Efficient production of bispecific antibodies—optimization of transfection strategy leads to high-level stable cell line generation of a Fabs-in-tandem immunoglobin

Bispecific antibodies (bsAbs) are often composed of more than two component chains, such as Fabs-in-tandem immunoglobin (FIT-Ig) comprising three different component chains, which bring challenges for generating a high proportion of the correctly assembled bsAbs in a stable cell line. During the CHO-K1 stable cell line construction of a FIT-Ig, we investigated the FIT-Ig component chain ratio in transfection, where two sets of expression vectors were designed. Both designs utilized two vectors for co-transfection. Multiple transfections with plasmid ratio adjustment were applied, and the resultant minipools were evaluated for expression titer and quality of produced FIT-Ig. The results suggested that abundant outer Fab short chains (twofold chain genes versus other chains) can promote complete FIT-Ig assembly and therefore reduce the fragmental impurities of FIT-Ig. This adjustment of the component chain ratios at the beginning is beneficial to FIT-Ig stable cell line generation and brings favorable clones to process development.