Key Analytical Methods for Ensuring Safety & Efficacy in Therapeutics – Part I : RNA

May 19th, 2025

As your end-to-end CDMO partner specializing in RNA, Exothera is committed to supporting the development and manufacturing of high-quality RNA-based therapeutics. Ensuring the safety and efficacy of these products requires a robust and tailored analytical approach, from starting materials to final release.

In today’s blog, we’ll explore key analytical challenges and solutions associated with RNA manufacturing, starting with the fundamental role of in vitro transcription (IVT) and the critical impurities that must be controlled.

Ensuring Quality from the Start: mRNA Manufacturing Fundamentals

The backbone of mRNA therapeutics production lies in in vitro transcription, a process where RNA polymerase enzymes synthesize long chains of ribonucleotides from a linear DNA template. Among these, T7 RNA polymerase is widely favored for its efficiency and fidelity. However, this process is highly sensitive to the quality of its components, particularly the DNA plasmid template.

The integrity of the mRNA product is inherently tied to the purity of the plasmid used during IVT. Even when sourced from trusted suppliers, R&D-grade plasmids can sometimes exhibit low purity, affecting transcription yields and mRNA integrity. Agarose gel electrophoresis can help detect contaminants like genomic DNA smears, often linked to residual nuclease activity. These nucleases degrade nucleic acids and compromise the IVT process, emphasizing the need for thorough quality control, even when using so-called high-grade plasmids.

Another critical aspect related to template DNA is its enzymatic degradation using DNase. A common method to evaluate the effectiveness of this treatment is the quantification of residual DNA fragments, typically performed by qPCR. Based on our experience, targeting multiple regions of the plasmid—rather than relying on a single sequence—is essential for a comprehensive assessment of process performance. Moreover, as part of the clinical development of mRNA therapeutics, additional characterization will be required to demonstrate the absence of intact copies of antibiotic resistance genes, which are often present on recombinant plasmids and may raise safety concerns.

Managing a Hidden Risk: Double-Stranded RNA (dsRNA) Impurities

One of the most critical impurities in mRNA manufacturing is double-stranded RNA (dsRNA), a by-product formed due to intrinsic limitations of T7 RNA polymerase. Despite its high fidelity, this enzyme can inadvertently generate dsRNA via several mechanisms, each producing structurally diverse and immunologically active molecules. 

The presence of dsRNA in therapeutic mRNA formulations is a significant concern, as it can activate innate immune receptors such as TLR3, RIG-I, and MDA5. These interactions may lead to inflammation, interfere with translation, and trigger undesirable immune responses, ultimately reducing the therapeutic’s efficacy and safety.

Detecting and quantifying dsRNA presents its own set of challenges, especially due to the intrinsic heterogeneity of the molecules. Current assays, typically based on anti-dsRNA antibodies (dot-blot or ELISA formats), suffer from variability in binding affinity, potential matrix interferences, and difficulties in assay calibration. As a result, developing a reliable and specific control strategy for dsRNA remains a top priority for RNA manufacturers. 

Building a Comprehensive Analytical Strategy

Ensuring the safety and efficacy of RNA therapeutics requires more than monitoring for dsRNA. A holistic analytical strategy includes:

• High-Quality Starting Materials: DNA plasmids and IVT reagents must meet stringent quality standards to reduce the risk of impurities and batch variability.
• Critical Quality Attributes (CQAs): These include identity, purity, potency, and structural integrity of the mRNA molecule, all of which must be precisely characterized and consistently monitored throughout manufacturing.
• Advanced Analytical Technologies: Characterization efforts rely on state-of-the-art equipment and robust methodologies to deliver comprehensive insight into product quality.
• Defined Release Specifications: Analytical methods must be validated to support product release for clinical use, ensuring safety and consistency.
• Shelf-Life Studies: Extensive sampling and stability testing strategies, including accelerated and real-time conditions, are essential to accumulate the data required for toxicology studies and Phase I release.

At Exothera, We Understand What’s at Stake

With RNA-based therapeutics playing an increasingly central role in modern medicine, analytical rigor is more important than ever. At Exothera, we bring deep expertise in RNA process development and analytics to support the full lifecycle of your therapeutic, from preclinical development to commercial readiness.

Let’s work together to optimize your RNA program. Contact us today to learn how our analytical solutions can support your product’s success.

Authors: Pol Harvengt, Senior Platform Manager, 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.