Process Development for Transient Gene Expression in Mammalian Cells at The 3 L Scale: 10–50 MG of R-Protein in Days

Meissner, Petra; Girard, Philippe; Kulangara, A.; Tsao, Mei-Lin; Jordan, Martin; Wurm, Florian Μ.

doi:10.1007/0-306-46875-1_76

Cited by 4 publications

(8 citation statements)

References 5 publications

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“…Even if transient transfection methods using 293 cells under SF conditions have been successfully transposed to a bioreactor scale for the production of recombinant proteins (Meissner et al 1999;Schlaeger and Christensen 1999 wtAd ∆PSAd Figure 7. Comparison of wtAd and AdDPS, as helper viruses.…”

Section: Discussionmentioning

confidence: 99%

“…In order to scale up the production size, the culture surface can be augmented in different ways: (a) the automation of transfection based processes in roller bottles (Liu et al 2003) or the use of large surfaces such as provided by the CellCube (Brown et al 1998), (b) microcarriers that support high density growth of adherent cells (Griffiths 2001), and (c) suspension cultures developed in bioreactors. Although a lot of improvements have been realised in order to develop protocols to transfect mammalian cells in suspension in a bioreactor, for the production of recombinant proteins (Meissner et al 1999;Schlaeger and Christensen 1999;Wurm and Bernard 1999;Durocher et al 2002), these techniques cannot be easily adapted to the production of viral vectors.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of a Serum-free Medium for the Production of rAAV-2 using HeLa Derived Producer Cells

et al. 2005

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During the last decade, recombinant AAVs have become of increasing interest for gene therapy. Clinical trials have been conducted following promising in vivo evaluations, thus leading laboratories to adapt their production systems for larger and higher quality demands. Classical transfection protocols seem difficult and cumbersome to adapt to a bioreactor scale. The use of stable producer cells appears as an attractive alternative, as this system requires only a single infection step to induce rAAV production. Furthermore, the switch to a serum-free medium is an interesting strategy to increase the biosafety level to satisfy clinical grade requirements for gene therapy products. Here, we have combined both approaches and evaluated different rAAV producer clones in a serum-free medium. We first evaluated the cell growth in a serum-free medium and then did a partial optimisation of the medium composition to obtain vector yields as close as possible to the yields obtained in a classical serum containing medium. Different helper viruses, multiplicity of infection, times of infection and harvest have been compared in small scale cultures in order to determine the optimal settings which were then transferred and evaluated in suspension cultures in spinner flasks. The yields obtained in this system were similar to or at most 2 times lower than those obtained in a serum-containing medium. The scale-up of such a production system as well as the use of high cell density perfusion culture systems will probably lead to considerably higher yields than those obtained in a classical process.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Evaluation of a Serum-free Medium for the Production of rAAV-2 using HeLa Derived Producer Cells

et al. 2005

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“…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%

“…The medium should support good cell growth and transfection efficiencies above 80%. Calcium phosphate mediated transfection in HEK-EBNA cells, as described by Meissner et al (1999), was successful only if the cells were transferred into DMEM/F12 medium supplemented with serum prior to transfection, although the cells could be grown in various serumfree culture media (Meissner et al 1999). PEImediated transfection, as described by Geisse and Henke (2005) is insensitive to the presence of serum (Geisse and Henke 2005).…”

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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“…The transfection complex must be dissolved, otherwise its toxic effects (Jordan et al, 1996) will override the beneficial effect of a longer incubation time that would allow the cells to take up transfection particles over an extended period. Adding extra medium and lowering the pH can readily achieve this goal (Meissner et al, 1999).…”

Section: Liquid Handling and Transfectionmentioning

confidence: 99%

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et al. 2002

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This is the first report of two successful 100 l scale transienttransfections in a standard stirred bioreactor. More than half a gram of a monoclonal antibody (IgG) were produced in less than 10 days using a technology called large-scale transient gene expression(LS-TGE). Suspension adapted HEK 293 EBNA SF cells were transfectedwithin a 150 l (nominal) bioreactor by a modified calcium phosphateco-precipitation method with more than 75 mg of plasmid DNA per run.A mixture of three different plasmids, one encoding for the heavychain of a human recombinant immunoglobulin, the other for the corresponding light chain and a third one for the green fluorescent protein (GFP, 2-4% of DNA in transfection cocktail)were co-transfected. The GFP vector was chosen to monitor transfection efficiency. Expression of GFP could be registered asearly as 20 h after DNA addition, using fluorescence microscopy. We demonstrate that transient transfection can be done at the100 l scale, thus providing a new tool to produce hundreds of milligrams or even gram amounts of recombinant protein. Akey advantage of LS-TGE resides in its speed. In the presentedcases, the entire production process for the synthesis of halfa gram of a recombinant antibody, including DNA preparationand necessary expansion of cells prior to transfection, wasexecuted in less than a month. Having an established transfection/expression process allows to run productioncampaigns for any given protein, within one facility, with onesingle host cell line and therefore only one single seed train. Without any need to create and maintain stable cell lines, expression of new r-proteins is not only faster and more economical but also more flexible.

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Process Development for Transient Gene Expression in Mammalian Cells at The 3 L Scale: 10–50 MG of R-Protein in Days

Cited by 4 publications

References 5 publications

Evaluation of a Serum-free Medium for the Production of rAAV-2 using HeLa Derived Producer Cells

Evaluation of a Serum-free Medium for the Production of rAAV-2 using HeLa Derived Producer Cells

Development of a generic transient transfection process at 100 L scale

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