Recombinant protein production from stable mammalian cell lines and pools

We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced.

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

Study of transposon-mediated cell pool and cell line generation in CHO cells

Sowmya Balasubramanian

The goal of this thesis was to evaluate the use of artificial transposon systems for the generation of recombinant cell pools and cell lines with Chinese hamster ovary (CHO) cells as the production host. Transposons are naturally occurring genetic elements present in the genome of most organisms. Several transposons of metazoan origin have been engineered to facilitate the integration of one or more recombinant genes (transgenes) into the genome of the host cell. In this thesis, three of the most commonly used transposons, piggyBac (PB), Tol2, and Sleeping Beauty (SB), were used for transgene delivery into CHO-DG44 cells. The transposon systems described here involves co-transfection of the host cells with a helper plasmid for the transient expression of the transposase, and a donor plasmid coding for the transgene and a gene for the selection, both positioned between two transposon repeat sequences that are required for DNA transposition. Through the optimization of various selection parameters we showed that PB-mediated cell pools can be generated with selection duration of as little as 5 days in the presence of puromycin. All three transposon systems, PB, Tol2, and SB, resulted in cell pools with similar volumetric TNFR:Fc productivities that were about 9 times higher than those generated by conventional plasmid transfection. Transposon-mediated cell pools had 10 - 12 transgene integrations per cell. However, we demonstrated that some the integration events occurred via DNA recombination rather than transposition. We also isolated clonal cell lines from cell pools. As expected, the average volumetric TNFR:Fc productivity of transposon-derived cell lines was higher than that of cell lines generated by conventional transfection. In 14-day fed-batch cultures, protein levels up to 900 mg/L and 1.5 g/L were obtained from transposon-mediated cell pools and cell lines, respectively. The stability over time of the volumetric productivity of cell pools was determined by maintaining the cells in culture for 3 months in the absence of selection. In general, the productivity decreased to 50 % its initial level over the first 7 weeks in culture and then remained constant for the following 5 weeks. In contrast, the volumetric protein yield from transposon-mediated cell lines remained constant for up to 4 months in the absence of selection. We also showed that the three transposon systems could be used for cell pool generation with CHO-K1 and CHO-S with similar volumetric productivities as observed with CHO-DG44 cells. Finally, we utilized the PB transposon system for generating cell pools co-expressing up to 4 different transgenes, enhanced green fluorescent protein (EGFP), secreted alkaline phosphatase (SEAP), and the light and heavy chains of an IgG1 monoclonal antibody, by simultaneous transfection of all four transgenes. We showed that PB-mediated cell pools had increased volumetric productivity of each of the proteins compared to those generated by conventional co-transfection. The use of the PB transposon system increased the percentage of cells in the pools that were co-expressing all four proteins as compared to the results with cell pools generated by conventional transfection. In conclusion, the transfection of CHO cells with the PB, Tol2 or SB transposon system is a simple, efficient, and reproducible approach to the generation of cell pools and cell lines for the rapid production of recombinant proteins.

EPFL2015

Establishment of recombinant mammalian cell lines

Madiha Derouazi

Recombinant proteins are gaining in importance for therapeutic applications. The proteins are expressed in stable cell lines, within which recombinant DNA has incorporated into the host cells genome. Identification and isolation of extremely high producers from transfected cell populations is a tedious and time-consuming effort. We proposed two different approaches for the establishment of recombinant cell lines: affinity cell sorting and microinjection. Even with the help of single cell sorting and analysis systems (flow cytometry) rarely more than thousand different cell clones, stably expressing a desired gene can be evaluated. We are considering a new approach in which polyclonal cell populations will be established which co-express a protein of interest and an epitope-tagged cell surface receptor. A microbead fixed antibody specific to the epitope tag is hoped to assist in the isolation of rare, but highly productive cells from large suspensions of transfected cells. For this reason we have also developed an efficient method for the bulk transfection of CHO cells. On the side of vector-development for this approach, we have chosen to investigate the utility of a mouse serotonin 5-HT3 receptor as a cell surface marker. This receptor can be expressed at very high levels (up to 1 x 107 receptors per cell) in heterologous systems. The receptor is detectable on the cell surface by means of fluorescent ligands, which allows the determination of transfection efficiencies in suspension cultures. A Flag-tag was fused to the amino terminal end of the receptor without adverse effects on receptor function or ligand binding. Only 5 % of receptor DNA in the transfection cocktail was sufficient to detect positive cells in a population. The specific interaction of the epitope-tagged receptor with the cognate antibody fixed to microbeads was tested in comparison to non-transfected control cells using laser scanning confocal microscopy. A 80% enrichment of cells expressing GFP and the 5-HT3 receptor was achieved in 30 minutes. We have investigated microinjection as a gene transfer method for the establishment of recombinant mammalian cell lines. The conditions for the injection of plasmid DNA into either the nucleus or the cytoplasm of CHO-DG44 cells were established using pMYK-EGFP, an expression vector with the enhanced green fluorescent protein (EGFP) gene under the control of the murine CMV promoter and the puromycin resistance gene for selection. To estimate plasmid copy number during microinjection, it was first necessary to determine the injection volume by fluorescence quantification of injected FITC-dextran. For microinjection into the cytoplasm the volume ranged from 200 to 350 fl and for nuclear microinjection it was about 180 fl. We then investigated the impact of DNA concentration and injection pressure on transient EGFP transient expression following microinjection into the cytoplasm or nucleus. As a general rule, we observed that concentration of the DNA solution injected into the cells was the most important parameter regardless of the pressure or the volume of injection. Recombinant cell lines expressing intracellular EGFP were established following microinjection into the nucleus and the cytoplasm with linear and circular DNA. Selection with puromycin, clonal expansion, and cell banking were completed within three to four weeks post-microinjection. In parallel, recombinant cell lines were established after calcium-phosphate transfection with linear and circular DNA. The number of plasmid copies integrated was determined by Southern blot and the number and localization of integration sites was determined by fluorescence in-situ hybridization (FISH). Growth of the cells and stability of EGFP expression were investigated by cultivation over a two-month period. This approach presents several advantages over other DNA delivery methods such as control of the quantity and localization of DNA delivered into the cell and speed of recovery of recombinant cell lines. This approach may be relevant for the establishment of recombinant cell lines from cells that are not easily transfected by classical methods.

EPFL2005

Transient gene expression for rapid protein production

Natalie Muller

EPFL2005

Study of transposon-mediated cell pool and cell line generation in CHO cells

Sowmya Balasubramanian

EPFL2015

Establishment of recombinant mammalian cell lines

Madiha Derouazi

EPFL2005