Overcoming RNA Bottlenecks

March 31, 2025

Ensuring the quality, purity, and potency of RNA-based therapeutics requires a sophisticated analytical and quality control (QC) strategy. Given the inherent complexity and sensitivity of RNA, robust analytical methods are essential for characterizing critical quality attributes (CQAs), supporting regulatory filings, and maintaining consistency from development through commercial production. 

As your end-to-end CDMO partner specializing in RNA, we recognize that traditional analytical techniques must be adapted to meet the unique challenges of RNA therapeutics.

In today’s blog, we explore key analytical and QC challenges in RNA manufacturing and the innovative solutions that enable reliable characterization and quality assurance.

Key Analytical and QC Challenges in RNA Manufacturing

1. RNA Integrity and Purity Assessment

Ensuring high-quality RNA with minimal impurities is critical for therapeutic efficacy and safety. RNA degradation, residual DNA, and truncated transcripts can all impact performance.

Solution:

• Utilize capillary electrophoresis (CE) and high-resolution liquid chromatography (LC) to assess RNA integrity and detect degradation products.
• Implement qPCR and digital droplet PCR (ddPCR) to quantify residual DNA and ensure compliance with regulatory limits.
• Leverage next-generation sequencing (NGS) to confirm transcript integrity and identify potential sequence variants.

2. Potency and Functional Characterization

RNA therapeutics must demonstrate consistent biological activity, but assessing potency is complex due to variable translation efficiency and cellular responses.

Solution:

• Characterize the Mechanism of Action and develop several assays to detect molecular attributes relevant for biological function of the product.
• Develop in vitro cell-based potency assays to evaluate RNA functionality and transfection efficiency.
• During assay development focus on the quantitative assay, which will be possible to interpret during stability studies and be most representative for Mechanism of Action

3. Lipid Nanoparticle (LNP) Characterization

For RNA therapies delivered via lipid nanoparticles (LNPs), precise characterization of particle size, encapsulation efficiency, and stability is essential.

Solution:

• Use dynamic light scattering (DLS) or nanoparticle tracking analysis (NTA) to monitor particle size distribution and uniformity.
• Employ reverse-phase HPLC and fluorescence-based assays to measure RNA encapsulation efficiency within LNPs.
• Conduct forced degradation and stability studies to evaluate LNP robustness under storage conditions.

4. Immunogenicity and Contaminant Testing

RNA therapeutics must be free from endotoxins, dsRNA contaminants, and other impurities that could trigger unwanted immune responses.

Solution:

• Implement double-stranded RNA (dsRNA) ELISA assays to quantify dsRNA impurities, which can trigger innate immune responses.
• Conduct limulus amebocyte lysate (LAL) assays and pyrogen testing to ensure endotoxin levels meet regulatory thresholds.

5. GMP-Ready QC Method Development and Validation

Transitioning analytical methods from R&D to GMP-compliant QC workflows requires rigorous validation and regulatory alignment. 

Solution:

• Develop and validate phase-appropriate analytical methods in compliance with ICH guidelines.
• Implement automation and digital analytics to enhance reproducibility and reduce human error in QC testing.
• Establish reference standards and comparability protocols to ensure consistency across batches and manufacturing sites.

Building a Robust Analytical and QC Framework for RNA Therapeutics

Developing effective RNA-based medicines requires a proactive approach to analytical method development and quality control. By leveraging advanced technologies, implementing stringent QC processes, and aligning with evolving regulatory expectations, companies can ensure the safety, efficacy, and consistency of their RNA products.

At Exothera, we specialize in developing and validating state-of-the-art analytical and QC methods for RNA therapeutics. Our expertise ensures that your RNA products meet the highest quality standards, from early-stage development to commercial manufacturing.

Let’s collaborate to advance the next generation of RNA therapeutics. Contact us today to discuss your analytical and QC needs.

Authors: Dmitrii Sorokin, Head of CMC, Exothera and Hanna Lesch, Chief Technology Officer, Exothera

About Exothera

Exothera is a specialized CDMO partner delivering preclinical and clinical process development, manufacturing optimization and CMC support for virus and RNA based therapeutics and vaccines.

With headquarters in Belgium, at the heart of Europe, we assist clients worldwide from start to scale, up to commercial manufacturing, including analytical testing and regulatory consultation. Thanks to our optimal technologies and experienced team, we help bring innovative therapies from bench to patient with quality, at speed and cost-effectively.