Cell 2025

Join our workshop on pDNA, mRNA and LNPs

Speakers:

• Klemen Božič, Scientist, Process Development mRNA | pDNA
• Tjaša Leban, Associate Scientist

Date and Time: Monday, November 10, 2025 at 3:00 PM
Location: London, UK

Register Now!

Part 1: Fast and Robust Plasmid DNA Purification Process to Support Advanced Therapy development

Plasmid DNA is a key raw material in advanced therapies, particularly for viral gene therapies and mRNA-based therapies. With a rapid increase in clinical trials approvals of new advanced therapies, the demand for pDNA is rising significantly, necessitating efficient, reliable, and cost-effective production processes. Plasmid DNA production faces a challenge in preserving the integrity of supercoiled isoform and achieving high recovery rates from alkaline lysis through to final filtration. We will present a purification process that achieves >98% supercoiled isoform and >64 % overall DSP process recovery and will cover continuous alkaline lysis, clarification and chromatography. Selective hydrophobic interaction chromatography (SHIC) approach to remove residual impurities and enrich supercoiled pDNA will be presented. A key feature of process development approach is the use of PATfix® pDNA analytical platform which enables rapid feedback loop used to control each step of the process.

Part 2: Advanced Analytics Unlocks Hidden Opportunities to Increase Yield of mRNA Manufacturing

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. IVT 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 ug-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.

Part 3: Chromatography Purification Combined With Innovative Analytics of LNP-Based Biopharmaceuticals

Lipid nanoparticle (LNP)-based biopharmaceuticals are transforming nucleic acid delivery technologies but face manufacturing and characterization challenges. Innovative solutions to improve drug product integrity, recovery rates, and characterization are being introduced. A new purification method for encapsulating purified RNA into LNPs replaces traditional tangential flow filtration (TFF) and dialysis techniques. This process effectively removes ethanol and unencapsulated RNA, resulting in higher recoveries, more uniform particles, and increased protein production activity, as measured by a luciferase reporter protein assay. PATfix platforms ensure rigorous product quality monitoring, especially for multi-cargo LNPs in CAR-T, CRISPR, and vaccine applications.