Impact of Cistron Topology and Bicistronic Vector Design in the piggyBac Transposon System for Improved Antibody...

The use of transposon-based technologies in cell line development has gained significant traction over the past decade. This study investigates the piggyBac transposon system combined with bicistronic vectors for monoclonal antibody expression, helping address the complex demands of multi-specific antibody formats that require multiple cistrons. We systematically evaluated bicistronic vector modifications within the piggyBac framework, including promoter sequences, reporter placements, and cassette configurations, and compared performance to piggyBac/single-gene vector co-transfection methods. Our findings demonstrate that double human CMV promoter configurations driving both heavy and light chain genes significantly enhanced pool productivity (2 to 2.5-fold) and reporter expression compared to separate promoter designs. Among tested cassette arrangements, the Light chain-GS-Heavy chain configuration yielded optimal productivity (1.5 to 6-fold) and superior heavy chain/light chain RNA transcript ratios (1.4 to 3.8-fold and 1.3 to 6.1-fold, respectively). While unfed batch conditions showed comparable productivities between optimized piggyBac/bicistronic and piggyBac/single-gene systems, the bicistronic approach exhibited superior productivity performance under fed-batch conditions (1.5-fold), with the resulting clones demonstrating significantly high productivity (top clone 9.6 g/L). This represents the first reported use of LC-selection marker-HC topology for monoclonal antibody expression, establishing a foundation for improved therapeutic biologic production through multicistronic vector systems combined with the piggyBac transposon strategy.