2nd Annual mRNA Conference Europe 2025

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Speaker: Rok Sekirnik, Head Process Development for mRNA and pDNA, Sartorius BIA Separations
Time and Date: Tuesday June 17, 2025 | 12:00 PM
Title: Quality by Design in mRNA Manufacturing – At-Line Monitoring Coupled With DOE for Higher IVT Yield and Efficiency of Purification

Abstract:

mRNA is produced through a cell-free in vitro transcription (IVT) process, where RNA polymerase catalyzes the formation of mRNA guided by a DNA template. This production can scale from micrograms to multi-grams, with rapid at-line monitoring of nucleoside triphosphates (NTPs) consumption and mRNA production. This allows for quick adjustments to IVT reaction parameters.

Due to the catalytic, multicomponent nature of the IVT reaction, optimization is a multi-factorial problem, ideally suited to design-of-experiment approach for optimization and identification of design space. A data-driven model of process yield (in g/L), including impact of nucleoside triphosphate (NTP) concentration and Mg:NTP ratio on reaction yield can be derived to optimize reaction cost drivers (e.g. RNA polymerase and DNA template), while minimizing dsRNA formation, a critical quality attribute in mRNA products. The yield of the IVT reaction can reach 25 g/L in batch.

A high-throughput purification train optimization is performed by coupling multiparallel (96 well) and spin-based purification devices at mg-scale with Design-of-Experiment methodology. mRNA purification is achieved with affinity chromatography selective for polyadenylated mRNA (Oligo dT) coupled with reverse-phase chromatography used to remove IVT components (NTPs, DNA, T7), and IVT by-products, in particular dsRNA, a major immunogenic impurity which activates dsRNA-dependent enzymes and leads to inhibition of protein synthesis. Elimination of dsRNA improves translation and minimizes the activation of innate immune response. In the advent of personalized, mRNA-based therapies, such as neoantigen mRNA vaccines, which require multiple milligram administrations, minimization of innate immune response may be critical to clinical success of mRNA therapeutics.

High-level takeaways:

• Present novel concept of at-line analytics to monitor IVT reaction to produce mRNA
• Demonstrate how coupling PAT with DOE increases yield of IVT up to 25 g/L
• Advanced use of affinity chromatography for isolation of mRNA with concomitant dsRNA removal
• Demonstrate removal of dsRNA using classical (reverse-phase) and novel (aqueous) conditions

Location:  Berlin, Germany

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