100 Liter Transient Transfection

Abstract

This is the first report of a successful 100 Liter scale transient transfection in a standard stirred bioreactor. More than half a gram of a monoclonal antibody (IgG) was produced in less than 10 days using a technology called large-scale transient gene expression (LS-TGE). Suspension adapted HEK 293 EBNA cells were transfected within a 150 I (nominal) bioreactor by a modified calcium phosphate co-precipitation method using a total of 76.3 mg of plasmid DNA. A mixture of three different plasmids, one encoding for the heavy chain of' a human recombinant immun-oglobulin, the other for the corresponding light chain and a third vector for the green fluorescent protein (GFP, 4% of DNA in transfection cocktail) were co-transfected. The GFP vector was chosen to monitor transfeclion efficiency. Expression of GFP could be registered as early as 20 hours after DNA addition, using fluorescence microscopy. We demonstrate that transient transfection can be done at the 100-liter scale, thus providing a new tool to produce hundreds of milligrams or even gram amounts of recombinant protein. A key advantage of large scale TGE lies in its speed. In the presented case, 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 transfectionlexpression process allows to run production campaigns for any given proteins 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.