Generation of high‐expressing transposon‐based stable pools to produce (1 + 1) and (2 + 1) bispecific common light chain antibodies

Abstract Bi‐ and multispecific antibodies can deliver effective protein‐based therapeutics by enabling novel biology. Transient expression of these complex asymmetric antibody formats may become a bottleneck in providing sufficient amounts of high‐quality material for preclinical discovery and developability assessment to support candidate selection activities. In contrast, transposase‐based stable cell line generation is being recognized as a robust and rapid path to enable the evaluation of product quality from stable pool derived material. Transposase‐based systems feature a semi‐targeted integration of multiple copies of the gene(s) of interest in the host cell's genome resulting in the rapid generation of homogeneous high‐expressing (up to g/L range) stable pools after 1–3 weeks of selection phase. Here, we evaluated the Leap‐In® transposase technology for the expression of IGI's 1 + 1 and 2 + 1 bispecific, common light chain antibodies, based on IGI's proprietary BEAT® (Bispecific Engagement of Antibodies based on T cell Receptor) platform. To generate stable CHO pools expressing IGI's bispecific molecules, transposon‐based constructs expressing the common light chain and the two heavy chain genes were co‐transfected with the Leap‐In® transposase mRNA. Based on the proportion of the different chain combinations detected in the harvests, their expression levels were further optimized through vector engineering to facilitate heterodimer assembly and maximize productivity. The best pool displayed a titer of 2.33 g/L in 14‐day fed‐batch cultures, and heterodimer represented 93.8% of the expressed species. The purified antibody displayed 97.4% purity, as determined by SE‐HPLC and binding to targets within range, as determined by SPR and cell‐based affinity.