Why Cell Therapy Developers Are Moving From Sanger to NGS for Identity & Purity Testing

As cell therapies move from experimental programs to licensed products, developers face growing pressure to verify product identity, purity, and genomic integrity with far greater precision than ever before. For years, Sanger sequencing was the standard tool for confirming transgene identity and detecting small sequence changes. But as vectors become more complex and regulatory expectations more rigorous, Sanger is no longer sufficient for many critical assays.

Next-generation sequencing (NGS) has rapidly emerged as the preferred method for identity and purity testing in cell therapy manufacturing. With higher sensitivity, broader detection capabilities, and the ability to interrogate entire populations rather than a single consensus sequence, NGS offers advantages that directly address the limitations of traditional Sanger workflows.

Here’s why developers are making the shift.

1. Sanger Misses Low-Frequency Variants That Matter

Sanger sequencing produces a single consensus read. If a subpopulation of cells contains a mutation or rearranged sequence, especially at frequencies below ~20%, the signal becomes undetectable in a Sanger trace.

For cell therapies, where heterogeneity is inherent and even rare variants can impact safety or potency, this is a critical blind spot.

NGS advantage:
NGS can detect variants down to 1% or even lower, depending on the assay design. That level of sensitivity is essential for identifying:

• Low-frequency indels introduced during CRISPR editing
• Minor contaminating cell clones with unintended integrations
• Subpopulations carrying truncated or rearranged transgenes
• Early signs of clonal drift during manufacturing

This ability to see the full diversity of the cell product makes NGS far more reliable for release and comparability testing.

2. Complex Vectors Require More Than a Single Read

Modern cell therapies depend on increasingly elaborate constructs: multi-domain CARs, polycistronic gene cassettes, regulatory element combinations, and safety switches, all packaged in lentiviral or transposon vectors.

Sanger sequencing can confirm short stretches of these sequences but cannot easily:

• Validate full-length transgenes
• Identify structural variations
• Confirm vector integrity after integration
• Detect partial or aberrant insertions

NGS advantage:
Long-read platforms like PacBio HiFi and Oxford Nanopore enable full-length sequence characterization, while short-read Illumina or AVITI data provides depth and accuracy. Combining both offers a complete picture of:

• Transgene structure
• Integration patterns
• UTR, promoter, enhancer, and polyA sequences
• Unexpected rearrangements or truncations

This level of detail is increasingly expected in IND submissions and regulatory filings worldwide.

3. Cell Therapy Products Are Heterogeneous — Sanger Can’t Capture That

Unlike plasmid DNA or purified viral vectors, cell therapy products are not uniform. They contain:

• Multiple integration events
• Different copy numbers across cells
• Subpopulations with varying editing outcomes
• Unique transcriptional signatures

Sanger, which provides only an averaged sequence, cannot profile these heterogeneous populations.

NGS advantage:
NGS captures the sequence landscape across millions of cells, enabling developers to understand:

• Editing efficiency and on-target profiles
• Integration site diversity
• Clonal dominance risk
• Vector copy number distributions
• Changes during cell expansion

For autologous and allogeneic therapies alike, these insights are essential for consistent manufacturing.

4. NGS Supports Regulatory Expectations for Genomic Characterization

Regulators are now asking for deeper, more comprehensive genetic analyses of cell therapy products. Agencies increasingly expect:

• Sensitive detection of off-target edits
• Evidence of vector integrity
• Characterization of integration patterns
• Population-level variant frequency data
• Confirmation of identity at the sequence level

Sanger often cannot satisfy these requirements without supplemental methods, which increase cost and complexity.

NGS advantage:
A single well-designed NGS assay can address multiple regulatory questions simultaneously, streamlining CMC documentation and accelerating development timelines.

5. NGS Enables Scalable QC as Programs Grow

Early-stage R&D may rely on Sanger because it’s fast and inexpensive. But as cell therapy programs advance to GMP manufacturing, the volume of required identity and purity testing increases dramatically.

Sanger’s manual workflows, primer design, cloning steps for mixed samples, chromatogram review, do not scale well.

NGS advantage:
NGS supports high-throughput, automated, and standardized testing that integrates seamlessly into:

• IND-enabling studies
• Lot release
• Comparability assessments
• Process and product characterization
• Long-term monitoring studies

This makes NGS a future-proof investment for growing pipelines.

Conclusion: NGS Is Becoming the New Standard for Cell Therapy Sequencing QC

As the field matures, developers need tools that match the complexity and safety requirements of modern cell therapies. NGS offers the depth, sensitivity, and scalability to thoroughly characterize cell products and meet evolving regulatory expectations, far beyond what Sanger can provide.

With multi-platform capabilities including: Illumina, Nanopore, PacBio, Element AVITI, and 10x Genomics, Avance Biosciences supports comprehensive NGS-based assays for identity, purity, integration, and genomic integrity testing across the entire development lifecycle.