Abstract:
Due to their ability to deliver larger cargo, transduce both dividing and non-dividing cells and maintain long-term and high expression levels, lentiviral vectors (LVV) are increasingly becoming a tool of choice in cell and gene therapy applications. This increases the pressure, particularly on CDMO’s, to meet the markets’ demand for safe, 3rd generation viral vectors with adaptable, scalable and high-yielding manufacturing processes. While continuing to develop new tools for LVV manufacture, such as a stable producer cell line, we are presenting here the results obtained for our proprietary HEK293T 2G7 suspension cell line for LVV production by transient transfection in serum-free, chemically-defined and animal component-free media and its subsequent downstream purification.
The process was developed using LVV-CD19 as model construct in addition to the commonly used LVV-GFP due to altered downstream performance observed by us when switching the gene of interest from GFP to a therapeutically relevant gene. The obtained process was confirmed repeatedly in a 3 L STR for both LVV-GFP and LVV-CD19, transferred to our process development department and tested there at 50 L scale using LVV-CD19, with an overall process recovery, including clarification, of 24 %.
The bulk drug substance (BDS) was analyzed for infective, genomic and capsid titer, as well as potency by T-Cell transduction and residual host cell DNA and host cell protein content.
Author: Jiasong Jiang, PhD, R&D Scientist