%N 2
%D 2024
%K lentivirus, mammalian cells, bioprocess, gene therapy, nuclease
%P 408-692
%T Engineering an autonucleolytic mammalian suspension host cell line to reduce DNA impurity levels in serum-free lentiviral process streams
%O This version is the author accepted manuscript. For information on re-use, please refer to the publisher's terms and conditions.
%A Geoffrey Howe
%A matthew Wasmuth
%A Pamela Emanuelle
%A Giulia Massaro
%A Ahad Rahim
%A sadfer Ali
%A Milena Rivera
%A john Ward
%A Elaheh Keshavarz-Moore
%A chris Mason
%A Darren Nesbeth
%J ACS Synthetic Biology
%I American Chemical Society
%L discovery10185876
%V 13
%X 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.