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.

Keywords: antibody production; bidirectional; monoclonal antibodies; promoters; upstream processing.

Figure 1

Schematic illustration of BiDi promoter system for antibody production. (A) The MD vector was designed to exhibit a 200-bp stuffer, flanked by Esp3I restriction sites (Esp3I sites A and B), adjacent to a SV40 polyA signal sequence and the regions encoding for hinge-CH2-CH3, terminated by a SV40 polyA signal sequence. (B) VL-CL and VH-CH1 amplicons can be inserted into MD by Golden Gate cloning utilizing Esp3I restriction sites (Esp3I sites A and B). The BiDi promoter can be chosen individually and is flanked by BbsI sites (BbsI sites A–D), compatible with the VL and VH sequences. (C) Golden Gate assembly results in a fully functional and re-circularized vector, with the light chain under the control of promoter I and the heavy chain under the control of promoter II. (D) Schematic representation of the resulting heterotetrameric IgG1 antibody using the same colour code as for the genetic elements.

Figure 2

Overview of the different bidirectional combinations tested. The 200-bp stuffer sequence is marked in red for each construct. Abbreviations: minimal CMV (minCMV), cytomegalovirus promoter (CMV), enhanced CMV (eCMV), major immediate early enhancer (MIE), human translation elongation factor 1 alpha (EF-1α), adenoviral tripartite leader sequence (TPL CDS), adenovirus major late promoter enhancer (MLP enh.), splicing donor site (SD), splicing acceptor site (SA), light chain (LC), heavy chain (HC).

Figure 3

Gene expression analysis of heavy and light chain genes after transient transfection of Durvalumab in Expi293-F cells. (A) Bar chart representing heavy (dark blue) and light (light blue) chain mRNA expression in the different constructs. Values are relative to the CMV-minCMV construct and normalised to housekeeping genes GAPDH and RPLP0. Error bars represent the standard error of the mean of technical triplicates. (B) Heat map representation of gene expression analysis. The relative normalised gene expression for light and heavy chain mRNA is shown on the right.

Figure 4

Gene expression analysis of heavy and light chain genes after transient transfection of Durvalumab in ExpiCHO-S cells. (A) Bar chart representing heavy (dark blue) and light (light blue) chain mRNA expression in the different constructs. Values are relative to the CMV-minCMV construct and normalised to housekeeping genes GAPDH and RPLP0. Error bars represent the standard error of the mean of technical triplicates. (B) Heat map representation of gene expression analysis. The relative normalised gene expression for light and heavy chain mRNA is shown on the right with their respective scales.

Figure 5

Protein quantification of Durvalumab in cell culture supernatants from transfected Expi293-F cells. (A) Table showing the mAb concentrations from 24-well transient transfections, listed according to their rank. The ranks 1–5 were set based on the mAb concentration of the different BiDi combinations. (B) Bar chart representation of BiDi mAb concentrations.

Figure 6

(A) Correlation of mAb concentration and Ct values for both heavy and light chain expression in Expi293-F for production of Durvalumab. (B) Quantification of antibody concentration for the production of Durvalumab and Avelumab using either a 2-plasmid reference or the BiDi 2xeCMV construct. Error bars represent the standard error of the mean of biological triplicates, while the symbols represent the individual measurements.