Recombinant AAV batch profiling by nanopore sequencing elucidates product-related DNA impurities and vector genome length distribution.

ABSTRACT: During production, recombinant adeno-associated virus (rAAV) capsids are equipped with heterogeneous genetic payloads including undesired DNA impurities as well as truncated vector genomes. Comprehensive analysis of encapsidated DNA by long-read next-generation sequencing is destined to guide platform optimization and provide crucial insights into safety of gene therapies. We used nanopore sequencing for in-depth profiling of an rAAV9 batch produced using our proprietary split two-plasmid system in a 50-L bioreactor. We compared three methods for single-strand to double-strand DNA conversion and their impact on the sequencing data. We observed a distinct library size profile but comparable impurity distribution. We contrasted recent nanopore sequencing advancements such as the V14 chemistr

SUBMITTER: Dunker-Seidler F 

PROVIDER: S-EPMC11850753 | biostudies-literature | 2025 Mar

REPOSITORIES: biostudies-literature