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Techniques & Tools Capillary Electrophoresis, Mass Spectrometry, Spectroscopy

Overcoming Oxidation

Adam Crowe

Despite promising preliminary studies, novel therapeutics and vaccines based on genetic material fail more often than we’d like. Modalities can alter their mode of action, safety, and efficacy during production and storage. Unfortunately, these modalities often go unnoticed in the absence of analytical techniques that provide detailed structural information. The consequences of this bottleneck are even more evident in gene-based products, where even a trace amount of impurity can affect the efficacy of the oligonucleotide.

Leading pharmaceutical companies – including Moderna and Pfizer/BioNTech – have used lipid nanoparticles (LNPs) as messenger-RNA (mRNA) carriers for COVID-19 vaccines. There are several advantages to LNPs, such as biocompatibility, high cellular uptake, permeability, enhanced protection of the drug, and relative ease of mass production (1). However, LNP components can also interact with mRNA and reduce its activity. These interactions are typically undetectable by conventional techniques used to assess mRNA purity.

Some impurities originate from ionizable lipids – one of the main components of all LNP-based therapeutics and vaccines. Pharmaceutical companies use ionizable LNPs thanks to chemical properties that help deliver genetic cargo and facilitate entry into cells. However, ionizable lipids are also susceptible to chemical alterations – predominantly oxidation. Site-specific oxidation can lead to the formation of reactive lipid species, generating mRNA-lipid adducts (2). Notably, even a small amount of oxidized lipids is enough to prevent proper mRNA function. In other words, the mRNA cannot trigger the expression of the encoded protein that prepares our immune response ahead of infection.

Capillary gel electrophoresis (CGE) is currently at the heart of mRNA analysis, measuring the integrity of mRNA throughout the R&D and manufacturing process. CGE analysis can help identify production steps that cause mRNA degradation, and it is also a common validated assay for quality control, ahead of batch release. Despite its strengths, CGE is unable to identify small amounts of impurities that do not significantly alter the size-to-charge ratio of mRNA, which means that mRNA-lipid adduct formation may go undetected.

Although quantifying impurities in mRNA is crucial, it does not guarantee its functionality. Rather than seeking mRNA-lipid adduct impurities in the final product, it would be better to assess the quality of the lipid starting material ahead of manufacture to detect the presence of oxidized lipids. This would be more efficient than trying to reverse the mRNA-lipid adduct formation process to get to the root cause, as that will take significant time and resources. Therefore, more efficient alternatives are needed to assess the quality of the lipid raw material before it is formulated with the mRNA.

Kirsten Pohl

Nuclear magnetic resonance (NMR) spectroscopy can be used to determine lipid molecular structure, but instrumentation is expensive and requires large amounts of very pure samples – not ideal for the detection of very low levels of impurities.

Mass spectrometry (MS) can be used for the elucidation of structure via fragmentation, typically using collision-induced dissociation (CID). Although CID has high efficiency in breaking different types of bonds, it cannot differentiate between oxidations occurring at different parts of the lipid with certainty. In the case of mRNA, such differentiation is vital – some species can cause the loss of mRNA function through adduct formation whereas other may not. And that’s why lipid and LNP providers – and pharmaceutical companies – are keen to explore MS methods with sufficient elucidation power and dynamic range to distinguish different types of oxidation in lipids at very low levels.

Enter electron-activated dissociation (EAD)! EAD is a newly emerging fragmentation mode that can help fill the gaps in LNP structural information. Specifically, it can elucidate structural information for lipids in very low abundance, revealing exact oxidation sites. A recent collaboration between Sciex and Precision NanoSystems revealed that EAD is able to comprehensively fragment lipids along the headgroup and fatty acid chains, enabling differentiation between several oxidized lipids. Most importantly, those oxidized lipids attributed to the loss of mRNA function could be clearly identified and quantified.

EAD has the potential to pave the way for accelerated and accurate analyses of lipids and LNPs to enhance LNP-based mRNA vaccines and therapeutics. Once LNP manufacturers locate and quantitate lipid oxidation based on EAD-derived data, they can deduce its potential harm, optimize lipid synthesis workflows and storage conditions, or conduct further purification.

That said, though EAD has demonstrated promising results, it should not replace but rather complement other methods in the mRNA-LNP analysis workflow; for example, CGE is still crucial for monitoring mRNA integrity from the unformulated phase through to the final product. Indeed, given the complexity of mRNA-LNP products, we need multilayered analyses to obtain all the data needed to cover the various important aspects of both raw materials and the formulated product. In short, we should not hesitate in using the most comprehensive approach to reduce risk, optimize efficacy, and decrease time to patient.

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  1. N Dhiman et al., “Lipid nanoparticles as carriers for bioactive delivery,” Frontiers in Chemistry, 9, 580118 (2021). DOI: 10.3389/fchem.2021.580118
  2.  M Packer et al., “A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems,” Nature Communications, 12, 1 (2021). DOI: 10.1038/s41467-021-26926-0
About the Authors
Adam Crowe

Kerstin Pohl

Senior Manager of Cell & Gene Therapy and Nucleic Acids at SCIEX, Framingham, US.

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