Activity in RNA-based drug research reached an all-time high thanks to the success of messenger RNA (mRNA) COVID-19 vaccines, which provided the pathway for clinical validation for this new modality. The great versatility of mRNA, where mRNA sequences can be easily tailored to encode any protein of interest, makes this approach valuable for rapid vaccine development against emerging pathogens and for novel RNA drugs to address a broad array of diseases. Key to their success is the delivery through lipid nanoparticles (LNPs), which protect the mRNA from nuclease degradation and facilitate entry of the mRNA across cell membranes and endosomes of the target cells to drive the desired responses in patients.
RNA-based drugs are distinct from small molecules and protein drug substances, presenting new analytical challenges for the industry. Proper characterization of mRNA during development and quality control (QC) phases is critical to ensure drug quality, efficacy and safety. And because mRNA is usually formulated within LNPs, additional analytics are needed to ensure the quality and safety of the lipid material used for nanoparticle formulation. As nucleic acids take center stage with next-generation medicine and the demand for mRNA-based therapeutics and vaccines grows, high-throughput and high-resolution analytical methods and the expertise to keep up with an ever-evolving regulatory environment is becoming vital to success.
Overview of the mRNA-LNP Process
The mRNA-LNP production process usually starts with linearized plasmid DNA (pDNA), which serves as the template for mRNA production and capping. Through in vitro transcription (IVT), the DNA template is transcribed into mRNA. Mature and biologically functional linear mRNA, however, needs specific 5’- and 3’-ends. The so-called 5’-capping can be added with a multi-step enzymatic reaction. The 3’ poly-A ends are already frequently encoded in the pDNA template, but sometimes are also added enzymatically. Considering this production process, a mixture of product variants can occur with different 5’-cap structures (i.e., G cap, cap 0 and cap 1), variable 3’-poly-A tail lengths, truncated mRNA transcripts and other impurities, such as double-stranded RNA molecules and residual plasmid DNA templates1.
Despite process optimization to increase yields of the desired product and purification steps to remove undesirable by-products, these impurities may persist, which is why proper characterization of mRNA products is crucial. However, identification and quantification of product quality attributes (PQAs) and critical quality attributes (CQAs) are more complex due to the large size and fragile nature of mRNA and the near-identical physical and chemical characteristics of mRNA molecules and their variants1. For example, mRNA transcripts with cap 0 and cap 1 on the 5’-end only differ by ~14 Da in their molecular masses, highlighting the need for high-resolution analytical technologies to resolve such subtle differences1.
After purification, mRNA can be encapsulated in LNPs. The mRNA-LNP formulation process is complex, requiring rapid yet precisely controlled mixing of the mRNA and lipid species to induce self-assembly of the encapsulated mRNA-LNPs. Since the particle size and polydispersity index (PDI) of the nanoparticles directly influence their in vivo function, advanced particle analytics are required to assess these attributes1. Furthermore, LNPs consist of different classes of lipids, adding up to the complexity of the final product. Key lipid species are ionizable lipids, which complex the negatively charged nucleic acid cargo, facilitate cellular uptake and are the center of intellectual property efforts.
It is evident there is a great need for comprehensive high-resolution analytical tools that can rapidly and accurately characterize raw materials, mRNA and LNPs to ensure drug product quality and safety. However, scientists are challenged by the diversity of compounds that need to be addressed and the downsides of existing analytical workflows in terms of complexity, data quality and method transferability. This overview shows key mRNA-LNP workflows, which are essential to the manufacture of high-quality LNP-based products.
High-Resolution, High-Quality mRNA Characterization
Nucleic Acid Profiling
Analysis of intact mRNA can be particularly challenging because of the size of the molecule of commonly several thousand nucleotides. While ion-pairing reversed-phase high-performance liquid chromatography (IP-RP-HPLC) is frequently used for short nucleic acids, such as anti-sense oligonucleotides (ASOs) and small interfering RNA (siRNA), it does not provide high-resolution power for larger nucleic acids, such as mRNA. Capillary electrophoresis (CE) systems, on the other hand, are fast, robust and offer high resolution to determine the size, integrity and purity of mRNAs by leveraging capillary gel electrophoresis (CGE).
This technical note highlights nucleic acid profiling of mRNA extracted from LNPs with the use of the RNA 9000 Purity & Integrity kit and the BioPhase 8800 system from SCIEX. After optimization of the extraction method, mRNA integrity and impurity profiling could be achieved rapidly and with good repeatability.
The recently commercialized multi-capillary BioPhase 8800 system is capable of high-throughput, sensitive analysis of nucleic acids using CGE with ultra-violet (UV) or highly sensitive laser-induced fluorescence (LIF) detection. This system, together with the RNA 9000 Purity & Integrity kit, enables a turnkey solution that provides all the necessary reagents to assess RNA integrity and purity and to estimate RNA size using CGE. Scientists can use the kit to analyze a wide variety of therapeutically relevant single-stranded nucleic acid classes, ranging from 50-9,000 nucleotides and beyond. The method is suitable for early process development to final product QC with seamless method transferability between the BioPhase 8800 system and the PA 800 Plus system from SCIEX, which can greatly shorten time to answers.
5’ mRNA Cap Structure
The addition of 5’ mRNA cap during IVT is necessary to maintain the biological function of the mRNA, helping prevent degradation and promoting its translation, making it an essential CQA for characterization during development. While CGE is the method of choice for monitoring intact mRNA integrity, differentiating the cap structures requires a different technique. High-resolution mass spectrometry workflows executed on the X500 QTOF series of systems or the ZenoTOF 7600 system from SCIEX can fill this analytical gap as the mRNA therapeutic pipeline continues to surge. After digestion of the mRNA, the 5’ ends can be assessed in detail and the capping structures (G cap, cap 0 and cap 1) and uncapped mRNA can be distinguished and relatively quantified. The flexible quantification options in SCIEX OS software enable the post-acquisition optimization of data analysis, for instance, through deconvolution of the multiply charged capping species.
Further details on the LC-MS 5’- capping workflow can be found in this technical note.
Electron Activated Dissociation (EAD) for Lipid Structural Characterization
As a non-viral drug delivery system, LNPs are highly effective, providing protection of the nucleic acid payload from degradation, and enabling cellular uptake and release of the genetic material in the cytoplasm of the target cells. LNPs are composed of a mixture of ionizable lipids, helper lipids, cholesterol and PEG-lipids, and it is the specific ratio of these lipid species that influence the physiochemical properties of the LNP, such as particle stability, tissue targeting, delivery efficacy, tolerability and biodistribution.
Some impurities can originate from ionizable lipids—the most important component of LNPs—which are susceptible to site-specific oxidation. Some of these impurities lead to the formation of reactive lipid species generating unwanted mRNA-lipid adducts that prevent proper mRNA function, inhibiting the expression of the encoded protein, even at very low abundancies of approximately 10 ppm2,3. Therefore, characterization of the lipid material, including low abundance oxidative species, is critical for the development of optimal LNP products that meet stability, safety and efficacy requirements.
Structural elucidation of lipids and lipid impurities, however, is a complex matter as they tend to produce only a limited number of fragments when applying MS methods. Alternative approaches, such as nuclear magnetic resonance (NMR), are cumbersome, requiring high expertise and a high amount of pure product, which inevitably is unsuitable for the detection of low abundance impurities. Electron activated dissociation (EAD), on the other hand, is a novel fragmentation mode available with the ZenoTOF 7600 system that is highly suitable for accelerated and accurate structural profiling of lipids for LNP characterization. In contrast to the more commonly used collision-induced dissociation (CID), EAD provides an abundance of unique fragment ions critical for complete lipid structural elucidation. Specifically, EAD was used to characterize an ionizable lipid commonly known as MC3 in this technical note and a the ionizable lipid ALC-0315 in this technical note. Several different impurities were found in the MC3 and ALC-0315 raw material, and diagnostic fragments derived from EAD could pinpoint exact sites of modification, enabling differentiation of N-oxides from other oxidation products. More information can also be found in this webinar.
Once impurities were identified, the SCIEX 7500 system, powered by SCIEX OS software, provides users with a highly sensitive and robust system to monitor critical components. The analysis using multiple reaction monitoring (MRM) offers MS/MS information with the highest quantitative performance while ensuring suitable specificity. The precise and robust engineering of the ion rail allows consistent and reproducible analysis, time after time, by focusing on the crucial ions of interest with the throughput and productivity needed to accelerate mRNA-LNP drug development. Moreover, the compliant-ready system is well suited for QC environments due to its ease of use and robustness.
Complementary Suite of Analytical Solutions Needed
As we have described in detail, there are numerous technological innovations in the analytical space that are helping to fill gaps and accelerate the development of RNA-based drugs, and SCIEX is at the forefront. The complexity of nucleic acids and LNPs in addition to the final drug product means that a suite of complementary analytical solutions is needed to cover the various important aspects of both raw materials and the formulated product at each stage of the mRNA-LNP development process. As a relatively new modality where the analytical and regulatory landscapes continue to evolve, it is prudent for industry stakeholders to utilize a multi-faceted approach to analytics. A complementary and comprehensive suite of analytical tools is required—one that forms a robust yet flexible framework that can adapt to ongoing changes in mRNA structures and final formulations while mitigating risk in the pursuit of novel RNA-based therapeutics and vaccines that are safe and efficacious.
For more information, please see SCIEX’s Resource center for developing better LNP-based genetic medicines
1. Cameau E, Zhang P, Ip S, Mathiasson L and Stenklo K. Process & analytical insights for GMP manufacturing of mRNA lipid nanoparticles. Cell & Gene Therapy Insights 2022; 8(4), 621–635 doi: 10.18609/cgti.2022.095.
2. Crowe A, Pohl K. Overcoming Oxidation. The Analytical Scientist. November 9, 2022. Accessed November 22, 2022. https://theanalyticalscientist.com/techniques-tools/overcoming-oxidation
3. Packer M, Gyawali D, Yerabolu R, Schariter J, White P. A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems. Nat Commun. 2021 Nov 22;12(1):6777. doi: 10.1038/s41467-021-26926-0. PMID: 34811367; PMCID: PMC8608879.