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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.