100-liter transient transfection

This is the first report of two successful 100 l scale transient transfections in a standard stirred bioreactor. More than half a gram of a monoclonal antibody (IgG) were produced in less than 10 days using a technology called large-scale transient gene expression (LS-TGE). Suspension adapted HEK 293 EBNA SF cells were transfected within a 150 l (nominal) bioreactor by a modified calcium phosphate co-precipitation method with more than 75 mg of plasmid DNA per run. A mixture of three different plasmids, one encoding for the heavy chain of a human recombinant immunoglobulin, the other for the corresponding light chain and a third one for the green fluorescent protein (GFP, 2-4% of DNA in transfection cocktail) were co-transfected. The GFP vector was chosen to monitor transfection efficiency. Expression of GFP could be registered as early as 20 h after DNA addition, using fluorescence microscopy. We demonstrate that transient transfection can be done at the 100 l scale, thus providing a new tool to produce hundreds of milligrams or even gram amounts of recombinant protein. A key advantage of LS-TGE resides in its speed. In the presented cases, the entire production process for the synthesis of half a gram of a recombinant antibody, including DNA preparation and necessary expansion of cells prior to transfection, was executed in less than a month. Having an established transfection/expression process allows to run production campaigns for any given protein, within one facility, with one single host cell line and therefore only one single seed train. Without any need to create and maintain stable cell lines, expression of new r-proteins is not only faster and more economical but also more flexible.