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Development of a generic transient transfection process at 100 L scale

Cytotechnology volume 56, pages 123–136 (2008)Cite this article

Abstract

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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Acknowledgements

We thank our colleagues at process R&D Anna Bogstedt, Ingrid Fagervall for ELISA support and Maud Lennmark for endotoxin measurements. We thank or colleagues Sebastian Baumann, Thomas Falkman, Ulrika Ritzén and Stefan Schmidt for their contribution and support to this work and Catherine Heddle and Ian W. Taylor for critical reading of the manuscript.

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  1. Global Protein Science & Supply, DECS, AstraZeneca R&D Södertälje, Building 841, 151 85, Sodertalje, Sweden

    Ola Tuvesson, Christina Uhe, Aleksei Rozkov & Elke Lüllau

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  1. Ola Tuvesson
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  2. Christina Uhe
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  3. Aleksei Rozkov
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  4. Elke Lüllau
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Correspondence to Elke Lüllau.

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Tuvesson, O., Uhe, C., Rozkov, A. et al. Development of a generic transient transfection process at 100 L scale. Cytotechnology 56, 123–136 (2008). https://doi.org/10.1007/s10616-008-9135-2

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  • DOI: https://doi.org/10.1007/s10616-008-9135-2

Keywords

  • Bioreactor
  • HEK293
  • Large scale
  • PEI
  • Transient transfection