Identity testing by NGS as a means of risk mitigation

Imagine that you just completed a manufacturing run for a mission critical gene therapy vector. All of your release testing is underway, and you are days away from releasing your product. Per regulatory guidelines, identity testing is performed to confirm the vector sequence is as expected. In order to obtain the full sequence of the vector, Next Generation Sequencing is used. The identity test results come back and to your dismay, the sequencing data reveals several unexpected sequence variants in the vector preparation! An insertion with a frequency of 7%, several substitutions with frequencies ranging from 2-8%, and a deletion with a frequency of 10%. The unexpected results lead to an investigation causing the release of the product to be significantly delayed

The situation described above is not hypothetical, and happens more that you think, costing valuable time and resources and introducing significant delays. Avoiding this situation is actually quite easy with an appropriate risk mitigation testing strategy at critical parts of the manufacturing process.

Viral-based vectors are commonly manufactured through the transfection of a series of plasmids into production cells. Plasmids used to produce vectors are manufactured to GMP compliance including a confirmation of the correct sequence. Adequate identification of low-level variants with an appropriately sensitive method is critical in ensuring the quality of the final product. Investigatory testing has shown that sequence variants present in starting materials (e.g., plasmids) are very likely to make their way to the final product. A risk-based testing strategy, in the context of identity, for viral vector manufacturing will be presented, focusing on key testing points. Next generation sequencing assays for identity and variant detection will be highlighted due to their extremely sensitive nature when compared to traditional approaches. Regulatory requirements in regard to identity testing will also be presented and discussed.