Reconciling pillars of transient gene expression: From DNA prep via media, reagent and cell line development to holistic process optimization
© Püngel et al. 2015
Published: 14 December 2015
Transient gene expression (TGE) is a multi-parametric process which is built upon four essential influencing pillars: First of all, it is imperative to have an easy to transfect cell line which allows high product titers and is cultivated in suspension . Secondly, only a few of the commercially available cell culture media allow / support transient transfection and production. Thus, it is highly recommended to select the appropriate medium to grow and transfect the favored cell line . Thirdly, the quality, source and backbone of the plasmid DNA which contains the genetic information for the protein of interest has a major impact . Last but not least, to introduce plasmid DNA into mammalian cells, selection of a suited transfection reagent plays an important role for high-yielding TGE processes.
In this work, we summarize our recent development of a novel TGE system for efficient transient transfection and expression in HEK cells. In cooperation with emp Biotech, InVivo BioTech Services developed a transfection reagent with very low cytotoxicity. A culture medium that can be used for transfection and production was designed in collaboration with Xell AG. The establishment of a TGE optimized HEK cell line (HEK-INV) and a method for large scale plasmid preparation of a corresponding vector complete the optimized production platform. It is easy applicable, scalable and supports large scale transfection for the production of gram quantities of IgG within a few days.
Materials and methods
Mammalian cells were cultivated and transfected in Xellvivo TM medium (Cat. No. 861-0001, Xell AG) under conditions of 37 °C, 5 % CO2 and 185 rpm agitation speed at 50 mm orbital diameter. For screening approaches 5x10E6 cells/mL were transfected with 2 pg DNA/cell and INVect transfection reagent (Cat. No. FK-0101-M001.0-001, emp Biotech GmbH) with INVect to DNA ratio of 6:1 (w/w) or 25 kDa L-PEI with PEI to DNA ration of 2:1 (w/w) in 8 mL culture volume in 50 mL bioreactor tubes. Expression of IgG1 was performed in 30 mL culture volume in 125 mL shake flasks or in 150 mL culture volume in 500 mL shake flasks respectively. Yields were quantified by proteinA affinity chromatography. Directed evolution was performed referring to Majors et al., 2009 . In detail, an iterative process of evolution rounds followed by analysis of favorable attributes, cell selection and recovery was implemented. The corresponding flow cytometry analysis was performed using a Bio-Rad S3 cell sorter.
Several E.coli strains and media were screened for high productivity, high quality and flexibility for DNA preparation in comparison to commercial kits in mini-preparation scale. A purification process was implemented using a reusable and scalable anion exchanger. For large scale plasmid preparation, 6 L suspension was lysed, clarified and purified using an Äktaprime chromatography system. DoE was used for TGE process optimization after combining all developed elements.
Transient expression of a monoclonal antibody in independent experiments with differing setups and scales.
Viable cell density
40 x106 cell/mL
5 x106 cell/mL
20 x106 cell/mL
In conclusion, InVivo's TGE system includes an advanced cell line and vector system, a novel transfection reagent as well as a unique and customized media, all synergistically optimized for highly efficient production of recombinant proteins. Applications of this streamlined process are in early development and lead identification as well as gram-scale production for pre-clinical trials. Furthermore, a pseudo-perfusion TGE process was developed for the production of toxic or labile products such as enzymes or vaccines. Lastly, a simplified procedure of the production process using a customized concentrated feed supplement was implemented which resulted in antibody titers up to 850 mg/L. Subsequent screenings of transfection enhancers show promising results and indicate potential for further improvements.
- Hacker DL, Kiseljak D, Rajendra Y, Thurnheer S, Baldi L, Wurm FM: Polyethyleneimine-based transient gene expression processes for suspension-adapted HEK-293E and CHO-DG44 cells. Protein Expr Purif. 2013, 92: 67-76.PubMedView ArticleGoogle Scholar
- Geisse S: Reflections on more than 10 years of TGE approaches. Protein Expr Purif. 2009, 64: 99-107.PubMedView ArticleGoogle Scholar
- Rozkov A, Larsson B, Gillstrom S, Bjornestedt R, Schmidt SR: Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol Bioeng. 2008, 99: 557-566.PubMedView ArticleGoogle Scholar
- Majors BS, Chiang GG, Betenbaugh MJ: Protein and Genome Evolution in Mammalian Cells for Biotechnology Applications. Mol Biotechnol. 2009, 42: 216-223.PubMedView ArticleGoogle Scholar
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