Characterizing RNA-Based Medicines

Historically, pharmaceutical advancements have occurred in distinct waves. The first focused on small molecule drugs such as acetaminophen, which typically involved chemically synthesized compounds acting on proteins or other cellular targets; the second focused on drug discovery and methodology; and the third wave saw the rise of protein and peptide-based therapies, including groundbreaking treatments such as recombinant human insulin. These therapies expanded the industry’s capabilities, especially in the realm of autoimmune diseases and cancer, where monoclonal antibodies became crucial tools.  

We are now firmly in a fourth wave of drug discovery and development, characterized by nucleic acid therapeutics — medicines that intervene directly at the genetic level. This wave includes the development of mRNA vaccines, ASOs (antisense oligonucleotides), siRNA, and CRISPR gene editing technologies. The unprecedented success of mRNA technology, especially highlighted by the COVID-19 vaccines, demonstrates the rapid scalability and potential these therapies hold for the future. 

Diverse modes of action
 

RNA therapeutics are fundamentally different to traditional drugs. Unlike small molecules that bind proteins or peptides that mimic biological activity, RNA-based drugs target the genetic code itself. This opens opportunities to modulate gene expression precisely and intervene in genetic disorders at their source.  

For instance, ASOs can bind to mRNA sequences, blocking their transcription or altering splicing, effectively silencing or modifying gene expression. Similarly, siRNA technology involves a duplex that targets specific mRNA for cleavage, preventing the translation of harmful proteins. mRNA therapies deliver synthetic instructions directly to cells, enabling them to produce therapeutic proteins. Finally, CRISPR-based gene editing allows precise edits within the genome, offering potential cures for monogenic disorders.  

Success stories are already emerging. Stoke Therapeutics, for example, is using ASOs to increase the expression of genes critical for neuronal function in Dravet's Syndrome, whilst Alnylam Pharmaceuticals’ siRNA-based approach targets amyloid production in rare heart diseases. Moderna’s mRNA technology is pioneering personalized cancer vaccines that harness the immune system to fight specific tumor mutations, and companies such as Beam Therapeutics are advancing CRISPR base-editing to correct genetic mutations causing metabolic diseases.   

An ecosystem of scientific collaboration
 

The RNA therapeutics sector is thriving thanks to an ecosystem full of academic contributors, biotech startups, pharmaceutical giants, regulatory bodies, and technology suppliers. Universities remain the breeding grounds for discovery, with medical schools and research centers driving early-stage innovation. Emerging startups, often spin-offs from academic research, are crucial in translating these discoveries into practical applications. However, they frequently require the partnership of larger pharmaceutical companies to navigate late-stage clinical trials and scale production.

CROs and CDMOs support these efforts, providing specialized expertise and additional capacity for drug development, whilst suppliers play a critical role by providing high-performance analytical tools that ensure the purity, potency, and efficacy of these new drugs. Regulatory agencies, including the FDA, also ensure that these innovations are safe and effective through rigorous review processes.

The role of characterization technologies
 

Chromatography and mass spectrometry technologies are crucial for characterizing and quantifying RNA molecules, ensuring their safety, stability, and efficacy throughout the development pipeline.  

High-performance liquid chromatography (HPLC) systems and columns optimized for nucleic acid separation are essential for measuring the integrity (intactness) of RNA therapeutics and reliably confirming their concentration. Like its use in mAb characterization, size exclusion chromatography (SEC) is particularly effective for this type of testing, allowing the detection of aggregates, monomers, and degradation products in mRNA drug substances, all critical attributes to be monitored. The size, charge, and complex chemical nature of RNA therapeutics have driven breakthroughs in column technology to provide the performance scientists have become accustomed to from traditional protein chromatography.   

These advances have led to the recent introduction of SEC columns capable of providing high-resolution separation of nucleic acids such as mRNA and guide RNA (gRNA), enabling precise measurement of their concentration and structural integrity. These columns are built with optimized pore size and surface chemistries that accommodate the delicate structure of RNA molecules. In even more recent work, SEC columns were shown to perform effectively with denaturing mobile phases, which aid in dissolving lipid nanoparticles (LNPs) structures to allow quick and accurate assessment of the RNA cargo in formulated samples.    

SEC platform methods are also under development, providing scientists with the ability to efficiently assess drug quality and give quick guidance during process development through complete measurements in one multi-attribute method when combined with a range of detectors, including UV, fluorescence, refractive index, and multiangle light scattering detectors.  

RNA sequencing, akin to bottom-up workflows for proteins and peptides, continues to pose challenges in industry because of the chemical nature of RNA and the complexity of LCMS data produced. To address and simplify these assays, new sample preparation enzymes and reagents are needed, including endoribonucleases and high purity mobile phase additives. These specialized tools allow for the digestion of RNA molecules to be analyzed by high sensitivity LC mass spectrometry, where analysts can tease apart and zoom in on the molecular details of the resulting puzzle to characterize and confirm the molecular composition.   

To this end recombinant, analytical grade enzyme reagents have been developed. These are designed to facilitate RNA digestion for LC-MS analysis, and are particularly beneficial for characterizing mRNA and single-guide RNA. Some reagents cleave primarily at the 5' end of uridine residues, generating fragments that enhance sequence coverage and modification identification, while others target the 3' end of cytidine residues, producing longer RNA digestion products. Used together, these enzymes provide overlapping fragments that improve sequence confidence and modification mapping.

The future of RNA
 

Even with advancements and immense market potential, the industry continues to face challenges. The complexity of RNA molecules demands precise analytical methods, and scaling these therapies to a global level requires substantial investments in both technology and manufacturing infrastructure. The industry must also navigate regulatory hurdles and ensure patient access while maintaining the high standards needed for personalized medicine.  

However, the stories of patients who received a custom ASO treatment for a genetic disorder, and others who underwent CRISPR-based therapy for sickle cell anemia, demonstrate the transformative potential of these therapies, highlighting how RNA drugs have already begun to provide life-changing outcomes.  

The future of RNA therapeutics is bright, with industry, academia, and technology providers working together to push the boundaries of what is possible in genetic medicine. As these treatments become more mainstream, the focus will be on making them affordable and accessible to all, ensuring that the promise of RNA therapy extends beyond the current limited market and becomes a global standard for healthcare across a more comprehensive set of diseases. As we continue to innovate and refine these technologies, one thing is clear: the RNA therapeutics industry is not just a wave – it's a sea change in medicine.