Sanger Sequencing

Sanger sequencing is a well-established, high-accuracy method for determining the nucleotide sequence of targeted DNA regions. In biopharmaceutical CMC, it is routinely used to confirm the identity of plasmid DNA (used as critical starting materials) and the vector genome (e.g., transgene cassettes in AAV products). Due to its single-read precision and ease of interpretation, it remains a gold-standard identity test in gene and cell therapy manufacturing.

• Legacy:
  Restriction Enzyme Mapping — used for structural confirmation of plasmid constructs; provides map-level but not base-level resolution.

• Established Standard:
  Sanger Sequencing by Capillary Electrophoresis — widely used for targeted identity testing of plasmid inserts and vector genomes. Supports ICH Q6B expectations for sequence-level characterization.

• Orthogonal/Characterization:
  Next-Generation Sequencing (NGS) — used for whole plasmid/vector sequencing, detection of minor variants, or challenging regions (e.g., ITRs).

  • Short-Read NGS (e.g., Illumina): Provides high-throughput, deep sequencing coverage, useful for detecting low-level variants or impurities.
  • Long-Read NGS (e.g., PacBio, Oxford Nanopore): Can sequence entire viral genomes in single, contiguous reads, making it exceptionally powerful for confirming full-length integrity and identifying complex structural variants.

• Sanger Sequencing: This method determines the sequence of a DNA fragment by generating copies of varying lengths. Each copy terminates at a specific base (A, T, C, or G) labeled with a distinct fluorescent dye. These fragments are then separated by size using capillary electrophoresis. A detector reads the color of the dye on each fragment as it passes, reconstructing the DNA sequence one base at a time.

• Output:

  • Nucleotide sequence file (e.g., .ab1 or FASTA)

  • Electropherogram (4-color chromatogram)

• Interpretation:

  • Sequence is aligned to reference using sequence analysis software

  • Critical regions (e.g., transgene cassette and regulatory elements) must match 100% to the expected reference sequence for identity confirmation.

  • Chromatograms are visually inspected for unambiguous base calls and signal quality

• High base-calling accuracy (≥99.9% over ~500–800 bp, sometimes extending to ~900 bp with optimal conditions)

• Simple data analysis and unambiguous interpretation

• Long regulatory track record; well-established validation pathways

• Short Read Length: Limited to ~800–1000 bases per reaction

• Low Throughput: Not suitable for high-volume or whole-genome applications

• Not Quantitative: Cannot detect low-frequency variants or mixed populations

• Accuracy: The primary parameter. The method must be shown to consistently generate the 100% correct sequence when tested against a certified, known reference standard (e.g., a well-characterized plasmid).
• Specificity: The sequencing primers used must be shown to bind only to the intended target DNA sequence and not to other potential DNA contaminants.
• Precision: The method must demonstrate high repeatability and intermediate precision, yielding the same correct sequence every time.

Standardization of Sanger sequencing relies on the consistent use of validated primers, high-quality sequencing reagents, and certified reference plasmids. Commercial DNA sequencing standards (e.g., well-characterized plasmids or synthetic DNA fragments) are routinely used to verify instrument performance and confirm accuracy. In a GMP environment, reference materials must be qualified, and sequencing controls (positive and negative) are included in every run to ensure traceability, reproducibility, and regulatory compliance.

• AAV Gene Therapy:
  Used to verify plasmid identity and transgene cassette sequence

• Plasmid-based Vaccines or RNA Therapies:
  Required to ensure plasmid integrity prior to IVT or vector packaging

• Cell Therapy:
  Confirms identity of lentiviral or retroviral vector genomes used for transduction