Background & Rationale
In the context of mRNA vaccine and therapeutic development, product quality is paramount. Conventional assays such as qPCR, gel electrophoresis, or HPLC offer partial insights and lack resolution for full-length, strand-specific, or modification-aware profiling.
This study introduces VAX-seq, a long-read nanopore-based RNA sequencing framework specifically optimized for IVT mRNA analysis. It was benchmarked on two model mRNA products: a ZIKV prM-E vaccine and a SARS-CoV-2 spike mRNA.
mRNA vaccine production and VAX-seq workflow.
Core Analytical Dimensions and Technical Findings
| Quality Attribute | Technique | Key Results & Interpretation |
|---|---|---|
| Plasmid Template Integrity | Nanopore DNA-seq | Accurate detection of plasmid length and rearrangements |
| Full-Length mRNA Structure | cDNA-PCR sequencing (VAX-seq) | Major product detected, truncated and fused variants quantified |
| Poly(A) Tail Length Distribution | Direct RNA-seq | Tail lengths consistently measured; batch variance observable |
| Contaminant Detection | Direct RNA-seq strand-aware mode | dsRNA, antisense RNA, and minor vector carryover detected |
| Modified Nucleotide Presence | Direct RNA-seq | Pseudouridine-modified bases inferred via signal dropout |
Interpretation and Industry Relevance
✅ 1. End-to-End Molecular Traceability
The study demonstrated a unified workflow covering:
- Plasmid QC
- IVT mRNA structure and truncation profiling
- Poly(A) length consistency
- Contaminant burden (dsRNA, antisense, template remnants)
- Modification validation
⏩ This sets a new benchmark for mRNA QC: a single sequencing strategy can replace multiple disjointed assays with higher resolution and scalability.
✅ 2. Regulatory-Grade Metrics
The quantitative outputs (e.g., % full-length, % truncation, tail length histogram) directly inform release criteria, stability testing, and regulatory filings (IND/BLA).
Implication: Early adoption of sequencing-based QC aligns mRNA pipelines with cGMP and ICH Q6B expectations.
✅ 3. Sensitivity to Process Variation
The approach was sensitive enough to detect:
- Subtle poly(A) tail variation between batches
- Increased fusion product levels in over-transcribed reactions
- dsRNA levels sensitive to purification stringency
Use Case: Manufacturers can use this method for process optimization, comparing IVT conditions, enzymes, or purification methods.
Strategic Takeaway for CRO/CDMO Providers
For service providers like CROs and mRNA CDMOs, integrating long-read RNA sequencing offers:
- Differentiation in quality reporting
- Competitive turnaround (single platform → multiple readouts)
- Flexibility across vaccine, therapeutic, and modified mRNA formats
- Seamless integration with regulatory documentation workflows
VAX-seq and Mana form a technically validated, modular solution ready for routine batch QC, troubleshooting, and
