A sensitive sample preparation pipeline for adventitious virus detection using nanopore sequencing

Abstract Recent regulatory guidance, such as the ICH Q5A(R2) and the Eur. Ph. 2.6.41 draft chapter, now encourage the use of sequencing as an alternative adventitious agent testing assay to lengthy compendial in vivo assays used for cell line qualification. A significant obstacle, however, is the abundance of background nucleic acid from the human or mammalian host. Using nanopore sequencing, MIT CBI has developed a new sample preparation workflow using concentration, nuclease treatment, and agnostic PCR methods to eliminate background signals and amplify contaminant reads, leading to a 3-log improvement in the limit of detection that is comparable to or better than compendial assays and short-read sequencing approaches. In addition, compared to short-read sequencing, long-read sequencing allows for improved alignment to a reference sequence, providing greater confidence of a potential contaminant. This approach could lead to more rapid and improved detection of viral contaminants in cell-culture manufacturing.  Biography Charley Swofford has been at the MIT Center for Biomedical Innovation since 2022, where he is the Assistant Director for Biomanufacturing Initiatives. In this role, he manages collaborative sponsored research projects, including investigating nanopore sequencing for adventitious agent detection. He also supports joint research projects among members of the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB), a pre-competitive biopharmaceutical industry consortium focused on identifying and sharing best practices to mitigate the risk of adventitious agent contamination in biopharmaceutical manufacturing.