Transient gene expression: Recombinant protein production with suspension‐adapted HEK293‐EBNA cells

Meissner, Petra; Pick, Horst; Kulangara, A.; Chatellard, Philippe; Friedrich, Kirstin; Wurm, Florian Μ.

doi:10.1002/bit.1179

Cited by 186 publications

(138 citation statements)

References 11 publications

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“…GFP was excited at 485 nm with a bandwidth of 10 nm, and the emission fluorescence was measured at 515 nm with a bandwidth of 10 nm. IgG concentration was determined by sandwich ELISA using a goat anti-human kappa light chain antibody for capture and alkaline phosphatase-conjugated goat anti-human IgG (BioSource, Lucerne, Switzerland) for detection (32).…”

Section: Methodsmentioning

confidence: 99%

Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells

Wulhfard

Tissot

Bouchet

et al. 2008

Biotechnology Progress

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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°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°C as compared to expression at 37°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).

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Section: Methodsmentioning

confidence: 99%

Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells

Wulhfard

Tissot

Bouchet

et al. 2008

Biotechnology Progress

View full text Add to dashboard Cite

show abstract

“…Although scalable transient expression using CHO cells is frequently reported (Derouazi et al 2004;Tait et al 2004;Kunaparaju et al 2005;Galbraith et al 2006) the human embryonic kidney 293 cell line (HEK293) is the most widely used cell line for transient protein expression. HEK293 cells readily take up DNA using various gene transfer vehicles such as calcium phosphate (Jordan et al 1998;Meissner et al 1999;Girard et al 2001;Meissner et al 2001;Durocher et al 2002;Baldi et al 2005), XtremeGENE Ro1539 (Schlaeger et al 1998;Schlaeger et al 2003) and PEI (Schlaeger and Christensen 1999;Durocher et al 2002;Pham et al 2003;Schlaeger et al 2003;Baldi et al 2005). Transient gene expression with PEI in a high-cell density perfusion culture was reported recently (Sun et al 2008).…”

Section: Introductionmentioning

confidence: 99%

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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“…transformed cells) to study a variety of cell biological (Thomas and Smart, 2005). It also showed that, the human cell line HEK 293 has been used extensively for transient gene expression as a result of its good transfectability and ability to grow in suspension to high cell density in serum free media (Durocher et al, 2002;Meissner et al, 2001;Schlaeger and Christensen, 1999).…”

Section: Hek 293 Cell Linementioning

confidence: 99%