Engineering an autonucleolytic mammalian suspension host cell line to reduce DNA impurity levels in serum-free lentiviral process streams
Author(s)
Type
Journal Article
Abstract
We engineered HEK293T cells with a transgene encoding tetracycline-inducible expression of a Staphylococcus aureus nuclease incorporating a translocation signal. We adapted the unmodified and nuclease-engineered cell lines to grow in suspension in serum-free media, generating the HEK293TS and NuPro-2S cell lines, respectively. Transient transfection yielded 1.19 × 106 lentiviral transducing units per milliliter (TU/mL) from NuPro-2S cells and 1.45 × 106 TU/mL from HEK293TS cells. DNA ladder disappearance revealed medium-resident nuclease activity arising from NuPro-2S cells in a tetracycline-inducible manner. DNA impurity levels in lentiviral material arising from NuPro-2S and HEK293TS cells were undetectable by SYBR Safe agarose gel staining. Direct measurement by PicoGreen reagent revealed DNA to be present at 636 ng/mL in lentiviral material from HEK293TS cells, an impurity level reduced by 89% to 70 ng/mL in lentiviral material from NuPro-2S cells. This reduction was comparable to the 23 ng/mL achieved by treating HEK293TS-derived lentiviral material with 50 units/mL Benzonase.
Date Issued
2024-02-16
Date Acceptance
2024-01-18
Citation
ACS Synthetic Biology, 2024, 13 (2), pp.466-473
ISSN
2161-5063
Publisher
American Chemical Society (ACS)
Start Page
466
End Page
473
Journal / Book Title
ACS Synthetic Biology
Volume
13
Issue
2
Copyright Statement
© 2024 The Authors. Published by American Chemical Society. This publication is licensed under CC-BY 4.0 .
License URL
Publication Status
Published
Date Publish Online
2024-01-24