A Comprehensive Evaluation of Analytical Method Parameters Critical to the Reliable Assessment of Therapeutic mRNA Integrity by Capillary Gel Electrophoresis

Introduction

• Messenger RNA (mRNA)-lipid nanoparticle (LNP) therapies are an innovative biopharmaceutical approach with quickly adaptable production methods. However, evaluating their quality requires specific analytical techniques that consider the unique challenges of analysing large, encapsulated RNA molecules.

• Existing integrity tests – mainly capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ion-pair reversed-phase liquid chromatography (IP-RPLC) – were primarily developed using naked mRNA, without LNP components or proprietary formulations. This can hide important impurities and complex interactions present in real drug products.

• This study shows how key factors like the type and amount of denaturant, LNP disruption methods, dye levels, and sample prep significantly influence mRNA integrity results. Optimal CGE-LIF conditions involve isopropanol precipitation, high urea levels, avoiding formamide as a diluent, and using high dye concentrations.

• Using a commercial mRNA-LNP sample (~4200 bases) relevant to biotherapeutics and rigorous experimental design, the research highlights gaps in current mRNA analytical methods. It also lays the groundwork for creating reliable reference standards and standardised protocols for the growing field of mRNA therapeutics.

Key Learning Outcomes

• Understand why traditional RNA analysis methods based on DNA and small oligonucleotides may fall short in biotherapeutic mRNA quality control. This includes issues with denaturant choice, strategies for disrupting lipid nanoparticles (lnps), and peak integration techniques.

• Learn how different denaturants like urea and formamide produce distinct impurity profiles and influence method performance through different mechanisms – formamide in-gel reduces late-migrating smears, while high urea concentrations enhance shoulder resolution and improve the main peak’s sharpness.

• Gain practical knowledge on systematically optimising methods using design of experiments (DOE), focusing on balancing sensitivity, variability, and resolution, especially when dealing with velocity-corrected peak integration of heterogeneous fragment mixtures.

• Recognise the complementary advantages and limitations of CGE-LIF and IP-RPLC techniques for mRNA characterisation, understanding how each reveals different quality aspects, such as fragment-length resolution versus impurity detection.

About

Dr. Huixin Lu (Lulu) works as a Research Scientist in the Regulatory Research Division at Health Canada. She focuses on developing innovative methods to assess the physicochemical quality of complex biotherapeutics, including peptides, vaccines, and gene therapies. Her recent work involves using machine learning for reversed-phase chromatography analysis, marking a notable progress in vaccine quality assessment and providing scalable solutions for quick method optimisation during pandemics. Dr. Lu actively collaborates with international organisations to enhance the safety and effectiveness of biotherapeutics through global standardisation of analytical techniques and reference materials. Prior to her role at Health Canada, she gained industry experience as a Research Scientist at a CDMO, working on biomanufacturing processes and analytical characterisation of various biotherapeutic products. Her research integrates advanced data science with practical regulatory science, preparing analytical laboratories to face future quality control challenges.