010;Park et al 2011Park et al , 2014Park et al , 2015Lee & Ko 2014;Kim et al 2015;Qiao et al 2015;Lim et al 2015aLim et al , 2015bLee et al 2016). BEVS has many advantages, including scalability using high-density suspension cultures, capacity for expressing multiple genes at high expression levels, and the possibility of post-translational modifications (Deparis et al 2003;Malde & Hunt 2004;Kost et al, 2005).…”

“…BEVS has many advantages, including scalability using high-density suspension cultures, capacity for expressing multiple genes at high expression levels, and the possibility of post-translational modifications (Deparis et al 2003;Malde & Hunt 2004;Kost et al, 2005). In the present study, we used the BEVS technology to express diverse recombinant proteins, including anti-colorectal cancer monoclonal antibody CO17-1A (Song et al 2010;Park et al 2011Park et al , 2014Lee & Ko 2014 and anti-cancer vaccine candidate GA733-Fc (Kim et al 2015;Park et al 2015;Qiao et al 2015;Lim et al 2015aLim et al , 2015bLee et al 2016).…”

“…Optimization of infection conditions influences the expression of recombinant proteins, such as the monoclonal antibody CO17-1A (Park et al 2011), which recognizes the colorectal antigen GA733-2 (gastrointestinal tumorassociated antigen 2) (Strassburg et al 1992;Zaloudik et al 2002;Lu et al 2012;Kim et al 2015;Park et al 2015;Qiao et al 2015;Lim et al 2015aLim et al , 2015bLee et al 2016). Accurate viral stock titration is crucial for determining the multiplicity of infection (MOI) value, which represents the total number of virions added per cell during infection (Neutra et al 1992;Mulvania et al 2004;Janakiraman et al 2006) and ultimately determines recombinant protein expression (Radford et al, 1997).…”

“…Thus, in this study, the Sf9 cell line, which was derived from the parental Spodoptera frugiperda cell line, was used for virus titration (Lee & Ko 2014;Park et al 2014;Qiao et al 2015). We then investigated the effect of the KDEL sequence on the expression of the colorectal cancer vaccine candidate GA733-Fc [a human epithelial cell adhesion molecule (EpCAM)] fused to the human IgG Fc region.…”

Purification of anti‐colorectal cancer monoclonal antibody CO17‐1A from insect cell culture using a French press and sonication

“…These cells originated from IPLBSF-21, a cell line derived from the pupal ovarian tissue of the fall army worm, Spodoptera frugiperda, in cell suspension cultures (Vaughn et al 1977;O'Reilly 1997;Jarvis 2003). The anti-colorectal cancer therapeutic monoclonal antibody (mAb) CO17-1A, which recognizes the colorectal cancer surface protein GA733-2, has been expressed successfully in the Sf9 insect cell system (Song et al 2010;Park et al 2011) and its expression conditions were optimized in our previous studies (Song et al 2010;Park et al 2011Park et al , 2014Qiao et al 2015). After successful protein expression, insect cell lysis is an important step in the ensuing purification of the newly produced recombinant proteins (O'Shaughnessy & Doyle 2011).…”