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

Abstract

Monoclonal antibody (mAb) CO17-1A binds to GA733, which is a tumorassociated glycoprotein antigen highly expressed on the colorectal cancer cell surface. Thus, mAb CO17-1A is considered a useful biomolecule for diagnosis and treatment against colorectal cancer. Previously, we established a baculovirus-insect cell expression system for the production of mAb CO17-1A. In order to use mAb CO17-1A as a diagnostic and therapeutic tool, however, the antibody must be properly purified from the insect cells. In this study, our aim was to investigate effective purification processes of mAb CO17-1A expressed in Spodoptera frugiperda (Sf9) insect cells, using a French press and sonication for cell disruption. SDS-PAGE confirmed that both mAb CO17-1A and mAb CO17-1A fused to the KDEL endoplasmic reticulum (ER) retention signal (mAb CO17-1AK) were expressed clearly in Sf9 insect cells. Western blot analysis showed that detection levels of mAb CO17-1A and CO17-1AK were higher when the insect cells were disrupted two times by the French press and then sonicated, compared to only one French press disruption plus sonication. Optical microscopy confirmed that insect cells treated with both the French press and sonication were properly disrupted. Analysis of gene sequence information on mAb CO17-1A verified that a signal peptide is present but a transmembrane protein does not exist. These results suggest that cell disruption by the French press twice and sonication once is an effective method for improving purification efficiency.

In vitro wound healing: Inhibition activity of insect‐derived mAb CO17‐1A in human colorectal cancer cell migration

“…) and its purification (Lim et al . ), and confirmed its anti‐tumor activity using colorectal cancer cell invasion assay function (Moussavou et al . ).…”

“…However, the baculovirus‐mediated insect cell expression system has been considered an alternative for production of therapeutic recombinant glycoproteins, because of its advantages such as ease of production scale‐up, sufficient glycomodification, and absence of human pathogen contamination (Lim et al . ). In our previous study, we optimized the baculovirus insect cell expression system conditions for production of anti‐cancer mAb CO17‐1A (Song et al .…”

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).…”