Lentiviral vectors have been increasingly employed as gene delivery vehicles for the treatment of a variety of genetic, neurodegenerative and acquired diseases. As gene therapy treatments progress from pre-clinical to clinical phases, there is a growing demand for high purity and concentrated lentiviral vectors. However, the current manufacturing process presents major challenges such as low virus production titers and viral vector stability leading to low downstream recoveries. This work presents a workflow for robust, fast and scalable process development for LVV purification. Viral vector stability was first evaluated in different mobile phase conditions spanning a range of pH, ionic strength, and buffer species to identify the optimal conditions for the screening of chromatographic media. The stability results suggested that the vector was stable in a pH range of 6.5-7.5 and the stability was observed to decrease above or below this range. The effect of salt concentration on vector stability was also investigated and it was observed to reduce at higher ionic strength likely due to increased osmotic pressure. The results of this study helped to devise a robust purification strategy with the use of chromatographic resins, membranes and/or monoliths which could provide stable lentiviral vector. Process development stage included a high-throughput screening of resins in a bind-elute mode using a 96-well slurry plate format to minimize time and material requirements. Following this, the top candidates were evaluated in a miniature column setting for obtaining resolution/selectivity under different gradient and loading conditions and ranked based on capacity, yield, and impurity clearance criteria. A lab-scale column was performed to ensure the validity of these findings along with tracking the levels of process-related impurities like HCP and hcDNA. Finally, the employed screening strategies and resulting purification schemes aided to guide the development of a GMP process for the manufacturing of clinical-grade lentiviral vectors for gene therapy applications.

Development of downstream purification process for lentiviral gene therapy vector

ACS Fall 2020 Virtual Meeting

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