Characterizing oligonucleotide aggregates is increasingly necessary as large nucleic acids and lipid nanoparticles (LNP) move beyond the lab and into production. Unfortunately, existing technologies have limitations that have prevented the analysis of ultralarge size variants.

For example, several studies report that anion exchange chromatography (AEC) underestimated open-circular impurities in plasmid DNA (pDNA) samples. Likewise, for lipid nanoparticles, dynamic light scattering that measures particle size and polydispersity is not well suited for the determination of aggregates.

Ultrawide pore size exclusion chromatography (SEC) enables the characterization of ultra large nucleic acids and delivery system size variants. “The early implementation of this tool in the process could help identify the root cause formation of the aggregates and offer early mitigation opportunities,” Alexandre Goyon, PhD, principal scientist, Genentech, tells GEN.

New tool in the toolbox

Using a prototype column developed by Waters, for perhaps the first time, “plasmid DNA topological forms and multimers were separated by analytical SEC,” Goyon and colleagues wrote in a recent paper. The SEC column packed with ultrawide pore particles (1300A) helped to reveal aggregation in mRNA and protein-guided mRNA-LNP samples.

The scientists found large differences in aggregates for mRNA samples manufactured by different processes with content ranging from 17.8% to 59.7%. Thermally stressing the mRNA lowered the percentage of aggregates from 59.7% to 4.1%, suggesting that they were mostly noncovalent in nature.

Next, the scientists compared degradation trends of pDNA using AEC and SEC. The study reports “a multistep degradation process that includes the formation of open-circular forms, large multimers, and insoluble species and fragments.” Using only one characterization method would have caused those findings to have remained ambiguous, the scientists note.

The characterization of ultralarge size variants could inform on product efficacy and safety, Goyon posits. “Depending on the use of the nucleic acid modality and effect of ultra large impurities on the functional activity, aggregates often referred to as multimers may need to be controlled.

Applications include the verification of mRNA quality among batches and manufacturing processes. “While this work focused on aggregates and topological forms, additional impurities could be separated, such as host proteins, RNA, and DNA,” he says.

Ultimately, Goyon says, “Technologies enabling the sequencing of the impurities could provide insights leading to the improvement of the manufacturing process.”


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