Quality by Design in mRNA Manufacturing

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Speakers: Rok Sekirnik, Head Process Development for mRNA and pDNA, Sartorius BIA Separations

Date and Time: Thursday, September 26, 2024 | 10am EDT (NA) / 3pm BST (UK) / 4pm CEST (EU-Central)

Title: Quality by Design in mRNA Manufacturing

Abstract:

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.

Who Should Attend?
This webinar will appeal to Scientists/Managers in the following fields or those working in pharma, biopharma, biotech, CDMO companies or academia:

• Purification
• Downstream processing
• Scientist/Manager in PD
• mRNA purification
• Manager MSAT
• Director of Scientific Innovation

What You Will Learn
Attendees will learn about:

• Scalable mRNA Production: The mRNA workflow scales from micrograms to multi-grams, supported by rapid HPLC monitoring
• Optimization via Design-of-Experiment: The IVT reaction is optimized using a design-of-experiment approach, improving yield and minimizing dsRNA formation
• Efficient Purification: High-throughput chromatography tools for microgram-scale purification effectively remove IVT components and dsRNA
• Clinical Impact: Reducing innate immune response is critical for the clinical efficacy of mRNA therapies