100 Liter Transient Transfection

Girard, Philippe; Derouazi, Madiha; Baumgartner, G.; Bourgeois, M.; Jordan, Martin; Wurm, Florian Μ.

doi:10.1007/978-94-010-0369-8_10

Cited by 35 publications

(57 citation statements)

References 9 publications

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“…Our results showed that the HEK293-EBNA cell line expressed significantly higher IgG titres than the HEK293-T cells. The most successful cell line currently employed in transient expression is HEK293-EBNA (Durocher et al 2002;Girard et al 2002;Schlaeger et al 2003;Pham et al 2003;. One genetic feature is the presence of the Adenovirus-derived E1a protein in the cells driving and enhancing transcription from a CMV promotor Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Even though we used a sub optimal vector, we achieved expression levels up to 8 mg mL -1 , demonstrating that, in some cases, re-cloning of the gene of interest is not necessary. Previously reported yields for secreted proteins in stirred bioreactors are 2.5-28 mg L -1 (Durocher et al 2002;Girard et al 2002;Baldi et al 2005). Durocher et al (2002) have expressed various secretory proteins in either a 3 L bioreactor or a 14 L bioreactor.…”

Section: Discussionmentioning

confidence: 99%

“…A major breakthrough was achieved when PEI technology demonstrated its ability to produce milligram amounts of recombinant protein within a few days in large-scale suspension cultures (Jordan et al 1998). Calcium phosphate has been reported to support transient gene expression for 2-100 L scale (Jordan et al 1998;Girard et al 2002) and PEI has been used successfully up to 15 L scale (Durocher et al 2002;Pham et al 2003;Geisse and Henke 2005).…”

Section: Introductionmentioning

confidence: 99%

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Development of a generic transient transfection process at 100 L scale

et al. 2008

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We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium, optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding bioreactor and the production bioreactor. After 7-8 days the harvest and capture is performed in a one-step operation using a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L -1 IgG.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development of a generic transient transfection process at 100 L scale

et al. 2008

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“…This approach has made it possible to produce milligram-to-gram quantities of a recombinant protein within days or weeks (Wurm and Bernard 1999;Derouazi et al 2004). Its ability to generate proteins within a relatively short timeframe enables TGE to evaluate functionalities and toxicities much earlier and therefore provide valuable information about drug candidates (Girard et al 2002;Jordan et al 1996). Transiently expressed materials are ideal for preclinical and early clinical trials because their use represents a faster and less expensive approach than stable expression (Derouazi et al 2004).…”

Section: Introductionmentioning

confidence: 99%

PEI/DNA formation affects transient gene expression in suspension Chinese hamster ovary cells via a one-step transfection process

et al. 2012

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Polyethylenimine (PEI) has been used widely in transient gene expression studies of mammalian cells. We performed transient gene expression in suspension Chinese hamster ovary cells using a onestep transfection procedure in which DNA and PEI were simultaneously added to a cell culture in suspension without prior PEI/DNA complex incubation. To further understand the effect of PEI/DNA formation on the transfection and expression of exogenous gene in shaking state, we investigated the diameter and overcharge of the PEI/DNA complex. The results showed that the diameter of the complex was smaller with more positive charge when the PEI/ DNA ratio was higher. Moreover, DNA more easily penetrated cells and nuclei at higher PEI concentrations. The highest transcription level, transfection efficiency, and GFP expression were obtained when the PEI/DNA ratio was 5:1.

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“…Prior to evaluating the effect of carrier DNA, we tested the effect of these factors on culture productivity. DNA delivery agents such as calcium phosphate, liposomes and cationic polymers like polyethyleneimine (PEI) have been used to transfect 1 ml to 100 l suspension cultures of mammalian cells to transiently express recombinant proteins (Derouazi et al 2004;Girard et al 2002;Liu et al 2008;Schlaeger and Christensen 1999;Meissner et al 2001;Muller et al 2007;Schlaeger et al 2003). Polyethyleneimine is one of the widely used transfection agents used due to its ability to transfect a broad range of cell lines and low toxicity along with its cost effectiveness over commercially available reagents which could be an important consideration for scale up (Boussif et al 1995).…”

Section: Introductionmentioning

confidence: 99%

Effect of addition of ‘carrier’ DNA during transient protein expression in suspension CHO culture

Pradhan

Gadgil

2012

Cytotechnology

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Transient protein expression using polyethyleneimine as a transfection agent is useful for the rapid production of small amounts of recombinant proteins. It is known that an increase in extracellular DNA concentration during transfection can lead to a nonlinear increase in intracellular DNA concentration. We present an approach that hypothesizes that this nonlinearity can be used to decrease the amount of plasmid required for productive transfections. Through addition of non coding 'carrier' DNA to increase total DNA concentration during transfection, we report a statistically significant increase in protein (IgG) expression per unit plasmid used for transfection. This approach could be useful to increase protein yields for large scale transfections under conditions where plasmid availability is limited.Keywords Transient protein expression Á Carrier DNA Á Salmon sperm DNA Á pH excursion Á Medium equilibration with CO 2 Á Cell age Á DNA: polyethyleneimine particle size Background

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100 Liter Transient Transfection

Cited by 35 publications

References 9 publications

Development of a generic transient transfection process at 100 L scale

Development of a generic transient transfection process at 100 L scale

PEI/DNA formation affects transient gene expression in suspension Chinese hamster ovary cells via a one-step transfection process

Effect of addition of ‘carrier’ DNA during transient protein expression in suspension CHO culture

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