Huijuan Li, PhD, Moderna, Inc.
To begin, Dr. Huijuan Li looked at several mRNAs products and their considered their development. While speed, precision and flexibility are the main advantages of mRNA vaccines, there are also challenges with mRNA vaccines. For instance, chemical and structural diversity of mRNA molecules might require development of novel assays. Additionally, amino acids, the building blocks are encoded by three nucleotides implying that a larger protein is encoded by a larger mRNA leading to an increase in mass and complexity. Therefore, we need to have a comprehensive understanding to come up with specific methods to assess these molecules.
Li explained that a mature mRNA molecule is composed of 5’ cap, coding region and 3’ polyA tail. Missing any of these components disrupts mRNA integrity and will not translate any protein, meaning that degraded mRNA will not result in a degraded protein and eliminates safety concerns. Similarly, heat degradation also disrupts RNA integrity and reduces protein expression.
Dr. Li stated that sequence characterization is an effective strategy to assess critical quality attributes (CQA) of mRNAs. High performance liquid chromatography and liquid chromatography mass spectrometry (HPLC LC-MS) method can be used to do oligo profiling of mRNA which is analogous to the peptide mapping for protein therapeutics. It is useful for pinpointing specific areas of interest, examining the consistency of the batches, and determining the insertions, deletions, and contamination. After treatment with RNAse T1, the digestion profile of mRNAs on LC-UV chromatogram seem unique like a fingerprint. Pearson correlation coefficient R provides a quantitative measure of chromatogram similarity between duplicate digestions confirming uniformity of RNA sample.
She mentioned that RNA purity is another critical parameter which is exemplified by a study held by Moderna indicating that lipid impurities leaded to the loss of mRNA activity in LPN delivery systems. This finding was rooted to the studies aiming to find strategies to assess mRNA purity. In earlier times, capillary electrophoresis (CE) -- a size-based assay -- was utilized. Moderna further developed Reverse-Phase Ion-Pairing (RP-IP) method which resulted in an unexpected late eluting peak (LP) in addition to the main peak (MP). Analysis on fraction collections demonstrated an absorbance at 260 nm indicating that both were RNA-related molecules. While next generation sequencing, oligo mapping and nucleotide profiling resulted in identical profiles, analysis in the level of nucleosides revealed that the unique masses were exclusively found in the isolated lipid particles. Eventually, it has been confirmed that these modifications were degradants of lipid or impurities that were reacting with the base by MS/MS.
The next quality attributes regarding RNA integrity underlined by Dr. Li are encapsulation state and encapsulation efficiency. She explained that mRNA cannot be delivered naked, and lipid nanoparticles are used in vaccine and therapeutic development.
Moderna achieves encapsulation by several microfluidic mixing methods that enable the interaction between cationic lipids and negatively charged nucleic acids. There are also strategies to visualize the encapsulation. Among the diversity of the Cryo-EM images, Moderna’s motivation is to enhance the visualization of mRNA molecules in lipid particles. The team has used a dye named Thionine, added a contrast through cryo-EM imaging, and helped us locate and understand how RNA is encapsulated. Thionine, as an RNA binding dye, has enabled observing the transitions through some processes (i.e. freeze-thaw) and learn how these RNAs have changed and whether it is linked to the stability.
According to Dr. Li, analytical comparability is another essential issue in biomanufacturing processes. Thanks to analytical comparability assessments, Moderna can ensure dose-to-dose or batch-to-batch consistency. At the time of development of mRNA vaccines for COVID-19, the company already had many programs in clinical development and was aware of the opportunities such as large scale production, technical ease of manufacturing, being applicable to other diseases, quick design, and greater efficiency.
Therefore, Moderna considered their process evolution and sequence design evolution and started with a personalized vaccine unit. This meant that small-scale output was needed at the beginning of a clinical trial. At every step along the way, they did analytical comparability to ensure the outputs from different scales (Research scale < personalized vaccine unit < clinical < commercial).
A comparability assessment has multiple focuses such as extended characterization, stability profile, impurity characterization, and these were done together to support the release testing. The testing includes multiple orthogonal method that ensure your release panel is the right assay and has the capability to detect from small-scale to commercial scale. Results of 23 lots of commercial scale drug product manufactured from four sites indicated that their material is consistent and their data gives confidence as long as they carefully test those CQAs.
