The talk will present case studies of leveraging the power of at-line HPLC monitoring for process development to increase purity and yield of mRNA production. Approaches to optimize plasmid purification and linearization, IVT reaction and mRNA purifi cation will be described, including purification methods to achieve >95% supercoiled plasmid purity, optimize linearization reaction, and increase of IVT reaction yield from 3-5 mg/mL to 10-12 mg/mL. Further, use of at-line anayltics to monitor automated, continuous production in single use bioreactors to produce multi-gram amounts of mRNA will be demonstrated. Oligo dT-based affinity purification will be described with approaches to optimize dynamic binding capacity to reach up to 6 g/L. Multimodal chromatography and reverse-phase chromatography cases will be discussed for capture of non-polyadenylated RNA, and removal of dsRNA.

Speaker:

Rok Sekirnik
Head Process Development mRNA|pDNA
Sartorius BIA Separations

Immuno-oncology is a rapidly growing area for therapeutic development. With the adoption of various autologous cell therapies and an increased focus on more broadly applicable allogeneic treatments and multi-specific therapies, the complexity of these development activities is also increasing. With this drive to increased complexity and the inherent risks associated with potential failure in Phase 1 clinical trials, it is critical to have a solid scientific foundation for the development of these next-generation immunotherapies. This talk will focus on how to overcome the limitations associated with traditional viral vector based recombinant TCR gene expression to build hundreds of candidates a week, resulting in improved confidence in screening and lead optimization. With the direct use of mRNA to drive transient modulation of cellular phenotypes, potential candidates can be screened quickly and cost-effectively. Come and learn how immuno-oncology researchers are automating synthetic biology on-site to unlock speed and efficiency in adoptive T-cell therapies and vaccine discovery workflows.

Speaker:

David Weiss
Senior Global Product Manager
Telesis Bio

Circular RNA is a type of single-stranded RNA with a continuous loop closed by covalent bonds, which confers resistance to exonuclease-mediated degradation. Currently, available methods have limitations in circularization efficiency, particularly the large size of the target RNA and contamination with linear RNA, which causes immunogenicity and consequently reduces target protein expression. We have developed a novel method to produce circular RNAs with stable protein expression using ribozymes and RNA ligases. This approach has significantly higher circularization efficiency compared to Group I permuted intron-exon (PIE) systems, especially for long genes. This high circularization efficiency allows for the production of high yields of circular RNA, which potentially reduces the time and cost of producing circular RNA-based medicines. Using firefly luciferase, we observed that the activity of the protein in the circular RNA was 45-fold and 7.6-fold higher than that of the mRNA ex vivo and in vivo, respectively. We have demonstrated the therapeutic potential of circular RNA through neoantigen cancer vaccines and type I diabetes. Taken together, these results suggest that the circulating RNA generated by the new approach could be applied to treat a variety of diseases, including cancer, autoimmune and degenerative diseases. In conclusion, Levatio’s circular RNA platform technology could lead the way for the next generation of RNA-based drug development.

Speaker:

Hyun-Bae Jie
CEO
Levatio Therapeutics

The RNA platform technology has emerged as one of the most promising and strategic technologies for rapid global vaccination, infectious disease control, biotherapeutic development and preparedness for future healthcare challenges. A form of production process template such as using and computationally processing disease agnostic-prior knowledge, based on the platform nature of both the RNA vaccine manufacturing process and of the QbDD framework. Audience members will learn about:

• Next generation continuous mRNA manufacturing processes
• Development of analytical methods for quantification of yield e.g., NTP uptake, RNA integrity and others
• Production process modelling
• Techo-economic analysis

Speaker:

Zoltan Kis
Assistant Professor, Biotechnologist & Bioprocess Engineer
University of Sheffield

• mRNA background and anatomy
• Raw material quality and their impact on producing mRNA
• Analytical consideration for analysis and characterization of mRNA

Speaker:

Khaled Yamout
Senior Director, Analytical Services and Quality Control
TriLink BioTechnologies

Development approaches for a complex drug product such as mRNA/LNP formulations will be outlined. The control strategies and the quality attributes for release and stability testing of mRNA lipid nanoparticle drug products will be summarized. Furthermore, delving into the analytical challenges, opportunities and strategies on how to leverage platform knowledge during development phases will be discussed. In addition, the main impurities and degradation pathways to consider will be described as well as showcasing key methods for mRNA LNP analysis.

Speaker:

Gunilla Nilsson
Associate Director, Advanced Drug Delivery
AstraZeneca