Two quality and stability indicating imaged CIEF methods for mRNA vaccines

Abstract

Two imaged capillary isoelectric focusing methods were developed to provide insight into the quality and stability of messenger ribonucleic acid (mRNA) vaccines, specifically, mRNA encapsulated in lipid nanoparticles (LNPs). A variety of stressed and lipid composition‐modified samples were measured and detected by their UV absorption. The results were supported by the data of an encapsulation assay and particle sizing. One method, using 9 M urea as an additive, shows two broad and jagged peaks in which the peak shape offers detailed information. The summed peak area of both peaks showed RSDs from 2% to 8% when one batch was measured in triplicate and apparently depends on the size of the LNPs. In the second method, a combination of 5.5 M urea and 2 M N‐ethylurea was used. This method is characterized by a high repeatability of the isoelectric point (pI, <0.5%). The repeatable peak area (RSD of 2%–7%) correlates linearly with the mRNA content, which also applies to the first method, and added stress is evident by the change in pI and peak area. Furthermore, experiments with the addition of a fluorescent dye were performed (fluorescence detection), which tremendously increased the sensitivity of the methods. Both methods can be used to characterize the stability of mRNA‐loaded LNPs, for example, when investigating various storage times at different temperatures and freeze–thaw cycles, as well as the ability of the methods to distinguish lipid compositions and measure batch‐to‐batch variability.

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“…A 24-h temperature stress demonstrated this in the range of 8-60 • C. This demonstrates the usefulness of iCIEF, especially for the stability analysis and sample characterisation of mRNA LNPs [148]. Using this publication, Krebs et al [149] developed the iCIEF method further to adjust it to another mRNA vaccine for stability and quality investigations. They settled for two slightly different methods, in which method A is better for stability information and may possibly show different LNPs or LNPs with different mRNA loading.…”

“…Using this publication, Krebs et al. [149] developed the iCIEF method further to adjust it to another mRNA vaccine for stability and quality investigations. They settled for two slightly different methods, in which method A is better for stability information and may possibly show different LNPs or LNPs with different mRNA loading.…”

“…Method B, on the other hand, enables high reproducibility p I measurement (<0.5%), and the peak area also correlates well with the mRNA loading, even in stressed samples. In addition, the peak area here is less influenced by particle size than method A [149].…”

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“…Exemplary applications include, but are not limited to, monoclonal antibodies [192,[209][210][211][212][213][214][215], bispecific antibodies [216][217][218], ADCs [219,220], PEGylated proteins [221,222], glycoproteins, in particular erythropoietin [213,223,224], and fusion proteins [213,225]. Because this subsection is part of the former "proteins" section, which was extended to all biopharmaceuticals, it should not go unmentioned that the range of applications has been expanded beyond proteins and includes oligosaccharides [226], LNPs and related mRNA vaccines [227][228][229][230], virus-like particles [231,232], poliovirus [230,233], adeno-associated virus [230,[234][235][236][237], papillomavirus [238], and bacteria and yeast cells [230]. Figure 4 shows a very exemplary iCIEF electropherogram of the hACE2/His receptor, which provides a clear representation of the signals of the individual isoforms of a protein.…”

“…However, several other substances have proven their usefulness. The list includes (without claim to completeness): ethylurea [229,269,270], formamide [271], dimethyl sulfoxide [272], sulfobetaines [273,274], glycerol [275], taurine [273,274], sucrose [273,276], sorbitol [273], and polysorbate 20 [267,270,277].…”