Quality Control of the Quality Control: Improving the Understanding of Analytical Separation Techniques for the Assessment of mRNA Integrity in a Biotherapeutic Context

Despite the growing interest in mRNA-based therapeutics, the platform’s novelty, and lack of published knowledge regarding mRNA quality attributes and assessment pose significant challenges in establishing guidelines and references for therapeutic mRNA analysis. As bioassays and in vitro translation assays are yet to be widely accepted to assess efficacy, physicochemical assays such as capillary electrophoresis and ion-pair reversed phase liquid chromatography serve as surrogates for quality assurance by measuring transcript length. While both are accepted bioanalytical techniques to assess transcript length, we reveal that they are not interchangeable and have significant resolution differences. In this work, we demonstrate how various factors such as method parameters, product-specific sample preparation, and excipients can easily influence mRNA integrity measurements. We emphasize the importance of understanding the influence of method parameters on mRNA integrity assessment and impurity detection by capillary gel electrophoresis and ion-pair reversed phase liquid chromatography, especially in a biotherapeutic context where mRNA is modified and encapsulated. Such knowledge is imperative in making accurate comparisons between mRNA products and designing practical reference standards. We anticipate that this work will be valuable for not only biopharmaceutical professionals but also for regulatory bodies. By providing insight into the performance of different analytical methods for assessing mRNA integrity, our study can facilitate the development of more optimized analytical methods and contribute to the overall quality assurance of mRNA-based therapies.