Journal Article

In-Depth Comparison of Adeno-Associated Virus Containing Fractions after CsCl Ultracentrifugation Gradient Separation

Dolinar, Ana, Jakomin, Tjaša, Žnidarič, Magda Tušek, Zevnik, Kaja, Zentilin, Lorena, Kutnjak, Denis, Štalekar, Maja, Janc, Mojca, Bačnik, Katarina, Fedorov, Dmitrii, Dobnik, David

2024

Overview

Recombinant adeno-associated viruses (rAAVs) play a pivotal role in the treatment of genetic diseases. However, current production and purification processes yield AAV-based preparations that often contain unwanted empty, partially filled or damaged viral particles and impurities, including residual host cell DNA and proteins, plasmid DNA, and viral aggregates. To precisely understand the composition of AAV preparations, we systematically compared four different single-stranded AAV (ssAAV) and self-complementary (scAAV) fractions extracted from the CsCl ultracentrifugation gradient using established methods (transduction efficiency, analytical ultracentrifugation (AUC), quantitative and digital droplet PCR (qPCR and ddPCR), transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA)) alongside newer techniques (multiplex ddPCR, multi-angle light-scattering coupled to size-exclusion chromatography (SEC-MALS), multi-angle dynamic light scattering (MADLS), and high-throughput sequencing (HTS)). Suboptimal particle separation within the fractions resulted in unexpectedly similar infectivity levels. No single technique could simultaneously provide comprehensive insights in the presence of both bioactive particles and contaminants. Notably, multiplex ddPCR revealed distinct vector genome fragmentation patterns, differing between ssAAV and scAAV. This highlights the urgent need for innovative analytical and production approaches to optimize AAV vector production and enhance therapeutic outcomes.

• Publisher: MDPI AG,MDPI
• Subject: analytical methods / analytical ultracentrifugation (AUC) / Centrifugation / Centrifugation, Density Gradient - methods / Cesium / Cesium - chemistry / Chemical properties / Chlorides / Chromatography / Clinical trials / Comparative analysis / Contaminants / CsCl ultracentrifugation gradient / Dependoparvovirus / Dependovirus - genetics / Dependovirus - isolation & purification / Dependoviruses / digital droplet PCR (ddPCR) / droplets / Enzyme-linked immunosorbent assay / FDA approval / gel chromatography / Genetic aspects / Genetic disorders / Genetic Vectors - genetics / genome / Genomes / HEK293 Cells / Humans / Impurities / Infectivity / Light scattering / Mass spectrometry / Medical innovations / Methods / Next-generation sequencing / pathogenicity / plasmids / Protein purification / Proteins / recombinant adeno-associated viruses (rAAVs) / Scientific imaging / Single-stranded DNA / Software / therapeutics / Transduction, Genetic / Transmission electron microscopy / transmission electron microscopy (TEM) / Ultracentrifugation / Virion - genetics / Virion - isolation & purification