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.
Highlights
With the growing interest in monoclonal antibodies for therapeutic applications, advances in antibody production have improved drastically over the last decades
To produce full-length antibodies in a bidirectional manner, we first designed the respective vector in silico
In order to allow the flexible use for a variety of binders, the fragment antigen binding (Fab), which can be of the κ or λ isotype, was not encoded on the plasmid
Summary
With the growing interest in monoclonal antibodies (mAbs) for therapeutic applications, advances in antibody production have improved drastically over the last decades. The largest part of transient production of mAbs is carried out using a two-plasmid system, known as co-transfection, for the expression of lc and hc, with each gene driven by its own promoter and transcribed separately [17,18] These are carried out, for the most part, with an equimolar ratio of heavy chain and light chain genes. Andersen and co-workers have previously shown the ability of the CMV enhancer to control two core CMV promoters simultaneously, resulting in efficient antibody expression [22] With this basis, we sought to investigate different promoters in a bidirectional format to facilitate transient transfection, avoiding co-transfections. Plasmid DNA for transient transfection was isolated using the PureYield Plasmid Midiprep System (Promega, Walldorf, Germany)
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