PATfix with CIMac PrimaS method tracks real-time NTP consumption in mRNA production every 15 minutes
The device is straightforward with ready-to-use methods and SOPs, allowing immediate sample measurement, says Michael Daume
WACKER Biotech is part of WACKER, one of the largest chemical companies in Germany. As part of the Biosolutions division, WACKER Biotech is an end-to-end mRNA CDMO expert with a 20-year experience and multiple EMA- and FDA-approved sites. They offer a full suite of services starting at the gene level, covering pDNA and mRNA and formulation into lipid nanoparticles (LNPs). The production services range from live microbials to classical vaccines, therapeutic proteins, and products used in advanced therapies, including pDNA, mRNA and LNPs. With an R&D department (Munich) and manufacturing sites (Amsterdam, San Diego & Halle) dedicated to nucleic acids, WACKER is able to deliver R&D-grade and GMP-grade materials, contributing to Germany’s pandemic preparedness program.
Meet the Expert: Michael Daume
Michael Daume leads Downstream Process Development for Nucleic Acids at WACKER R&D, focusing on pDNA and RNA purification. After earning a PhD in Small RNA Biology in 2018, he returned to nucleic acid research at WACKER in 2023, where he drives innovation in downstream processing. Michael’s expertise in chromatography is central to advancing WACKER’s R&D efforts in nucleic acids, and he’s passionate about sharing analytical data from mRNA process development.
WACKER deploys a wide array of analytical methods in their processes, following the guidelines from US Pharmacopeia. Along these methods, they introduced PATfix for fast, real-time insights into production and purification. They were able to obtain great results in just a few months: “The device is really straightforward. It comes with fully developed methods and foolproof SOPs that really minimize your developmental activities, so you can basically start measuring your samples right away,” added Michael Daume.
Accurate Analysis of Template pDNA Linearization Using PATfix
Quantifying linear and circular pDNA can be challenging with conventional methods like AGE, often leading to errors. PATfix pDNA analytics, however, streamlines this process using the CIMac pDNA method for precise analysis. It separates open circular (OC), supercoiled (SC), and linear pDNA isoforms, making it easy to quantify the amount of linearization within the reactions. WACKER Biotech successfully achieved 100% linearized pDNA using this method. Remarkably fast, PATfix delivers initial results in just 10 minutes, making it ideal for at-line analytics, providing feedback on linearization status, and lowering production costs and times significantly.
Optimizing mRNA Analytics with PATfix
- In-Depth Insights into IVT Reactions Using PATfix
WACKER utilized the CIMac PrimaS method to track their in vitro transcription (IVT) reactions in real-time, offering precise monitoring of nucleotide triphosphates (NTPs) in Cas9 and Fluc mRNA production. By analyzing samples every 15 minutes, they observed how NTPs were consumed over time as they were incorporated into the growing mRNA strands. This method proved to be even faster than the CIMac pDNA method.
They have observed some differences in Cas9 and Fluc mRNA production. After 105 minutes, Cas9 mRNA production stopped due to ATP depletion, making the PATfix method ideal for fed-batch processes where replenishing ATP could extend production. In contrast, Fluc mRNA production continued, supported by a steady presence of all NTPs. This highlights the importance of understanding the IVT reaction dynamics, as PATfix allows for easy adaptation and optimization for specific constructs.
- Monitoring Oligo dT Purification via CIMac PrimaS
Using the CIMac PrimaS method, WACKER closely monitors the oligo dT purification process during DSP steps. While AGE and CGE methods give a basic overview of mRNA quality, they don’t capture small components like NTPs. With the PrimaS chromatogram, WACKER identifies remaining NTPs in the IVT load, observes their removal during washing, and ensures the final elution contains pure mRNA free from contaminants, ensuring optimized purification.
- Integrity Analysis of mRNA Using CIMac SDVB
Integrity analysis of mRNA using CIMac SDVB versus CGE methods highlights the strengths of both techniques. CIMac SDVB chromatography, an ion-pair reversed-phase method, effectively separates RNA by size, such as the High Range RNA Ladder (200–6,000 nucleotides), and differentiates various mRNAs like Fluc, eGFP, and Cas9. However, for precise size determination, capillary gel electrophoresis (CGE) provides more accurate results. A study for integrity analysis and comparing the SDVB method versus CGE method showed that results aligned very well for Fluc and eGFP mRNA, with Cas9 mRNA showing an acceptable 8% variation. This variation is consistent with observations mentioned in CGTI paper, published by Sartorius BIA Separations.
Summary
WACKER Biotech employs advanced methods like CIMac pDNA and PrimaS to streamline mRNA production. These techniques enable rapid analysis of plasmid forms and real-time monitoring of nucleotide consumption, optimizing production efficiency and reducing costs. The PrimaS method also ensures the purity of mRNA by effectively removing contaminants. For mRNA integrity, CIMac SDVB provides reliable size separation, complementing traditional methods. With extensive experience, WACKER Biotech delivers high-quality nucleic acids for advanced therapeutics.
Watch how WACKER develops novel manufacturing technologies and processes for both RNA and DNA manufacturing.
