by Kerstin Pohl, Senior Global Marketing Manager, Gene Therapy & Nucleic Acid, SCIEX
Rapid approval of the Moderna and Pfizer/BioNTech vaccines against COVID-19 highlighted the benefits of established mRNA technology. Many companies, including both emerging biotech and established biopharma firms, are continuing to build on these recent successes and the past three decades of research in the mRNA field.
Most mRNA therapeutic and vaccine candidates are formulated as lipid nanoparticles (LNPs). LNPs are great delivery vehicles for mRNA active ingredients. They protect the mRNA from enzymatic degradation within the body and enhance cellular uptake. Their manufacturing process is also highly scalable.
However, a recent Moderna study revealed that impurities formed due to the oxidation of certain lipids used in the formation of LNPs can covalently bond to mRNA and disrupt its function.1 The formation of these undesirable mRNA-lipid adducts was not known prior to the recent study.
Capillary gel electrophoresis (CGE) techniques, which separate molecules based on size and charge, cannot detect these impurities that differ only slightly from desired mRNA products.1 Standalone liquid chromatography showed separation of these species based on hydrophobicity, however, was not able to determine their identity. Fortunately, a new fragmentation approach known as electron-activated dissociation (EAD) can provide the molecular detail needed over a wide dynamic range to enable detection of these low abundance lipid impurities.2
An Issue with Ionizable Lipids
Undesired impurities in vaccines have been attributed to ionizable lipids, which are used to facilitate mRNA’s entry into cells.1,3 Ionizable lipids, however, are susceptible to oxidation.2 The specific culprits in this case are oxidized tertiary amines.1 Degradation of the resultant N-oxide species generates aldehydes that react with mRNA to form lipid-mRNA adducts. Even at a relative abundance in the range of 10-5 (10 ppm), these lipid impurities prevent mRNA from triggering the expression of the encoded protein designed to generate an immune response.
Developers generally prefer LNPs that have been tailored to their specific mRNA molecule and intended application. Consequently, according to Dr. Adam Crowe, Analytical Development Manager at Precision NanoSystems Inc.*, scientists are exploring a vast array of ionizable lipid species with highly variable oxidation susceptibility levels. Not all oxidized species are likely to form covalent bonds with mRNA.1
“Developers need to be able to identify not only the presence, but also the type of oxidation that has taken place within the lipid to decipher the extent of damage it can do to the mRNA molecule in question,” he explains. “That requires advanced analytics.”
Limitations of Existing Analytical Techniques
CGE is the analytical methodology for mRNA integrity determination and is used during early-phase research, process development and manufacturing (quality control for batch release). Oxidized lipid-mRNA adduct impurities are not detected by CGE,1 however, due to the minimal differences in size and weight between such impurities and unmodified mRNA products. Even if 10 lipid adducts are formed on one mRNA molecule, the change in molecular weight of that mRNA, which is on the order of 1,000,000 Daltons (Da), would be just 1000 to 2000 Da.
Nuclear magnetic resonance (NMR) spectroscopy, while capable of elucidating lipid structures, is an expensive and involved analytical technique to collect and interpret, added Crowe. Moreover, it does not have the dyamic range required to detect oxidized lipids with a relative abundance of approximately 10 ppm.
The ideal approach, according to Dr. Crowe, would be to use fragmentation mass spectrometry to determine the type of oxidation in the lipid raw material and its percentage prior to bringing it in contact with the mRNA cargo. Most current MS instruments, however, rely on collision-induced dissociation, which is highly efficient at breaking bonds, but for lipids cannot provide the structural details necessary to identify specific oxidation sites—information needed to determine if a specific lipid has a risk of mRNA adduct formation.
EAD Supports Comprehensive Assessment and Derisks mRNA-LNP Development and Commercialization
EAD, on the other hand, can provide information on the specific oxidation sites for lipids, even when they are at very low abundance. This ability was demonstrated in a study performed by SCIEX in collaboration with Precision NanoSystems Inc. using the ZenoTOF 7600system, which leverages EAD technology and offers more than five orders of linear dynamic range.
Lipid impurities below 0.01% relative abundance of the main lipid species were detected and comprehensively fragmented along the headgroups and fatty acid chains. The obtained fragment information was used to differentiate between various oxidized species, including oxidized tertiary amines that can lead to mRNA adducts and contribute to loss of mRNA function.
MS analysis and EAD is a complementary methodology alongside CGE. CGE remains essential for mRNA integrity analysis from R&D through commercial manufacturing. Many other techniques are also used to characterize the mRNA, lipids and LNPs. EAD rounds out the mRNA analytical toolkit for assessing different quality and safety aspects.
Derisking mRNA Development and Commercialization
It can be highly beneficial to identify potential lipid-mRNA adduct impurities at the earliest development stages for mRNA therapeutics and vaccines formulated as LNPs. EAD provides a means for accurately analyzing a wide range of ionizable lipids and their oxidation products, thereby showing the potential for the formation of mRNA-lipid adducts that would interfere with mRNA function.
EAD can be employed practically from the outset of each mRNA development effort and with a smooth and fast workflow and industry-relevant throughput.
The information obtained from such analyses provides a new baseline for quality assessment of lipids used in LNPs and can be extended to new and proprietary ionizable lipids used for future therapeutics and vaccines. Potential problems can be addressed by modifying the lipid manufacturing process to avoid adduct generation or revising the downstream purification process to ensure its removal. As a result, optimized processes that afford mRNA products with greater efficacy can be realized more quickly and cost-effectively.
References
1. Packer, M., Gyawali, D., Yerabolu, R. et al. A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems. Nat Commun 12, 6777 (2021). https://doi.org/10.1038/s41467-021-26926-0
2. Distinguishing oxidative impurities from ionizable lipids used in LNP formulations using electron activated dissociation SCIEX technical note, RUO-MKT-02-14983-A.
3. Han, X., Zhang, H., Butowska, K. et al. An ionizable lipid toolbox for RNA delivery. Nat Commun 12, 7233 (2021). https://doi.org/10.1038/s41467-021-27493-0
*Precision NanoSystems, Inc. is a SCIEX affiliate through the Danaher network of companies.