Journal
JOURNAL OF BIOTECHNOLOGY
Volume 326, Issue -, Pages 11-20Publisher
ELSEVIER
DOI: 10.1016/j.jbiotec.2020.12.004
Keywords
Clarification; Filtration; Lentivirus; Downstream processing
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Funding
- Sartorius Stedim Biotech (Goettingen, Germany)
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Lentiviral vectors have shown great potential for gene modified cell therapy and gene therapy applications, but downstream processing remains a challenge due to the low stability of the virus. Researchers analyzed whether lentiviral vectors produced by transient transfection of HEK293 T/17 SF suspension cells can be efficiently clarified with a lab-scale method using diatomaceous earth (DE) and bioburden reducing membrane filters, achieving high recoveries. Experiment results showed that higher DE concentrations were beneficial for turbidity reduction and shorter filtration times, but with a decrease in LV titer. A DE concentration of 9 g/L was identified as a robust set-point for clarification.
Lentiviral vectors have proven their great potential to serve as a DNA delivery tool for gene modified cell therapy and gene therapy applications. The downstream processing of these vectors is however still a great challenge, particularly because of the low stability of the virus. Harvesting and clarification are critical and until now insufficiently characterized steps for lentivirus processing. To address this bottleneck, we analyzed whether lentiviral vectors produced by transient transfection of HEK293 T/17 SF suspension cells can be efficiently clarified with a lab-scale method with the filter aid diatomaceous earth (DE) and bioburden reducing membrane filters achieving high lentivirus recoveries. Using a design of experiment approach we found that higher DE concentrations are advantageous for a higher turbidity reduction and shorter filtration times, but at the same time LV titer decreases with increasing DE concentration. A DE concentration of 9 g/L was identified with a DoE as a robust set-point. Clarification with DE was compared with for lab-scale traditionally employed centrifugation and subsequent bioburden reduction filtration of viral vectors. The use of DE allows to perform a harvest and clarification process, which does not only facilitate faster and safer virus handling, but enables a lower material consumption due to the extremely increased filter capacity, thus representing an efficient and robust lab-scale clarification process.
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