A simple plasmid-based transient gene expression method using High Five cells

The High Five (H5) cell line, derived from the lepidopteran Trichoplusia ni, is one of the major insect cell hosts for the production of recombinant proteins using the baculovirus expression vector system (BEVS). Here, we describe a simple polyethylenimine (PEI)-based transient gene expression (TGE) process for the rapid production of recombinant proteins from suspension-adapted H5 cells. The method was optimized using two model proteins, enhanced green fluorescent protein (EGFP) and human tumor necrosis factor receptor-Fc fusion protein (TNER-Fc). After screening several promoter and enhancer combinations for high levels of TNER:Fc production, an expression vector containing the Autographa californica multicapsid nucleopolyhedrovirus immediate early 1 (ie1) promoter and homologous region 5 (hr5) enhancer was selected. Cells were transfected at a density of 2 x 10(6) cells/mL by direct addition of DNA and PEI. Under optimized conditions, a 90% transfection efficiency (percentage of EGFP-positive cells) was obtained. In addition, we observed volumetric TNER-Fc yields over 150 mu g/mL within 4 days of transfection. The method was found to be reproducible and scalable to 300 mL. This plasmid-based transient transfection process is a simple and efficient alternative to the BEVS for recombinant protein production in H5 cells. (C) 2015 Elsevier B.V. All rights reserved.

Virus-free transient protein production in Sf9 cells

Lucia Baldi Unser, David Hacker, Xiao Shen, Florian Maria Wurm

A method for virus-free transient gene expression from suspension-adapted Sf9 insect cells was developed with the gene of interest being expressed from a plasmid carrying the homologous region 5 enhancer (hr5) and the immediate early 1 (ie1) promoter from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Under the optimal conditions described in the study, cells were transfected at a density of 30 x 10(6) cells/mL with 0.9 mu g DNA and 1.35 mu g of linear 25 kD polyethylenimine (PEI) per million cells. Following transfection, the culture was diluted to 4 x 10(6) cells/mL for the protein production phase. The volumetric yield of tumor necrosis factor receptor (ectodomain) fused to an Fc domain (TNFR-Fc) was about 100 mu g/mL for cultures at volumes up to 300 mL. As expected, the molecular weight of the dimeric TNFR-Fc produced from Sf9 cells was about 6 kDa less than that produced from a recombinant Chinese hamster ovary (CHO) cell line due to differences in glycosylation between the two hosts. Transient transfection provides an alternative to the baculovirus expression vector system (BEVS) for the rapid production of recombinant proteins from Sf9 cells. (C) 2013 Elsevier B.V. All rights reserved.

Elsevier2014

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

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.