Quality by Design in mRNA Manufacturing

The COVID-19 pandemic triggered an unprecedented surge in development of mRNA-based vaccines and other therapeutics, such as protein replacement therapy and cancer. mRNA is produced by a cell-free process based on in vitro transcription (IVT) reaction, a RNA-polymerase-catalyzed polycondensation of NTPs into a nascent mRNA chain guided by DNA template. mRNA production workflow is adaptable to production from mg to multi-g scale, supported by on rapid at‐line high pressure liquid chromatography (HPLC) monitoring of consumption of nucleoside triphosphates (NTPs) with concomitant production of mRNA, with a sub‐3 min read‐out, allowing for adjustment of IVT reaction parameters with minimal time lag. 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.

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