Overcoming RNA Bottlenecks

March 6, 2025

The promise of RNA-based therapies is undeniable. From groundbreaking mRNA vaccines to innovative gene therapies, RNA is transforming modern medicine. However, with great potential comes significant challenges, particularly in ensuring consistent formulation and long-term stability. As an end-to-end CDMO partner specializing in RNA, we understand the complexities that can hinder successful RNA development. In today’s blog, we explore the key bottlenecks in RNA formulation and stability and how to overcome them with concrete, actionable solutions.

Understanding RNA’s Unique Challenges

Unlike small molecules or even traditional biologics, RNA is inherently unstable and highly sensitive to environmental factors such as temperature, pH, and enzymatic degradation. This fragility presents obstacles at every stage of development, from early research through to commercialization. For any biotech, pharma, or research organization working on novel RNA therapies, overcoming these hurdles is critical for ensuring reliable and scalable production.

Key Bottlenecks and How to Overcome Them

1.                  Construct Design and PolyA Tail Integrity

RNA stability is inherently linked to its construct design, with key elements such as sequence composition, chemical modifications, and PolyA tail length playing a crucial role in degradation resistance and translational efficiency. A well-optimized construct can enhance stability and minimize susceptibility to exonuclease degradation.

Solution:

• Optimize PolyA tail length to ensure proper mRNA stability and translation efficiency, balancing degradation resistance with effective protein expression.
• Introduce sequence modifications, such as N1-methylpseudouridine, to enhance resistance to innate immune recognition and enzymatic degradation.
• Utilize codon optimization strategies to improve mRNA half-life while maintaining efficient protein synthesis.
• Implement advanced capping strategies, such as CleanCap or ARCA, to enhance RNA integrity and reduce susceptibility to degradation.
• Apply rigorous in silico and in vitro screening methods to assess construct stability before large-scale production.

2.                  Raw Material Variability

RNA manufacturing begins with high-purity raw materials, yet variability in quality can lead to inconsistencies in drug substance. Ensuring tight control over raw material sourcing, qualification, and testing is essential to minimizing batch-to-batch variations.

Solution:

• Partner with experienced and validated suppliers who adhere to stringent quality control measures.
• Implement a robust raw material testing strategy, including nucleoside purity analysis, endotoxin testing, and contaminant screening.
• Establish a supplier qualification program with routine audits to ensure consistency in raw material quality over time.
• Ensure raw material quality aligns with final formulation requirements, as excipient purity and composition play a crucial role in RNA stability and overall drug product quality.

3.                Scalable and Reproducible Manufacturing

Scaling from research-grade RNA to GMP-compliant production without losing consistency is a major hurdle. Variability in synthesis, purification, and final formulation can lead to inconsistencies in product performance.

Solution:

• Standardize RNA synthesis protocols using automated in vitro transcription (IVT) systems to ensure reproducibility – such as the Ntensify® equipment used at Exothera; the world’s first continuous-batch RNA manufacturing platform.
• Optimize purification strategies, such as high-resolution chromatography and tangential flow filtration, to achieve high purity and yield at scale. At Exothera, we use magnetic beads to achieve a one step purification process.
• Utilize process analytical technology (PAT) tools for continuous monitoring of RNA integrity, yield, and purity throughout the manufacturing process.
• Establish well-defined tech transfer protocols to facilitate seamless scaling from lab-scale to commercial production while maintaining quality attributes.

4.               Optimizing Lipid Nanoparticle (LNP) Encapsulation

For RNA therapeutics, LNPs are the gold standard for delivery, but even slight variations in the encapsulation process can impact product efficacy and stability. Poor formulation can lead to degradation, reduced encapsulation and consequently inferior therapeutic potency. Additionally, the quality and purity of lipid raw materials play a crucial role in ensuring consistent LNP performance.

Solution:

• Ensure rigorous material testing for lipids, including purity assessments using MS/NMR, to confirm consistency and eliminate contaminants that could affect RNA stability.
• Employ high-throughput screening methods to identify optimal lipid compositions and ratios for RNA payloads.
• Utilize microfluidic mixing technology for precise and reproducible LNP formulation at both small and large scales.
• Integrate real-time particle size and encapsulation efficiency monitoring during manufacturing to ensure consistency.
• Perform forced degradation studies to assess and mitigate potential risks to RNA stability within LNPs.

5.                Degradation Risks and Stability Optimization

RNA is particularly susceptible to hydrolysis and oxidation, which can compromise its function before it reaches the patient. Storage conditions and formulation buffers play a crucial role in mitigating these risks.

Solution:

• Develop optimized buffer systems that incorporate pH stabilizers and chelating agents to reduce metal ion-catalyzed degradation.
• Include cryoprotectants such as trehalose or sucrose to enhance freeze-thaw stability for LNP (DP).
• Store RNA formulations at ultra-low temperatures (-80°C or lower) to significantly slow degradation rates.
• Utilize lyophilization techniques to improve the long-term stability of RNA-based therapeutics.
• Implement stability-indicating analytical methods, such as LC-MS and qPCR, to detect early signs of degradation and adjust formulation strategies accordingly.

Collaboration: The Key to RNA Success

RNA development is complex, and no single organization can easily tackle all its challenges alone. True innovation happens when biotech, pharma, and academic research teams collaborate with specialized CDMO partners who understand the nuances of RNA formulation and stability.

At Exothera, we take a comprehensive, transparent, and partnership-driven approach to solving RNA bottlenecks. Whether you’re navigating early-stage formulation challenges or optimizing for commercial production, we provide the expertise, infrastructure and equipment to bring your RNA therapies to life; consistently, reliably, and at scale.

Let’s work together to accelerate the future of RNA medicine. Reach out to our team to learn how we can support your journey from discovery to delivery.

Authors: Hanna Lesch, Chief Technology Officer, Exothera and Ashiqul Haque, Technical Lead Vaccine Development, Univercells

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.