Client Challenge
A gene therapy company preparing for an IND submission required a validated, regulatory-compliant method to confirm the identity, purity, and sequence fidelity of its plasmid-based drug substance prior to batch release. In addition to standard QC, the client required high-resolution detection of low-frequency sequence variants, including substitutions and indels, and trace levels of contaminating DNA (e.g., host or microbial) to ensure the integrity of their therapeutic product.
Avance Biosciences’ Solution
Avance Biosciences implemented its validated Plasmid ID and Purity by NGS assay, designed to meet FDA expectations for genetic fidelity and contamination control. The workflow is based on the Illumina MiSeq platform currently utilizing the TruSeq PCR-Free or PCR-based workflows and is transitioning to PacBio SMRT sequencing for long-read applications. The assay detects low-frequency DNA variants and exogenous DNA contamination with high sensitivity and specificity.
- Plasmid prep and QC: DNA was purified from client cell banks using validated SOPs and quantified by spectrophotometry (Nanodrop or equivalent), ensuring accurate input into library prep.
- Shearing and library prep: DNA was mechanically sheared to ~550 bp (Covaris or equivalent) and prepared with the TruSeq PCR-Free kit to minimize amplification bias.
- Sequencing and mapping: Paired-end sequencing (2×150 bp) was performed. Reads were aligned to the plasmid reference sequence and screened against human and coli genomes to assess potential host or microbial DNA contamination. PhiX was included as a sequencing control.
- Variant Detection: A custom-validated bioinformatics pipeline detected single-nucleotide variants and indels with a validated limit of detection (LOD) of 1% variant allele frequency.
- Sequence Fidelity Verification: A consensus sequence was assembled de novo and compared to the reference to confirm 100% sequence identity and structural integrity (absence of structural variants or rearrangements).
Qualification Protocol
To validate assay performance, QC samples were prepared at defined variant frequencies (1–50%) by mixing the drug substance (wild-type plasmid) with variant plasmids containing defined mutations. This enabled verification of assay linearity, reproducibility, and sensitivity for rare variant detection.
Customer Outcome
The client integrated the NGS-based assay into their lot release strategy for GMP plasmid production. Avance’s data package supported regulatory filings by demonstrating:
- Sensitive and quantitative detection of low-frequency variants (≥1%)
- Reliable identification and quantification of contaminants (e.g., host DNA, microbial, ect.)
- High specificity and reproducibility for lot-to-lot consistency
- A detailed data package suitable for regulatory submission
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