Overcoming Challenges To The Commercialization Of Lentiviral-Based Therapies: A Scalable And Robust Production Process Using A Suspension Transient Transfection Platform

By Jiasong Jiang, Leila M. Aneke, F. Michael Haller, Vijetha Bhat, Chien-Ting Li, Victor Paredes, Peng Wang, Miguel Betancourt, Thanh Dang, Phi Le, Aaron Bedee, Ares Ayandokon, Peng Lu, Shuo Lu, Mariya Viskovska

Due to their capacity for delivering larger cargo, transducing both dividing and non-dividing cells, and maintaining high expression levels over extended periods, lentiviral vectors (LVVs) are increasingly favored for cell and gene therapy applications. This growing demand places significant pressure, particularly on contract development and manufacturing organizations (CDMOs), to provide safe, third-generation viral vectors manufactured through adaptable, scalable, and high-yielding processes. While we continue developing new LVV manufacturing tools, such as stable producer cell lines, this paper presents results obtained using our proprietary HEK293T 2G7 suspension cell line for LVV production via transient transfection in serum-free, chemically-defined, and animal component-free media, followed by downstream purification.

The process was developed using LVV-CD19 as a model construct, in addition to the commonly used LVV-GFP, due to observed differences in downstream performance when switching the gene of interest from GFP to a therapeutically relevant gene. The established process was consistently replicated in a 3 L stirred-tank reactor for both LVV-GFP and LVV-CD19, transferred to our process development department, and subsequently tested at a 50 L scale using LVV-CD19, achieving an overall process recovery of 24% including clarification.

The bulk drug substance (BDS) was analyzed for infectious, genomic, and capsid titer, potency via T-cell transduction, and residual host cell DNA and protein content.