Effective gene therapy vector manufacturing processes result in high titer, potency, and purity to promote stable transgene expression and prevent unwanted immunogenic responses. However, many vector production processes are often expensive, lack scalability, and require multiple purification steps with low yield to achieve desired product quality. Traditional purification methods that incorporate multiple filtration and polishing steps exacerbate viral product loss, especially for labile Lentiviral vector (LVV). Thus, it is vital to minimize downstream unit operations, without sacrificing impurity clearance.<br/>Nuclease digestion is a scalable impurity removal step that maintains constant volume and conserves titer. Endonuclease degrades plasmid and host cell genomic DNA impurities via enzymatic reaction without causing significant product inactivation. This presentation provides a case study of the evolution of nuclease digestion characterization and process understanding for multiple gene therapy viral vector clinical programs.<br/> <br/>Characterization was completed for two vector products utilizing a staggered platform approach under aggressive CMC timelines. Characterization for the first product involved screening studies to establish fundamental knowledge such as identification of significant process parameters and insight into potential interaction effects. Leveraging the first data set, the second approach incorporated a higher resolution design to create a more predictive model of future process behavior. Experiments were conducted using mammalian cell-culture derived LVV particles produced under controlled conditions. Multiple scale-independent process parameters were purposely varied in Design of Experiment (DoE) studies, and impact to titer and impurity levels were assessed. Results showed significant influence over DNA removal, yet minimal adverse impact to LVV titer was observed. Overall, the increased process understanding gained through systematic studies led to robust process performance and provided opportunities for further optimization. Finally, since enzymatic digestion is not product-specific, results from these two campaigns are directly applicable to other gene therapy processes.<br/>

Rapid development and characterization of a nuclease digestion step for multiple gene therapy viral vector manufacturing processes: A case study

2019 Spring National Meeting

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