Comparison of Three Transposons for the Generation of Highly Productive Recombinant CHO Cell Pools and Cell Lines

Several naturally occurring vertebrate transposable elements have been genetically modified to enable the transposition of recombinant genes in mammalian cells. We compared three transposonspiggyBac, Tol2, and Sleeping Beautyfor their ability to generate cell pools (polyclonal cultures of recombinant cells) and clonal cell lines for the large-scale production of recombinant proteins using Chinese hamster ovary cells (CHO-DG44) as the host. Transfection with each of the dual-vector transposon systems resulted in cell pools with volumetric yields of tumor necrosis factor receptor-Fc fusion protein (TNFR:Fc) that were about ninefold higher than those from cell pools generated by conventional plasmid transfection. On average, the cell pools had 10-12 integrated copies of the transgene per cell. In the absence of selection, the volumetric productivity of the cell pools decreased by 50% over a 2-month cultivation period and then remained constant. The average volumetric TNFR:Fc productivity of clonal cell lines recovered from cell pools was about 25 times higher than that of cell lines generated by conventional transfection. In 14-day fed-batch cultures, TNFR:Fc levels up to 900mg/L were obtained from polyclonal cell pools and up to 1.5g/L from clonal cell lines using any of the three transposons. (C) 2015 Wiley Periodicals, Inc.

Lentiviral Vector-Mediated Gene Delivery for the Generation of Chinese Hamster Ovary Cell Lines with Improved Productivity and Stability

Agata Oberbek

Recombinant cell line generation by standard transfection techniques is a time-consuming and labor-intensive process often leading to an unpredictable outcome as transgene integration is a rare, random event. Consequently, the population of cells obtained after standard gene delivery is heterogeneous in terms of specific growth and recombinant protein productivity. Therefore, genetic selection is usually applied to the population for up to 3 weeks to eliminate non-recombinant cells. A large number of the surviving cell lines must be screened to find a few which stably produce the recombinant protein at the desired level. These must be maintained in culture for 2-4 months in the absence of selection to assess the stability of protein production. In contrast, lentiviral vectors (LVs) deliver the recombinant gene to target cells through infection and a controlled integration process, which allows the inserted transgene to become a permanent genetic element of the transduced cell. Moreover, lentiviruses tend to integrate into transcriptionally active sites of the host cell genome. Hence, LV-mediated gene transfer was expected to increase the probability of generating a stable high-producing clone in comparison to the standard methods based on plasmid transfection. Therefore, the objective of this PhD thesis was to improve the efficiency of stable cell line generation with the use of LVs. For this purpose, LVs carrying the enhanced green fluorescent protein (eGFP) gene under the control of either the cytomegalovirus immediate early promoter/enhancer (CMV) or the elongation factor-1 alpha promoter (EF-1α) were generated and used to infect Chinese hamster ovary (CHO) cells. Two days after gene delivery at a multiplicity of infection (MOI) of 2, up to 98% of infected cells were eGFP-positive. Following isolation by fluorescence-activated cell sorting (FACS), CHO cell pools and clonal cell lines were analyzed for the stability of eGFP expression over time. In these studies, the EF-1α promoter was found to yield more stable and homogeneous protein expression compared to the CMV promoter. For comparison, cell pools and cell lines were generated by transfection with the LV transfer plasmid bearing the eGFP gene. The level and stability of eGFP expression was greater in LV-generated pools and cell lines than in those established by transfection. Subsequently, an LV expressing the tumor necrosis factor receptor-Fc (TNFR-Fc) fusion gene and the eGFP gene from a bicistronic mRNA under the control of the EF-1α promoter (LV_EF-TNFR) was generated and used to infect CHO cells. Based on selective FACS gating for high eGFP expression, stable CHO clones showing volumetric productivities of up to 100 mg/L of TNFR-Fc in a non-optimized batch process could be isolated within 2 days of infection at an MOI of 2. The level of expression of the recombinant protein remained constant for 3 months in serum-free suspension culture and in the absence of chemical selective agents. Next, CHO cells were infected at 3 higher MOIs with the LV_EF-TNFR and clonal cell lines with high eGFP-specific fluorescence were recovered by FACS at 2 d post-infection. These cell lines had volumetric TNFR:Fc productivities ranging from 100 to 400 mg/L in 4-day cultures with cell-specific secretion rates up to 100 pg/cell/day. Moreover, recombinant cell lines developed by the LV-based technology could be successfully cultured in bench-scale bioreactors, yielding up to 1.5 g/L of TNFR:Fc under batch mode operation. The increase in integrated TNFR:Fc copy number was found to correlate with the MOI, and resulted in improved protein productivity. However, this relationship was found to be non-linear: cell saturation in terms of protein productivity and integrated transgene copy number was observed at an MOI of 100. In order to determine the shortest time-frame necessary for the production of satisfactory quantities of a recombinant product, the high MOI-infection approach was tested on pools of transduced cells. Without a priori FACS isolation, recombinant pools of CHO cells expressing on average 500 mg/L of TNFR:Fc within 8 days of inoculation at 3 × 105 cells/ml could be obtained. This strategy provided a simple protocol for high titer recombinant protein production, executable within 4 weeks from LV batch preparation to product harvest. Since large-scale production of LVs would be needed for industrialized bioprocesses, a method for LV production in serum-free suspension culture was developed. However, the best titers generated by this system were still approximately 20-fold lower than the yields obtained regularly from the adherent cultures, thus leaving room for further optimization. In conclusion, LV-mediated gene transfer provided an efficient alternative to plasmid transfection for the generation of stable and high-producing recombinant cell lines. Moreover, it proved amenable to large-scale manufacture and could yield several hundred milligrams per liter of the recombinant product without the need for any kind of genetic selection. In addition, using the LV technology described here it seems possible to target the number of copies of the delivered transgene to a specific range which balances favorable growth with good productivity characteristics.

EPFL2010

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

Development of plasmid-based expression technology for rapid and sustained production of recombinant proteins in insect cells

Xiao Shen

Due to the high demand for heterogeneous protein production in insect cells, we have established efficient methods for plasmid-based transient gene expression (TGE) in various insect cell lines, including commonly used Spodoptera cell lines Sf9 and HighFive™ (H5) and the Diptera cell line Schneider S2 from Drosophila melanogaster, as a high-yield protein production platform. The cells were grown in suspension in orbitally shaken containers at working volumes of 5-300 mL. Rapid cell growth and high cell densities were obtained. Among the various transfection agents tested, polyethylenimine supported high transfection efficiencies in all three cell lines. Intensive optimizations of TGE processes for the three cell lines were executed. For example, various promoter and enhancer elements were compared. Using an optimal TGE process, transfection efficiencies (percentage of transfected cells) of over 85% were obtained for all three cell lines. Yields of the secreted protein tumor necrosis factor receptor-Fc fusion (TNFR-Fc) ranged from 100 – 200 mg/L for the three cell lines using a gene optimized for expression in insect cells. The molecular weight of the Sf9-derived dimeric TNFR-Fc was about 6 kDa less than the equivalent product synthesized by Chinese hamster ovary cells due to differences in glycosylation. In addition, several difficult-to-express proteins, that failed to be delivered at sufficient quantity using other expression systems, were successfully produced in S2 cells by TGE. A human serotonin receptor was expressed at 20-60 mg/L; empty and peptideloaded MHC II tetramer complexes were expressed at up to 150 mg/L; neutrophil serine proteases were expressed at levels over 50 mg/L; and protein tyrosine kinases were expressed at levels over 2 mg/L. These yields were obtained within 5 days of transfection at volumetric scales up to 300 mL. We also developed methods for the generation of stable polyclonal pools of recombinant S2 cells based on plasmid cotransfection in the context of the Piggybac transposon. After two weeks of genetic selection of recombinant cells, the recombinant pools remained stable for at least 100 days in the absence of selection with volumetric TNFR-Fc productivity over 40 mg/L in a 4-day batch culture. Our data highlight the use of plasmid-based expression systems in insect cells to meet the demand for rapid and sustained production of proteins in sufficient quantities for biomedical research and for the biotechnology industry.

EPFL2014