Size exclusion chromatography used for characterizing large mRNA and LNP aggregates
Messenger RNA (mRNA) lipid nanoparticle (LNP) vaccines have successfully curbed the COVID-19 pandemic. However, the potential effects of mRNA and LNP aggregates on drug safety and efficacy remain unclear, highlighting the need for new technologies to accurately characterize these types of impurities. A recent Analytical Chemistry publication describes the development of an ultrawide pore size exclusion chromatography (SEC) column for determining different size variants of large nucleic acid and LNP aggregates. Using this method, the authors show the aggregate content of a model mRNA to vary considerably between manufacturers, as well as reveal differences in the types of aggregates that are formed, both of which are key considerations when developing mRNA-LNP therapeutics. Importantly, this study represents the first reported use of SEC for mRNA and LNP aggregate characterization.
Existing technologies have limited utility for mRNA-LNP characterization
Current methods for assessing mRNA-LNP therapeutics include capillary gel electrophoresis (CGE) for determining mRNA integrity, and dynamic light scattering (DLS) for monitoring LNP size and polydispersity. A limitation of CGE is that it cannot detect noncovalent aggregates, which dissociate under the harsh denaturing conditions required, while DLS is unable to accurately resolve bimodal populations. Critically, neither of these methods looks specifically at LNP-mRNA aggregation assemblies, making it impossible to tell how these might impact drug safety and efficacy.
Construction of an ultrawide pore SEC column
The SEC column used for mRNA and mRNA-LNP characterization was developed by silanizing 100% silica particles to minimize non-specific binding. Key physicochemical particle attributes were then determined, including the pore size (1275 Å), pore volume (0.66 mL/g), and average particle diameter (2.47 μm), as well as the degree of silanization. Next, the particles were packed into a low adsorption column designed to minimize electrostatic interactions and hydrophobicity, prior to SEC analysis.
Characterization of mRNA aggregates
The ultrawide pore SEC column was initially used to compare EGFP mRNAs (980 – 996 nucleotides) from two different manufacturers, TriLink and GenScript. Both mRNAs were modified with 5-methoxyuridine residues, a 3′ poly(A) tail, and a 5′ Cap1 structure, of which the latter was added to the TriLink mRNA using our proprietary CleanCap®, mRNA capping technology. The resultant SEC-UV profiles showed the TriLink EGFP mRNA to have a significantly increased absolute peak area corresponding to intact mRNA elution, as well as fewer aggregates (17.8% vs 59.7%) and nucleotide fragments compared to the GenScript product.
Next, the nature of the aggregates was investigated by heating each of the EGFP mRNAs at 70oC for 2 minutes before performing the SEC analysis. While the intact mRNA peak area remained similar for the TriLink mRNA, a 3.3-fold increase was observed for the GenScript mRNA accompanied by a significant reduction of aggregates (59.7% to 4.1%). These data suggest the GenScript mRNA aggregates to be predominantly noncovalent in nature and demonstrate the value of using a nondenaturing method for aggregate quantification.
Resolution of free mRNA from mRNA-LNP samples
To explore the utility of the ultrawide pore SEC column for characterizing mRNA-LNP samples, TriLink’s CleanCap® Cre mRNA (1350 nucleotides) was encapsulated in a series of LNP formulations, including several fragment antigen-binding (Fab)-guided LNPs, and subjected to SEC analysis. Key findings included the observation that the ultrawide pore SEC columns were capable of resolving free mRNA from intact LNPs and the discovery that the Fab-functionalized mRNA-LNPs had a higher tendency to aggregate. In combination, these data show ultrawide pore SEC as an important enabling technology for determining multiple critical attributes of mRNAs and complex mRNA-LNP samples.
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Article reference: Goyon A, Tang S, Fekete S et al. Separation of Plasmid DNA Topological Forms, Messenger RNA, and Lipid Nanoparticle Aggregates Using an Ultrawide Pore Size Exclusion Chromatography Column, Anal. Chem. 2023, https://doi.org/10.1021/acs.analchem.3c02944