mRNA therapies have been impactful and common in our industry with many examples and health authority trends available. FDA manufacturing and regulatory expectations are also better understood through the various inputs in submissions and interactions with the Agency, as well as guidance published.

Our research tackles a key issue in RNA biology: predicting RNA secondary structures accurately, especially for long RNAs like mRNAs and ncRNAs. We developed eFold, a novel deep learning approach trained on our expansive RNAndria database, featuring over 2,500 complex RNA structures. eFold shows superior accuracy in predicting long RNA structures, marking a substantial advancement in RNA biology and computational modeling.

In the rapidly evolving landscape of mRNA-based vaccines and therapeutics, the demand for robust quality control (QC) measures and advanced analytical capabilities is key. This presentation will discuss our strategy in overcoming some of the QC challenges and strengthening analytical capabilities in support of mRNA Drug Substance and Lipid Nanoparticle product and process development. It will delve into the development of some of the traditional analytics used within Quality Control as well as emerging trends in analytical techniques for more in-depth characterization to ensure safety, efficacy, and quality of our products. The goal is to share insights and to foster open dialogue to collectively advance within the field and ultimately benefit global public health.

The biophysical properties of mRNA such as size, diffusivity, density, and elasticity is not well understood. This in-depth knowledge of these properties is important to optimize purification processes in a rational way. An overview of the current information of these properties are provided and how they can be used to design chromatography and filtration processes.

Current RNA manufacturing generates dsRNA impurities that must be removed, along with enzyme(s) and DNA, in purification. Functional co-immobilization of enzyme and DNA to a solid support prevents the formation of dsRNA, and eliminates costly purification. This allows a single-use chip, continuous flow reactor for a single-path workflow from NTPs to highly pure RNA of any length. New analytics allow for real-time quality and yield optimizations in long continuous production runs at all scales.

The adoption of mRNA for novel vaccines and therapeutics has presented new CMC challenges for drug developers. A successful mRNA drug candidate requires a manufacturing process that is robust, well-controlled, and cost-effective. Importantly, developers must also consider the specific requirements of their drug product indication, from global vaccines to gene therapies. This presentation highlights important strategies, tools, and examples to accelerate mRNA process development from research through regulatory submission.

In recent years, lipid nanoparticle-based delivery of mRNA therapeutics in infectious diseases has shown great clinical and commercial success. However, in contrast to protein therapeutics, mRNA-LNP drug product manufacturing involves complex steps that pose a number of challenges in building a robust process. Hence, it is critical to develop a good understanding of mRNA-LNP drug product unit operations to ensure a successful manufacturing scale-up and tech transfer. The purpose of this talk is to inform on process considerations to develop high-quality mRNA-LNP drug products.

We developed a multiclade VLP-forming HIV-1 env-gag mRNA vaccine that encompasses sequential immunizations with germline bNAb-engaging Envs followed by repeated heterologous Env boosts. The platform was further optimized by inclusion of the viral protease (pro) to yield mature VLPs. Preclinical studies in macaques documented efficient early priming with the recruitment of bNAb precursors against the CD4-binding site and, eventually, elicitation of heterologous tier-2 neutralization and protection from heterologous SHIV challenge.

Double-stranded (dsRNA) RNA impurities pose a significant challenge in mRNA-based therapeutics due to their potential immunogenicity and off-target effects. Droplet Digital PCR has emerged as a sensitive and precise tool for the detection and quantification of nucleic acid impurities. In this study, we present a robust ddPCR-based method for the detection of dsRNA impurities in mRNA drug substance samples. The process involves cDNA generation using target-specific RT primer, followed by ddpcr. The antisense RNA in the region of interest is amplified using a tagged RT primer that ensures specificity towards the antisense strand only. 

With several FDA-approved products and >1600 ongoing clinical trials in the USA, gene therapy has emerged as a modality to challenge a wide variety of diseases. Consequently, CMC has become front-and-center to the development of gene therapies. This talk covers key elements for developing phase-appropriate CMC strategies for gene therapies, including the critical role of early FDA interactions in developing a robust reg-CMC strategy, keeping up with evolving regulatory guidance, and balancing resources and timelines.

Critical Quality Attributes (CQAs) are crucial in the development and manufacturing of biotherapeutics and vaccines, including biologics and mRNA-LNP formulations. The strategies and analytical tools for assessing CQAs differ between mRNA as a drug substance and traditional biologics. Timely and precise assessment of CQAs for mRNA-LNPs is essential for identifying potential CMC issues and mitigating the risk of delays. This presentation will also explore a case study on the impact of supercoiled DNA on mRNA CQAs and highlight the importance of establishing percentage supercoiled DNA as part of In-Process Control (IPC) measures.

The emergence of mRNA technology as a modality for treatment viruses, infectious disease, and various therapies has ushered a new era of medicine. As the regulatory landscape continues to evolve to ensure mRNA-based products are of high quality, safe and effective. To meet these requirements, suitable testing methodologies are needed to properly measure critical quality attributes such as identity, content, purity and functionality. As such, we will discuss new and enhanced analytical trends for the characterization of mRNA.

Sequencing technologies combined with computational tools can enhance our understanding of nucleic acid therapeutics. This talk will provide an overview of these methods that we have developed for heightened characterization of mRNA.

Introducing Sail Biomedicines' platform and discussing various methods for the analysis of circular RNAs. The presentation will identify key challenges in the analytical development of high-quality Endless RNA (eRNA). The discussion will focus on purity evaluation of circular RNAs and a novel AEX-HPLC analytical method used for eRNA.

The presentation will outline critical quality attributes of mRNA lipid nanoparticle (LNP) drug products, with a focus on early clinical phases. It will also address potential impurities and degradation pathways pertinent to mRNA LNP formulations. A selection of key analytical methods essential for quality control and analytical characterization will be showcased. 

• Quality attributes for mRNA LNP products will be outlined
• Impurities/degradation pathways of mRNA LNP products to consider
• A snapshot of some key analytical methods?