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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 (TNFR-Fc). After screening several promoter and enhancer combinations for high levels of TNFR: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 2×10(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 TNFR-Fc yields over 150μg/mL within 4 days of transfection. The method was found to be reproducible and scalable to 300mL. This plasmid-based transient transfection process is a simple and efficient alternative to the BEVS for recombinant protein production in H5 cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Here we describe a simplified method for transient gene expression (TGE) in suspension-adapted Chinese hamster ovary (CHO) cells using polyethylenimine (PEI) for DNA delivery. Both the transfection and production phases of the bioprocess were performed at a density of 4 × 10⁶ cells/mL at 31 °C. In addition, the amounts of both PEI and plasmid DNA were reduced up to 50% on a per cell basis compared to previously published protocols from this laboratory, resulting in higher cell viability after transfection and higher volumetric recombinant protein yields. In batch cultures of up to 14 days, reproducible recombinant antibody yields up to 300 mg/L were achieved at small scale (5 mL) and up to 250 mg/L at large scale (500 mL). The simplicity and improved yields are expected to increase the utility of CHO cells for the rapid production of recombinant proteins at larger scales by TGE., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.

Large-scale transient gene expression (TGE) in mammalian cells is a rapid method to generate recombinant proteins, but the volumetric productivity for secreted proteins is still more than an order of magnitude lower than the yields typically achieved with recombinant cell lines. Here transient recombinant protein production in Chinese hamster ovary cells transfected with linear 25 kDa polyethylenimine was significantly enhanced by incubation of the cells at temperatures ranging from 29 to 33 degrees C after DNA delivery. With this approach, transient recombinant antibody yields of 60-80 mg/L were achieved within 6 days of transfection. The increase in TGE correlated with the accumulation of cells in the G1 phase of the cell cycle, increased cell size, higher cell viability, higher steady-state levels of transgene mRNA, reduced consumption of nutrients, and decreased accumulation of waste products. The enhancement of TGE was not vector-dependent, but the presence of the woodchuck hepatitis virus post-transcriptional regulatory element in the 3' untranslated region of the transgene mRNA increased transient recombinant antibody expression more than 3-fold at 31 degrees C as compared to expression at 37 degrees C. The yields achieved by the low-temperature enhancement of TGE in CHO cells makes this technology feasible for the rapid production of gram amounts of secreted recombinant proteins at large scale (up to 100 L).

Cell expansion, gene transfer and protein production were all executed with a single serum-free, animal protein-free commercial medium designed for suspension-adapted Chinese hamster ovary cells (CHO DG44). This is a most important process to consider for clinical production of recombinant proteins. The transfection with polyethylenimine (PEI) was shown here to be scalable using both stirred-tank bioreactors of 3- and 150-l and novel agitated cultivation vessels (50 ml ventilated centrifuge tubes and 1-l square-shaped glass bottles) that lack any instrumentation. The transient transfections spanned a range of working volumes from 2 ml to 80 l. The maximum transient recombinant antibody yield was 22 mg/l, the highest ever reported for a multiliter transfection in CHO. The transiently expressed protein had the same extent of glycosylation as the same antibody produced from a stably transfected recombinant CHO cell line.

The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials. Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2-10 days. Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time--weeks. With "classical" approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line. This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies.

Intracellular lipid droplets (LDs) can accumulate in response to inflammation, metabolic stresses, and other physiological/pathological processes. Herein, we investigated whether spike proteins of SARS-CoV-2 induce LDs in human peripheral blood mononuclear cells (PBMCs) and in pulmonary microvascular endothelial cells (HPMECs). PBMCs or HPMECs were incubated alone or with endotoxin-free recombinant variants of trimeric spike glycoproteins (Alpha, Beta, Delta, and Omicron, 12 µg/mL). Afterward, cells were stained with Oil Red O for LDs, cytokine release was determined through ELISA, and the gene expression was analyzed through real-time PCR using TaqMan assays. Our data show that spikes induce LDs in PBMCs but not in HPMECs. In line with this, in PBMCs, spike proteins lower the expression of genes involving lipid metabolism and LD formation, such as SREBF1, HMGCS1, LDLR, and CD36. On the other hand, PBMCs exposed to spikes for 6 or 18 h did not increase in IL-1β, IL-6, IL-8, MCP-1, and TNFα release or expression as compared to non-treated controls. Thus, spike-induced LD formation in PBMCs seems to not be related to cell inflammatory activation. Further detailed studies are warranted to investigate in which specific immune cells spikes induce LDs, and what are the pathophysiological mechanisms and consequences of this induction in vivo. [ABSTRACT FROM AUTHOR]

Heterogenous populations of recombinant cells (cell pools) stably expressing 1-4 transgenes were generated from Chinese hamster overy (CHO) cells with the piggyBac (PB) transposon system. The cell pools produced different combinations of three model proteins-enhanced green fluorescent protein (EGFP), secreted alkaline phosphatase (SEAP), and a monoclonal IgG1 antibody. Each transgene was present on a separate PB donor plasmid with either the same or a different selection gene. In both cases, we obtained PB-derived cell pools with higher recombinant protein yields than from cell pools generated by conventional gene delivery. In PB-derived cell pools generated using a single selection agent, both protein production and the number of integrated copies of each transgene declined as the number of transfected transgenes increased. However, the total number of integrated transgenes was similar regardless of the number of different transgenes transfected. For PB-derived cell pools generated by selection of each transgene with a different selection agent, the total number of integrated transgenes increased with the number of transfected transgenes. The results suggest that the generation of cell pools producing multiple recombinant proteins is feasible and that the method is more efficient when each individual transgene is selected with a different marker. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1308-1317, 2016 [ABSTRACT FROM AUTHOR]

Although the protein yields from transient gene expression (TGE) with Chinese hamster ovary (CHO) cells have recently improved, the amount of plasmid DNA (pDNA) needed for transfection remains relatively high. We describe a strategy to reduce the pDNA amount by transfecting CHO-DG44 cells with 0.06 lg pDNA/106 cells (10% of the optimal amount) in the presence of nonspecific (filler) DNA and various polar solvents including dimethylsufoxide, dimethyl formamide, acetonitrile, dimethyl acetamide (DMA), and hexamethyl phosphoramide (HMP). All of the polar solvents with the exception of HMP increased the production of a recombinant antibody in comparison to the untreated control transfection. In the presence of 0.25% DMA, the antibody yield in a 7-day batch culture was 500 mg/L. This was fourfold higher than the yield from the untreated control transfection. Mechanistic studies revealed that the polar solvents did not affect polyethylenimine-mediated pDNA delivery into cells or nuclei. The steady-state transgene mRNA level was elevated in the presence of each of the polar solvents tested, while the transgene mRNA half-life remained the same. These results indicated that the polar solvents enhanced transgene transcription. When screening a panel of recombinant antibodies and Fc-fusion proteins for production in the presence of the polar solvents, the highest increase in yield was observed following DMA addition for 11 of the 12 proteins. These results are expected to enhance the applicability of high-yielding TGE processes with CHO-DG44 cells by decreasing the amount of pDNA required for transfection. [ABSTRACT FROM AUTHOR]

Transient gene expression (TGE) in human embryonic kidney (HEK-293) and Chinese hamster ovary (CHO) cells is a well-established technology for the rapid generation of recombinant proteins. Although the maximum TGE yields have reached 1 g/L or more, the amount of plasmid DNA (pDNA) required for transfection remains high. Although greater than 103 copies of pDNA are present per transfected cell, protein yields are still lower than those achieved in recombinant cell lines with only one or a few copies of the transgene. This indicates a clear limitation to TGE in terms of the maximum level of recombinant protein production. In this study, we investigated the limitations to high-yielding TGE processes with CHO and HEK-293E cells using a monoclonal antibody as a model protein. For either cell host, both the intracellular and intranuclear pDNA levels increased linearly with the amount of pDNA added to the culture. In contrast, transgene mRNA accumulation reached a plateau as the intranuclear pDNA amount increased, suggesting a limitation in pDNA transcription. A post-transcriptional limitation to TGE yields was revealed by calculating the amount of antibody produced per transgene mRNA (mRNA utilization). For both hosts the transgene mRNA utilization decreased dramatically when transfected pDNA amounts increased beyond the level giving the maximum protein yield. The post-transcriptional limitation did not appear to be due to bottlenecks in antibody assembly or secretion, suggesting that transgene mRNA translation may be limiting. The results show that TGE yields are not limited by pDNA delivery into the nuclei, but in pDNA and transgene mRNA utilization. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:541-549, 2015 [ABSTRACT FROM AUTHOR]

Abstract: Mammalian cells in bioreactors as production host are the focus of this review. We wish to briefly describe today’s technical status and to highlight emerging trends in the manufacture of recombinant therapeutic proteins, focusing on Chinese hamster ovary (CHO) cells. CHO cells are the manufacturing host system of choice for more than 70% of protein pharmaceuticals on the market . The current global capacity to grow mammalian cells in bioreactors stands at about 0.5 million liters, whereby the largest vessels can have a working volume of about 20,000l. We are focusing in this article on the upstream part of protein manufacturing. Over the past 25years, volumetric yields for recombinant cell lines have increased about 20-fold mainly as the result of improvements in media and bioprocess design. Future yield increases are expected to come from improved gene delivery methods, from improved, possibly genetically modified host systems, and from further improved bioprocesses in bioreactors. Other emerging trends in protein manufacturing that are discussed include the use of disposal bioreactors and transient gene expression. We specifically highlight here current research in our own laboratories. [Copyright &y& Elsevier]

The exposure of CHO DG44 cells to an osmotic shock, after DNA uptake, results in a cellular volume decrease of approx. 55%. Repetitive osmotic shocks targeted different sub-populations of cells as was demonstrated using two different fluorescent reporter genes. Also the exposure of a calcium phosphate–DNA coprecipitate to high osmolarity in vitro caused the release of the DNA from the precipitate. The results demonstrate the importance of the osmotic shock on the efficient delivery of plasmid DNA to the nucleus of CHO cells following calcium phosphate-mediated transfection. [ABSTRACT FROM AUTHOR]

The article presents study of the improved Sf9 transient gene expression process. Topics discussed include use of insect cells for the production of recombinant proteins, studying efficiency of polyethylenimine (PEI) for plasmid delivery in Sf9 cells, cell culture involving transfection of Sf9 cells and estimation of plasmid copy number, cell cycle analysis, and measuring plasmid delivery efficiency in Sf9 cells. It reveals no improvement in plasmid delivery.

An abstract of the article "Transient gene expression with CHO cells in conditioned medium: a study using TubeSpin bioreactors," by Yashas Rajendra and colleagues is presented.

An abstract of the article "Transposon mediated co-integration and coexpression of transgenes in CHO-DG44 cells," by Mattia Matasci and colleagues is presented.

An abstract of the article "The use of filler DNA for improved transfection and reduced DNA needs in transient gene expression with CHO and HEK cells," by Divor Kiseljak and colleagues is presented.

Abstract: The purpose of this review is to describe the current status and to highlight several emerging trends in the manufacture of recombinant therapeutic proteins in cultivated mammalian cells, focusing on Chinese hamster ovary cells as the major production host. Over the past 25 years, specific and volumetric productivities for recombinant cell lines have increased about 20-fold as the result of improvements in media and bioprocess design. Future yield increases are expected to come from further developments in gene delivery and genetic selection for more efficient recovery of high-producing cell lines and in high-throughput cultivation systems to simplify medium design and bioprocess development. Other emerging trends in protein manufacturing that are discussed include the use of disposal bioreactors and transient gene expression. We specifically highlight current research in our own laboratories. [Copyright &y& Elsevier]

Abstract: 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 30×106 cells/mL with 0.9μg DNA and 1.35μg of linear 25kD polyethylenimine (PEI) per million cells. Following transfection, the culture was diluted to 4×106 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 100μg/mL for cultures at volumes up to 300mL. As expected, the molecular weight of the dimeric TNFR-Fc produced from Sf9 cells was about 6kDa 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. [Copyright &y& Elsevier]

Highlights: [•] A brief overview of principles of TGE using mammalian cells. [•] Description of TGE processes for HEK293 and CHO cells. [•] Description of orbitally shaken bioreactors for suspension cell cultivation. [•] Description of polyethylenime-based transfection processes. [Copyright &y& Elsevier]

Abstract: Although respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in infants and adults at risk, no RSV vaccine is currently available. In this report, efforts toward the generation of an RSV subunit vaccine using recombinant RSV fusion protein (rRSV-F) are described. The recombinant protein was produced by transient gene expression (TGE) in suspension-adapted human embryonic kidney cells (HEK-293E) in 4L orbitally shaken bioreactors. It was then purified and formulated in immunostimulating reconstituted influenza virosomes (IRIVs). The candidate vaccine induced anti-RSV-F neutralizing antibodies in mice, and challenge studies in cotton rats are ongoing. If successful in preclinical and clinical trials, this will be the first recombinant subunit vaccine produced by large-scale TGE in mammalian cells. [Copyright &y& Elsevier]