Jessica P. Tran, Jun Gao, Casey Lansdell, Barry Lorbetskie, Michael J. W. Johnston, Lisheng Wang, Xuguang Li, Huixin Lu
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Currently, capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ion-pair reversed-phase liquid chromatography (IP-RPLC) are techniques of choice for mRNA integrity analysis. However, most methods proposed for biotherapeutic analysis have been developed using naked mRNA without LNP components or proprietary buffer formulations, which can obscure undiscovered impurities or complex interactions between mRNA and the sample matrix. In this study, we addressed these methodological challenges by using a biotherapeutically relevant commercial mRNA-LNP sample (approx. 4200 b) to refine and optimize a customizable CGE-LIF method currently under consideration for mRNA-LNP biotherapeutic analysis. We systematically characterized how critical method parameters—such as denaturant type, concentration, and usage—and LNP disruption protocols can interfere with accurate mRNA integrity analysis in CGE-LIF and IP-RPLC. 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A Comprehensive Evaluation of Analytical Method Parameters Critical to the Reliable Assessment of Therapeutic mRNA Integrity by Capillary Gel Electrophoresis
In recent years, messenger ribonucleic acid (mRNA)-lipid nanoparticle (LNP) biotherapeutics have demonstrated significant promise in disease treatment and prevention given their rapidly modifiable production processes and considerable capacity to adapt to complex or low-yielding proteins of interest. As a result, many products are currently being developed in this space. Critically, well-characterized and appropriately designed assays are required to monitor purity and integrity in order to maintain the efficacy and consistency of these novel products. Currently, capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ion-pair reversed-phase liquid chromatography (IP-RPLC) are techniques of choice for mRNA integrity analysis. However, most methods proposed for biotherapeutic analysis have been developed using naked mRNA without LNP components or proprietary buffer formulations, which can obscure undiscovered impurities or complex interactions between mRNA and the sample matrix. In this study, we addressed these methodological challenges by using a biotherapeutically relevant commercial mRNA-LNP sample (approx. 4200 b) to refine and optimize a customizable CGE-LIF method currently under consideration for mRNA-LNP biotherapeutic analysis. We systematically characterized how critical method parameters—such as denaturant type, concentration, and usage—and LNP disruption protocols can interfere with accurate mRNA integrity analysis in CGE-LIF and IP-RPLC. We found that optimal conditions for CGE-LIF assay sensitivity, variability, and resolution included sample precipitation by isopropanol, high urea concentrations, no formamide as a sample diluent, and high concentrations of dye. Finally, the advantages and disadvantages of both CGE-LIF and IP-RPLC are highlighted, and a discussion of key considerations when using or designing methods for mRNA integrity assessment is presented.
期刊介绍：
ELECTROPHORESIS is an international journal that publishes original manuscripts on all aspects of electrophoresis, and liquid phase separations (e.g., HPLC, micro- and nano-LC, UHPLC, micro- and nano-fluidics, liquid-phase micro-extractions, etc.).
Topics include new or improved analytical and preparative methods, sample preparation, development of theory, and innovative applications of electrophoretic and liquid phase separations methods in the study of nucleic acids, proteins, carbohydrates natural products, pharmaceuticals, food analysis, environmental species and other compounds of importance to the life sciences.
Papers in the areas of microfluidics and proteomics, which are not limited to electrophoresis-based methods, will also be accepted for publication. Contributions focused on hyphenated and omics techniques are also of interest. Proteomics is within the scope, if related to its fundamentals and new technical approaches. Proteomics applications are only considered in particular cases.
Papers describing the application of standard electrophoretic methods will not be considered.
Papers on nanoanalysis intended for publication in ELECTROPHORESIS should focus on one or more of the following topics:
• Nanoscale electrokinetics and phenomena related to electric double layer and/or confinement in nano-sized geometry
• Single cell and subcellular analysis
• Nanosensors and ultrasensitive detection aspects (e.g., involving quantum dots, "nanoelectrodes" or nanospray MS)
• Nanoscale/nanopore DNA sequencing (next generation sequencing)
• Micro- and nanoscale sample preparation
• Nanoparticles and cells analyses by dielectrophoresis
• Separation-based analysis using nanoparticles, nanotubes and nanowires.
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