BioProcessing Network 2023

At-Line Analytical Chromatography for mRNA Process Development: From pDNA to LNPs

Speaker: Blaz Bakalar, Sartorius BIA Separations
Abstract:

Production of mRNA via the in vitro transcription (IVT) reaction has become a widespread approach in both academia and industry over the past few years. A key starting point of the IVT reaction is the DNA template, usually in the form of a linearized plasmid DNA, from which mRNA is produced. 
Processing pDNA from E. coli paste to a linearized template involves five or more process steps, each with specific considerations. At-line analytics using the CIMac pDNA monolith column provide precise information about pDNA purity, quantity, isoform percentage, as well as, effectiveness of removal of impurities such as RNA. Using this information, individual process steps are optimized to achieve desired pDNA purity and yield in the subsequent IVT reaction.
In the IVT reaction, NTPs and capping reagent are the building blocks that are consumed during polymerization which produces a single stranded mRNA according to the pDNA template sequence. PrimaS analytical column enables separation and quantification of these nucleic acid species in less than three minutes.
Using such multimodal column technology within the PATfix analytical platform enables determination of reaction kinetics, identification of bottlenecks, and rapid optimization of the IVT process parameters, resulting in higher mRNA yields and lower mRNA production costs.
Lipid nanoparticle (LNP) encapsulation is the final step in the production of the mRNA drug product. We present a new approach to monitor mRNA encapsulation efficiency, a critical encapsulation process parameter. In contrast to existing methods, our new approach does not require extensive sample preparation or usage of dyes.

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