The kinetics of polyethylenimine-mediated transfection in suspension cultures of Chinese hamster ovary cells

The kinetics of polyethylenimine (PEI)-mediated gene transfer at early times after transfection of Chinese hamster ovary (CHO) cell in suspension were investigated using a novel in vitro assay. Addition of an excess of competitor DNA to the culture medium at various times after the initiation of transfection inhibited further cellular uptake of PEI-DNA particles. Using this approach, a constant rate of particle uptake was observed during the first 60 min of transfection at a PEI:DNA ratio of 2:1 (w/w) and a cell density of 2 x 10(6) cells/ml under serum-free conditions. The uptake rate declined considerably during the next 2 h of transfection. Both the rate and the level of PEI-DNA uptake in serum-free minimal medium were found to be dependent on the PEI-DNA ratio, the cell density at the time of transfection, and the extent of particle aggregation. These studies of the early phase of PEI-mediated transfection are expected to lead to further opportunities for optimization of gene transfer to suspension cultures of mammalian cells for the purpose of large-scale transient recombinant protein production.

Transient gene expression for rapid protein production

Natalie Muller

Recombinant proteins are important for biomedical research and for the treatment of human disease. Therefore it is necessary to develop reproducible bioprocesses to rapidly produce proteins of adequate quality and quantity. Expression in mammalian cells is preferred if the proteins are to be properly folded and post-translationally modified. For the rapid production of milligram to gram quantities of a protein in mammalian cells, large-scale transient gene expression is an attractive option. The two most important components of this technology are the cell cultivation system and the gene delivery method. For this reason the goal of this thesis was to develop novel cost-efficient cell cultivation systems and gene delivery methods for the large-scale transfection of mammalian cells in chemically defined media free of any animal-derived components including serum. Cultivation of mammalian cells in suspension is essential for large-scale transient gene expression. A superior system for the cultivation of mammalian cells based on the agitation of cells in "square-shaped" glass bottles (square bottles) was developed. It is an inexpensive but efficient means to grow cells to a high density without instrumentation. The growth and viability of cultures of both human embryo kidney (HEK) 293E and Chinese hamster ovary (CHO) DG44 cells in agitated one-liter square bottles exceeded that in spinner flasks, reaching cell densities 1.5 times higher on average. Furthermore, an efficient and reproducible method to transfect cells in agitated square bottles was developed for both HEK 293E and CHO DG44 cells. A novel gene delivery method termed calfection that relies on the addition of DNA and CaCl2 directly to a suspension culture was developed. Calfection has a number of advantages over existing gene delivery methods such as calcium phosphate-DNA coprecipitation in that there are no time-dependent steps. The DNA/CaCl2 solution can be stored for long periods and is filterable. It is suitable for both large-scale transfections in bioreactors and for high-throughput transfections in microtiter plates. One drawback, however, is its dependence on the presence of serum in the culture medium. A second gene delivery method, serum-free transfection with polyethylenimine (polyfection), proved to be highly efficient for the transfection of both HEK 293E and CHO DG44 cells. A reproducible method for polyfection in microtiter plates, 50 ml centrifuge tubes, agitated square and round bottles, spinner flasks, and bioreactors was optimized for these two cell lines. Polyfection proved to be a simple and successful gene transfer method in both instrumented and non-instrumented cultivation systems. Importantly, it could be performed in serum-free chemically defined media. With HEK 293E cells, 80 mg/l of antibody was produced in agitated square bottles and with CHO DG44 cells, more than 2 g of antibody were produced in one week in a 150-liter bioreactor.

EPFL2005

Calfection: a novel gene transfer method for suspension cells

Philippe Girard, Martin Jordan, Natalie Muller, Florian Maria Wurm

We have developed a novel method called Calfection for gene delivery to and protein expression from suspension-cultivated mammalian cells. Plasmid DNA was simply diluted into a calcium chloride solution and then added to the cell culture for transfection. We evaluated and optimized this approach using suspension-adapted HEK293 cells grown in 12-well plates that were shaken on an orbital shaker. Highest expression levels were obtained when cells were transfected at a density of 5x10(5) cells/ml in the presence of 9 mM calcium and 5 microg/ml of plasmid DNA while maintaining a culture pH of 7.6 at the time of transfection. Suspension-adapted BHK 21 and CHO DG 44 cells could also be transfected using this method. Calfection differs from the widely known calcium phosphate coprecipitation technique. The physico-chemical composition of the DNA interacting complexes is not yet known. The transfection cocktail, DNA in a calcium chloride solution, remained highly efficient during long-term storage at temperatures ranging from room temperature to -80 degrees C. In contrast, calcium phosphate-DNA cocktails are only efficient for gene transfer when prepared fresh. Furthermore, passing the calcium-plasmid DNA mixture through a 0.2-microm filter did not compromise protein expression, whereas calcium phosphate-DNA coprecipitates were retained by the filter. High protein expression levels, a limited number of manipulations and the possibility to filter the cocktail make the Calfection approach suitable for both large-scale transfection in bioreactors and for high-throughput transfection experiments in microtiter plates.

2004

Accelerating the development time of recombinant mammalian cell lines producing high titers of protein therapeutics

Sébastien Chenuet

The forthcoming arrival on the market of numerous protein therapeutics that require high clinical doses will foster the need for high-producing mammalian cell lines. Furthermore, pressures to reduce the development time for new therapeutics has meant more emphasis on efforts to accelerate the process that yields such cell lines. The objective of this thesis work was to develop methods to decrease the time needed to generate stable and high producing mammalian cell lines. This could be achieved by overcoming some of the major limitations that hamper the process of cell lines development. The host systems chosen for this study were Chinese hamster ovary (CHO DG44) and baby hamster kidney (BHK-21). The recombinant proteins used as reporters were the green fluorescent protein (GFP) and a human anti-Rhesus D IgG. One limitation hampering the development of cell lines producing high recombinant protein titers is the extensive variability of transgene expression within a transfected population as a result of the gene delivery process. This decreases the reproducibility of the generation of high producing cell lines. A direct comparison of two gene chemical delivery methods, polyethylenimine (PEI)- and calcium phosphate (CaPO4)-mediated transfection, with a mechanical method (microinjection) provided some insights into ways to overcome this limitation. Indeed, a correlation was observed between the amount of plasmid DNA delivered into cells and the specific productivity of the recovered recombinant cell lines. Hence, by increasing the amount of DNA delivered per cell, IgG-producing clones with specific productivities up to 22 pg/cell/day were recovered with a high efficiency. A second limitation is the time needed to assure the stability and clonality of cell lines by current analytical methods. This was overcome by using GFP as a reporter for the level of production of a co-expressed therapeutic protein. The co-transfection of plasmids individually carrying the GFP and IgG heavy (Hc)- and light chain (Lc) genes, resulted in integration of all three plasmids at the same site in the cell's genome. As a consequence, the stability of GFP expression correlated with that of IgG expression. This proved to be advantageous finding and eliminating unstable cell lines early in the process of selection. As a consequence, it was possible to derive in serum-free medium clones with a specific productivity almost 6 times higher than that of clones derived from transfection performed with the IgG Hc and Lc vectors only. A third limitation to the rapid recovery of stable cell lines is the time devoted to the cultivation of transfected cells in selective medium, normally one month. Here, the time of selection was shortened to one week by increasing the stringency of the selective pressure. The results from studies of plasmid integration kinetics by fluorescence in situ hybridization (FISH) showed that the increased stringency of selection decreased the time needed to enrich a transfected population with recombinant cells. Furthermore, stable clones were shown to be present in this population after one week of selection and could be uncovered by identifying patterns of GFP expression predictive of production stability. By limiting the screening to clones exhibiting these stable patterns of GFP expression, the number of clones needed in order to isolate stable cell lines producing high IgG titers could be dramatically reduced. In conclusion, these results combined together provided a new procedure to isolate within a week of the start of selection, stable and high-producing clones. 30-40% of the clones isolated using this procedure were stable for at least three months and their average specific productivity was 29 pg/cell/day.