Rapid and cost-effective development of stable clones for the production of anti-Ebola monoclonal antibodies in HEK293T cells

Abstract

The Ebola virus (EBOV) disease has caused serious and recurrent epidemics in recent years, resulting in a fatality rate of nearly 50%. The most effective experimental therapy against the EBOV is the use of monoclonal antibodies (mAbs). In this work, we describe the development of HEK293T cells engineered for the transient and stable expression of mAb13C6, a neutralizing anti-EBOV monoclonal antibody. We transfected the HEK293T cells with a tricistronic vector to produce the heavy and the light chain of the antibody 13C6 and intracellular Green Fluorescent Protein (GFP) using Lipofectamine 3000. We then selected the transfected cells using puromycin pressure, dilution cloning, and cloning disks. This integrated strategy generated mAb-producing cells in 7 days with a transient expression of ~1 mg/L. Stable pools were produced after 4 weeks, with expression levels of ~0.8 mg/L. Stable clones with expression levels of ~1.8 mg/L were obtained within 10 weeks. The produced antibodies exhibited the expected functionality; they recognized the GP glycoprotein of the Ebola virus in both ELISA assays and cell binding experiments using HEK293T cells engineered to express the EBOV GP at their membrane surface. By the combined use of GFP and the set of selection techniques here described, we drastically reduced the time from transfection to stable clone generation without resorting to costly equipment. In outbreaks or emergencies, this platform can significantly shorten the development of new biopharmaceuticals and vaccines.

Efficient Production of Human Hyaluronidase PH20 in HEK293T Cells Using Site Specific Integration

“…They are employed for both transient and stable protein expression, and can be cultured in both suspension and a monolayer. HEK cells are transfected easily with a variety of methods and have the capacity of a cell factory for manufacturing a large amount of recombinant proteins [27,28]. The important nding in our experiments was that the expressed rhPH20 could not cause damage of HEK cells due to this fact that the cells are semi-adherent in comparison to adherent CHO cells.…”