Recombinant Antibody Production Using a Dual-Promoter Single Plasmid System

Abstract

Monoclonal antibodies (mAbs) have demonstrated tremendous effects on the treatment of various disease indications and remain the fastest growing class of therapeutics. Production of recombinant antibodies is performed using mammalian expression systems to facilitate native antibody folding and post-translational modifications. Generally, mAb expression systems utilize co-transfection of heavy chain (hc) and light chain (lc) genes encoded on separate plasmids. In this study, we examine the production of two FDA-approved antibodies using a bidirectional (BiDi) vector encoding both hc and lc with mirrored promoter and enhancer elements on a single plasmid, by analysing the individual hc and lc mRNA expression levels and subsequent quantification of fully-folded IgGs on the protein level. From the assessment of different promoter combinations, we have developed a generic expression vector comprised of mirrored enhanced CMV (eCMV) promoters showing comparable mAb yields to a two-plasmid reference. This study paves the way to facilitate small-scale mAb production by transient cell transfection with a single vector in a cost- and time-efficient manner.

Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells

“…Due to the physical linkage with the yeast Gal1,10 promoter, heavy chain—light chain coupling is maintained. In the next step, this yeast-specific promoter is exchanged by Bbs I-mediated Golden Gate Assembly for the 2xeCMV promoter, which was recently demonstrated to be the optimal bidirectional promoter for mAb expression ( Carrara et al, 2021b ). An overview of the entire procedure is depicted in Figure 1 .…”

“…By Sap I-mediated GGC, VH and VL sequences, previously amplified from cDNA (see below) were subcloned into the destination vector, similar to the approach described previously ( Rosowski et al, 2018 ). The mammalian destination (MD) vector was recently described in detail ( Carrara et al, 2021b ). In brief, the MD vector encodes a stuffer sequence flanked by Esp3 I sites, adjacent to coding sequences comprising partial hinge-CH2-CH3 domains.…”

“…The 2xeCMV promoter combination was shown to be the most suitable for recombinant antibody production and was thus used herein. The sequence of this promoter construct is available from ( Carrara et al, 2021b ).…”

“…To continue the production of IgG molecules in mammalian cells and avoid the cumbersome reformatting steps, we have developed a novel two-pot, two-step cloning procedure in order to facilitate the transition of hit candidates from a YSD-display vector to a mammalian bidirectional (BiDi) expression vector. Initial studies were carried out to analyse the most suitable BiDi promoter for both κ- and λ-isotype antibodies ( Carrara et al, 2021b ). On top of simplifying and facilitating the transition between display on yeast cells to production in mammalian cells, VH and VL pairing is also preserved.…”

Strategies and Considerations for Improving Recombinant Antibody Production and Quality in Chinese Hamster Ovary Cells

“…The heavy- and light-chain genes of antibodies use different vectors and then enter the host cells by co-transfection. This method has very low requirements on the vector, but the transfection efficiency is high, which is a common strategy for recombinant antibody expression ( Carrara et al, 2021 ). However, the main defect of this strategy lies in the random integration of light- and heavy-chain genes into chromosomes, and the insertion positions and numbers are different, resulting in uncontrollable expression levels of light and heavy chains ( Ahmadi et al, 2016 ).…”