Next Generation Sequencing For Sequence Identity Confirmation And Variant Detection

The use of Next Generation Sequencing-based assays in the biosafety testing space has grown dramatically over the past decade with the advent of advanced instrumentation. Once considered to be cutting edge R&D-only methods, instrumentation and systems have been refined in such a way that lends themselves to compliant regulated testing.

As a leading service provider, we have pioneered the use of NGS to expand our portfolio of GMP service offerings. One such offering is the use of NGS to confirm the nucleotide sequence identity of various product types. NGS is particularly well suited for genomic characterization, including nucleotide variant or sub-population detection. For this type of analysis, the sequence data set is aligned against one or more defined reference sequences. In instances where sufficient depth of coverage is achieved across the genome or region of interest (ROI), sequence variant detection is possible, enabling the detection of sub-populations within a test sample. Furthermore, NGS is also well suited to sequence complex regions such as the inverted terminal repeat regions (ITRs) found in adeno-associated viruses (AAV).

The regulatory expectations for virus seeds, vectors, and plasmids that are used in therapeutic product manufacturing is to ensure the correct genetic sequence. For most viruses and viral vectors, the recommendation is that each are fully sequenced with a complete annotation of the entire vector. It is also recommended to evaluate and detail the significance of differences between the expected sequence and the experimentally derived sequences. Furthermore, for vectors produced by transient transfection of plasmids the guidelines outline full plasmid sequencing as well as sequencing of the resulting vector to demonstrate sequence consistency during manufacturing.