RNA-based therapeutics are rewriting the future of medicine. Vaccines, gene therapies, and treatments that can correct or modulate cellular function all hold immense potential to change how we treat disease. But that promise brings pressure. Developers need detailed, data-driven insights into RNA quality to confidently support regulatory filings.

However, the analytical methods used to assess RNA quality haven’t evolved as quickly as the science. Traditional characterization assays, once sufficient for simpler molecules, can no longer keep up with the complexity of today’s RNA modalities. As regulatory expectations increase, relying on legacy techniques risks leaving critical quality attributes unseen, unresolved, and unoptimized.

Developers need data that reflect the full molecular reality and capture how identity, integrity, purity, stability, potency, and safety interconnect. Sequencing-based characterization provides that level of visibility, giving teams the assurance needed to support both optimization and regulatory readiness.

The limits of traditional RNA characterization

Standard analytical tools like gel electrophoresis, HPLC, and mass spectrometry have been the workhorses of nucleic acid profiling for decades.

While these methods deliver useful snapshots, such as verifying approximate size, assessing purity, or confirming the presence of full-length transcripts, they often fall short when developers need deeper insight.

Surface-level data can’t capture molecular complexity. Traditional assays reveal what’s visible: overall size, yield, or bulk purity. What they miss are the subtle but significant molecular events that can make or break therapeutic efficacy, like sequence misincorporations, structural misfolding, or fragmentation. These defects may be invisible to conventional QC but can lead to truncated proteins, poor translation efficiency, or immune activation in vivo.

They’re not built for emerging RNA modalities. With mRNA, saRNA, and circRNA now in the pipeline, the structural and functional diversity of RNA therapeutics has exploded. Circular and self-amplifying constructs bring new challenges in structure, replication dynamics, and intracellular behavior that require the establishment of new, empirically-driven quality control thresholds.

They don’t meet today’s regulatory demands. Agencies are increasingly asking developers to demonstrate a comprehensive understanding of critical quality attributes (CQAs): identity, integrity, purity, stability, potency, and safety. Legacy assays can only partially address these factors. As a result, developers face blind spots that can slow filings, require rework, or trigger follow-up requests during regulatory review.

Outdated methods create data gaps and obscure the insights that can guide process optimization and ensure consistent performance from early development through manufacturing.

How sequencing-based characterization delivers a complete view of RNA quality

Sequencing-based technologies have opened new possibilities for understanding RNA therapeutics at single-nucleotide resolution. Rather than inferring quality from indirect measurements, these approaches observe it directly and capture the molecular fingerprints that define how an RNA behaves, performs, and persists.

Here’s what sequencing-based characterization can reveal that traditional assays cannot:

Identity: Confirm the right RNA, every time

Regulators expect the drug substance to match its intended design in nucleotide sequence and secondary structure. Sequencing provides direct confirmation that every base is correct and that folding patterns remain consistent across manufacturing lots. Developers can also detect rare misincorporations or structural deviations early and prevent the production of incorrect or misfolded proteins that compromise efficacy.

Integrity: Detect fragmentation before it impacts translation

RNA’s inherent instability means breakage can occur during transcription, purification, or storage. While commonly used assays show gross degradation, they can’t pinpoint where breaks occur or why. Sequencing maps fragmentation sites to reveal patterns linked to secondary structure or process conditions. This data allows developers to identify vulnerable regions and improve RNA design or manufacturing steps before they affect translation.

Purity: Map impurities at their source

Double-stranded RNA (dsRNA) impurities are among the most concerning for developers because they can activate innate immune responses and reduce therapeutic potency. Traditional antibody-based assays measure total dsRNA levels but can’t specify which regions of the RNA form these structures or why. Conversely, sequencing-based methods can directly identify dsRNA species and trace them back to their sequence origins—offering a roadmap for reducing impurity generation through data-informed design changes.

Stability: Predict degradation and performance

Stability defines how well an RNA therapeutic endures. Sequencing reveals which bases are most prone to hydrolysis maps their susceptibility under different conditions or formulations. It also connects structure to stability to clarify how modifications, buffers, or LNP encapsulation can affect RNA longevity.

Potency: See beyond expression levels

Traditional potency assays often measure protein output as a proxy for success, but they don’t capture why some constructs outperform others. Sequencing-based profiling tracks the complete translational journey from cellular uptake and endosomal escape to ribosome engagement and pausing. Gleaning such insights enables developers to refine sequence design, codon optimization, and delivery strategies for stronger, more predictable protein production.

Safety: Understand cellular responses in full

Even when an RNA product appears functional, off-target effects can arise at the transcriptional or translational level. Sequencing provides a comprehensive view of how cells respond after exposure, identifying gene expression or translation changes that may indicate unwanted immune or stress responses. Detecting these early supports safer candidate selection before clinical studies.

Building regulatory confidence through comprehensive data

Sequencing-based RNA characterization strengthens the evidence base that teams need to demonstrate control and consistency in regulatory submissions.

Alignment with CQA expectations

Regulatory agencies increasingly emphasize detailed characterization of the drug substance and drug product. Sequencing-based methods provide the robust datasets needed to demonstrate control over each CQA (identity, integrity, purity, stability, potency, and safety).

Consistency and traceability

Developers can compare lot-to-lot consistency at the molecular level, which builds confidence in reproducibility and control. Such a level of traceability supports comparability studies during process optimization and scale-up.

Risk mitigation and process optimization

Sequencing insights help teams detect issues early and correct design or manufacturing problems before they become regulatory roadblocks. This accelerates development timelines while reducing the likelihood of rework or additional testing during review.

A foundation for continuous improvement

Comprehensive RNA data not only satisfy regulators but also drive smarter development. Insights into structure-function relationships empower R&D and manufacturing teams to optimize performance and stability throughout the therapeutic’s lifecycle, enabling robust quality by design.

A new standard for RNA therapeutic characterization

As RNA medicines advance, a complete picture of molecular quality is no longer optional. Legacy assays can show what an RNA looks like, but can’t always explain how it behaves. Comprehensive characterization platforms like eMERGE use sequencing-based analyses to show how potency, stability, and safety influence therapeutic performance. The results inform decisions at every stage of development and manufacturing.

Whether you’re preparing for regulatory submission or refining your approach to RNA characterization, contact us to learn how our solutions can help.