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Abstract

This is the first report of two successful 100 l scale transienttransfections 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 transfectedwithin a 150 l (nominal) bioreactor by a modified calcium phosphateco-precipitation method with more than 75 mg of plasmid DNA per run.A mixture of three different plasmids, one encoding for the heavychain 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 asearly as 20 h after DNA addition, using fluorescence microscopy. We demonstrate that transient transfection can be done at the100 l scale, thus providing a new tool to produce hundreds of milligrams or even gram amounts of recombinant protein. Akey advantage of LS-TGE resides in its speed. In the presentedcases, the entire production process for the synthesis of halfa gram of a recombinant antibody, including DNA preparationand necessary expansion of cells prior to transfection, wasexecuted in less than a month. Having an established transfection/expression process allows to run productioncampaigns for any given protein, within one facility, with onesingle 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.

Optimization of transfection mediated by calcium phosphate for plasmid rAAV‐LacZ (recombinant adeno‐associated virus–β‐galactosidase reporter gene) production in suspension‐cultured HEK‐293 (human embryonic kidney 293) cells

“…Increasing the shaking or stirring speed could control the size of cell aggregates [32], but enhanced shear stress would also lead to lower cell viability in suspension culture. Some researchers have reported that the chelation of extracellular Ca 2+ induced disassembly of tight and adherent junctions [29,33].…”

Transient Gene Expression in Mammalian Cells: Promises and Challenges for Medical Biotechnology

“…While large-scale TGE has been carried out in a variety of instrumented and noninstrumented cultivation vessels, the largest transfection reported to date has been in a 150-L stirred tank bioreactor with a working volume of 110 liters with HEK 293 cells (104). The largest transfection with CHO cells has been 80 L (99).…”

“…The largest transfection with CHO cells has been 80 L (99). Overall, secreted r-protein yields in the range of 2-22 mg/L have been achieved in instrumented bioreactors ranging in nominal volume from 3-to 150-l using either batch or fed-batch approaches with either HEK 293 or CHO cells (83,94,96,99,104). The Wave bioreactor with disposable plastic bags has also proven to be well-suited for TGE with the largest volume reported to be 10 L with cultures of HEK 293 cells (105).…”

Strategies for the efficient synthesis of stimuli-responsive ss-DNA bioconjugates for capture and transfection of plasmid DNA

“…However, fairly large amounts of plasmid DNA are needed for the transient transfection of bioreactors at large scale [41]. Under such circumstances the efficiency of the DNA transfection as well as the cost of the plasmid are of major concern, while the purity of the plasmid per se is only of secondary importance as long as an adverse effect of the putative impurities (toxicity, introduction of adventitious agents) can be excluded.…”

“…The methods of choice for the transient transfection used in our laboratory are the DNA-calcium phosphate coprecipitation technique [41] and the Lipofectamine 2000 method (GibcoBRL). It was not possible to use the calcium phosphate transfection protocol with plasmid DNA still linked to the AML (protein expression was unsatisfactory, data not shown).…”