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Process optimization of large-scale production of recombinant adeno-associated vectors using dielectric spectroscopy

  • Biotechnological Products and Process Engineering
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Abstract

A well-characterized manufacturing process for the large-scale production of recombinant adeno-associated vectors (rAAV) for gene therapy applications is required to meet current and future demands for pre-clinical and clinical studies and potential commercialization. Economic considerations argue in favor of suspension culture-based production. Currently, the only feasible method for large-scale rAAV production utilizes baculovirus expression vectors and insect cells in suspension cultures. To maximize yields and achieve reproducibility between batches, online monitoring of various metabolic and physical parameters is useful for characterizing early stages of baculovirus-infected insect cells. In this study, rAAVs were produced at 40-l scale yielding ~1 × 1015 particles. During the process, dielectric spectroscopy was performed by real time scanning in radio frequencies between 300 kHz and 10 MHz. The corresponding permittivity values were correlated with the rAAV production. Both infected and uninfected reached a maximum value; however, only infected cell cultures permittivity profile reached a second maximum value. This effect was correlated with the optimal harvest time for rAAV production. Analysis of rAAV indicated the harvesting time around 48 h post-infection (hpi), and 72 hpi produced similar quantities of biologically active rAAV. Thus, if operated continuously, the 24-h reduction in the production process of rAAV gives sufficient time for additional 18 runs a year corresponding to an extra production of ~2 × 1016 particles. As part of large-scale optimization studies, this new finding will facilitate the bioprocessing scale-up of rAAV and other bioproducts.

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Acknowledgments

The authors wish to acknowledge the support of J. Shiloach and L. Trinh from NIDDK for facilitating the 40-l bioreactor, L. Yang from NHLBI for providing the baculovirus, and S. Cecchini from NHLBI for the valuable discussions.

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Authors and Affiliations

  1. Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, US National Institutes of Health, 10 Center Drive, NIH, Building 10, Room 7D05, Bethesda, MD, 20892, USA

    Alejandro Negrete & Robert M. Kotin

  2. FOGALE Nanotech, Nimes, 30900, France

    Geoffrey Esteban

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  1. Alejandro Negrete
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  2. Geoffrey Esteban
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Correspondence to Robert M. Kotin.

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Negrete, A., Esteban, G. & Kotin, R.M. Process optimization of large-scale production of recombinant adeno-associated vectors using dielectric spectroscopy. Appl Microbiol Biotechnol 76, 761–772 (2007). https://doi.org/10.1007/s00253-007-1030-9

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