Application of Size Exclusion Chromatography with Multiangle Light Scattering in the Analytical Development of a Preclinical Stage Gene Therapy Program.

Abstract

To provide safe recombinant adeno-associated viruses (rAAV) to patients, scalable manufacturing processes are required. However, these processes may introduce impurities that impact the performance and quality of the final drug product. Empty rAAV capsids are product-related impurities. Regulatory guidance requires that accurate analytical methods be implemented early in product development to measure the level of empty capsids. A process confirmation vector, produced from 200 L production, was used to develop and optimize a size exclusion chromatography with UV and multiangle light scattering (SEC-MALS) method. Vector produced from a 500 L production was used to assess the full-to-empty ratio using the following analytical methods: sedimentation velocity analytical ultracentrifugation (SV-AUC), droplet digital PCR (ddPCR) with capsid enzyme-linked immunosorbent assay (ELISA), bulk absorbance at 260/280 nm, cryogenic electron microscopy, and SEC-MALS. This test article was used for a 30-day, non-Good Laboratory Practices animal study that assessed biodistribution of the product (STRX-330). SEC-MALS outperformed the other methods and correlated well with SV-AUC values of full-to-empty particles. In addition, SEC-MALS agreed with ddPCR and ELISA measurements for vector genomes/mL and capsid particles/mL, respectively. SEC-MALS was linear, accurate, and precise while achieving chromatography quality control (QC) recommendations. Compared to other stability-indicating assays, SEC-MALS performed similarly to ddPCR, capsid ELISA, and infectivity assays in accelerated stress studies. In response to alkaline, but not acidic stress, SEC-MALS indicated distinct changes in the DNA content of the monomer Adeno-associated viruses (AAV) peak for STRX-330, which was supported by ddPCR data. Conversely, acidic treatment resulted in more aggregated vector, but did not impact the DNA content. This work indicates that SEC-MALS is a valuable analytical tool in the analytical development and QC testing of AAV. In addition, this work suggests that SEC-MALS can provide fundamental understanding of AAV in response to environmental stress. This may impact steps of the manufacturing process to minimize conditions that reduce performance.