Liter-scale manufacturing of shelf-stable plasmid DNA/PEI transfection particles for viral vector production

Yizong Hu; Brendan A. Eder; Jinghan Lin; Sixuan Li; Yining Zhu; Tza-Huei Wang; Ting Guo; Hai-Quan Mao

Molecular Therapy: Methods & Clinical Development (Mar 2024)

Liter-scale manufacturing of shelf-stable plasmid DNA/PEI transfection particles for viral vector production

Yizong Hu,
Brendan A. Eder,
Jinghan Lin,
Sixuan Li,
Yining Zhu,
Tza-Huei Wang,
Ting Guo,
Hai-Quan Mao

Affiliations

Yizong Hu: Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21218, USA; Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA
Brendan A. Eder: 2seventy bio, Inc., Cambridge, MA 02142, USA
Jinghan Lin: Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA; Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA
Sixuan Li: Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA; Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA
Yining Zhu: Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21218, USA; Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA
Tza-Huei Wang: Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21218, USA; Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA
Ting Guo: 2seventy bio, Inc., Cambridge, MA 02142, USA; Corresponding author: Ting Guo, 2seventy bio, Inc., Cambridge, MA 02142, USA.
Hai-Quan Mao: Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21218, USA; Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA; Corresponding author: Hai-Quan Mao, Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA.

Journal volume & issue: Vol. 32, no. 1
p. 101194

Abstract

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The transfection efficiency and stability of the delivery vehicles of plasmid DNA (pDNA) are critical metrics to ensure high-quality and high-yield production of viral vectors. We previously identified that the optimal size of pDNA/poly(ethylenimine) (PEI) transfection particles is 400–500 nm and developed a bottom-up assembly method to construct stable 400-nm pDNA/PEI particles and benchmarked their transfection efficiency in producing lentiviral vectors (LVVs). Here, we report scale-up production protocols for such transfection particles. Using a two-inlet confined impinging jet (CIJ) mixer with a dual syringe pump set-up, we produced a 1-L batch at a flow rate of 100 mL/min, and further scaled up this process with a larger CIJ mixer and a dual peristaltic pump array, allowing for continuous production at a flow rate of 1 L/min without a lot size limit. We demonstrated the scalability of this process with a 5-L lot and validated the quality of these 400-nm transfection particles against the target product profile, including physical properties, shelf and on-bench stability, transfection efficiency, and LVV production yield in both 15-mL bench culture and 2-L bioreactor runs. These results confirm the potential of this particle assembly process as a scalable manufacturing platform for viral vector production.

Published in Molecular Therapy: Methods & Clinical Development

ISSN: 2329-0501 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Biology (General): Genetics; Science: Biology (General): Cytology
Website: http://www.cell.com/molecular-therapy-family/methods/latest-content

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