Building a robust and scalable lentiviral vector purification platform

Abstract

Background & Aim Lentiviral vectors (LVV) are common vehicles of genetic delivery for cell and gene therapies. LVV commercial manufacturing is costly and downstream processing (DSP) is a primary driver for the high costs due to challenges in maintaining viral infectivity. Additionally, these processes are not standardized across the industry and have poor process definition, resulting in large batch-to-batch variability, limited robustness, performance, and scalability. In collaboration with CCRM, the GE Fast Trak Centre for Advanced Therapeutic Cell Technologies (CATCT) has developed a DSP platform that can process LVV from multiple upstream systems; suspension and serum-free cultures from both stable producer and transient-transfection modalities. The different characteristics of these systems (transient vs. inducible, cell viability and density at harvest, amount of host cell contaminants, etc.) showcase the compatibility of our process with various upstream sources. Our semi-closed, single-use, single-day, room temperature LVV purification process yields infectious recoveries and contaminant clearances that are on par with current industry standards, while maintaining robustness and scalability. Methods, Results & Conclusion The final full process consists of nucleic acid digestion inside the bioreactor, clarification using filtration, ultrafiltration and diafiltration using tangential flow filtration, chromatography with multi-modal Capto™ Core 700, an optional secondary concentration, and finally 0.2 µm sterile filtration. Overall infectious titre recoveries and final concentrations of 13.7% ± 6.2% and 5.2E7 TU/mL (stable) and 30.8% ± 2.4% and 1.4E8 TU/mL (transient) have been achieved, with a ≥ 2-log removal of host cell protein and DNA in both. Additionally, a 25 L pilot run was completed with similar results, demonstrating the scalability of the process. Supported by continual gains of process knowledge and understanding, we have built a robust, consistent, and scalable LVV purification platform.