The Syntax of the Selection Marker Within an Antibody Producing Plasmid can Affect Productivity

• Derek Consula, Chemical Engineering, University of Delaware

• Topher Pirner, Chemical Engineering, University of Delaware

There is an emerging need for advanced biotherapeutics, particularly in the monoclonal antibody market, which is expected to increase twofold by 2034. Chinese hamster ovary (CHO) cells have been demonstrated to be the ideal vessel for antibody production, exhibiting high rates of production and secretion, human-like post-translational modifications, and easy-to-scale cultures. To develop antibody-expressing CHO cell lines, three genes must be integrated in the CHO cell: an antibody light chain, an antibody heavy chain, and a selection marker. Previously, we have established that the relative position of all three genes can impact the titer and specific productivity of CHO cells. However, recent studies have also emphasized the importance of gene orientation when developing expression plasmids for mammalian cells. This experiment seeks to build upon our previous studies by also determining the importance of gene orientation in tandem with relative gene position. 

Six plasmids were developed, consisting of light and heavy chain antibody fragments expressed with strong promoters, and a selection marker, glutamine synthetase (GS), expressed with a weak promoter. Each configuration varied the position and orientation of the GS gene relative to the antibody fragments​. The plasmids were co-transfected with the JumpIN transposase into CHO via electroporation. Additionally, the cells were supplemented with MSX during selection, as preliminary testing revealed that cell lines selected in MSX recorded a three-fold increase in light and heavy chain copy number. Initial growth data show that placing the GS gene in the middle, regardless of direction, worsened selection behavior. These data suggest that GS selection is more stringent, which may indicate an increase in light and heavy chain expression and copy number.

Subsequent analysis upon each cell line will include qPCR to determine copy numbers and RNA transcripts, alongside fed batch cultures to quantify antibody titers.