709. Characterization of Nanoparticles in Lentiviral Vector Preparations

Abstract

The potential of lentivirus-based gene therapy vectors for the treatment of severe genetic diseases using genetically modified CD34+ cells and hematological malignancies using chimeric antigen receptor T-cells (CAR-T) is supported by recent positive data in clinical trials showing promising therapeutic benefits and safety. The progress from early-to-late stage clinical development requires enhanced characterization of the purified lentivirus vector product. Lentiviral vector preparations are complex in nature and contain a heterogeneous mixture of transduction-competent as well as transduction-deficient virus particles. In addition, lentiviral vector production utilizes host cells that can produce not only the viral particles of interest, but a variety of closely-related impurities that include exosomes and microvesicles. These cell-derived impurities can overlap key biophysical and biochemical attributes of the lentiviral vector, including size, net charge and composition, making them challenging to analyze. We used a variety of analytical tools to further characterize lentiviral vector preparations in terms of size distribution, particle counts and to determine the total particle (viral or non-viral) to infectious particle ratio. These tools included Nanoparticle Tracking Analysis (Nanosight), Field-Flow Fractionation coupled to Multi-Angle Light Scattering (FFF-MALS), and p24-ELISA. Particle protein composition was evaluated by several standard orthogonal protein quantitation assays. Characterization of nanoparticle subpopulations is important as it can support the refinement of manufacturing processes. The impact of variation of these parameters on lentivirus vector performance still remains largely unknown, so interpretations of the results must be carefully assessed and further study is warranted.