Helen Maunder, PhD
Principal Scientist, Oxford BioMedica plc
The Use of Automation in the Development of
Lentiviral Vector Packaging and Producer Cell Lines
Abstract
Large-scale production of lentiviral vectors (LV) for therapeutic applications in gene therapy is challenging with current transient transfection processes. The development of packaging (PaCL) and producer cell lines (PCL) enabling the generation of large quantities of vectors will not only reduce the cost of raw materials but provide consistency between manufacturing runs and downstream processing. To generate PaCLs and PCLs, isolation of stably transfected clones by cloning by limiting dilution (LDC) in antibiotic selective media is required. Oxford BioMedica (OXB) has developed a bespoke Automated Cell Screening System (ACSS) which uses state of the art automation. The ACSS enables the isolation of up to 3000 clones by automated LDC in either 96- or 384-well format. Furthermore, the ACSS can perform routine passage and high-throughput (HTP) clonal LV production and evaluation of clonal LV productivity using various screening methods.
HIV-1 PaCLs were generated at OXB by stably transfecting an HEK293T.TetR cell line which constitutively expresses the Tet Repressor (TetR) protein, with inducible plasmids encoding HIV-1 GagPol, HIV-1 Rev, VSV-G envelope, and selectable antibiotic resistance makers. Following antibiotic selection, more than 1000 HIV-1 PaCL clones were isolated and screened for their ability to produce LV using the ACSS. The best LV producing candidate PaCL clones were selected and expanded, then were screened further to select the best five clones. The best candidate PaCL clones underwent a second round of LDC to ensure monoclonality and were further screened using the ACSS before scale-up and selection of the best sub-clones. The best sub-clones produced higher titre LV than obtained using the manual clone selection process, and also demonstrated higher titres than those achieved using the current HEK293T transient transfection process. In conclusion, the isolation and screening of larger numbers of clones (>1000) using the ACSS leads to the selection of higher titre LV producing clones than previously achieved when screening smaller numbers of clones (100–200) manually.
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