A Scalable And Robust Lentiviral Platform For GMP Manufacturing

By James Xin, James Fasano, Chyan-Jang Lee, Theresa Dao, Daniel Kennedy, Briana Orlando, Nhi Tran, Anthony Leyme, Tam Nguyen, Sheyla Mirabal, Miranda Williams, Christine Beaudry, Lorenz Ponce, Johnny Tran, Josh Sorafine, Dawn Maier, Mike Paglia, Mercedes Segura, and Bojiao Yin

Lentiviral vectors (LV) are a potent tool in the growing field of cell and gene therapy as they enable efficient delivery of genetic material into cells for therapies such as CAR-T and HSC-based gene therapies. With the increasing number of clinical applications and interest in the field, robust, scalable, and cost-effective platforms for GMP manufacturing of high-quality lentiviral vectors are urgently needed.

We describe here a well-established platform process for LV production based on transient transfection of serum-free cells grown in suspension. Cell growth and transfection production parameters were determined using DoE studies to achieve optimal vector yields. The crude LV harvest from upstream operations shows a high infectious titer (> 1E7 TU/ml) and a low particle-to-infectious titer ratio (< 1000 particles/TU). More importantly, the robustness and scalability of upstream process has been demonstrated in different reactor configurations (e.g., Ambr® 250, bench-top and pilot scale bioreactors). The downstream unit operations have been established and optimized for both small and large-scale production requirements. The purified and highly concentrated LV final product shows efficient and potent transduction of T cells and remarkable reduction in the host cell protein (HCP), host cell DNA and plasmid DNA impurity content.

Aiming to provide a robust, scalable and GMP compatible process, this platform also focused on timeline acceleration and seamless transition from PD to GMP manufacturing in the following aspects: ability to produce LVs coding for different genes of interest (GOI) including CAR/TCR constructs, employment of the single-use technologies, employment of non-animal-derived raw materials and alignment of equipment, protocols, and data collection tools between PD labs and GMP manufacturing suites.