Identity tests are a crucial part of product testing packages submitted to regulatory agencies during the drug development and approval lifecycle. These tests include verifying the identity of plasmids and/or viral vectors that are critical raw materials, drug products (DP), or drug substances (DS) for cell and gene therapy manufacturing. These tests serve to both verify that the gene of interest (or therapeutic gene) has been successfully inserted into the plasmid and/or viral vector and that the inserted sequence is identical to the expected sequence of the inserted gene of interest. These tests are critical prior to releasing the plasmid or viral vector for downstream processes, including manufacturing.
Non-high throughput methods such as Sanger sequencing can be employed, but these are limited in the sequence coverage and depth, which compromises the sensitivity of the assay. Next Generation Sequencing (NGS) is a high throughput platform enabling rapid, cost-effective, parallel sequencing of DNA and RNA. Unlike Sanger sequencing, which analyses one fragment at a time, NGS sequences millions of fragments simultaneously.
NGS is the method of choice for identity testing as it provides the depth and sensitivity that are needed to identify the presence of any variants in a plasmid and/or viral vector nucleotide sequence. NGS also offers the ability for multiplex sequencing where samples can be batched and sequenced simultaneously, saving both costs and time, which is ideal for start-ups. Validated, GMP methods are ideal for release testing.
Eurofins BPT in Lancaster, PA, recently added GMP Sequence Identification Testing (SIT) by NGS to its testing portfolio. The method utilises short read sequencing performed on an Illumina platform. The sequence obtained is compared to a client-provided reference sequence, using bioinformatic analysis to identify any mismatches or variants, including insertions or deletions in the plasmid or viral vector sequence. The GMP method for Sequence Identity Testing has been validated in accordance with ICH Q2 (R2) as an identity method with additional attributes challenge to ensure the robustness of the method for detection of variants.
NGS provides high quality, regulatory-accepted results to assure product safety and accelerate getting key materials into manufacturing and medicines to patients. NGS is part of a complete biosafety program, enabling clients to meet or exceed product milestones and deliver critical therapies to patients faster than traditional methods.