Targeted dual selection to optimize transposon stable pool generation of multispecifics

Growing knowledge around disease states has led to opportunities within research to make designer molecules with improved specificity and broader efficacy. These next-generation molecules frequently take advantage of multispecific targeting and controlled mechanisms of action by utilizing four unique peptide chains as seen in many bispecific or trispecific antibodies. However, with all the opportunities these multispecifics offer, their increased biological complexities come with increased challenges during expression and purification to produce high-quality material. Lower yields accompanied with a high degree of mispairing after the initial capture purification step are often limiting factors. Developing new methods for stable pool expression can offer a strong advantage for progressing these molecules through research toward development. Here, we implemented optimized stable cell pools using targeted dual selection (TDS), a novel approach that combines specified selective pressure with transposon-guided semi-targeted gene integration. By utilizing key analytical data obtained during early-stage high-throughput transient productions, we can predict improved vector configurations for the generation of optimized TDS stable pools. We demonstrate that this design can improve molecule quality at the initial capture purification step in two Y-shaped bispecific molecules and two cross-over dual variable trispecific molecules by achieving up to four-fold increase in protein of interest yields while maintaining product quality. Use of this strategy in research can enable simplified purification strategies as well as increased production yields required for successful and timely project progression.